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Analysis of hematopoietic progenitor cell cycle control in the myeloproliferative disorders 1986

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J ANALYSIS OF HEMATOPOIETIC PROGENITOR CELL CYCLE CONTROL IN THE MYELOPROLIFERATIVE DISORDERS by JOHANNE CASHMAN B.Sc, McGill University, 1975 M.Sc, University of British Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1986 ®Johanne Cashman, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pathology The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date March 12, 1986 DE-6(3/81) i i ABSTRACT The myeloproliferative disorders (MPD) comprise an interesting group of hematological neoplasms in which clonal expansion is initiated at the level of the pluripotent stem cell compartment but differentiation proceeds essentially normally. Although evidence for the involvement of specific genetic changes exist in one of these diseases (chronic myeloid leukemia, CML), the nature of the lesion that permits the progeny of a single stem cell to dominate the mature cell compartment has not been elucidated. Application of clonal assay systems to the study of the MPD has provided information about the numbers, proliferative capacity, physical properties and the responsiveness to regulatory factors of hemopoietic progenitors from a l l cell lineages. However, clonal assays can offer only limited information about the processes that underly stem cell regulation. Some of the limitations imposed by these assays may be overcome by the use of long-term cultures in which primitive and pluripotent progenitors may be maintained for at least 2 months. The purpose of this thesis was to determine if consistent alterations in cell cycle activity were characteristic of progenitors in MPD patients, and to evaluate the potential of the long-term culture system for further investiga- tions of any changes observed. The proliferative behaviour of clonogenic pro- genitors from the blood and bone marrow of a large number of MPD patients was compared with that of normal individuals using the ^H-thymidine cell suicide technique. These experiments showed that a l l progenitor classes in the blood and marrow of patients with CML and polycythemia vera (PV), which in normal individuals are quiescent, had a significant component of cycling cells. In addition, a consistent association of this abnormality in cycling control with expression of erythropoietin (EP)-independence in patients with essential thrombocytosis (ET) was revealed. Further studies were then undertaken to i i i determine if these abnormalites could be reproduced in vitro. Experiments with normal marrow showed that the most primitive progenitor classes located in the adherent fraction of standard long-term cultures undergo cyclic changes of pro- liferative activity with each weekly addition of new growth medium. These stu- dies suggested that the proliferative activity of normal primitive hemopoietic cells may be both positively and negatively regulated by close range interac- tions with marrow stromal elements. In contrast, in similar experiments with long-term cultures established with PV marrow, where maintenance of neoplastic cells could be documented, analogous primitive progenitor cells in the adherent layer failed to return to a quiescent state and remained continuously in cycle. From previous experiments with CML patients, it was already known that Ph1 -positive progenitors usually disappeared rapidly in long-term cultures established with CML marrow. Therefore, as an alternative approach CML peripheral blood cells were seeded onto preestablished normal marrow adherent layers, since preliminary studies had suggested that this would allow sufficient numbers of primitive Phi-positive progenitors to be maintained for cycling studies. Analysis of such cultures together with studies of appropriate normal controls, revealed the same lack of cycling regulation in CML as previously shown for PV. In addition, studies of control cultures showed that in the absence of an adherent layer, normal peripheral blood progenitors cycle continuously, again suggesting that one regulatory function of the adherent layer is to maintain normal progenitors in a quiescent state. These studies demonstrate that consistent abnormalities of cell cycle con- trol characterize the primitive progenitor compartments in MPD patients, and that these abnormalities can be reproduced in vitro. Initial findings with the long-term marrow system suggest that these abnormalities may be due to the in- sensitivity of primitive neoplastic cell types to respond to factors that ar- rest their normal counterparts from further progression through the cell cycle. iv TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES vi LIST OF FIGURES v i i LIST OF ABBREVIATIONS ix ACKNOWLEDGMENTS xi Chapter I THE HEMOPOIETIC SYSTEM 1) Regulation of Hemopoiesis 1 A) Hemopoietic progenitor assays 2 B) Stem cell regulation 15 C) Hemopoietic growth factors 20 2) The Cell Cycle 28 A) Description of the cell cycle 30 B) Factors controlling cell proliferation 33 C) Models of cell regulation 39 D) Principles of the ^H-thymidine cell suicide assay 41 3) Long-Term Bone Marrow Cultures 47 A) Description of the system 49 B) Role of the adherent layer 56 4) The Myeloproliferative Disorders 62 A) Chronic myeloid leukemia 65 B) Polycythemia vera 75 C) Essential thrombocytosis 80 5) Thesis Objective 82 References 85 Chapter II MATERIALS AND METHODS 1) Marrow and Peripheral Blood Preparation 118 2) Hemopoietic Colony Assays 118 3) Long Term Bone Marrow Cultures 121 4) Long Term Peripheral Blood Cultures 123 5) Enzymatic Detachment of Adherent Cells 125 6) The 3H-Thymidine Cell Suicide Assay 126 7) Cytogenetic Methods 128 References 130 V Chapter III ANAYLSIS OF THE PROLIFERATIVE ACTIVITY OF HEMOPOIETIC PROGENITORS IN THE MYELOPROLIFERATIVE DISORDERS 1) Introduction 131 2) Results A) Patients 132 B) ̂ H-thymidine dose response curve 134 C) Cell cycle status of hemopoietic progenitors from marrow and blood 136 D) Time course experiments 143 3) Discussion 143 References 146 Chapter IV REGULATED PROLIFERATION OF PRIMITIVE HEMOPOIETIC PROGENITOR CELLS IN LONG-TERM HUMAN MARROW CULTURES 1) Introduction 148 2) Results A) Patients 149 B) Gellularity and composition of the adherent layer 149 C) ̂ H-thymidine cell suicide assay 151 3) Discussion 156 References 158 Chapter V UNREGULATED PROLIFERATION OF PRIMITIVE HEMOPOIETIC PROGENITORS IN LONG TERM POLYCYTHEMIA VERA MARROW CULTURES 1) Introduction 159 2) Results A) Patients 160 B) Cellularity and composition of PV long-term cultures 160 C) Erythropoietin-independence in long-term PV cultures 162 D) ̂ H-thymidine suicide assay of hemopoietic progenitors from long-term PV cultures 162 3) Discussion 165 References 167 Chapter VI UNREGULATED PROLIFERATION OF PRIMITIVE CML PROGENITORS IN THE PRESENCE OF NORMAL MARROW ADHERENT CELLS 1) Introduction 168 2) Results A) Patients 169 B) Culture of CML marrow on normal marrow adherent layers 169 C) Culture of CML peripheral blood on normal marrow adherent layers 175 D) Culture of normal peripheral blood on normal marrow adherent layers 179 3) Discussion 183 References 186 Chapter VII CONCLUSION AND FUTURE DIRECTIONS 188 vi LIST OF TABLES Page TABLE 1 Percent k i l l for peripheral blood progenitors from normal individuals and patients with myeloproliferative disorders. 137 TABLE 2 Percent k i l l for marrow progenitors from normal individuals and patients with myeloproliferative disorders. 138 TABLE 3 Percent k i l l for peripheral blood progenitors from patients with essential thrombocytosis 139 TABLE 4 Percent k i l l for marrow progenitors from patients with essential thrombocytosis. 140 TABLE 5 The effect of varying temperature and time of specimen storage on the percent k i l l of hemopoietic progenitors. 142 TABLE 6 Effect of different feeding procedures on thymidine suicide values (% kil l ) of primitive hemopoietic progenitors in the adherent layer of normal long-term human marrow cultures. 155 TABLE 7 Thymidine suicide (% kill) of hemopoietic progenitors in PV long-term marrow cultures. 164 TABLE 8 Enhancing effect of feeders on primitive progenitor numbers maintained in long-term CML marrow cultures and assessed after 4 weeks. 173 TABLE 9 Primitive hemopoietic progenitor numbers and their cycling status at week 4 in long-term marrow cultures from an anomalous CML patient whose Ph^-cells were maintained. 174 TABLE 10 Thymidine suicide measurements (X kill) of nonadherent progenitors in CML blood cultures. 177 TABLE 11 Thymidine suicide measurements (% kil l ) of adherent progenitors in CML blood cultures 178 TABLE 12 Cytogenetic analysis of CML peripheral blood cultures with and without feeder layers. 180 TABLE 13 Thymidine suicide measurements (% kill) of hemopoietic progenitors from the adherent fraction of normal blood cultures. 182 v i i LIST OF FIGURES Page FIGURE 1 Photographs of granulocyte colonies grown in methylcellulose culture. As with erythroid colonies the differences in colony size reflects the plating of progenitors at varying stages of differentiation (X80). 7 FIGURE 2 Different sizes of colonies generated in methylcellulose cultures by erythroid progenitors at different stages of differentiation (X80). 9 FIGURE 3 A mixed granulocytic/erythroid colony photographed in a methylcellulose culture after 18 days of incubation. The cell of origin of this colony is termed a CFU-G/E (X120). 12 FIGURE 4 Diagrammatic representation of the hierachy of hemopoietic progenitor compartments currently identified by colony assay procedures. 14 FIGURE 5 Three types of stem cell transitions where regulatory mechanisms may act to influence stem cell behaviour. 17 FIGURE 6 The cell cycle. Terminally differentiated cells are incapable of further division while cells in GQ may be quiescent for varying periods of time before responding to an appropriated stimulus to enter the cell cycle and divide again. 29 FIGURE 7 Schematic illustrating thymidine incorporation into DNA. 43 FIGURE 8 Schematic illustrating the most critical effects of 3 H -thymidine incorporation into DNA. 46 FIGURE 9 In situ staining of a putative cobblestone area in the adherent layer of a normal long term culture at 3 weeks of incubation (May-Grunwald-Giemsa staining, X160). 50 FIGURE 10 Morphological appearance of a PV long term culture adherent layer. These photographs were taken of the same culture dish at varying times after initiation. 52 FIGURE 11 Cytospin preparations stained by the May-Grunwald-Giemsa procedure illustrating the morphological appearance of cells from a normal long term culture 3 weeks after initiation (X400). 55 FIGURE 12 Metaphase chromosomes from a CML patient. In this particular case the Phi chromosome (circled) was formed by the transfer of part of chromosome 22 to chromosome 16. 64 v i i i FIGURE 13 FIGURE 14 FIGURE 15 FIGURE 16 FIGURE 17 FIGURE 18 FIGURE 19 FIGURE 20 FIGURE 21 FIGURE 22 FIGURE 23 FIGURE 24 FIGURE 25 Schematic diagram discribing the methylcellulose assay system for the growth of hemopoietic cells 120 Schematic representation of the 3-layer composition of a long-term culture after 2-3 weeks of incubation. 122 Schematic diagram demonstrating the application of the tritiated thymidine cell suicide assay to the study of hemopoietic cell kinetics. 127 ^H-thymidine dose response curve for erythroid and granulocytic progenitors from normal marrow. 135 The cellularity and progenitor content of the adherent (solid line) and nonadherent (dotted line) fractions of normal long-term marrow cultures assessed at varying incubation times. 150 Thymidine suicide values for primitive BFU-E present in normal long-term marrow cultures assessed at various times after feeding. 152 Thymidine suicide values for CFU-C present in normal long-term marrow cultures assessed at various times after feeding. 153 Total number of nucleated cells, erythroid colony- forming cells and granulocyte progenitors in the adherent and nonadherent fractions of PV marrow cultures assayed at varying times after initiation. 161 Assessment of BFU-E number in the adherent and nonadherent fractions of PV long-term cultures at varying periods of incubation. 163 The adherent layer of a reconstituted CML long-term blood culture 3 weeks after initiation (X160) 171 Comparison of total cell and progenitor content of a long-term CML marrow culture initiated with (solid symbols) or without (open symbols) a pre-established normal marrow feeder. 172 Comparison of total cell and progenitor content of long-term CML PBL cultures initiated with (solid symbols) or without (open symbols) a pre-established normal marrow feeder. 176 Comparison of total cell and progenitor content of long-term normal PBL cultures initiated with (solid symbols) or without (open triangles) a pre-established normal marrow feeder. 181 i x LIST OF ABBREVIATIONS MPD myeloproliferative disorders CML chronic myelogenous leukemia PV polycythemia vera ET essential thrombocytosis Ep erythropoietin, a glycoprotein required for in vitro and in vivo erythropoiesis Phi Philadelphia chromosome CFU-S spleen colony-forming unit, pluripotent stem cells capable of macroscopic spleen colony formation in irradiated mice CFU-C culture colony-forming unit, granulopoietic progenitors that generate colonies of >20 granulocytes and/or macrophages in culture CFU-E erythroid colony-forming unit, relatively late erythropoietic progenitor cells that generate single or double clusters of 8- 50 erythroblasts in culture BFU-E erythroid "burst"-forming unit, erythropoietic progenitors more primitive than CFU-E, that generate colonies containing multiple erythroblast clusters in culture (mature BFU-E: 3-8 clusters, Primitive BFU-E:>8 or >16 clusters) CFU-M megakaryocyte colony-forming unit, progenitors that generate colonies of >2 megakaryocytes CFU-GEMM mixed colony-forming unit, progenitors that generate colonies of granulocytes, erythrocytes, macrophages, and megakaryocytes CSF colony stimulating factor (also referred to as colony stimulating activity, CSA) a family of glycoprotein molecules required for in vitro granulocyte/macrophage colony growth IL-3 interleukin-3, glycoprotein molecule that stimulates a l l types of early clonogenic myloid precursors BPA burst-promoting activity (probably identical to interleukin-3) PGE prostaglandin E PDGF platelet derived growth factor tritium GAG glycosaminoglycans, components of the extracellular matrix G6PD glucose-6-phosphate dehydrogenase DNA deoxyribonucleic acid S-phase DNA synthetic phase of the cell cycle LCM leukocyte-conditioned medium PHA phytohemagglutinin BSA bovine serum albumin FCS fetal calf serum HS horse serum PBL peripheral blood xi ACKNOWLEDGMENTS I would like to express my gratitude: To Dr. Allan Eaves, my research supervisor, for his encouragement and support, and for obtaining the patient material used in this study; To Dr. Connie Eaves, my teacher and mentor, for her constant guidance and remarkable patience, and for her critical evaluation of this thesis; To Dr. Nellie Auersperg, Dr. Don Brooks and Dr. Don Brunette, members of my graduate committee for their interest and advice; To Dr. Dagmar Kalousek and Gloria Shaw for the cytogenetic analysis presented in Chapter VI; To Dr. Gerry Krystal, Dr. Fumio Takei, Dr. Keith Humphries, and the other staff and students of the Terry Fox Laboratory for the practical assistance and many helpful discussions over the years; To Dr. Louis Gaboury, for his assistance with the control experiments presented in Chapter VI; To Don Henkelman, for his continual efforts in helping me deal with the computer, and for the statistical analysis of the data; To Marjorie Hutchison, Dianne Reid and Agnes Leung for expert technical assistance; To Michele Coulombe and Judy Waite for secretarial assistance; And finally, to my husband Fred and my daughter Genevieve for their patience, encouragement and their belief in me. 1 C H A P T E R I THE HEMOPOIETIC SYSTEM 1) REGULATION OF HEMOPOIESIS The hemopoietic system has two main components; 1) a variety of free interstitial elements composed of hemopoietic cells in various stages of maturation and; 2) a fixed stromal compartment which provides the specialized environment necessary for their maintenance and proliferation. Normal hemopoiesis is dependent on the effective interplay between these two components. The mature blood cells - i.e the lymphocytes, granulocytes, monocytes, platelets and red blood cells - a l l possess specialized intracellular machinery that permit expression of their various unique biological functions. Most represent end cells that are incapable of further proliferation and are also short lived in relation to the lifespan of the individual. Thus production of new blood cells is a continuous process. The mature blood cells are maintained by the proliferative activity of less differentiated cells. The most primitive of these that are restricted in their differentiative potential retain a considerable proliferative capacity but appear to possess l i t t l e capacity for self-renewal. Maintainance of their numbers is therefore thought to be dependent upon continual replenishment from a common pluripotent stem cell compartment whose members are capable of extensive self-renewal as well as differentiation down any one of the myeloid cell pathways. The hemopoietic stroma is believed to provide both mechanical support and regulatory signals important for the growth and maintenance of blood- forming cells. However, it has only been in the last few years that 2 a t ten t ion has focussed on the d e f i n i t i o n of the c e l l types involved in these processes and the development of appropriate assays and probes to analyze the mechanisms invo lved . Much of our present knowledge regarding the ro le of hemopoiet ic-stromal c e l l i n te rac t ions in hemopoiesis s t i l l dates from in v ivo experiments in mice. Nevertheless, the p r a c t i c a l l i m i t a t i o n s imposed by such approaches and the d i f f i c u l t i e s in d e r i v i n g analogous data in man has encouraged the development of appropriate in v i t r o systems. During the past twenty years a wide va r i e ty of hemopoietic colony assay systems have been developed. These permit the enumeration of var ious c lasses of hemopoietic progeni tors in a given sample. Observations of the s i z e , morphology and c e l l u l a r composition of the c o l o n i e s , coupled with appropriate manipulat ions of the cu l tu re condi t ions have led inves t iga tors to theor ize about the funct ion ing and regula t ion of var ious stages of hemopoiesis. By the i r very nature however, colony assay systems do not permit examination of the int imate contact in te rac t ions between hemopoietic and stromal c e l l s that may be of importance in v i v o . Recent ly , an in v i t r o marrow cu l ture system has been developed which o f f e r s an opportunity to explore r e l a t i o n s h i p s between hemopoietic and stromal elements in a c losed environment in which hemopoiesis may proceed for severa l weeks and which may a lso be manipulated. Th is d o c t o r a l project i s concerned with the a p p l i c a t i o n of both types of in v i t r o cu l tu re systems to the study of the c e l l cyc le con t ro l of p r i m i t i v e normal and n e o p l a s t i c hemopoietic c e l l s . A. Hemopoietic Progenitor Assays The spleen colony assay and the concept of a hemopoietic stem c e l l The f i r s t a p p l i c a t i o n of c l o n a l assay techniques was developed by T i l l and McCulloch in 1961. They in jec ted l i m i t i n g numbers of hemopoietic c e l l s from donor mice in to heav i ly i r r a d i a t e d histocompat ible r e c i p i e n t s . Within 8 3 to 14 days macroscopic nodules were observed on the spleens of the recipients. When examined microscopically the nodules were seen to consist of recognizable cells of the various myeloid series. Usually only a single lineage was found to predominate in each colony although, "mixed" colonies containing erythropoietic, granulocytic, and megakaryocyte cells were also found (McCulloch, 1963; Fowler et al, 1966; Chen and Schooley, 1968). A linear relationship between the number of spleen colonies formed and the number of nucleated cells injected suggested that each colony arose from a single cell. Direct evidence for this hypothesis came with karyotypic analysis of metaphases from individual colonies generated in mice injected with mixed populations of radiation-induced chromosomally marked marrow cells. Unique karyotypes were demonstrated in 95-100% of the cells from individual colonies, therefore providing strong evidence for their single cell origin. (Becker et al, 1963; Wu et al, 1967). The entity capable of giving rise to a colony was termed a "colony forming unit-spleen" (CFU-S). Once the clonal nature of spleen colonies had been established certain properties of CFU-S could be inferred from analysis of the cellular composition of the colonies to which they gave rise. The extensive proliferative capacity of CFU-S and its pluripotentiality was evidenced by the large size of the colonies and the presence of several cell types within individual colonies. The self-renewal ability of CFU-S was demonstrated by serial transplantation experiments in which the progeny of a single colony was injected into a secondary recipient. Production of secondary colonies comparable in size and of mixed or different cellular composition indicated that primary colonies contain cells which can also be characterized as CFU-S (Siminovitch et al, 1963; Lewis and Trobaugh, 1964; Juraskova and Tkadlecek, 1965). These properties of CFU-S - pluripotentiality, extensive 4 proliferative capacity and self-renewal appear to f u l l f i l l the minimum criteria for stem cells. Although considerable cytogenetic and isoenzyme studies have indicated that a common ancestral cell exits for both the myeloid and lymphoid lineages in both mouse and man (Wu et al, 1967; Abramson et al, 1972; Sacker-Walker and Hardy, 1975; Fialkow, 1978; Prchal et al, 1978; Dick et al, 1985) no direct cytological evidence exists for the presence of lymphocytes in spleen colonies. Despite the considerable contribution that applications of the spleen colony assay have made to our knowledge of the hemopoietic system, several problems associated with its use remain unresolved. The CFU-S represent only those stem cells which develop in the spleen, and are therefore only a proportion, referred to as the "f" fraction of the total of such cells originally present in the inoculum. Not a l l of the cells capable of forming colonies reach the spleen (Siminovitch et al, 1963); a proportion will seed in other parts of the body, notably the bone marrow and lungs. Of the stem cells that reach the spleen, a number may be expelled (Playfair and Cole, 1965), or may not generate colonies. Though retransplantation studies have been used to estimate the f fraction, the values obtained vary widely (Siminovitch et al, 1963; Hendry, 1971). An accurate estimation of CFU-S number is made more difficult by the demonstration, using cytogenetic markers, that more than one colony may arise from a single cell (Barnes et al, 1968). Furthermore, recent studies have demonstrated that "early" spleen colonies which develop 7 to 10 days after transplantation are transient, and are composed of the progeny of a more developmentally restricted cell than the colonies which may develop a few days later (Magli et al, 1982) and cells capable of long-term hemopoietic cell repopulation may differ again from even day 14 CFU-S (Phillips et al, 1984). Finally the spleen colony assay suffers 5 from many of the problems that plague most in vivo assays. It is d i f f i c u l t to modify the culture conditions to permit examination of the effects of environmental and exogenous factors on the proliferation and differentiation of stem cells, nor can an investigator be completely aware of a l l the endogenous influences in the system which may affect the experimental results. Some of these problems could be overcome by cultivating hemopoietic cells in a r t i f i c i a l media outside the body. Accordingly, much effort has been expended in attempts to develop an appropriate in vitro culture system that permits the proliferation and differentiation of the most primitive hemopoietic cells under more controlled conditions. Clonal assay systems and the concept of committed progenitors The concept of an intermediate compartment of hemopoietic progenitors was f i r s t suggested by in vivo studies of Ep stimulated red c e l l production in polycythemic mice. These experiments showed that the Ep responsive cells (ERC) detected by this assay were more primitive than the f i r s t morphologically recognizable c e l l , the proerythroblast, but further along the differentiation pathway than CFU-S. These "committed progenitors" were envisaged as lineage restricted, incapable of self-renewal, but s t i l l possessing a considerable proliferative capacity (Krantz and Jacobson, 1970). Granulocyte/macrophage colony forming cells The f i r s t in vitro hemopoietic colony assay system was developed independently in the mid- sixties by two different groups (Bradley and Metcalf, 1966; Pluznick. and Sachs, 1965). In both instances culture conditions were employed that permitted the formation of discrete colonies containing 50 to several hundred cells of the granulocyte/macrophage (GM) series from suspensions of mouse marrow cell s . The c e l l of origin was named the CFU-C for colony forming unit-culture. Shortly thereafter human granulocyte/macrophage colonies were 6 a l s o s u c c e s s f u l l y c u l t u r e d from human bone marrow (Senn et a l , 1967; Pike and Robinson, 1970) and p e r i p h e r a l blood c e l l s (Kurnick and Robinson, 1971) by analogous procedures (Figure 1). Subsequently c u l t u r e c o n d i t i o n s were developed that allowed the d e t e c t i o n of committed progenitors f o r a l l hemopoietic pathways i n a v a r i e t y of species i n c l u d i n g man (see review by Metcalf, 1977: Eaves and Eaves, 1984). In each instance hemopoietic c e l l s are immobilized i n a s e m i - s o l i d or viscous medium composed of agar, methycellulose or plasma c l o t , c o n t a i n i n g e s s e n t i a l n u t r i e n t s and growth f a c t o r s . Under such c o n d i t i o n s the c l o n a l progeny of a s i n g l e precursor are held immobilized i n c l o s e proximity to one another, thus p e r m i t t i n g the enumeration of i n d i v i d u a l c o l o n i e s and examination of t h e i r morphological c h a r a c t e r i s t i c s . In such c l o n a l assay systems the s t a t e of d i f f e r e n t i a t i o n of the progenitor c e l l i s u s u a l l y c o r r e l a t e d w i t h the length of the incubation period required f o r the production of recognizably d i f f e r e n t i a t e d progeny and the s i z e of the mature colony obtained (see review by Eaves and Eaves, 1984). The s i n g l e c e l l o r i g i n of a l l types of i n v i t r o c o l o n i e s has been e s t a b l i s h e d by s e v e r a l i n v e s t i g a t o r s using a v a r i e t y of techniques i n c l u d i n g 1) d i r e c t microscopic observation of the formation of a colony from a s i n g l e c e l l by r e p l a t i n g experiments (Paran and Sachs, 1969; Johnson and M e t c a l f , 1977; Nakahata and Ogawa, 1982), p h y s i c a l i s o l a t i o n by p l a s t i c r i n g s ( P l u z n i c k and Sachs, 1966), or photography (Cormack, 1976); b) isoenzyme s t u d i e s of i n d i v i d u a l c o l o n i e s ( P r c h a l et a l , 1976; Singer et a l , 1979a) and c) mixing experiments with male and female c e l l s and demonstration by Y chromosome a n a l y s i s of the homogeneity of i n d i v i d u a l c o l o n i e s (Strome et a l , 1978; Fauser and Messner, 1978, Dube et a l , 1981). 7 A B Figure 1. Photographs of granulocyte colonies grown in m e t h y l c e l l u l o s e c u l t u r e . As with ery thro id co lonies the d i f f e r e n c e s in colony s i z e r e f l e c t s the p l a t i n g of progeni tors at vary ing stages of d i f f e r e n t i a t i o n (X80). A. A large granu locy t ic colony (>500 c e l l s ) from an e a r l y , p r i m i t i v e CFU-C with a high p r o l i f e r a t i v e p o t e n t i a l . B. A smal l g ranu locy t ic colony (<500 c e l l s ) from a l a t e , mature CFU-C. Such a progenitor has a low p r o l i f e r a t i v e capaci ty and i s fur ther down the d i f f e r e n t i a t i o n pathway than the p r i m i t i v e CFU-C. 8 Erythroid colony-forming cells Recognition of the differences in culture conditions required to support the growth of large and small erythroid colonies and demonstration of differences in the properties of their precursors (Axelrad et al, 1974, Gregory, 1976), led to the concept of a hierarchy of erythropoietic progenitor cell classes. These subpopulations of committed progenitors within the erythroid pathway could be identified by the number of clusters present in each colony and their sequential appearance in the culture dish (Figure 2). For example, the most differentiated type of erythroid colony progenitor, the colony forming unit-erythroid (CFU-E) in man yields a colony within 9 days of 1 or 2 clusters, each consisting of 8 to 64 erythroblasts. In contrast, the most primitive burst forming unit-erythroid (BFU-E) is defined in man as a progenitor capable of producing a colony of 8 or more clusters and up to 10̂  cells after 18 days of incubation (see review, Eaves and Eaves, 1984). A number of intermediate colony types composed of varying numbers of clusters and order of appearance can also be recognized (Gregory and Eaves, 1977). The number of clusters present in a burst thus provides a convenient measure of colony size which in turn appears to be predetermined by the stage of differentiation of the progenitor cell that gave rise to i t . The precursor:progeny relationship of primitive BFU-E, mature BFU-E, and CFU-E has been validated in a number of ways including successive changes in responsiveness to Ep, cell volume, proliferative capacity and cell cycle activity (Axelrad et al, 1974; Gregory, 1976; Gregory and Eaves, 1978). Megakaryocyte colony-forming cells Megakaryocyte colony forming progenitors were first detected by Nakeff and his associates (1975) using bone marrow cells from vinblastine treated mice, cultured in agar over feeder layers of mouse embryo fibroblasts. Larger colonies, containing up to 80 9 Figure 2. D i f fe ren t s i zes of co lonies generated in methy lce l lu lose c u l t u r e s by e ry th ro id progenitors at d i f f e r e n t stages of d i f f e r e n t i a t i o n (X80). A. A large ery thro id burst , conta in ing >16 c l u s t e r s , produced by a p r i m i t i v e BFU-E. B. A smal l e ry thro id burs t , c o n s i s t i n g of 3-8 c l u s t e r s , from a mature BFU-E. C. An ery thro id colony, conta in ing 2 c l u s t e r s , from a CFU-E. 10 cells were obtained by the addition of pokeweed mitogen stimulated spleen cell conditioned medium (Metcalf et al, 1975). Initially the progeny of the megakaryocytic colony-forming cell (CFU-M) were identified by morphological means, until the development of a plasma clot assay permitted the in situ cytochemical staining of colonies for the presence of acetylcholinesterase (McLeod et al, 1976; Nakeff et al, 1976). Platelet formation from megakaryocytes in colonies has been observed in plasma clot cultures (McLeod et al, 1976). A plasma clot culture system for the growth of human CFU-M was initially described by Vainchenker and his associates (1979a,b). Subsequently an immunochemical method for the identification of human megakaryocytes was developed, utilizing a highly specific antibody against human platelet glycoproteins (Mazur et al, 1981). More recently larger, compact megakaryocyte colonies have been grown in methylcellulose cultures containing human plasma and PHA-stimulated human leukocyte conditioned media and the cells identified by a positive reaction with antibodies directed against human factor VIII antigen (Messner et al, 1982). A linear relationship between the number of cells plated and the number of colonies formed is consistent with the clonal origin of each colony (Metcalf et al, 1975; Nakeff et al, 1976). Additional evidence of clonality was obtained from studies utilizing cells from patients with the Tn polyagglutinability syndrome. When examined for the Tn phenotype, megakaryocytes in individual colonies were either Tn+ or Tn-, providing considerable support for the single cell origin of each colony (Vainchenker et al, 1982). Multi-lineage colony-forming cells Similar modifications in culture conditions also resulted in the production of mixed colonies containing more 11 than one lineage of mature cell (Figure 3). Erythroid-megakaryocyte colonies were first described from mouse bone marrow (McLeod et al, 1976). Subsequently larger mixed colonies, containing cells from a l l three myeloid cell lines were recognized in cultures of murine fetal liver cells, (Johnson and Metcalf, 1977), and shortly thereafter in cultures of adult mouse bone marrow though at a lower frequency (Hara and Ogawa,1978; Humphries et al, 1979a; Metcalf et al, 1979). Such colonies were shown to contain CFU-S (Humphries et al, 1979b) and to be derived from cells capable of self-renewal (Humphries et al, 1981). Other mixed colony types, containing different combinations of myeloid lineages have also been described more recently (Nakahata and Ogawa, 1982b; Suda et al, 1983). Comparable mixed colonies of human origin have been obtained in cultures of human bone marrow, peripheral blood, cord blood and fetal liver. In ini t i a l studies a bipotent progenitor was described, capable of giving rise to colonies composed of cells of the granulocytic and erythroid lineage (Fauser and Messner, 1978). The cell of origin was termed a CFU-G/E. Subsequently these authors were able to obtain mixed colonies in human bone marrow cultures in which megakaryocytes and macrophages were present in addition to granulocytes and erythroid cells, and a new term, CFU-GEMM was coined (Fauser and Messner, 1979). In the human system, as in the mouse system, the presence of stimulatory factors present in mitogen stimulated conditioned media was shown to be necessary for the optimal growth of such colonies (Fauser and Messner, 1978; Johnston and Metcalf, 1977). Comparisons between CFU-S and CFU-GEMM have demonstrated several characteristics in common. These include similarities in proliferative activity of the progenitor compartment in both steady state conditions and perturbed bone marrow, proliferative capacity and some, albeit limited 12 Figure 3 . A mixed granulocytic/erythroid colony photographed i n a methylcellulose culture a f t e r 18 days of incubation. The c e l l of o r i g i n of this colony i s termed a CFU-G/E (X120). 13 ability to self-replicate (Messner and Fauser, 1980; Ash et al, 1981). Recently T lymphocytes (Messner et al, 1981; Fauser and Lohr, 1982; Lim et al, 1984a) and also B cells (Hara, 1983) have been described in a small proportion of multi-lineage myeloid colonies. However, these studies remain controversial and unconfirmed. Normal blast colony forming cells Another type of colony has recently been described in cultures of normal mouse bone marrow and spleen cells after 16 days of incubation (Nakahata and Ogawa, 1982b). These small colonies, tentatively named stem cell or blast colonies were characterized by the complete absence of terminal differentiation prior to 16 days and their ability, upon replating, to generate large numbers of pure and mixed secondary colonies. These authors suggested that the progenitors of the primary colonies may be located prior to day 9 CFU-S and CFU-GEMM in the hierachy of stem cell differentiation. Subsequently these investigators demonstrated the presence of progenitors in human umbilical cord blood which gave rise to comparable small undifferentiated colonies after 25 days of incubation (Nakahata and Ogawa, 1982c). Summary - the hieracherial structure of the hemopoietic system The myeloid component of normal hemopoiesis as defined by in vivo and in vitro clonal assays of cells produced in vivo is typically subdivided into the following three stages of cellular development, each stage demarcated by major differences in proliferative capacity and differentiation potential (Figure 4). The most primitive compartment consists of pluripotent cells some of which are also capable of generating pluripotent daughter cells. The second compartment consists of progenitors committed to a specific pathway. These cells are the progeny of pluripotent cells in which determination or total lineage restriction has occurred. Committed progenitors may, however, 14 ULTIMATE STEM CELL iv. • * Lymphopoiesis Figure 4. Diagrammatic representation of the hierachy of hemopoietic progenitor compartments currently identified by colony assay procedures. Colonies are grown in semisolid media, such as methylcellulose or agar, with the addition of serum, essential nutrients and appropriate stimulatory growth factors. According to this model, the state of differentiation of the colony forming cell determines the size of the colony it produces in vitro. From reference (Eaves and Eaves, 1983); used with permission. 15 s t i l l possess extensive p r o l i f e r a t i v e capaci ty .The th i rd and la rgest compartment i s composed of c e l l s that are morphological ly recognizab le . These c e l l s are very l i m i t e d in the i r p r o l i f e r a t i v e p o t e n t i a l with only a porpor t ion of the compartment undergoing a small number of terminal d i v i s i o n s . Once f u l l y mature these c e l l s are released into the c i r c u l a t i o n to f u l f i l l the i r b i o l o g i c a l ro le before they die and are replaced by new c e l l s . The grouping of hemopoietic c e l l s into three compartments i s a convenient method of descr ib ing the h i e r a r c h i a l s t ruc ture of the system. However, considerable heterogeneity e x i s t s wi th in a l l compartments and the borders between them may be somewhat b lurred when many parameters are examined s imul taneously . In add i t ion the existence of bipotent p rogen i to rs , such as CFU-G/E or CFU-E/M ind ica tes that intermediate populat ions of c e l l s may e x i s t between the p lur ipotent stem c e l l and the var ious committed progeni tor compartments; i . e . l ineage r e s t r i c t i o n may occur (apparently randomly) in a s i n g l e or in mul t ip le steps (Nakahata et a l , 1982; Suda et a l , 1983, 1984 a , b ; Lim et a l , 1984b; Leary et a l , 1984, 1985). B) Stem C e l l Regulat ion The nature of the mechanisms c o n t r o l l i n g c e l l u l a r p r o l i f e r a t i o n and d i f f e r e n t i a t i o n i s a fundamental problem in b io logy . In the hemopoietic system our understanding of such regulatory mechanisms a f f e c t i n g the most p r i m i t i v e c e l l s i s considerably hampered by our i n a b i l i t y to to observe and study the c e l l s of in te res t d i r e c t l y because they represent rare elements in the e n t i r e marrow populat ion and are not p h y s i c a l l y l o c a l i z e d in a fashion analogous to the stem c e l l populat ions of other t i s s u e s . However, c e l l c y c l e s tud ies coupled with c y t o l o g i c a l examination of the progeny produced by stem c e l l s in v ivo suggest three b i o l o g i c a l l y important types of stem c e l l 16 decisions that may or may not be independently regulated. These are; 1) decisions affecting changes in proliferative states; 2) decisions to self- renew or differentiate; 3) decisions affecting further restriction of differeniation capacity (Figure 5). Decisions affecting changes in proliferative status Lajtha and his asociates (1963) were the first to postulate a model in which the majority of the stem cells were envisaged to be in a non- proliferative resting state, termed GQ, but which could be "triggered" into a cycling state by perturbations in more differentiated cell compartments. Experiments utilizing -^-thymidine or other cycle active drugs provided considerable support for this model. Thus, although the majority of the CFU- S in a normal intact mouse are in a resting state, this changes within a few hours after sublethal irradiation (Becker et al, 1965; Eaves and Bruce, 1974), the administration of cycle active cytotoxic drugs ( Bruce et al, 1966; Blackett et al, 1968; Vassort et al, 1973; Eaves and Bruce, 1974; Hodgson et al, 1975), endotoxin (Eaves and Bruce, 1972) or bleeding (Duplan and Feinendegen, 1970). Furthermore, while CFU-GEMM in normal humans are predominently quiescent, the majority are in S-phase during hemopoietic regeneration following marrow transplantation (Fauser and Messner, 1982). These findings indicate that primitive hemopoietic cell populations are capable of changing their proliferative state in response to stimuli associated with terminal cell depletion. In certain experimental situations, as for example in mice after phenylhydrazine administration (Rencricca et al, 1970; Wright and Lord, 1977) or in experiments using partial body irradiation (Gidali and Lajtha, 1972) the CFU-S in one site may be in active cell cycle, while in another part of the body the CFU-S are quiescent. Such findings may suggest local A B E G 1 1 © m M 1 Figure 5. Three types of stem cell transitions where regulatory mechanisms may act to influence stem cell behaviour. A. Control of stem cell proliferation B. Self-renewal versus differentiation C. Restriction of differentiation capacity 18 regulatory mechanisms in at least some aspects of the control of CFU-S proliferation. However, it has also been shown that depletion of stem cells in one part of the body can result in an increased proliferative activity in an unaffected site, implicating long range humoral control mechanisms (Croizat et al, 1970; Gidali and Lajtha, 1972). Although the size of the local CFU-S compartment appears to be a common feature associated with stem cell proliferative status, the significance of this association is s t i l l not known. A diffusible factor from damaged cells (Frindel et al, 1976) as well as a thymic factor (Lepault et al, 1980) have been suggested as possible long range stimulatory factors. Local negative feedback inhibitors and promoters of cell proliferation have also been described and will be discussed in a later section. Decisions affecting loss of self-renewal capacity and lineage restriction By definition commitment involves loss of the capacity to generate new pluripotent daughter cells. Both deterministic and stochastic models have been proposed for the mechanism initiating this restriction process. The deterministic model postulates an inductive effect by external factors on the choice between self-renewal and differentiation. According to this model, originally proposed by Trentin and his associates (1968, 1970) commitment is regulated by the specific inductive microenvironment surrounding each individual stem cell and the spectrum of differentiated daughter cell types produced will thus vary according to local variations in the microenviroment. The ability of individual pluripotent cells to form colonies of different sizes and composition both in vivo (Gregory, 1976) and in vitro (Humphries, 1979a) provide strong evidence against such a model and lent support to the stochastic model proposed first by T i l l and his associates (1964). According to this model the choice between self-renewal or commitment is made at 19 random, with a f ixed p r o b a b i l i t y for each event ( T i l l et a l , 1964; Korn et a l , 1973; T i l l and McCulloch, 1980). This model was o r i g i n a l l y proposed on the bas is of the marked heterogeneity found upon ana lys is of the c e l l u l a r content of spleen c o l o n i e s , p a r t i c u l a r l y in the number of CFU-S contained i n i n d i v i d u a l co lon ies (S iminov i t ich et a l , 1963). An extension of th is model was proposed by Suda and h is assoc ia tes (1983). They suggested that stem c e l l commitment i s a progressive and s t o c h a s t i c process r e s u l t i n g in the gradual r e s t r i c t i o n of the d i f f e r e n t i a t i o n p o t e n t i a l of the stem c e l l . Th is model i s based on the demonstration of var ious combinations of bipotent progeni tors which can express colony formation in v i t r o (Nakahata et a l , 1982; Leary et a l , 1984; Lim et a l , 1984b). Such f ind ings a lso argue against the stem c e l l competit ion model pathway in which s p e c i f i c inducers such as e r y t h r o p o i e t i n (Ep) and var ious colony s t imula t ing fac to rs (CSF) were thought to act upon a common stem c e l l to e f fec t d i f f e r e n t i a t i o n in to a s p e c i f i c pathway (Van Zant and Goldwasser, 1977). This model p r e d i c t s that competing demands made upon the stem c e l l resu l ted in an increase i n one c e l l l i n e at the expense of the other . Although evidence for th is was claimed with the demonstration that increas ing concentrat ions of Ep could i n h i b i t g r a n u l o c y t i c colony formation (Van Zant and Goldwasser, 1977; 1979), such an e f f e c t occurred only when high c e l l concentrat ions were p l a t e d , suggest ing that i t was a secondary phenomenon. Other inves t iga to rs have not observed stem c e l l compet i t ion. Rather, the add i t ion of increas ing concentrat ions of Ep to mixed e ry thro id colonies resul ted in an increase in the mean number of e ry th ro id c e l l s per colony, without a f f e c t i n g other hemopoietic l ineages (Metcalf and Johnson, 1979). Other s tudies have shown that a s t imulatory e f f e c t on stem c e l l s r e s u l t s in a general increase in a l l p r i m i t i v e c e l l types (Gregory and Henkelman, 1977). 20 However, evidence of some heterogeneity in the self-renewal capacity of different CFU-S and other progenitor cell populations due to non-stochastic processes has also been obtained. Subpopulations of stem cells differing in self-renewal capacity have been demonstrated by experiments using physical separation (Worton et al, 1969), alkylating agents (Schofield and Lajtha, 1973; Morley and Blake, 1974; Botnick et al, 1976; Rosendaal et al, 1979), irradiation (Schofield et al, 1980), serial transplantation (Siminovitch et al, 1964; Micklim and Ogden, 1976; Schofield, 1980), and adherence separation (Mauch et al, 1982 Kerk et al, 1985). Variation in self-renewal capacity have also been found between found between stem cells in the blood and those in the marrow (Micklen and Odgen, 1974). Such findings have led some authors to postulate an age structured stem cell population whose members demonstrate variations in self-renewal capacity based on their proliferative history (Hellman et al, 1978). In addition, environmental factors, such as exogenous CSF concentrations have been shown to modulate the proliferative capacity exhibited by granulocytic/macrophage progenitors (Metcalf, 1985). Whether such mechanisms may also play a role in limiting stem cell behaviour has yet to be determined. C) Hemopoietic Growth Factors In a normal individual, regulatory mechanisms operate to maintain the numbers of a l l circulating mature cell types within narrowly defined limits. Since terminal hemopoietic cells are not capable of division, perturbations which require increased production of mature cells must in some manner affect the status of the more primitive cell compartments. Much of our present knowledge regarding the role of growth factors on the proliferation and survival of hemopoietic cells has been obtained from in vitro clonal assay systems where a requirement for these factors for colony formation was 21 demonstrated. The heterogenous nature of the various stimulatory factors that could interact with target progenitor cells in the bone marrow would considerably increase the flexibility of the hemopoietic regulatory system, permitting the precise control of mature cell production. However, except for Ep, the in vivo significance of these factors has been highly controversial. More recently, with the availability of pure recombinant growth factors for assessment of in vivo effects (Lee et al, 1985; Rennick et al, 1985; Wong et al, 1985) and the recognition of associations between growth factor independence and the acquisition of a neoplastic phenotype (Cochran et al, 1983; Kelly et al, 1983; Rapp et al, 1985) the significance of most of these factors has assumed a new respectability. Colony stimulating factors (CSFs) In the in i t i a l description of the in vitro cloning assay for granulocytic progenitors the presence of a cell feeder system was required for colony formation (Bradley and Metcalf, 1966; Pluznick and Sachs, 1966). Subsequently the presence of colony stimulating factor was demonstrated in the medium conditioned by a variety of cells (Bradly and Metcalf, 1966; Austin et al, 1971; Iscove et al, 1971; Parker and Metcalf, 1975; Aye, 1977). More recently purification studies have indicated that the CSFs represent a family of glycoprotein molecules which must be continually present for GM colony growth to proceed (Paran and Sachs, 1968; Stanley et al, 1975; Burgess et al, 1977). The members of the CSF family include 1) M-CSF (or CSF-1) which supports primarily macrophage colony formation (Stanley and Heard, 1977); 2)G-CSF which stimulates almost exclusively granulocytic colony formation in the mouse (Williams et al, 1978a); 3) GM-CSF, originally thought to be specific for granulocyte/macrophage colony progenitors (Burgess et al, 1977); and 4) interleukin-3 (IL-3) or multi-CSF, a factor that stimulates a l l 22 types of clonogenic myeloid cells (Metcalf and Johnson, 1978). Besides eliciting responses from different target cell populations, these four CSFs are heterogenous with respect to their molecular weight, carbohydrate content, amino acid sequence and production source (Metcalf, 1985). More recently the genes for human CSF-1, murine and human GM-CSF, murine IL-3 and a human G-CSF like factor have been cloned and the activities of their purified products analyzed both in vivo and in vitro. These and other studies with pure "natural" CSF's have established that none are totally lineage-specific in their effects in vivo and that in both mouse and man GM-CSF and G-CSF can stimulate pluripotent and a variety of lineage restricted progenitor cell types (Gough et al, 1984; Fung et al, 1984; Lee et al, 1985; Hapel et al, 1985; Kawasaki et al, 1985; Rennick et al, 1985; Wong et al, 1985). Other studies indicate that the CSFs may have similar biochemical effects to other growth factors. For example, the addition of murine CSF-1 to resting target cells results in an increase in DNA synthesis within 10-12 hours (Tushenski and Stanley, 1983), stimulation of the rate of protein synthesis and an inhibition of the rate of intracellular protein degradation. Furthermore GM-CSF also stimulates RNA synthesis and this effect is independent of protein synthesis (Burgess and Metcalf, 1977; Price et al, 1975). Erythropoietin (Ep) The concept that a humoral factor could regulate red cell production in response to tissue oxygen demands and perturbations in hemostasis was developed early in this century (Carnot and Deflandre, 1906). In the mid 1950s a target cell for this factor was identified by studies in polycythemic mice (Jacobson et al, 1957) and termed the Ep-responsive cell. The demonstration that maturing erythroblasts disappear rapidly from the 23 marrow and spleen when Ep is removed, but reappear within 48 hours after Ep administration identified the ERC as a fairly mature progenitor cell of the erythroid series. Ep has been purified to homogeneity and studies on its biochemistry have characterized the molecule as an acidic glycoprotein with a molecular weight of about 34,000 daltons (Jacobs et al, 1985). The gene has recently been cloned and the expressed product shown to have the same activity in vivo and in vitro initially ascribed to the naturally produced material. Ep is normally made by the kidney and during fetal life is also produced in the liver. When in vitro colony assays for erythroid progenitors were developed a critical role for Ep in the regulation of erythropoiesis was reaffirmed by the strict dependency of CFU-E colony formation on the presence of Ep in the culture (Stephanson, 1971). Reduction of Ep levels in mice resulted in a substantial decrease in the number of CFU-E in both spleen and marrow, and this effect could be reversed by Ep administration (Gregory et al, 1973; Axelrad et al, 1974). Ep dose response studies have demonstrated that CFU-E are the most sensitive erythroid progenitor class and can respond to very low levels of the hormone (Gregory et al, 1973). Other studies have indicated that the immediate precursors of CFU-E, the mature BFU-E may also be regulated in part by Ep. Mature BFU-E colony formation is also dependent on the presence of Ep, although a considerably higher concentration of the hormone is required for optimal cloning efficiency in comparison to CFU-E (Gregory et al, 1976). The molecular mechanisms of Ep induced red cell differentiation are not well known. Since the cycling activity of residual CFU-E is not altered in polycythemic mice (Iscove, 1977) it has been suggested that Ep acts to 24 promote the survival of CFU-E thus permitting the completion of their predetermined differentiation program (Eaves et al, 1979a). Other studies using radioactive cloned globin gene fragments have shown that Ep may directly induce globin gene transcription (Bondurant et al, 1985). A mitogenic effect of Ep at the level of mature BFU-E has been suggested by the demonstration that some of these cells can respond to erythropoietic stimulation by increasing their cell cycle activity (Adamson et al, 1978). Other factors A number of inhibitory factors have also been implicated in the regulation of granulopoiesis. Colony inhibitory activity (CIA) derived from polymorphonuclear neutrophils is a specific inhibitor of CSF production by monocytes in vitro (Bruch et al, 1978; Broxmeyer et al, 1977a). Subsequently this glycoprotein was identified as lactoferrin (Broxmeyer et al, 1978). In addition to its supressive effects on the release of CSF, lactoferrin may inhibit monocytic and macrophage production of acidic isoferritins (Broxmeyer et al, 1984a). Lactoferrin is present in the plasma at a much higher concentration than that required to inhibit CSF production in vitro, making its in vivo significance unclear (Bennett et al, 1978). However, in vivo administration of purified, endotoxin-free lactoferrin was shown to decrease the number of multipotential, erythroid and granulocytic precursors in murine marrow and spleen and to arrest the turnover of these normally cycling progenitor cells (Broxmeyer et al, 1984b; Lu et al, 1983). Prostaglandins of the E series appear to have an inhibitory effect on CFU-C number in vitro (Pelus et al, 1979; Williams, 1979). Macrophages elaborate PGE in response to CSF stimulation in vitro, suggesting an interplay between CSF and PGE may have a role in granulocyte regulation (Kurland et al, 1978). Other inhibitors of CFU-C colony formation include the adenosine nucleotides (Kurland et al, 1977; Taettle and Mendelson, 1980), 25 i n t e r f e r o n (McNeil and Fleming, 1971, Greenberg and Mosney, 1977; Kimpel et a l , 1982;) and l i p o p r o t e i n s (Douay et a l , 1983) but in these cases no experimental assessment of the i r ro le in normal granu lopoies is has been repor ted . Although l a c t o f e r r i n has no e f fec t on e r y t h r o p o i e s i s , an i n h i b i t o r y e f f e c t of a c i d i c i s o f e r r i t i n on BFU-E p r o l i f e r a t i o n has been demonstrated (Lu et a l , 1983). In contrast to i t s e f fec t on g r a n u l o p o i e s i s , PGE st imulates the growth and d i f f e r e n t i a t i o n of BFU-E (Chan et a l , 1980; Rossi et a l , 1980; Degowin and Gibson, 1981). PDGF has a lso been shown to increase the p r o l i f e r a t i o n of e r y t h r o p o i e t i c c e l l s in v i t r o (Dainiak et a l , 1983). Wright and h is assoc ia tes have descr ibed two fac tors that were capable of s p e c i f i c a l l y i n h i b i t i n g or s t imula t ing the p r o l i f e r a t i v e a c t i v i t y of CFU-S (Wright and Lord , 1977; 1978). The st imulatory a c t i v i t y was extracted from regenerat ing murine bone marrow where a high proport ion of CFU-S were in S phase (Lord et a l , 1977a), while the i n h i b i t o r y substance was obtained from normal bone marrow when the majori ty of CFU-S were quiescent (Lord et a l , 1976). Subsequently these inves t iga tors demonstrated the presence of both fac to rs in numerous other hemopoietic t issues of human and murine o r i g i n . In each instance the r e l a t i v e proport ion of each fac tor was cor re la ted with the p r o l i f e r a t i v e status of the CFU-S in the t i ssue (Wright and Lord , 1979). In a d d i t i o n , each fac tor could be used in competit ion with the other to r e v e r s i b l y a l t e r the p r o l i f e r a t i v e a c t i v i t y of the stem c e l l i n e i t h e r a p o s i t i v e or negative fashion (Lord et a l , 1977b). These authors suggested that the r e l a t i v e concentrat ion of the two fac tors in v ivo could regulate the l e v e l of stem c e l l p r o l i f e r a t i o n (Lord and Wright, 1982). Extensive work in Byron's laboratory has demonstrated that ^-adrenergic agents (Byron, 1972, Byron, 1975), c h o l i n e r g i c agents (Byron, 1975) and 26 histamine (Byron, 1977a) could increase the proportion of stem cells in S-phase in vitro, and their effects appear to be mediated through the cyclase system. Since the addition of cyclic nucleotides will also increase CFU-S cycling (Byron, 1971) a regulatory role for phosphodiesterase in maintaining stem cells in a quiescent state was postulated. Androgens (Byron, 1970, 1971, 1972), prostaglandin E 2 (Feher and Gidali, 1974; Lu et al, 1984. and PDGF (Michalevicz et al, 1985) have also demonstrated a stimulatory effect on stem cell cycling. In contrast, interferon appears to decrease the clonogenic efficiency of CFU-GEMM (Neumann and Fauser, 1982). Role of the stroma The main site for hemopoiesis in the adult is the bone marrow where numerous cell types such as endothelial, adventitial reticular cells, adipocytes, fibroblasts and macrophages form a complex three-dimensional matrix. Morphological studies have shown that hemopoietic cells in various stages of differentiation and maturation form specific interactions with various of these components of the marrow stroma. For example, areas of active erythropoiesis are found in close proximity to the sinusoidal endothelium, while granulocytic cells are observed in association with the extravascular reticulum cells (Sorrell and Weiss, 1980). In rodent marrow erythroid progenitors have been associated with a macrophage-like acid phosphatase positive cell (Westin and Bainton, 1979; Ben-Ishay and Yoffey, 1972) and granulocyte precursors with an alkaline phosphatase positive reticulum cell (Westin and Bainton, 1979). In addition, adipocytes are often found in areas of active granulopoiesis (Weiss, 1980). Several experimental observations provided indirect evidence for a regulatory role of the bone marrow stroma in vivo. For example, hemopoietic stem cells circulate freely (Everett and Perkins, 1979) but only proliferate in certain organs such as the bone marrow and spleen ( T i l l and McCulloch, 27 1961). Hemopoiesis in bone marrow that has been i r r a d i a t e d or mechanical ly damaged only resumes a f te r a func t iona l stromal a rch i tec tu re i s r e - es tab l i shed (Tavasso l i and Crosby, 1968). A s i m i l a r s i t u a t i o n i s seen in the regenerat ing marrow of leukemic pat ients a f te r marrow t ransp lanta t ion or fo l lowing extensive chemotherapy (C l ine et a l , 1977). In these pat ients hemopoiesis resumes as d i s c r e t e f o c i of regenerat ing hemopoietic precursors in s p e c i f i c micro-anatomical r e l a t i o n s h i p s with stromal elements comparable to those observed during f e t a l development (Islam et a l , 1980, 1984). S imi la r observat ions led Trent in and h is co-workers to propose the concept of a hemopoietic induct ive microenviroment (HIM) which could induce the d i f f e r e n t i a t i o n of p lur ipotent stem c e l l s along s p e c i f i c committed c e l l l i n e s (T ren t in , 1970, 1971). They found that in i r r a d i a t e d rec ien ts of hemopoietic c e l l suspensions colony formation in the spleen was predominently e r y t h r o p o i e t i c , while g ranu locy t ic co lonies were more common in the bone marrow. S imi la r e r y t h r o p o i e t i c / g r a n u l o c y t i c r a t i o s were a lso found in secondary r e c i p i e n t s whether the in jec ted c e l l suspension was obtained from an e ry th ro id or a g ranu locy t ic colony. According to th is concept mixed co lon ies arose when a colony enlarged and encountered new areas of stroma with d i f f e r e n t microenviromental in f luences (Curry and T r e n t i n , 1967). Indeed, d i r e c t v i s u l i z a t i o n of the induct ive e f fec t of stroma on the morphology of spleen co lon ies was seen in experiments where plugs of bone marrow stroma were implanted within the sp leen. In such sp leens , when i n d i v i d u a l co lon ies were located at the junc t ion of marrow and spleen stroma, that por t ion of the colony within the spleen stroma was predominently e r y t h r o i d , while the por t ion of the same colony in a s s o c i a t i o n with marrow stroma was predominently g ranu locy t ic (Wolf and T r e n t i n , 1968). However, in s tud ies where the contents of i n d i v i d u a l spleen co lonies were replated in 28 v i t r o the heterogenous nature of the secondary co lonies produced suggest that these apparent e f f e c t s of the stroma on stem c e l l d i f f e r e n t i a t i o n may be explained by s e l e c t i v e a m p l i f i c a t i o n of l a t e r c e l l types (Gregory, 1973: Gregory et a l , 1974). An e x t r i n s i c ro le of the microenvironment on hemopoiesis has a lso been suggested by s tudies on the g e n e t i c a l l y anemic S l / S l ° mouse. Stem c e l l s from S l / S l d mice are capable of forming spleen co lonies when in jec ted in to +/+ normal syngeneic mice, but transplanted stem c e l l s from +/+ l i t t e rmates do not form spleen co lon ies in S l / S l d mice (McCulloch et a l , 1965), suggest ing that the microenviroment in S l / S 1 Q mice may be responsib le for the p r o l i f e r a t i v e f a i l u r e of the transplanted normal stem c e l l growth. The macrocytic anemia present in these mice can be cured by t ransplant of normal spleen t i s s u e , but not by in fus ions of normal marrow c e l l s . The normal implanted spleen t issue d isp lays areas of ac t i ve hemopoiesis in contrast to the ad jo in ing S l / S l d stroma which remains in a quiescent s ta te (Wolf, 1978). 2) THE CELL CYCLE Much of our knowledge of the c e l l cyc le and growth k i n e t i c s of homogeneous c e l l populat ions was i n i t i a l l y obtained from l a b e l l i n g s tud ies u t i l i z i n g t r i t i a t e d thymidine, p a r t i c u l a r l y in conjunct ion with the technique of autoradiography. More recent ly , flow cytometry has enabled more rap id and prec ise measurements of c e l l cyc le parameters for homogeneous c e l l suspensions or suspensions in which the c e l l populat ion of in te res t can be i d e n t i f i e d in the flow cytometer by some other unique property . In the case of hemopoietic c e l l suspensions where the c e l l s of in te res t cannot yet be i d e n t i f i e d by such means, i n h i b i t i o n of colony formation has been used to d i s t i n g u i s h S-phase c e l l s exposed to l e t h a l S-phase s p e c i f i c agents such as high s p e c i f i c a c t i v i t y t r i t i a t e d thymidine or var ious drugs. 2 9 Figure 6. The cell cycle. Terminally differentiated cells are incapable of further division while cells in G Q may be quiescent for varying periods of time before responding to an appropriate stimulus to enter the cell cycle and divide again. 30 A) Descr ip t ion and Regulat ion of the C e l l Cycle The c e l l cyc le concept or ig ina ted in the ear ly 1950's when i t was found that the c e l l made new DNA at a d i s c r e t e time between d i v i s i o n s , subsequently re fe r red to as "S" phase (Howard and P e l c , 1953). A f te r S there i s a "gap" of vary ing length G2> during which the c e l l has a 4n DNA content , before proceeding to the next phase, termed M ( for mi tos is ) or D ( for d i v i s i o n ) . Fol lowing c e l l d i v i s i o n , the c e l l enters the phase. This part of the c e l l c y c l e may vary extens ive ly in length , not only between d i f f e r e n t c e l l types, but between members of the same c e l l populat ion. Cer ta in c e l l s , such as blastomere c e l l s , do not enter a G^ phase (G^~ c e l l s ) but proceed d i r e c t l y in to S phase a f t e r d i v i s i o n (Prescot t , 1982). In most c e l l s however, the four phases G^,S,G2 and M const i tue the c e l l cyc le (see review by Wheatly, 1982). Despite the popular nomenclature however, i t must be kept in mind that the processes of growth, synthesis and d i v i s i o n are not t r u l y c y c l i c a l , as considerable v a r i a b i l i t y may occur in sucessive d i v i s i o n s . G_i The c e l l enters G^ as soon as d i v i s i o n i s completed. Some of the ear ly events in G^ include the synthesis of membrane components, such as phosphol ip ids , g l y c o p r o t e i n s , and s i a l i c a c i d . As G^ proceeds, the c e l l may develop many f inger l i k e m i c r o v i l l i , which can be re t racted at d i v i s i o n to provide the extra surface cover ing capaci ty required when a s p h e r i c a l object d i v i d e s in to two approximately equal par ts . Most G^ c e l l s undergo steady l i n e a r growth u n t i l the time at which S phase i s i n i t i a t e d . Cer ta in enzymes necessary for the process of DNA s y n t h e s i s , such as thymidine k inase , thymidylate synthetase and others increase sharply in c e l l s during the l a s t part of G]_. I n h i b i t o r s of p ro te in or RNA synthesis w i l l block the formation of these enzymes and prevent the c e l l s from enter ing S. Once a c e l l has synthesized a l l the bas ic 31 requirements for DNA replication it has passed the point of commitment, but the nature of the trigger that permits progression into the next phase is unknown. S The mammalian cell contains approximently 2000 initiation sites where the two strands of parental DNA are separated to permit template formation. The sequence of initiation from site to site follows a rigid pattern that is repeated with remarkable consistancy cycle after cycle. The rate of DNA synthesis accelerates at the beginning of S-phase, reaches a maximum and then decreases. Once initiated DNA synthesis normally proceeds until completion. During S-phase the cell continues to grow and synthesis of subcellular organelles occurs, along with changes in surface morphology. In addition replication of DNA requires synthesis of histones, highly basic proteins rich in lysine and arginine which are characteristically associated with DNA. G_2 G2, the period from S phase to division is poorly understood. Both protein and RNA synthesis are necessary for an interval after the start of G2« Soon after, RNA synthesis is no longer needed, and then closer to mitosis, protein synthesis ceases. Once the cell has progressed into G2, it will reach division within a fairly well proscribed period of time. A certain minimum span of time is required for most cell types however, suggesting that certain definite preparations must be undertaken before mitosis can begin. Current opinion suggests the concurrence of a number of discrete proccesses, such as absolute size of the cell, right concentration of important molecules, and the assembly of macromolecules into specific structures are necessary before division begins. A second possibility is that the cell requires a certain amount of time between S-phase and division to edit the newly synthesized DNA and to remove any errors in copying. 32 Mitosis C e l l d i v i s i o n i n eukaryotes i s mechanically a very complex process. F i r s t , the chromosomes must l i n e up, separate, and then move to the opposite sides of the c e l l . Then, during the process of cytokinesis the c e l l must divide i n such a manner as to ensure an equal d i s t r i b u t i o n of a l l necessary cytoplasmic constituents and organelles. The c e l l u l a r machinery responsible for this complex task i s the mitotic apparatus. The process of mitosis i s divided into f i v e stages; prophase, metaphase, anaphase, telophase and cytokinesis. During prophase the d i f f u s e chromatin of interphase condenses into well defined chromosomes, each con s i s t i n g of two s i s t e r chromatids joined by a centromere. The nucleolus, nuclear membrane and cytoskeleton disassemble, and construction of the mitotic apparatus begins. The c e n t r i o l e s , r e p l i c a t e d during S phase now separate, with each pair and i t s r a d i a l array of microtubules moving apart to form a bipo l a r m i t o t i c spindle. As metaphase i s reached the chromosomes are aligned at the metaphase plate by the i n t e r a c t i o n of their kinetochore f i b e r s with the mit o t i c spindle. Anaphase begins when the chromosomes begin to move apart, and l a s t s only for a few minutes. As the chromosomes move apart, the kinetochore f i b e r s shorten, the spindle f i b e r s elongate, and the two poles of the polar spindle move further apart. The sudden and dramatic movement of chromosomes appears to require l i t t l e energy and may be due to disassembly of the microtubules at the polar end of the mitotic spindle. During telophase many of the events occuring i n prophase are reversed; the kinetochore f i b e r s disappear, the spindle disperses, a new nuclear envelope forms around each group of daughter chromatids, the condensed chromatids expand, and n u c l e o l i begin to reassemble. The process of cleavage begins i n lat e anaphase when a c o n t r a c t i l e r i n g forms i n the region of the c e l l periphery around the area of the metaphase plate. This c o n t r a c t i l e r i n g consists of a thickened area of 33 filamentous cytoplasm, containing fibronectin and actin, just beneath the surface of the cell membrane. As it contracts, a cleavage furrow is formed, which continues until only the pole to pole fibers of the mitotic spindle are contained within the ring, forming the midbody. Eventually the bridge breaks apart, and two new separate daughter cells are formed. The Gn state Some normal cells may make a choice between proliferation or quiescence when they are in the phase of the cell cycle. According to one view (Lajtha, 1963), quiescent cells withdraw from the cell cycle to enter a qualitatively distinct G0 state. Definite biochemical differences do exist between G0 and Ĝ  cells, such as polyribosome content (Becker et al,1973), chromatin template activity (Rovera and Baserga, 1973), and membrane transport (Saunder and Pardee, 1972). Cell fusion studies suggest that quiescent cells may be in a distinct arrested state because they contain an inhibitor of DNA synthesis not present in cycling cells (Yanishivsky and Stein, 1980). Cells emerging from the GD state are more sensitive to certain drugs than are actively cycling cells (Yen et al, 1978) and require a longer lag time to initiate S-phase suggesting that specific biochemical events are required to leave the G0 state. Cells may enter GQ under a variety of adverse conditions, including serum limitation, nutrient deprivation, high cell density, and certain drugs. In the intact animal quiescent cells may serve as a reserve population which can enter the proliferating pool when required, as for example, hemopoietic stem cells. B) Factors Controlling Cell Proliferation The different cell populations in most multicellular animals show different rates of turnover. Some cell types, such as neurons, skeletal muscle cells and red blood cells cannot divide at a l l . Other cell populations such as the epithelial stem cells of the skin and the gut appear 34 to consist almost entirely of cells that divide rapidly and continuously during the entire lifespan of the individual. Most cell populations however, fa l l between these extremes. In every tissue a balance must exist between the rate of cell turnover and the rate of cell loss, not only cell loss due to differentiation but also cell loss due to natural or injury related death processes. Various factors have been implicated in maintaining this balance. These factors may induce quiescence, result in withdrawal from the G0 state, play a role in regulation of Ĝ -S transition, or simply serve to maintain cell viability throughout the cell cycle Density dependent inhibition Normal anchorage-dependent cells in culture will cease mitotic activity at confluence. The final cell density is a function of the concentration of mitogens in the growth medium (Scher et al, 1979) or the serum concentration (Holley, 1975). A number of mechanisms have been attributed to this phenomenon of density dependent inhibition, including cell-cell contact (Bunge et al, 1979; Lieberman and Glaser, 1981), changes in cell shape (Fox et al, 1979), the accumulation of inhibitors (Holley, et al, 1978) and depletion by the cells of media components, such as necessary nutrients or growth regulatory factors, or their restriction to the cell as a consequence of crowding (Dulbecco and Stoker, 1970; Stoker and Piggott, 1974). Recent studies have focussed on the role of membranes in producing density dependent inhibition. Addition of partially purified membrane fractions from confluent 3T3 cells to growing 3T3 cells resulted in GQ arrest of 50% of the cycling cells but did not inhibit growth of SV40 transformed 3T3 cells (Whittenberger et al, 1977). In contrast, no inhibition of DNA synthesis was seen when the surface membrane fraction from proliferating 3T3 were used. Further studies indicated that the inhibitory component was a 35 heat l a b i l e , nondia lyzable f r a c t i o n of the membrane (Whittenberger et a l , 1978, 1979). Growth i n h i b i t o r s on the c e l l surface have been descr ibed by severa l other groups (Yeh and F i s h e r , 1969; Natraj and D a l t r a , 1978; Lieberman et a l , 1981). Data from these experiments are compatible with the hypothesis that receptor molecules on the c e l l surface mediate c e l l - t o - c e l l contact , and these receptor molecules may be released into the media. Pro te in metabolism Studies u t i l i z i n g i n h i b i t o r s of prote in synthes is i n cu l tured mammalian c e l l s have shown that cont inuing prote in synthesis i s necessary to maintain progression through the c e l l c y c l e . A decrease in the rate of p ro te in accumulation r e s u l t s in extension of the G^ per iod (Brooks, 1977; Baxter and Stamma, 1978). Conversely , quiescent c e l l s show l i t t l e or no net accumulation of prote in (Castor , 1977; Sta iners et a l , 1977a), but w i l l increase prote in synthesis when st imulated to regrow. In c e r t a i n mammalian c e l l s that don' t have a Gj_, treatment with i n h i b i t o r s of p ro te in synthes is delays entry in to S-phase, thereby creat ing a G^ i n t e r v a l (L iskay et a l , 1980). These data would support the hypothesis that mammalian c e l l s must s a t i s f y a growth/protein re la ted requirement for entry in to S-phase. C y c l i c nuc leot ides The ro le of c y c l i c nucleot ides in the regu la t ion of c e l l growth has been studied extensive ly in recent years . An ear ly hypothesis associa ted cAMP and cGMP as antagonists c o n t r o l l i n g c e l l p r o l i f e r a t i o n where cAMP acted to a r res t c e l l growth, whereas cGMP st imulated growth (Goldberg et a l , 1974). However, although c y c l i c nuc leot ides may have an important r o l e in regu la t ing other c e l l u l a r processes, more recent work does not support th is hypothesis ( Z e i l i g and Goldberg, 1977). Nevertheless the l e v e l of cAMP does f lucuate depending on the growth s ta te of the c e l l (Pastan et a l , 1975) and the stages of the c e l l cyc le (Burger et a l , 1972; Costa et a l , 1976) i n d i c a t i n g that cAMP may regulate s p e c i f i c stages in the 36 growth cycle. The role of cGMP i n c e l l growth regulation however, remains unclear. Polyamines and ornithine decarboxylase The polyamines putrescine, spermine and spermidine are involved i n several aspects of nucleic acid and protein metabolism. Increased l e v e l s of polyamines and of ornithine decarboxylase, the key enzyme regulating their synthesis, occur very soon a f t e r stimulation of c e l l s (Pardee et a l , 1978; Rothstein, 1982). In some c e l l s increased l e v e l s of spermine and spermidine are correlated with passage through and entry into S and may be involved in DNA synthesis (Heby et a l , 1975; Boyton et a l , 1976) Prior addition of i n h i b i t o r s of ornithine decarboxylase to quiescent cultures prevents the entry of c e l l s into S-phase induced by serum stimulation (Boyton et a l , 1976). Conversely, agents that stimulate c e l l growth, such as hormones (Nissley et a l , 1976), tumor promoting chemicals (Yuspa et a l , 1976), and tumor viruses (Gazdar et a l , 1976) increase ornithine decarboxylase a c t i v i t y . However, although numerous experiments have shown that in some c e l l s a c e r t a i n minimum l e v e l of polyamines i s necessary for progression through the c e l l cycle, increased l e v e l s of polyamines and heightened ornithine decarboxylase a c t i v i t y are not always associated with enhanced mitotic a c t v i t y (Niskanen et a l , 1983). Therefore any true regulatory role for polyamines in c e l l growth remains unclear. Ions Calcium, and i t s ubiquitous binding protein calmodulin have been implicated in the regulation or modulation of various stages i n c e l l p r o l i f e r a t i o n including d i v i s i o n (Welsh et a l , 1979) and i n i t i a t i o n of DNA synthesis (MacManus et a l , 1978). Calmodulin and calcium may act by regulating in c e r t a i n c e l l s , the fluxes of other ions l i k e K +, which i n turn a f f e c t g l y c o l y s i s , and protein and nucleic acid synthesis (Durham, 1978). 37 Many mitogens such as serum and growth factors may act to mobilize cations in cells (Ralph, 1983). In calcium free media, serum refeeding has no effect, but stimulation will occur when calcium is added (Lobue and Lobue, 1984). Increases in the intracellular levels of calcium could regulate the second messenger function of cAMP, either by inhibiting adenylate cyclase, or stimulating cyclic nucleotide phosphodiesterase (Ross and Gilman, 1980). An increase in DNA synthesis also occurs when zinc, cadium and mercury are added to cultured cells (Rubin, 1975a) while magnesium deprivation induces quiescence in rapidly dividing chick embryo fibroblasts (Rubin, 1975b). A regulatory role for magnesium has been proposed in those metabolic pathways where the rate limiting reactions are transphosphorylation ones. Growth factors and hormones Since the early days of cell culture serum has been a necessary component of culture systems. Attempts to define factors in serum that induce DNA synthesis in fibroblasts has led to the discovery of a number of specific polypeptide growth factors. These include insulin-like growth factors I and II, somatomedins C and A, epidermal growth factor (EGF) and platelet derived growth factor (PDGF) (see review by Antoniades and Owen, 1982). These factors initiate their effects by binding to high affinity membrane receptors. This event is closely followed by ion movements associated with changes in membrane permeability. The steps leading from these preliminary events to the initiation of DNA synthesis and mitosis are obscure, although in the case of EGF and PDGF the activation of protein kinase, resulting in the phosphorylation of tyrosine residues of membrane proteins appears to be a critical event. Other events observed within minutes after binding of the factor include phospholipase A2 activation, effects on cyclic nucleotide concentrations, and increased uptake of amino acids, phosphate and glucose. Growth factors activate the machinery 38 for protein synthesis, and transcription, RNA processing, polysome assembly and the formation of polypeptide chains are also stimulated after treatment with these factors (Antoniades and Owen, 1982). The prereplicative phase (G0/G^) of the cell cycle has been suggested as the primary site of growth factor action (Pardee, 1978). Proliferation is initiated by "competence" factors which permit quiescent cells to enter the cell cycle and to respond to "progression factors" which enable the competent cell to progress toward the S-phase of the cell cycle. In the absence of competence factors cells are unable to proliferate in response to progression factors (Pledger et al, 1977). Competence and progression are two functionally distinct subphases of GQ/GJ. Growth factors such as PDGF, fibroblast growth factor, and macrophage-derived growth factor do not function well as progression factors. (Stiles et al, 1979, Wharton et al, 1982). Competence is transferable from one cell to another by fusion suggesting that the competent state may require the activation or synthesis of a cytoplasmic factor (Smith and Stiles, 1981). In contrast, progression factors like EGF, must be present continuously until S-phase is reached (Lembach, 1976). The recent demonstration that the amino acid sequence of PDGF shows considerable homology to that of the transforming protein (p28 s l s) of Simian sarcoma virus (Waterfield et al, 1983) implies that growth factors play an important role in alterations in cell proliferation behaviour. In some cell lines viral transformation may abrogate the requirement for competency perhaps by directly activating genes required for the induction of the competent state. For example, PDGF has been shown to be capable of inducing the competence genes c-myc, the cellular homologue of the avian transforming oncogene v-myc (Cochran et al, 1983; Kelly et al, 1983) and c-fos, the 39 cellular homologue of v-fos, the transforming gene of the FBJ sarcoma virus (Cochran et al, 1984). In addition, infection of hemopoietic cells by recombinant murine retrovirusis expressing v-myc oncogenes has been shown to result in the abrogation of the requirement for IL-3 (Rapp et al, 1985). C) Models of Cell Cycle Regulation In most cell culture systems the M, S and G2 phases of the cell cycle are fairly consistent in length. The phase however, is quite variable even between individual cells in the same population. Adverse culture conditions can greatly extend the Gj period. Most models dealing with the control of cell proliferation therefore propose Ĝ  as the most likely part of the cell cycle where growth factors act. Transition probability model According to this model the cell cycle is divided into two parts. A cell at birth is in the A state, where its activity is not directed towards replication (Smith and Martin, 1973, 1974). The cell remains in the A state until a transition occurs upon which it enters B state, and proceeds to mitosis through the usual sequence of stages Ĝ , S, G2, and M. Once initiated the cell in the B state progresses towards completion within a defined time Tg. The transition from A to B state occurs at random so that the probability of the transition occuring in unit time is constant. Environmental factors influence proliferation rate by altering the transition probability (Shields and Smith, 1977). Quiescent cells are described by this model as cells in the A state which have a very low K̂ . As originally proposed, this model takes as implicit the constancy of the B phase, although other investigators have demonstrated that variability in T B can account for a significant part of the total variability in the intermitotic time (Shields and Smith, 1977). In addition the original transition model could not account for the long lag period, on average ten AO hours, that occurs between s t imula t ion of the c e l l s and the i n i t i a t i o n of DNA s y n t h e s i s . To accomodate these f i n d i n g s , Brooks et a l (1980) postulated the two t r a n s i t i o n model. In th is vers ion quiescent c e l l s are located in an indeterminate state Q, and pass from th is s tate to A by completing a lengthy process L, of durat ion T L , which occupies most of the lag time. The process L i s i n i t i a t e d at random with a rate constant KQ and i s the f i r s t random t r a n s i t i o n . Once L i s completed the c e l l s enter the A s t a t e , which as in the previous model they leave at random with a rate constant to enter the B s t a t e . Both KQ and K^ depend on the environmental c o n d i t i o n s . The i n i t i a t i o n of c e n t r i o l e r e p l i c a t i o n and the separat ion of the mother and daughter centers have been proposed as p o t e n t i a l candidates for the two random t r a n s i t i o n s (Brooks, 1981). R e s t r i c t i o n point model From the k i n e t i c s with which quiescent c e l l s enter S-phase a f t e r the reversa l of adverse environmental c o n d i t i o n s , Pardee (197A) proposed that a s i n g l e major regulatory event must occur in i f a c e l l i s to progress to S-phase. This r e s t r i c t i o n or R point corresponds to the f i r s t of a s e r i e s of biochemical events, st imulated by environmental f a c t o r s which leads to the i n i t i a t i o n of DNA syn thes is . According to th is model, a c e l l w i l l pass through the R point i f condi t ions are favourable . Once the R point i s past , the c e l l i s committed to complete the rest of the c y c l e . Under adverse condi t ions a c e l l cannot pass through R, and must then cease p r o l i f e r a t i o n , remaining with a G^ DNA content. In th is model only one c o n t r o l point e x i s t s and i t i s responsive to many environmental f a c t o r s . A recent add i t ion to the model suggests that the R point may cons is t of the synthes is of a l a b i l e regulatory prote in to a c r i t i c a l threshold neccessary for commitment to reproduct ion (Rossow et a l , 1979). A p o s s i b l e candidate for th is R prote in i s a cytoplasmic inducer present in S-phase 41 cells which can stimulate nuclei from non-S cells to synthesize DNA (Roa and Johnson, 1970; Yanishevsky and Prescott, 1978). More recently, the demonstration that the 53 kilodalton T protein originally found in SV40 transformed cells is also present in high amounts in rapidly proliferating normal human cells (Dippold et al, 1981) has led to the hypothesis that it may be the R protein (Campiri et al, 1982). D) Principles of the ^H-Thymidine Cell Suicide Assay Since thymidine is a specific precursor of DNA, damage to the DNA is the cause of the detrimental effects of tritiated thymidine incorporation, which can result in cell killing, mutation, and loss of reproductive capability. Incorporation of radionuclides exposes DNA to radiation and/or transmutation effects, and therefore interferes with DNA replication, and affects to DNA structure in a manner that alters genetic function. The energy from incorporated tritium (̂ H), like that of other beta emitters is absorbed over a defined, relatively short path from the decay event. The radiation effects are thus produced in close proximity to this path. In the case of ^H-thymidine, the radiation is limited to•the site of radionuclide incorporation within the cell, i.e. the nucleus. The number of cells at risk in a cell suspension from exposure to solutions containing high specific activity ^H-thymidine will therefore be determined by the number of cells synthesizing nucleic acids at the time the labeled precursor is present (see NCRP report, 1969). Characteristics of tritium and thymidine 3 H 3 H has a half-life of 12.26 years and decays to 3He by emitting beta particles with a mean energy of 5.7 kev and a maximum energy of 18.6 kev. In water the mean range of the beta particle is .69 ym. 3̂  has been used to trace label a wide variety of organic molecules by exchange reactions in which some of the hydrogen atoms in the molecules are replaced by 3H. 42 Thymidine In the adult organism about 20g of DNA is synthesized in one day, mostly in the GI tract, skin, and bone marrow, resulting in a daily requirement of about 4g of thymidine. This amount is provided by the body by de novo synthesis from deoxyuridylic acid and by reutilization of thymidine from dead cells (Figure 7). Thymidine may be labelled with at carbon 6, or in the methyl group attached to carbon 5. When cells in S-phase are exposed to ̂ H-thymidine, either in vivo by injection, or directly in vitro, the labeled nucleoside easily crosses the membrane in an energy requiring process controlled by thymidine kinase (Cleaver, 1967). B) Modes of radiation injury The decay of 3 H incorporated into DNA produces the following two main types of effects: Transmutation When decays it is converted to helium, a process referred to as transmutation. The newly formed nuclei require a set of balance electrons different from those in the old nuclei. Transmutation will therefore cause a sudden disturbance in the orbital electrons followed by a rearrangement to form a stable configuration. This rearrangement may then inflict damage to the molecule containing the decayed atoms (Carsten, 1979). Transmutation effects are localized to the immediate site of nuclear decay, and are limited to those molecules to which the radionuclide is attached. Since the daughter nuclide of is a noble gas, it will not form a stable chemical bond. Hence transmutation of attached to a carbon results in the loss of the daughter nuclide and production of a reactive carbonium ion. The effects of decay have been studied in cultured Chinese hamster cells (Cleaver, 1977) and it was shown that transmutation of % to 3ffe in the carbon 6 position in thymine in the DNA resulted in single-strand and double- 4 3 0 O H N HN CH Deoxyuridylic acid (dUMP) ~ CH I d e o x y r i b o s e - P j6 10 dTN - m e t h y l e n e - tetrahydrofolate H0CH2 / ° rt^C CH N Thymidine THYMIDYLATE SYNTHETASE H H OH H cell membrane THYMIDINE KINASE dihydrof olate jm K A D P deoxythymidylic acid (dTMP) O ;—CH, d e o x y r i b o s e - P TMP KINASE A T P NJ ADP-4 dTDP dATP DNA template formation dGTP dCTP ATP \ | ADP̂ TI dTTP H NUCLEOSIDE DIPHOSPHATE KINASE DNA replication DNA POLYMERASE DNA Figure 7 . Schematic i l l u s t r a t i n g thymidine incorporation into DNA. 44 strand breaks. However, the contribution by transmutation to DNA strand breakage in mammalian cells is minor compared to the effects from the beta particle, though this latter effect may be important when mutations are considered. Irradiation by emitted beta particles A beta particle from a decayed atom reacts with other portions of the molecule and/or with other nearby molecules by direct and indirect action. The beta particle ejected from % decay produces a densely ionized column with an average track length of 1 urn and with an average of 160 ionizations along the track. The ionizing effect of incorporation therefore, is not restricted to the molecular site but to an area of about 1 ym in radius. Beta particles produce damage primarily by the formation of free radicals which are neutral atoms or molecules having an unpaired electron. When 3{j incorporated into the DNA of a cell nucleus decays, the electron emitted reacts with the surrounding water to form two types of free radicals: e- + H20 .̂ H- + OH- (hydrogen) (hydroxy) which recombine to yield H- + H- > H2 OH- + OH- *H 20 2 Organic free radicals are formed when organic molecules (RH) combine with the hydroxy free radical RH + OH- ^R- + H20 Most of the free radicals formed have a short lifespan, generally less than lO-lO seconds. Since they contain unpaired electrons they are very reactive, and can oxidize or reduce the biological molecules within the cell (see review by Spinks and Marks, 1976). 45 In the presence of oxygen the emission of beta particles can create energy excited oxygen species known as singlet or triplet oxygen. When this process occurs, certain electrons in the oxygen atom are raised to higher energy or excited states. This excitation energy can be transferred from one part of a molecule to another, or between molecules, altering normal chemical bonds within the DNA helix (Peak, 1981; Piette et al, 1981; Houba-Herin et al, 1982). Biological effects of ^H-thymidine incorporation Chromosomal aberrations The first report on Ĥ induced chromosome breaks was published in 1958 (Taylor, 1958). Many authors have described qualitative similarities between the effects of ^E decay on chromosomes and those produced by external ionizing radiation such as X-rays. The production of chromosome aberrations in cultured mammalian cells by incorporated 3H- thymidine has been studied in Chinese hamster cells (Dewey et al, 1965, 1967; Brewer and Olivieri, 1973) and in monkey cells (Hung et al, 1973). More recently these studies have been extended to human leukocytes (Vig et al, 1968; Vig, 1974; Bosian et al, 1977; Hori and Nakai, 1978) and fibroblasts (Nalarajan and Meyers, 1979). Some of the effects induced by decay include double and single strand breaks, base destruction, chromatid gaps, breakage of glycoside-phosphate ester linkages, chromatid interchanges, and cross linking. The efficency of incorporated methyl ^H-thymidine for producing DNA strand breaks has been calculated as 2.1 single strand breaks for 1 decay (Cleaver et al, 1972). In addition, the production of chromosomal aberations by incorporated ^E is an essentially linear function of the number of decays occuring in the labeled nucleus (Dewey et al, 1965,1967). • 4 6 Figure 8. Schematic illustrating the most critical effects of ^H-thymidine incorporation into DNA. Tritium decays to helium3 by emission of a beta particle which can react with other portions of the molecule or with other nearby molecules. The action of the beta particle may be direct or indirect, as with the interaction of the particle with a molecule of water resulting in the formation of free radicals. The most common of many types of DNA damage are shown here: A) single or double strand breaks in the DNA helix; B) loss of a base; C) cross-linking between two DNA strands. 47 Impairment of gene function Incorporation of 3H-thymidine results in damage to the DNA which may impair several critical aspects of gene function. Single or double-strand cuts in the replicating forks of DNA may interfere with the unwinding and winding actions of replicating DNA (Cairns and Daverns, 1966). The induction of specific enzymes by bacteria was shown to be impaired after exposure to 3H-thymidine (Rachmeler and Pardee, 1963). Other studies have demonstrated the mutagenic efficiency of 3H-thymidine in bacteria (Person et al, 1976), in Drosophila (Olivieri and Olivieri, 1965) and in the mouse (Greulich, 1967; Bateman and Chandley, 1962). Since the capacity of the mammalian cell to reproduce depends on the replicating ability of its DNA, such extensive damage to chromosomes would result in the inability of the cell to divide. Ultimately however, the damage to DNA that occurs as a result of 3H decay may depend as much on the effectiveness of the repair processes as it does on the nature of the original lesion. When the cell attempts to rejoin chromosome breaks for example, the broken ends from one break may be joined incorrectly with those from another. Such breaks may be recombined to form various types of chromosome aberrations, such as translocations, inversions, rings, and other types of structural rearrangements (Upton, 1982). In this way even simple breaks may be lethal to the parent cell or its immediate progeny (Figure 8). 3) LONG TERM BONE MARROW CULTURES The first successful culture system for the prolonged proliferation of hemopoietic stem cells was described by Dexter and his associates (1977) using murine bone marrow. The essential feature of this culture system was the prior establishment of a marrow derived adherent cell layer, upon which after 2 or 3 weeks a second inoculum of marrow cells could be seeded. 48 Maintenance of substantial numbers of hemopoietic cells was dependent on the formation of an adequate adherent layer in which numerous foci of large, lipid-containing cells were present. In turn the development of these fat cells was contingent upon a few selected lots of horse serum. However, Greenberger (1978a) reported that addition of corticosteroids could overcome the deficiency in unsuccessful lots of horse serum by increasing the number of lipid-containing cells. Furthermore, supplementation of serum with corticosteroids permitted the establishment of long term cultures with a single marrow inoculum, a procedure which had limited success previously. Removal of the nonadherent cells on a weekly basis permitted their assay in conventional methylcellulose culture systems. In such a manner the continuous long term production of CFU-S (Dexter et al, 1977b) as well as committed progenitors of the granulocytic/macrophage, erythroid (Testa and Dexter, 1977; Gregory and Eaves, 1978) and megakaryocytic lineage (Williams et al, 1978b) could be demonstrated for several months. In addition the cumulative numbers of progenitors removed from the system at each weekly medium change may be greater than the number of cells present in the original innoculum suggesting de novo production of progenitors from the adherent layer (Eaves et al, 1983a). Further evidence for a progenitor:progeny relationship between the adherent and nonadherent cells is the demonstration that CFU-S in the adherent layer exhibit a higher self-renewal capacity than those in the growth medium (Mauch et al, 1980) and the demonstration of maintenance of a hemopoietically active non-adherent fraction with the removal at each week of a l l of the cells in that fraction (Greenberger et al, 1979). .Initial attempts to apply the principles of murine long term culture to human marrow were disappointing. Although recent improvements have permitted 49 the survival and perhaps limited proliferation of primitive granulocytic and erythroid progenitors in the nonadherent fraction of human cultures for several weeks (Greenberger et al, 1979; Gartner and Kaplan, 1980; Coulombel et al, 1983a) progenitor yields are not comparable to those obtained with the murine system, and most investigators would agree that existing culture conditions are not yet optimal. Nevertheless long term culture techniques have been usefully applied to marrow from normal subjects (Eaves et al, 1983; Coulombel et al, 1983a), from leukemic patients (Coulombel et al, 1983b) and from patients with myeloproliferative disorders (Powell et al, 1982; Coulombel et al, 1983b) and have offered new insights into normal and neoplastic hemopoietic mechanisms. A) Description of the System Upon initiation of mouse or human cultures there is an ini t i a l attachment and subsequent proliferation of marrow cells over a period of 2 to 3 weeks. A confluent multilayer is formed, composed of a variety of hemopoietic and nonhemopoietic cells of marrow origin (Dexter et al, 1977a; Coulombel et al, 1983a). Formation of "cobblestone" areas occur which are believed to represent foci of primitive hemopoietic cells (Figures 9 and 10) (Gartner and Kaplan, 1980; Greenberg et al, 1981). Above the adherent cells a layer of pseudo-adherent cells are found, with the largest concentration of these highly refractile cells often located just above the hemopoietic islands. These cells appear to be enmeshed in a viscous layer of material and are capable of only limited movement upon gentle agitation of the dish. They are not removed when the growth medium is changed. Hemopoietic cells The progress of the long term culture can be assessed by monitoring the total cell and progenitor cell content of the nonadherent fraction. During the initial 2 to 3 weeks of culture there is a dramatic 50 Figure 9. In s i t u s t a i n i n g of a putat ive cobblestone are in the adherent layer of a normal long term cul ture at 3 weeks of incubat ion (May-Grunwald-Giemsa s t a i n i n g , X160). 51 fa l l in the number of a l l cell types in the nonadherent fraction, followed by a plateau phase of several weeks duration when an equilibrium is reached between the rate of production and loss of the hemopoietic cells. The majority of the supernatant population is composed of granulocytes in various stages of differentiation up to mature neutrophils and macrophages. The mature cells produced in human cultures are comparable to fresh peripheral blood granulocytes in physiological properties and retain many biological functions associated with bactericidal capacity in vivo (Greenberg et al, 1981). Though primitive BFU-E can be detected in the supernatant for several weeks, morphologically recognizable erythroid cells rapidly disappear and are not found thereafter unless culture conditions are specifically modified to allow their production in vitro. Simularly, CFU-E and mature BFU-E can rarely be detected in the nonadherent fraction after 2 or 3 weeks (Dexter et al, 1978; Gregory and Eaves, 1978; Eaves et al, 1983) B and T cell precursors at a primitive stage of differentiation are present in murine long term cultures, but as with erythropoiesis, culture conditions must be modified to permit production of their mature progeny in vitro (Schrader and Schrader, 1978; Jones-Villeneuve et al, 1980; Whitlock and Witte, 1982; Dorshkind and Phillips, 1983). The lymphoid potential of human long-term cultures has not yet been extensively investigated. The development of a technique for the suspension of the adherent layer from human cultures for assessment in clonal assay systems has permitted the evaluation of their hemopoietic progenitor content (Coulombel et al, 1983a). Such studies have shown that after 4 weeks of culture the majority of BFU-E and CFU-C present in the cultures are located in the adherent layer. In addition more primitive progenitors, those capable of forming mixed colonies 52 Figure 10. Morphological appearance of a PV long term cu l tu re adherent l a y e r . These photographs were taken of the same cu l tu re d ish at vary ing times a f te r i n i t i a t i o n . A. Adherent is lands present a f te r 4 days of incubat ion (X80). B. Another area of adherent is lands 2 weeks a f t e r i n i t i a t i o n . At th is time the majori ty of the red blood c e l l s have lysed (X80). C. The adherent layer a f te r A weeks of incubat ion showing the presence of numerous fat laden c e l l s (X160). D. The adherent layer 7 weeks a f t e r i n i t i a t i o n . A few fa t g lobules from an adherent is land are s t i l l present (X160). 54 with granulocytic and erythroid elements (CFU-G/E) as well as CFU-C with very high proliferative capacities are located exclusively in the adherent layer after 2 to 3 weeks of culture. What determines such discrepancies in the growth of hemopoietic cells between the adherent and nonadherent fraction of long term cultures is not known, but may indicate a role for close interactions with stromal elements. Stromal elements The composition of the stromal layer is controversial. In mouse cultures three main cell types were originally described on the basis of electron microscopy (Allen and Dexter, 1978): flat endothelial-like "pavement" cells, macrophages, and large lipid-containing cells. The presence of comparable cell types in human long term cultures has also been observed (Figure 11). In particular the appearance of fat-laden cells was seen in the in i t i a l attempts at culturing human marrow, and as in the murine system, an association of such cells with the more successful cultures was noted (Gartner and Kaplan, 1980). Recently investigators have attempted to identify cell types in the adherent layer of murine cultures by delineating their secretion products. One laboratory reported finding collagen types I and III, and fibronectin, indicating the presence of fibroblasts, although no evidence for the presence of endothelial cell products was found by these authors (Bentley and Foidart, 1980; Bentley, 1982; Bentley and Tralka, 1982). However other laboratories have demonstrated the secretion of collagen types I, III, and IV, fibronectin, and lamimin in the adherent layer, suggesting the presence of both fibroblast and endothelial cell types in murine and human long term cultures (Castro-Malaspina et al, 1981; Keating and Singer, 1983; Zuckerman and Wicha, 1983; Zuckerman et al, 1983). Endothelial cells have been further characterized in human cultures as possessing a well developed submembranous 55 Figure 11. Cytospin preparations stained by the May-Grunwald-Giemsa procedure i l l u s t r a t i n g the morphological appearance of c e l l s from a normal long term culture 3 weeks a f t e r i n i t i a t i o n (XAOO). A. C e l l s from the nonadherent f r a c t i o n . B. C e l l s from the adherent f r a c t i o n , a f t e r detachment by t r y p s i n . 56 microfilament layer, reactivity to Factor VIII antibody (Keating and Singer, 1983) and Weibel-Palade bodies, which are specific endothelial organelles (Allen, 1981). These cells may function in culture in a similar manner to their counterparts in vivo; mature granulocytes have been observed migrating through endothelial monolayers leading to an in vitro "transmural passage". Macrophages, though hemopoietically derived are also part of the adherent layer and may be considered as part of the stroma in vivo. Cells which exhibit properties common to macrophages, such as esterase positivity and the presence of receptors are routinely observed in long-term cultures, though the F c receptor, an essential feature of mononuclear phagocytes, was not demonstrable (Bentley and Foidart, 1980). Recently another cell type, termed "blanket cells" have been characterized in long term cultures (Dexter et al, 1984). These are large, well spread alkaline phosphatase positive cells which are found overlaying numerous tightly packed macrophages and granulocytes in the cobblestone areas of the cultures, and may have a functional role in granulopoiesis. D) The Role of the Adherent Layer The importance of the adherent layer in the maintenance of hemopoiesis can be inferred from a number of studies. Stem cells are poorly maintained in siliconized cultured flasks where cell adherence is prevented. Cultures showing poor development of adherent cells are unsuccessful in producing stem cells for any extended period of time. Perhaps the most conclusive evidence for the role of adherent cells in in vitro hemopoiesis is given by experiments with genetically anemic mice (Dexter and Moore, 1977). The defect in the hemopoietically inefficient microenviroment characteristic of the Sl/Sl d mouse may be reproduced in long-term cultures. Defective hemopoiesis results when norma 1, Sl/Sl d stem cells are cultured on a Sl/Sl d 57 adherent layer, although S1/S1U stem cells could be maintained on a normal adherent layer. These studies suggest that interactions between competent stem cells and an appropriate microenviroment are critical for the continuous in vitro proliferation of hemopoietic cells. Cell to cell interactions The role of cell to cell interactions in murine long-term cultures was investigated by Bentley (1981) who placed hemopoietic cells in a diffusion chamber which in turn, was placed over a long term culture adherent layer along with free-floating cells. The rate of loss of CFU-S from the diffusion chamber where no direct interaction with stromal elements could occur, was greater than the rate of loss from the free floating population. Successful hemopoiesis in vitro is correlated with the presence of large cobblestone areas where granulocyte development occurs. All the stromal cell types previously described are found here in intimate contact with the developing hemopoietic cells. Junctional membrane complexes between the hemopoietic and stromal elements may be indicative of intercellular metabolic cooperation. Extensive interdigitive coupling of blast cells to macrophages has also been observed (Lambertsen, 1984). In addition, numerous coated vesicles present in the closely opposed regions of membrane between the developing hemopoietic cells and macrophages are suggestive of the short- range release and receptor-mediated endocytosis of regulatory factors (Allen and Dexter, 1982). Such intimate opposition of large areas of membrane also occur extensively between adjacent macrophages across the entire cobblestone area. Though normal long-term cultures do not support the further differentiation of erythroid progenitors, the addition of anemic mouse serum or Ep coupled with a mechanical stimulus will result in full erythroid 58 maturation (Allen and Dexter, 1982). Modifications in the adherent layer occur with the induction of erythropoiesis, and erythroblastic islands consisting of synchronously maturing cohorts of erythroid cells in close association with a central macrophage appear. The developing erythroid cells have large areas of closely opposed membrane, gap junctions, and possible reciprocal vesicular activity, indicative of cell to cell cooperation. Simular associations between clusters of normoblasts and erythrocytes with macrophages have been observed in human long term cultures (Hocking and Golde, 1980). Regulatory factors Despite the continuous proliferation of granulocytic cells in vitro initial studies indicated that CSF was not present in long term murine or human cultures (Dexter et al, 1977b; Toogood et al, 1980). Furthermore the addition of anti-CSF antiserum on a weekly basis to long term cultures did not reduce the production of granulocytes or other hemopoietic cells (Dexter and Shadduck, 1980). A marked decrease in granulocyte production occured when a crude source of CSF was added Dexter et al, 1977b), although more recent experiments have shown that this effect could be attributed to impurities in the CSF preparation as the addition of a more purified CSF had no effect (Dexter and Shadduck, 1980; Williams and Burgess, 1980). Other studies have provided evidence for CSF production in long-term cultures by demonstrating colony formation by fresh hemopoietic cells immobilized in agar overlaying the adherent layer (Gualtieri et al, 1982). The observation of an inverse relationship between the number of hemopoietic cells and the levels of CSF in long-term cultures led to the suggestion that a local regulatory mechanism exists resulting in feedback inhibition of CSF production (Gualtieri et al, 1984) or the consumption of CSF by differentiating myeloid cells (Gualtieri et al, 1982; Heard et al, 1982). 59 The development of a sensitive radioimmunoassay for CSF has permitted its detection in virtually a l l long term media examined (Shadduck et al, 1983). In addition, the removal of inhibitory substances, or the concentration of supernatant has resulted in the routine detection of biological activity in media previously considered inactive (Shadduck et al, 1983). Recently a number of cell lines have been generated from the adherent layer which are active producers of CSF providing further evidence that CSF is involved in the control of cell production in this system (Harigaya et al, 1981; Cronkite et al, 1982; Lanotte et al, 1982). The production of erythropoietic factors in long term culture is less well documented. The presence of Ep is not demonstrable in this system. Attempts to measure levels of burst promoting factor were not successful, as conditioned media from the cultures was found to be strongly inhibitory to burst formation (Eliason et al, 1982). A lack of BPA in this system might be suggested by the absence of erythroid development. However, this does not proceed when BPA is added to the cultures. On the other hand, subcultures of adherent cells are capable of producing BPA, which has led to the hypothesis that in the intact system other cells in the culture may block BPA production (William et al, 1979). The effect of anemic mouse serum in supporting erythropoiesis in these cultures (Dexter et al, 1981) could result from inhibition of those cells which in turn prevent the production of BPA. The demonstration that the removal of certain cell types from the cultures can greatly increase the yield of BFU-E provides additional evidence for the presence of inhibitory cells (Williams et al, 1979). In an analysis of stem cell cycling in murine long term cultures Toksoz and his associates (1980) provided evidence that the adherent layer produces factors that can modify the proliferative activity of stem cells. In earlier 60 experiments u t i l i z i n g ^H-thymidine (Dexter et a l , 1977b) these i n v e s t i g a t o r s had reported that CFU-S p r o l i f e r a t i v e a c t i v i t y in long term cu l tu res fo l lows a c y c l i c pattern re la ted to the feeding regime. Within 1 to 2 days a f t e r replacement of the growth medium and demipopulation of the cu l tu res a high proport ion of the CFU-S were in S phase. However, wi th in 4 to 7 days a f t e r the cu l tu res were fed , the proport ion of CFU-S in ac t ive c e l l cyc le p rogress ive ly decreases u n t i l a majori ty are quiescent . Fol lowing a fur ther feed the pat tern of s t imula t ion of c y c l i n g a c t i v i t y and eventua l ly revers ion to a noncycl ing s ta te could be repeated. During th is e n t i r e per iod CFU-C remained in a constant high c y c l i n g s t a t e . Subsquently these authors demonstrated that th is c y c l i c pattern of CFU-S p r o l i f e r a t i o n was cor re la ted with the re lease in to the medium of st imulatory and i n h i b i t o r y fac to rs simular to those descr ibed prev ious ly (Lord et a l , 1976, 1977) in normal and regenerat ing bone marrow. When CFU-S were in ac t ive c e l l cyc le one day a f t e r feeding the c u l t u r e s , the concentrat ion of the st imulatory mater ia l increases r e l a t i v e to the i n h i b i t o r , while the reverse s i t u a t i o n i s found severa l days l a t e r when the CFU-S are quiescent . In a d d i t i o n , the p r o l i f e r a t i v e a c t i v i t y of CFU-S, whether in an ac t ive or noncycl ing state could be modif ied by the add i t ion of the appropriate f a c t o r s . In fur ther experiments these authors concluded that the add i t ion of f resh media or the absolute c e l l number was not the primary st imulus for the product ion of these fac tors (Dexter et a l , 1980). Simple mechanical a g i t a t i o n or the replacement of spent media could reproduce the c y c l i c pat tern of CFU-S p r o l i f e r a t i o n . This f ind ing led to the formulat ion of a model in which the proximity of CFU-S to the adherent layer i s the c r i t i c a l s t imu lus . A stem c e l l wi th in the adherent layer could s i g n a l i t s presence lead ing to the production of i n h i b i t o r . Removal of the stem c e l l , by 61 mechanical means, or by d i f f e r e n t i a t i o n , death, or migrat ion would r e s u l t i n s t imula tor product ion. It i s of in te res t that in th is system the p r o l i f e r a t i o n of CFU-S r e s u l t s in stem c e l l d i f f e r e n t i a t i o n , not s e l f - renewal, as there i s no increase in the t o t a l number of CFU-S in the c u l t u r e . Role of the e x t r a c e l l u l a r matrix (ECM) In teract ions between parenchymal c e l l s and the e x t r a c e l l u l a r matrix have a cen t ra l ro le in d i f f e r e n t i a t i o n in a v a r i e t y of c e l l systems (Grobstein , 1975). Recent i n t e r e s t therefore has focussed on the p o s s i b i l i t y that hemopoietic stem c e l l d i f f e r e n t i a t i o n may be in f luenced in a s i m i l a r manner by in te rac t ions with connective t i ssue components. Examination of the d i s t r i b u t i o n of glycosaminoglycans (GAGs) i n long-term cu l tures has shown a constant pattern of sulphated GAGs assoc ia ted with the adherent l a y e r , where heparan sulphate i s a main component, and the medium, where chondro i t in sulphate predominates (Spooner et a l , 1983). Add i t ion of 3 - D - x y l o s i d e s resul ted in a dramatic increase in chondro i t in sulphate content in the growth medium and had a marked st imulatory e f f e c t on hemopoiesis in suboptimal cu l tures by increas ing the porport ion of CFU-S in S phase. Other inves t iga to rs have demonstrated that high concentrat ions of chondro i t in sulphate and heparin sulphate could block the response of CFU-E to the add i t ion of Ep while other sulphated GAGs had no e f fec t (Ploemacher et a l , 1978). On the other hand, low concentrat ions of chondro i t in sulphate had a s t imulatory e f fec t on CFU-E p l a t i n g e f f i c i e n c y . A comparable e f f e c t on g r a n u l o c y t i c colony formation was not observed. Other inves t iga to rs have invest igated the ro le of f i b r o n e c t i n in c e l l adhesion in long term c u l t u r e s . F ib ronec t in has been detected on var ious adhesion s i t e s , inc lud ing substratum attachment surfaces of adherent c e l l s , between f i b r o b l a s t o i d elements and at s i t e s of i n te rac t ions between hemopoietic c e l l s and adherent c e l l s (Bentley and T r a l k a , 1982). 62 4) THE MYELOPROLIFERATIVE DISORDERS The term "myeloproliferative disorders" (MPD) was introduced by Dameshek (1951) to describe a group of closely related syndromes - polycythemia vera (PV), essential thrombocytosis (ET), chronic myeloid leukemia (CML) and myeloid metaplasia with myelofibrosis - a l l of which share a number of clinical and pathological features. The most outstanding unique characteristic of each of these diseases is the elevation of a single hemopoietic cell lineage (i.e. RBC in PV, platelets in ET, granulocytes in CML). However, the common involvement of the lineages suggested that each originated from the disordered growth of a pluripotent stem cell, although conclusive evidence for the existence of such a cell was not obtained until ten years later. Evidence that these diseases represent clonal neoplasms was also established at about the same time with the consistant demonstration in CML patients of the Philadelphia (Phi) chromosome in multiple hemopoietic cell lines (Figure 12) (Nowell and Hungerford, 1961; Whang et al, 1963; Golde et al, 1977). Another approach to the study of clonality in the MPD has made use of a different genetic marker system, the isoenzymes of glucose-6-phosphate dehydrogenase (G6PD). The gene for G6PD is on the X chromosome. Since one X chromosome in each cell in women is inactivated at random early in embryogenesis and the choice is then fixed in a l l subsequent progeny, most tissues in G6PD female heterozygotes are composed of equal numbers of cells synthesizing one or the other isoenzyme type (Lyon, 1961). The isoenzyme product of the usual gene Gd^ and of some variants, such as Gd̂ , may be separated by electrophoresis. Using this marker system only one isoenyzme type was seen in the circulating erythrocytes, granulocytes and platelets of G6PD heterozygotes with Phi negative and Phi positive CML (Fialkow et al, 63 1967, 1977; Douer et al, 1981), PV (Prchal et al, 1976), ET (Fialkow et al, 1981) and myeloid metaplasia with myelofibrosis (Jacobson et al, 1978) even though in each case mosiacism was demonstrated in their skin fibroblasts. When individual hemopoietic colonies were tested, direct evidence of early progenitor involvement in these neoplasms was also obtained (Aye et al, 1973; Moore and Metcalf, 1973; Singer et al, 1979; Dube et al, 1981). Recently the demonstration of clonality in multilinage colonies has confirmed the involvement of pluripotent cells (Douer et al, 1981; Dube et al, 1984a). Nevertheless, the mechanism responsible for the selective growth advantage of the progeny of the abnormal clone remains obscure. Typically, the MPD exhibit a prolonged chronic steady state which may persist for a decade or more with minimal intervention, although progression to an acute leukemia (blast crisis) commonly occurs within 3 to 5 years in patients with CML. Despite the extensive number of progeny produced by the single abnormal pluripotent stem cell, essentially normal, functional mature cells are formed. However, a number of hematological abnormalites may be found to a varying degree in each disease. These include abnormalities of red cell, leukocyte and platelet counts, extramedullary hemopoiesis, abnormal leukocyte alkaline phosphatase values and elevations in serum B-̂  and B^2 binding capacity. In addition, a number of investigators have shown the presence of erythroid progenitors capable of terminal differentiation in vitro in the absence of added Ep in CML (Eaves and Eaves, 1979), PV (Prchal and Axelrad, 1974; Eaves and Eaves, 1978) and ET (Prchal and Axelrad, 1974; Eaves et al, 1983b). Though diagnostic for PV (Eaves et al, in press) the expression of Ep independent growth is much more variable in CML and ET but s t i l l suggests a common biological abnormality in a l l three disorders (Eaves et al, 1980). 64 Figure 12. Metaphase chromosomes from a CML patient. In this particular case the Ph^ chromosome (circled) was formed by the transfer of part of chromosome 22 to chromosome 16. This metaphase was obtained from an erythroid colony in methylcellulose culture established with cells from the nonadherent fraction of a 5 week old reconstituted long term blood culture. 65 A) Chronic Myeloid Leukemia The most outstanding clinical feature of CML is the elevation in the number of circulating white blood cells (WBC) which may rise to as much as 100 times normal values. In addition to mature granulocytes, immature forms are also found. The age of onset of CML is variable, but the disease is most frequent above the age of 40. Typical symptoms at presentation include fatigue, low grade fever, weight loss, pallor, and discomfort due to an enlarged spleen or liver (Wintrobe, 1976). Laboratory studies indicate that a l l stages of the neutrophilic series, from myeloblasts to segmented neutrophils are present, usually in normal ratios. Anemia is often present at diagnosis. Approximately half of the patients will have some degree of thrombocytosis, and in some cases this may be severe with platelet counts of over 10̂ /mm3. Most of these symptoms can be reduced or elimimated in the chronic phase by treatment with cytotoxic agents, such as busulfan or hydroxyurea. CML usually terminates in a blast crisis with many of the characteristics of acute myeloid leukemia. The onset of blast crisis in relation to time of diagnosis is quite variable, but most series indicate a median time to transformation of about 3 years. Despite new and more agressive therapeutic modalities this median time has not changed in 50 years (Minot et al, 1924). During blast crisis the WBC may rise to extreme levels, but the more important feature is the rapid accumulation of immature blasts. These appear to resemble immature members of the neutrophil series in many cases although a l l lineages may be involved including cells of the B lymphoid series. As the blast phase proceeds normal blood elements decrease in number, recurrent infection and hemorrhage occur, and these are usually the cause of death. Remissions are rare, and even when they occur tend to be of short duration, seldom lasting more than 6 months. There are no well 66 documented cases of cure with chemotherapy, though some success has been obtained with bone marrow transplantation (Armitage et al, 1984). Chromosomes in CML The majority of CML patients (80-95%) have a unique chromosomal marker, the Philadelphia chromosome (Ph^) (Nowell and Hungerford, 1960). Though initially described as an abnormally small G group chromosome, the development of banding techniques enabled Rowley (1973) to identify the Ph1 chromosome as a balanced translocation between the long arm of chromosome 9 and the long arm of chromosome 22 - t(9;22)(q34;qll). Most patients will have the classic Ph-*- chromosome as the sole cytogenetic abnormality at least in the chronic phase of their disease. Occasionally, a complex translocation involving several chromosomes, particularly no. 17 may be found (Rowley, 1980). An atypical Pĥ - chromosome does not appear to influence survival as the clinical course of these cases does not differ significantly from patients with the typical 9;22 translocation (Sandberg, 1980). Additional changes in karyotype occur at blast crisis in about 75-80% of a l l Phi positive patients. These secondary aberrations are superimposed on the Ph1 cell line, and are nonrandom. In order of frequency about 80% of the cases will involve one or more of the following: 1) double Ph*; 2) additional no. 8; 3) abnormalities of 17q; 4) additional no. 19; 5) additional no. 21 (Rowley, 1976; Pearson et al, 1983). In some patients these additional chromosomal aberrations may be present weeks or months before blast crisis suggestive of an "accelerated phase" of progressive genetic change finally leading to disruption of normal differentiation potential (Sandberg, 1980). New interest in the relationship of the Pĥ  chromosome to the the abnormal control of hemopoietic differentiation in CML has arisen with the demonstration that the translocation of the human cellular homologue of the transforming sequence of Abelson murine leukemia virus (c-abl) from 67 chromosome 9 to a specific region on chromosome 22q (de Klein et al, 1982) is a consistent feature in Phi positive cells, even when more complex translocations are involved (Bartan et al, 1983). Subsequently it was shown that the fusion region of chromosome 9 to chromosome 22 was contained within a 5.8 kilobase (kb) segment. This region on chromosome 22 was designated bcr (breakpoint cluster region) (Groffen et al, 1984). The translocation fuses the c-abl oncogene to a bcr gene on the Phi chromosome (Heisterkamp et al, 1985; Shtivelman et al, 1985) resulting in the formation of a hybrid mRNA of about 8 kb (Collins et al, 1984; Gale and Canaani, 1984). In turn, the novel mRNA codes for an altered 210K molecular weight polypeptide which has been identified in the leukemic cells of essentially a l l CML patients and CML cell lines examined (Collins et al, 1982; Blick et al, 1984; Konopka et al, 1984). The bcr-abl polypeptide from CML cells was shown to possess tyrosine kinase activity, unlike its normal 145K counterpart (Konopka et al, 1984; Konopka et al, 1985). Differentiation potential of the CML stem cell The Phi chromosome has been found in 90-100% of bone marrow metaphases in CML patients early on, indicating its presence in both granulocytic and erythroid lineages ( Whang- Peng et al, 1963). The presence of the marker in metaphases of cells capable of hemoglobin synthesis as measured by radioactive iron uptake was more direct evidence for erythroid cell involvement in the abnormal clone (Rastrick et al, 1968). In addition metaphases of cells from individual erythroid (Dube et al, 1981), granulocytic (Chervenick et al, 1971) and macrophage (Golde et al, 1977) colonies grown in vitro have also demonstrated the Phi chromosome. Megakaryocytic involvement in the neoplastic clone has been confirmed by the presence of the Phi marker in tetraploid and octaploid bone marrow cells (Tough et al, 1963; Whang-Peng et al, 1968). 68 Early reports indicated that the Pĥ  chromosome was restricted to cells of the myeloid lineages. However, a minority of patients with CML progress to a blast phase in which the predominant cell types have lymphoid characteristics, including a cellular content of a unique enzyme, terminal deoxynucleotidyl transferase (TdT) previously identified in the lymphoblasts of patients with acute lymphoblastic leukemia (ALL) (McCaffrey et al, 1973, 1975). These blast cells may share certain antigenic features with ALL lymphoblasts (Janossy et al, 1976), contain cytoplasmic immunoglobulin (LeBien et al, 1979) and demonstrate immunoglobulin gene rearrangements typical of pre-B cells (Bakhshi et al, 1983). Relatively early it was postulated that a myeloid-lymphoid stem cell was the site of the lesion in CML (Boggs, 1981). Considerable evidence has now been presented for the involvement of the B cell lineage even in chronic phase CML. The Ph^ chromosome has been detected in cells which also demonstrated the presence of surface immunoglobulin, a marker for B cells (Bernhiem et al, 1981). Cytogenetic studies of Epstein Barr virus (EBV) transformed lymphoid cell lines from a CML patient heterozygous for G6PD isoenyzmes has also demonstrated the presence of the Pĥ  chromosome in some of these (Martin et al, 1980). In other studies Pĥ - positive B cells were found in about 25% of CML patients after transformation with EBV (Nitta et al, 1985). The possible involvement of T lymphocytes in the malignant clone remains controversial. PHA stimulated T lymphocytes have consistently been found to be cytogenetically normal even in a patient in chronic phase CML of long duration (Kearney et al, 1982; Nitta et al, 1985). Isoenzyme studies have shown T lymphocytes to be polyclonal, whereas in the same patient the B lymphocytes demonstrated only one isoenzyme type (Fialkow et al, 1978). On the other hand, the presence of Phi-positive T lymphocytes in chronic phase 69 CML has been reported by two groups (Shabtai et al, 1980; Itani and Hashino, 1982). In addition, several examples of lymphoblastic transformation of CML have been reported in which T cell involvement was established by the presence of T cell specific surface antigens on Phi-positive cells (Janossy et al, 1978; Hernandez et al, 1982; Griffin et al, 1983; Herrman et al, 1984; Jacob and Greaves, 1984). Moreover, Ph1 positive T lymphoblasts have been demonstrated in the lymph nodes of CML patients, without detectable preceeding or concurrent lymphoblastic transformation in the marrow (Palutke et al, 1982; Jacob and Greaves, 1984). Although the majority of investigators have not been able to demonstrate T cell involvement in CML the possibility remains that since T lymphocytes are very long lived cells, they may be progeny of the abnormal stem cell which antedate the development of the malignant lesion in that cell. Residual normal stem cells in CML Since the goal of chemotherapy in CML is eradication of the abnormal clone, the question of residual normal hemopoiesis in these patients is of considerable therapeutic importance. Early studies indicated the presence of the Phi chromosome in a l l dividing hemopoietic cells examined, even in treated patients whose counts have been returned to normal levels. In addition, application of the in vitro colony assay system demonstrated the a l l CFU-C had the same isoenyzme type as the abnormal clone in several patients initially studied in this way (Fialkow et al, 1978b; Singer et al, 1979b). In other studies however, some Ph1 -negative granulocyte colonies were seen in 2 out of 4 CML patients (Chervenick et al, 1971). More recently, in serial cytogenetic studies, 10 of 41 Phi-positive CML patients demonstrated Phi-negative cells in their early chronic phase though the percentage of such cells decreased during the course of the disease and were absent in the acute phase (Sonia and Sandberg, 1978). In 70 vitro studies on the progeny of colony-forming cells also revealed the presence of Phi-negative progenitors in a number of CML patients whose WBC counts were within normal limits (Dube et al, 1984a). The presence of Phi- negative cells in Phi positive CML has been correlated with increased survival time (Sakurai et al, 1975) though this has been disputed by others (Sokal, 1980). A more aggressive chemotherapeutic protocol has resulted in the appearance of Phi-negative cells in two other studies (Cunningham et al, 1979, Goto et al, 1982) but in each case the reduction in Phi-positive cells was temporary, and did not affect the survival of the patients. In one patient heterozygous for G6PD alleles, the emergence of Phi-negative cells was paralleled by the return of both isoenzyme types, indicating that the Phi -negative cells were not clonal in origin (Singer et al, 1980a). More recently, the use of long term marrow cultures has offered a more sensitive assay for residual normal cells in CML. Coulombel and her associates (1983b) have shown that when marrow cells from newly diagnosed Phi CML patients were placed in culture, in the majority of cases a previously undetectable population of chromosomally normal hemopoietic cells of a l l myeloid lineages could be demonstrated within two to four weeks. The number of Phi positive cells rapidly declined but Phi-negative progenitors could be maintained for 8 to 12 weeks in such a culture system. In order to determine if these Phi negative progenitors were nonclonal, marrow cells from a mosaic Turner's syndrome patient with CML (46,XX/45,X,Phl) were placed in long term culture (Dube et al, 1984b). All the metaphases examined from fresh marrow preparations and in vitro granulocytic and erythroid colonies obtained from marrow progenitors were 45,X,Phi, while granulocytic colonies from the adherent layer of the long term cultures yielded only 46,XX metaphases. Since the patient had been diagnosed 5 years previously these studies provide 71 additional evidence of the long term persistence in vivo of normal, nonclonal progenitors in at least some CML patients. Cell culture studies in CML Numerous investigators have demonstrated a large increase in the number of CFU-C in the blood of patients with CML (Moore et al, 1973; Goldman et al, 1974; Eaves and Eaves, 1979). A relationship has been noted between the increase in blood CFU-C and the WBC count (Goldman et al, 1974, Olofsson and Olsson, 1976). The CFU-C concentration usually returns to normal when the WBC is brought down to within normal limits. In the marrow, the CFU-C concentration is more variable, with most investigators reporting a modest increase in CFU-C number (Goldman et al, 1974; Eaves and Eaves, 1979). In some CML patients therefore, the concentration of CFU-C per 10̂  cells in the blood exceeds the proportion in the marrow, unlike the normal situation. The morphology of the colonies produced in vitro during the chronic phase, however, appears to be normal, and the maturity and distribution of the cell types is typical of that seen in normal subjects. When blast crisis intervenes fewer normal sized colonies are seen. In some cases large numbers of small (<50 cell) colonies, composed of poorly differentiated cells as seen in acute leukemia, may be produced (Moore et al, 1973, Goldman et al, 1980) Eaves and Eaves (1979) reported that large increases in the CFU-E and BFU-E compartments also accompany, and parallel, the increase seen in CFU-C. Thus, the ratio of BFU-E:CFU-C numbers in the circulation of CML patients is constant and does not differ substantially from that seen in normal individuals (Eaves et al, 1980). This suggests that the large increase in mature granulocytes is not due to a preferential "channeling" of progenitors into the granulocytic pathway at the level of the pluripotent stem cell, but rather lack of terminal control of granulopoiesis post CFU-C. 72 The cell cycle characteristics of CFU-C are altered in CML. A number of investigators have noted that fewer marrow CFU-C are in S-phase in chronic phase CML compared to normal controls (Moore et al, 1973; Rickard et al, 1979; Singer et al, 1981) suggesting that a greater than normal number of CFU-C are in a noncycling state. Restoration of the cell cycle characteristics of CFU-C to normal was achieved after treatment (Moore et al, 1973). During blast phase, the proportion of CFU-C in S phase was found to be increased over chronic phase values, despite an increasing leukocytosis (Moore et al, 1973; Rickard et al, 1979). Pluripotent progenitors (CFU-GEMM) in chronic phase CML were also found to show alterations in cell cycle activity, In the blood and marrow of normal subjects these primitive progenitors are quiescent (Fauser and Messner, 1982a), but in CML CFU-GEMM were shown to be in active cell cycle (Messner et al, 1980; Lepine and Messner, 1983). Unlike the previous studies of CFU-C, the proliferative rate was not modulated in treated patients, thus indicating that such alterations in cycling kinetics may be fundamental to the disease process. An increase in the frequency of circulating CFU-GEMM, up to 600 fold over normal values, was noted by some investigators (Hara et al, 1981, Hibbin et al, 1983) but not by others (Messner et al, 1980; Lepine and Messner, 1983). Regulatory abnormalities Defects in negative feedback regulation have been suggested as a possible mechanism for the excess myelopoiesis seen in CML. For instance, colony formation by CFU-C from CML patients is not inhibited by concentrations of PGÊ  that inhibit normal CFU-C and this altered sensitivity persisted even after treatment (Pelus et al, 1980). In another study, neutrophils from CML patients were defective in production of colony inhibiting activity (CIA), subsequently identified as lactoferrin, 73 which decreases production and release of CSA from monocytes and macrophages (Broxmeyer et al, 1977). In addition, the CSA producing cells from CML patients were less sensitive than normal cells to inhibition with low concentrations of lactoferrin obtained from normal neutrophils. However, contradictory results were obtained by other investigators (Moberg et al, 1978). CFU-C colony formation in CML is comparable to normal colony formation in its strict requirement for CSF (Moore et al, 1973; Metcalf et al, 1974) although differences have been noted in the threshold sensitivity of the CFU- C. Levels of CSF in the serum and urine of chronic phase CML patients are normal or increased (Moore and Robinson, 1974), while in blast phase no activity is detectable (Moore et al, 1973; Golde et al, 1974). The colony stimulating activity of peripheral blood leukocytes from CML patients was found to be lower than normal by some authors (Goldman et al, 1974) but not by others (Moore et al, 1973). When abnormal, the colony stimulating activity returned to normal following treatment, suggesting that the decrease in CSF production by leukemic cells was due to the inhibitory effects of the large numbers of circulating granulocytes. Marrow samples from most patients were reported to have normal colony stimulating activity, but a few patients had consistently higher levels of activity (Bianchi Scarra et al, 1981). However, because of the lack of precision in these assessments and the fact that a number of different molecules are now known to be active in CFU-C colony stimulating assays, these studies are difficult to interpret. In summary, CML is a clonal disorder, with a prominent leukocytosis, terminating in a blast phase and subsequent death. The Phi chromosome is a consistent finding in the large majority of these patients. Other chromosomal abnormalities may also be seen, particularly in blast phase, but 74 the contribution of any of these to the clincal course of the disease has yet to be established. Recent evidence indicates that residual normal stem cells are found in a large porportion of CML patients, though their existence can often be documented only by in vitro assays. A number of other anomalies are seen on examination of the growth characteristics of CML cells. These included increases in progenitor cell number, differences in cell cycle activity, and possibly the altered production of some regulatory factors. At the present time, however, the role of each of these findings in the pathogenesis of CML remains unclear. B) Polycythemia Vera (PV) The outstanding feature of PV is an absolute increase in the red cell mass, which is often associated with simultaneous or sequential cytopathological proliferative changes in the marrow resulting in a panmyelosis of varying degree. The patient initially presents with symptoms resulting from increased blood volume and hyperviscosity. Cerebrovascular accidents or myocardial infarction may result if the disease is not treated. The usual age of onset of PV is in the middle or later years with a peak in the fifth or sixth decade (Wintrobe, 1976). The chronic phase of PV may last anywhere from 5 to 20 years. Therapy during this period may include phlebotomy, and/or treatment with radioactive phosphorus or cytotoxic drugs such as busulfan. With such treatment many of the symptoms usually disappear and the circulating cell mass may be brought down to within normal limits. With time the disease may evolve into a spent phase, characterized by extramedullary hemopoiesis, an increasing degree of anemia, and the development of myelofibrosis (Ward and Block, 1971). This phase has been called post polycythemic myeloid metaplasia (PPMM) and varies in frequency from 7-30% of cases (Silverstein, 1976). In 1-30% of patients, 75 the disease terminates in leukemic changes typical of acute myeloblastic leukemia with progressive anemia, thrombocytopenia and the appearance of blast cells (Ellis et al, 1975; Wasserman, 1954, Glasser and Walker, 1969). The contribution of therapy to the evolution of PV to acute leukemia is now well documented (Berk et al, 1981). Cytogenetics Though no specific chromosomal marker comparable to the Ph^ chromosome has been found in PV, studies have shown that up to 25% of untreated patients may demonstrate chromosomal abnormalities in the marrow at time of diagnosis (Westin et al, 1976; Wurster-Hill et al, 1976; Testa, 1980) The most common cytogenetic findings are hyperdiploidy, particularly trisomy 8,9,12, and 19. A more specific cytogenetic defect, in about 20% of patients is the 20qll deletion which is rarely found in other hematological disorders (Millard et al, 1968; Westin et al, 1976; Zech et al, 1976). Structural rearrangements such as deletions and translocations, aneuploidy, polyploidy and hypoploidy are also found (Kay et al, 1966; Westin et al, 1976; Wurtser- Hill et al, 1976). Karyotypic aberrations are more extensive in patients with a longstanding history of the disease, and in those who have been treated with 32p o r alkylating agents (Testa, 1980). The role of chromosomal changes in the etiology of PV is unknown. Although a higher incidence of karyotypic abnormalities is seen in treated PV patients when the disease transforms into acute leukemia (Testa, 1980), no direct correlation was seen between the presence of chromosomal abnormalities and progression to acute leukemia (Wurster-Hill and Mclntyre, 1978). Conversely, the presence of a normal karyotype in PV does not eliminate the possibility that the disease will terminate in a leukemic transformation. The prognostic value of cytogenetic findings in PV at this time appears to be very limited. 76 Ep-independence The presence of an increased cell mass without a concomitant increase in Ep levels led early on to the speculation that erythropoiesis in PV patients was autonomous, i.e. outside the regulatory control of Ep (Adamson, 1968). However, a number of in vivo observations indicate that some degree of normal responsiveness to erythropoietic stimuli may be present in PV patients. For example, both phlebotomy and hypoxia lead to an increased production of Ep (Gurney, 1973), and concomitant with the appearance of Ep in the urine, an increase in iron turnover and reticulocyte production occurs, indicative of augmented erythropoiesis (Adamson, 1968). In vitro studies utilizing the plasma clot (Prchal and Axelrad, 1974), agar (Horland et al, 1977) and methycellulose (Aye, 1977; Eaves and Eaves, 1978; Lacombe et al, 1980) have demonstrated the presence of erythroid progenitors in PV capable of colony formation in culture without the addition of Ep. Such "Ep-independent" erythroid colony formation is not seen in normal subjects, or in patients with secondary polycythemia if care is taken to ensure that levels of Ep in other components of the culture (e.g. in the fetal calf serum) are insignificant (Eaves et al, 1980). Controversy exists however, as to whether Ep-independent progenitors present in PV patients are truly autonomous to the normal requirement for continuing contact with Ep. From studies reporting the reversible inhibition of such colony formation by the addition of Ep and anti-Ep antiserum preparations (Weinberg, 1977; Zanjani et al, 1977) and the failure of Ep-independent erythroid colony growth in serum-free cultures (Casadevall et al, 1982), it has been suggested that erythroid progenitors in PV may be exquisitely sensitive to Ep and can satisfy their requirement from the trace amounts of the hormone contributed by the fetal calf serum in the culture medium. Other in vitro experiments have provided evidence that at least some erythroid precursors in PV retain a 77 normal Ep responsive mechanism. A stimulatory effect of added Ep on the number of colonies obtained in PV culture was first demonstrated by Prchal and Axelrad (1974) and has subsequently been confirmed by a number of investigators (Aye, 1977; Eaves and Eaves, 1978; Zanjani et al, 1979); Eaves et al, 1980). This suggests that in PV at least a proportion of erythroid progenitors may be subject to normal regulation by variable Ep levels in vivo. Ep dose-response studies have confirmed that two populations of erythroid progenitors exist in PV: 1) a phenotypically abnormal population capable of colony formation in culture containing less than .001 u/ml of Ep, and 2) a phenotypically normal population with a normal Ep responsiveness (Eaves and Eaves, 1978). The use of G6PD isoenzymes markers in female heterozygotes with PV have shown that a l l erythroid precursors capable of Ep independent colony formation are members of the abnormal clone (Prchal et al, 1978b). However, the ratio of isoenzyme types obtained from colonies cultured in the presence of optimal levels of Ep was indicative of a subpopulation of cells belonging to the abnormal clone but not capable of endogenous colony formation. Ep-independent growth can thus be considered a unique, but not consistent marker of the abnormal clone. This finding is supported by more recent experiments in which the distribution of Ep independent and Ep dependent colony forming cells amoung the progeny of single BFU-E was examined (Cashman et al, 1983). The data obtained from these experiments established that primitive BFU-E belonging to the neoplastic clone as shown by their ability to produce Ep-independent colony forming cells could also produce substantial numbers of Ep-dependent progenitors, and thus suggests that phenotypic expression of Ep-independence is not fixed prior to the BFU-E stage. 78 Ep-independence is not restricted to the MPD; cells from murine fetal liver (Johnson and Metcalf, 1977), neonatal lamb bone marrow (Roodman and Zanjani, 1979) and human cord blood (Tchernia et al, 1981) also variably express the potential for endogenous colony formation. We have suggested therefore that the capacity for Ep-independent growth associated with stem cell transformation may not be a "new" acquistion but may represent the reemergence of a fetal characteristic (Cashman et al, 1983). The results of a recent survey of a large number of unselected patients with PV demonstrate that unlike CML or ET, Ep-independence is a constant feature of a l l clones that lead to this disease regardless of treatment or time from diagnosis (Eaves et al, in press). It would be expected that low circulating levels of Ep in the PV patient would provide a considerable growth advantage for abnormal cells that could complete the erythropoietic program under such conditions, hence permitting them to dominate the mature cell compartment. However, the demonstration that only one isoenzyme type is found in the circulation, even when red cell levels (and hence presumably Ep levels) are brought down to normal in the treated patient, is at variance with Ep-independence being the sole pathological mechanism (Prchal et al, 1978b). Furthermore such a phenotypic advantage does not account for the increase in granulopoiesis and megakaryopoiesis usually seen in these patients. Cell culture studies In vitro studies of progenitors in PV have demonstrated a number of characteristics similar to those of CML cells, such as alterations in cell cycle activity (Fauser and Messner, 1981), elevated CSA levels (Metcalf, 1977), high porportions of buoyant light density colony- forming cells (Singer et al, 1980), and normal ratios of primitive erythroid to granulocytic progenitors, although in PV, unlike CML, the frequency of 79 these progenitors is not elevated (Eaves and Eaves, 1979). This latter finding would indicate that early commitment events are not altered in the MPD, but rather that the preferential amplification of the progeny of the abnormal clone occurs at later stages of maturation, perhaps by a mechanism that confers a growth advantage on clonal cells while suppressing the proliferation of normal progenitors. Several lines of evidence support the hypothesis that such a mechanism may operate in PV. Unlike the situation seen in CML, in vitro colony assays using cells from G6PD heterozygotes indicate that substantial numbers of non- clonal progenitors are present in PV patients (Prchal et al, 1978b). Since direct analysis of the peripheral blood could not detect the progeny of these normal cells, even in patients with normal counts (Adamson et al, 1976), their further maturation was blocked in vivo. The proportion of erythroid progenitors which are not members of the abnormal clone decreases substantially as cells progress down the differentiation pathway (Fialkow et al, 1978; Eaves and Eaves, 1979). This predominance of abnormal progenitors in later compartments has led to the suggestion that normal erythropoiesis may be suppressed at a differentiation step between BFU-E and CFU-E (Adamson et al, 1980). Analysis of the G6PD isoenzyme type of individual colonies before and after exposure to -^-thymidine demonstrated that a porportion of clonal CFU-C were in S-phase, while non-clonal CFU-C were quiescent (Singer et al, 1980). Since in normal marrow up to 40% of CFU-C are killed by exposure to ^H-thymidine (Metcalf et al, 1974), these data would suggest that in PV the proliferation of normal granulocytic progenitors may be suppressed. Alternations in the cell cycle regulation of hemopoietic progenitors in PV have been found by some authors, but not confirmed by others. No differences were noted in the ratio of normal and neoplastic BFU-E from G6PD 80 heterozygotes after exposure to -*H-thymidine (Singer et al, 1979c). Similar results were obtained by other investigators using Ep-independence as a marker for the abnormal clone (Mladenovic and Adamson, 1982). These findings contradict the observations of Fauser and Messner (1981) who found an increased fraction of circulating Ep-dependent and -independent BFU-E and CFU-GEMM in active cell cycle, and of Zanjani and his associates (1978) who demonstrated an increased sensitivity to ̂ H-thymidine suicide of endogenous CFU-E. The possible role of increased cell cycle activity in the numerical expansion of the abnormal clone in PV therefore remains obscure. In summary, PV is a disease of clonal origin, that may be described as a neoplastic disorder, in which the progeny of a single stem cell, insensitive to normal growth regulatory mechanisms and perhaps with a proliferative advantage, completely f i l l the mature cell compartments in vivo. Unlike CML residual normal stem cells have been readily demonstrable in vitro. However, as the disease progresses these may decrease in number (Adamson et al, 1980). Although chromosomal abnormalities are found in a minority of patients with PV, there is no direct evidence yet as to which genetic rearrangements might play a primary role in the origin and evolution of the neoplastic characteristics of the abnormal clone. C) Essential Thrombocytosis (ET) ET (primary, hemorrhagic or idiopathic thrombocythemia) is characterized by abnormal proliferation of the megakaryocytes resulting in increased platelet production. The most common clinical manifestations are hemorrhage and/or thrombosis. It occurs most frequently in the fifth or sixth decade of li f e , equally amoung males and females, though a second peak of onset occurs in younger adults with a strong female preponderance (Silverstein, 1985). 81 ET may be distinquished from PV or CML by a platelet count in excess of 106/mm3, with marked marrow megakaryocytic hyperplasia and absence of the Ph1 chromosome. The red cell mass is usually normal, with adequate iron stores if previous bleeding episodes have been kept under control. A modest leukocytosis is a common finding. Defects in platelet function have been reported with abnormalities in agreggation and adhesiveness, but platelet survival is normal. Hemorrhagic or thrombotic complications may become li f e threatening when the platelet count rises above lÔ /mm3, and may be lowered with myelosuppressive drugs, 3^p ; o r thrombocytopheresis (Wintrobe. 1976). ET was firmly established as a MPD when its clonal origin in a pluripotent stem cell was demonstrated (Fialkow et al, 1981). Clinical remission in ET is not accompanied by the emergence of nonclonal progenitors. A minority of patients undergo transition to another MPD, including PV, CML, or myelofibrosis, or may evolve into an acute leukemia (Silverstein, 1985). Karyotypic abnormalities are rare in ET. Only 5% out of 170 of ET analyzed during one study were considered to have a definite chromosomal abnormality (Third International Workshop on Chromosomes in Leukemia, 1981). No common anomaly was noted. In vitro studies in ET have demonstrated a number of similar stem cell characteristics with other MPD. Endogenous erythroid colony forming cells have been described in ET (Prchal and Axelrad, 1974; Wong and Tobin, 1979; Partenem et al, 1983; Eridani et al, 1983; Eaves et al, 1983b) and like the other MPD, addition of Ep results in an increase in the number and size of the erythroid colonies formed. An elevation in the number of circulating CFU-M has been found in ET (Hoffman et al, 1983; Hibben et al, 1984). The presence of endogenous megakaryocytic colonies (i.e. colonies formed independent of the addition of conditioned media) has been documented in MPD 82 with thrombocytosis suggesting that abnormalities in responsiveness to pathway specific regulators may be a common pathological mechanism in the MPD (Gerwitz et al, 1983). 5) THESIS OBJECTIVES This series of experiments was undertaken to elucidate the role of altered cell kinetics in the pathogenesis of the MPD. Though numerous authors have studied the proliferative characteristics of CFU-C in CML, comparable data for the erythroid lineage was not available at the time this study was intiated. More extensive studies had been attempted in PV with the examination of the cycling characteristics of erythroid and granulocytic progenitors but these had yielded contradictory results in different laboratories. At the present time an extensive review of the literature indicates that the cycling characteristics of hemopoietic progenitors in ET s t i l l have not been studied elsewhere. Since the possibility exists that the contradictory results obtained by various other groups were due to differences in culture conditions and progenitor cell classification, the need was obvious for a complete examination of the cell cycle characteristics of a l l classes of hemopoietic progenitors in each of the MPD by a single laboratory under identical conditions. Accordingly, progenitor cell cycle studies were undertaken on marrow and peripheral blood cells from patients with CML, ET, and PV and from normal controls. The results of these studies, which are detailed in Chapter III of this thesis, demonstrated consistent differences in the proliferative activity of primitive, normally quiescent hemopoietic progenitors in patients from a l l MPD catagories when compared to normal controls. These interesting 83 observations encouraged an extension of cell cycling studies in an in vitro system that might be amenable to manipulation and in which the basis of the cell cycle control of normal and neoplastic cells might be characterized and analyzed. For a number of years investigators in our laboratory have been able to maintain primitive clonogenic hempoietic progenitors in the adherent layer of normal long term marrow cultures for periods of up to 8-12 weeks (Coulombel et al, 1983). Application of the ^H-thymidine cell suicide assay to normal long-term marrow cultures demonstrated a regulated proliferation of the most primitive hemopoietic progenitor types in this layer (see Chapter IV). It was of interest, therefore, to examine the cycling characteristics of progenitors from long term cultures established with cells from patients with MPD in order to determine if the abnormalities in proliferative behaviour seen in vivo could be reproduced in vitro. Preliminary experiments had indicated that the long-term culture system could support the proliferation of marrow cells from PV patients. Such cultures were therefore established and evidence of persisting neoplastic cells sought. The cycling behaviour of primitive progenitors was then measured (Chapter V). On the other hand, previous application of long-term marrow cultures to the study of hemopoietic progenitors in CML had shown that with most CML patients, Ph^ positive cells could not be maintained under these conditions. Recently, a modification of the long term culture system, using CML peripheral blood added to pre-established normal marrow adherent layers proved effective in maintaining Pĥ  positive progenitors in vitro for an extended period of time (Eaves et al, 1983). Experiments were therefore designed to look for changes in the cycling characteristics of a l l classes of clonogenic erythroid and granulopoietic progenitors present in normal long term cultures as well as in control cultures initiated with normal blood progenitors on pre-established feeders. These then served as a basis of comparison for studies of neoplastic cells maintained under similar conditions (Chapter VI). 85 REFERENCES Abramson, S., Miller, R.G., Phillips, R.A: Identification of pluripotent and restricted stem cells of the myeloid and lymphiod systems. J. Exp. Med. 145: 1567-1579, 1972. Adamson, J.W: The erythropoietin/hematocrit relationship in normal and polycythemic man: Implications of marrow regulation. Blood 32; 597-609, 1968. Adamson, J.W., Fialkow, P.J., Murphy, S., Prchal, J.F., Steinmann, L: Polycythemia vera: Stem cell and probable clonal origin of the disease. N. Eng. J. Med. 295; 913-916, 1976 Adamson, J.W., Torok-Storb, B., Lin, W: Analysis of erythropoiesis by erythroid colony formation in culture. Blood Cells 4; 89-103, 1978. Adamson, J.W., Singer, J.W., Catalono, P., Murphy, S., Lin, N., Steinmann, L., Ernst, C., Fialkow, P.J: Polycythemia vera. Further in vitro studies of hemopoietic regulation. J. Clin. Invest. 66; 1363-1368, 1980. Allen, T.D: Hemopoietic microenviroments in vitro: Ultrastructural aspects. In: Microenviroments in Hemopoietic and Lymphoid Differentiation, Porter, R. and Whelan, J., eds. Ciba Foundation 84, Pitman Medical, London, pp. 38- 67, 1981. Allen, T.D. and Dexter, T.M: Cellular interrelationships during in vitro granulopoiesis. Differentiation 6; 191-194, 1978. Allen, T.D. and Dexter, T.M: Ultrastructural aspects of erythropoietic differentiation in long term bone marrow. Differentiation 21; 86-101, 1982. Antoniades, H.N. and Owen, A.J: Growth factors and regulation of cell growth. Ann. Rev. Med. 33; 445-463, 1982. Armitage, J.O., Klassen, L.W., Patil, R., et al: Marrow transplatation for stable phase chronic granulocytic leukemia. Exp. Hematol. 12; 717-719, 1984. Ash, R.C., Detrick, R.A., Zanjani, E.D: Studies of human pluripotential hemopoietic stem cells (CFU-GEMM) in vitro. Blood 58; 309-316, 1981. Austin, P.E., McCulloch, E.A., T i l l , J.E: Characterization of the factor in L-cell conditioned medium capable of stimulating colony formation by mouse marrow cells in culture. J. Cell Physiol. 77; 121-133, 1971. Axelrad A.A., McLeod, D.L., Shreeve, M.M., Heath, D.S: Properties of cells that produce erythrocytic colonies in vitro. In: Hemopoiesis in Culture, Robinson, W.A., Ed., DHEW Publication No. (NIH)74-205 pgs 226-237, 1974. Axelrad, A.A., McLeod, D.L., Suzuki, S., Shreeve, M.M: Regulation of population size of erythropoietic progenitor cells. In: Differentiation of Normal and Neoplastic Hemopoietic Cells, Clarkson, B., Marks., T i l l , J.E., (eds.). Cold Spring Harbor Laboratory, pp. 155-163, 1978. 86 Aye, M.T., T i l l , J.E., McCulloch, E.A: Cytological studies of granulopoietic colonies from two patients with chronic myelogenous leukemia. Exp. Hematol. 1; 115-118, 1973. Aye, M.T: Erythroid colony formation in cultures of human marrow: Effect of leukocyte conditioned medium. J. Cell Physiol. 91; 69-77, 1977. Bakhsi, A., Minowada, J., Jensen, J.P., Whang-Peng, J., Waldmann, T., Korsmeyer, S.J: Lymphoid blast crisis of chronic myelogenous leukemia represent stages in the development of B-cell precursors. N. Engl. J. Med. 309; 876-830, 1983. Barnes, D.W., Evans, E.P., Ford, C.E., West B.J: Spleen colonies in mice: Karyotypic evidence of multiple colonies from single cells. Nature 219; 518-520, 1968. Bartram, C.R., DeKlein, A., Hagemeijer A., et al: Translation of c-abl oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukemia. Nature 306; 277-280, 1983. Bateman, A.J. and Chandly, A: Mutations induced in the mouse with tritiated thymidine. Nature 193; 105-107, 1962. Baxter, G.C. and Stanners, CP: The effect of protein degradation on cellular growth characteristics. J. Cell. Physiol. 96; 139-146, 1978. Becker, A.J., McCulloch, E.A., T i l l , J.E: Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197: 452-454, 1963. Becker, A.J., McCulloch, E.A., Siminovitch, L., T i l l , J.E: The effect of differing demands for blood cell production of DNA synthesis by hemopoietic colony forming cells of mice. Blood 26; 296-308, 1965. Becker, H., Stanners, CP., Kudlow, J.E: Control of macromolecular synthesis in proliferating and resting Syrian hamster cells in monolayer culture. II Ribosome complement in resting and early G± cells. J. Cell Physiol. 77; 43-50, 1973. Ben-Ishay, Z. and Yoffey, J.M: Ultrastructural studies of erythroblastic islands of rat bone marrow. II. The resumption of erythropoiesis in erythropoietically depressed bone marrow. Lab. Invest. 26; 637-648, 1972. Bennett, R.M., Kokocinski, T: Lactoferrin content of peripheral blood cells. Brit. J. Hematol. 39; 509-521, 1978. Bentley S.A: Close range cell-cell interaction required for stem cell maintenance in continuous bone marrow culture. Exp. Hematol. 9; 308-312, 1981. Bentley, S.A: Bone marrow connective tissue and the hemopoietic microenviroments. Brit. J. Hematol. 50; 1-6, 1982. Bentley, S.A. and Foidart, J.M: Some properties of marrow-derived adherent cells. Blood 56; 1006-1012, 1980. 87 Bentley, S.A. and Tralka, T.S: Characterization of marrow-derived adherent cells: Evidence against an endothelial subpopulation. Scand. J. Haemat. 28; 381-388, 1982. Berk, P.D., Goldberg, J.D., Silverstein, M.N., et al: Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy. N. Eng. J. Med. 304; 442-447, 1981. Bernheim, A., Berger, R., Prud'homme J.L., Labaume S., Bussel, A., Barot- Ciorbaru, R: Philadelphia chromosome positive B lymphocytes in chronic myelocytic leukemia. Leuk. Res. 5; 331-339, 1981. Bianchi Scarra, G.L., Barresi, R., Sessaregao, M., Ajmar, F., Salvidio, E: Marrow colony stimulating activity in chronic myelogenous leukemia. Exp. Hematol. 9; 917-925, 1981. Blackett, N.M: Investigation of bone marrow stem cell proliferation in normal, anemic, and irradiated rats, using methtrexate and tritiated thymidine. J. Natl. Can. Inst. 41; 909-918, 1968. Blick, M., Westin, E., Gutterman, J., Wong-Staal, F., Gallo, R., McCredie, K., Keating, M., Murphy, E: Oncogene expression in human leukemia. Blood 64; 1234-1239, 1984. Bocian, E., Ziemba-Zak, B., Rosiek, 0., Sablinski, J: Chromosome aberrations in human lymphocytes exposed to tritiated water in vitro. Curr. Top. Radiat. Res. 12; 168-181, 1977. Boggs, D.R: Clonal origin of leukemia: Site of origin in the stem cell hierarchy and the significance of chromosomal changes. Blood Cells 7; 205- 215, 1981. Bondurant, M.C., Lind, R.N., Koury, M.J., Ferguson, M.E: Control of globin gene transcription by erythropoietin in erythroblasts from Friend virus- infected mice. Mol. Cell. Biol. 5; 675-683, 1985. Botnick, L.E., Hannon, E.C., Helmas, S: Limited proliferation of stem cells surviving alkylating agents. Nature 262; 68-70, 1976. Boynton, A.L., Whitfield, J.F.,. Isaacs, R.T: Calcium dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor promoting phorbol ester (PMA). J. Cell Physiol. 87; 25-32, 1976. Bradley, T.R., Metcalf, D: The growth of mouse bone marrow cells in vitro. Aust. J. Exp. Biol. Med. Sci. 44; 287-300, 1966. Brewen, J.G. and Oivieri, G: The kinetics of chromatid aberrations induced in Chinese hamster cells by tritiated thymidine. Radiat. Res. 28; 779-792, 1966. Brooks, R.F., Bennett, D.C, Smith, J.A: Mammaliam cell cycles need two random transitions. Cell 19; 493-504, 1980. Brooks, R.F: Continuous protein synthesis is required to maintain the probability of entry into S phase. Cell 12; 311-317, 1977. 88 Brooks, R .F: Random t r a n s i t i o n s and c e l l cyc le c o n t r o l . Prog. C l i n . B i o l . Res. Part A 593-601, 1981. Broxmeyer, H . E . , Moore, M . A . S . , Ralph, P: C e l l f ree granulocyte colony i n h i b i t i n g a c t i v i t y der ived from human polymorphonuclear n e u t r o p h i l s . Exp. Hematol. 5; 87-102, 1977a. Broxmeyer, H . E . , Mendelsohn, N, Moore, M.A.S: Abnormal granulocyte feedback regu la t ion of colony-forming and co lony -s t imula t ing a c t i v i t y producing c e l l s from pat ients with chronic myelogenous leukemia. Leuk. Res. 1; 3-12, 1977b. Broxmeyer, H . E . , Smithyman, A . , Eger, R .R . , Meyers, P . A . , De Sousa, M: I d e n t i f i c a t i o n of l a c t o f e r r i n as the granulocyte-der ived i n h i b i t o r of colony s t imu la t ing a c t i v i t y product ion. J . Exp. Med. 148; 1052-1067, 1978. Broxmeyer, H. E . , J u l i a n o , L . , Waheed, A . , Shadduck, R: Release from mouse macrophages of a c i d i c i s o f e r r i t i n s that suppress hematopoietic progeni tor c e l l s i s induced by p u r i f i e d L - c e l l colony s t imula t ing fac tors and suppressed by p u r i f i e d human l a c t o f e r r i n . Exp. Hematol. 12; 369 (abst) 1984a. ~~ Broxmeyer, H . E . , Cooper, S . , Wi l l iams, D. , G e n t i l e , P: Inf luence of p u r i f i e d human l a c t o f e r r i n (LF) in v ivo on mouse m u l t i p o t e n t i a l (CFU-GEMM), e r y t h r o i d , (BFU-E) , and granulocyte/macrophage (CFU-GM) progeni tor c e l l s . Blood 65 (Suppl . 1); 126a (abst ) , 1984b. Bruce, W.R., Meeker, B . E . , V a l e r i o t e , F .A: Comparison of the s e n s i t i v i t y of normal hemopoietin and transplanted lymphoma colony forming c e l l s to chemotherapeutic agents administered in v i v o . J . N a t l . Can. Ins t . 37; 233- 245, 1966. — Bruch, C , Kovas, P . , Ruber, E . , F l i e d n e r , J . M : Studies on the i n h i b i t o r y e f f e c t of granulocytes on human granulocytopoies is in agar c u l t u r e s . Exp. Hematol. 6; 337-345, 1978. Bunge, R., G l a s e r , L . , Lieberman, M.A. , Raben, D., Sa l ze r , J . , Whittenberger, B . , Woolsey, T .A : Growth cont ro l by c e l l to c e l l contact . J . Supramol. S t r u c t . 11; 175-187, 1979. Burger, M.M., Bombik, B .M. , Breckenridge, B . , Sheppard, J . R : Growth c o n t r o l and c y c l i c a l t e r a t i o n s of c y c l i c AMP in the c e l l c y c l e . Nature New B i o l . 239; 161-163, 1972. Burgess, A.W. and Metca l f , D: The e f fec t of colony s t imula t ing fac tor on the synthesis of r i b o n u c l e i c ac id by mouse bone marrow c e l l s in v i t r o . J . C e l l P h y s i o l . 90: 471-484, 1977. Burgess, A.W. , Camakaris, J . , Metcal f , D: P u r i f i c a t i o n and proper t ies of colony s t imu la t ing fac tor from mouse lung condit ioned medium. J . B i o l . Chem 252; 1998-2003, 1977. Byron, J.W: E f f e c t of s te ro ids on the c y c l i n g of hemopoietic stem c e l l s . Nature 228; 1204-1206, 1970. Bryon, J.W: In v i t r o e f fec t of s te ro ids and c y c l i c AMP on the s e n s i t i v i t y of hemopoietic stem c e l l s to ^H-thymidine. Nature 234; 39-40, 1971. 89 Byron, J.W: Evidence for a beta-adrenergic receptor initiating DNA synthesis in hemopoietic stem cells. Exp. Cell Res; 71; 228-232, 1972. Bryon, J.W: Manipulation of the cell cycle of the hemopoietic stem cell. Exp. Hematol. 3; 44-53, 1975. Bryon, J.W: Mechanism for histamin H2 receptor induced cell cycle changes in the bone marrow stem cell. Agents Actions 7; 209-213, 1977. Cairns, J. and Davern, C.I: Effect of 32p decay upon DNA synthesis by a radiation sensitive strain of Escherichia coli. J. Mol. Biol. 17; 418-427, 1966. Camprisi, J., Medrano, E.E., Morreo, G., Pardee, A.B: Restriction point control of cell growth by a labile protein: Evidence for increased stability in transformed cells. Proc. Natl. Acad. Sci. U.S.A. 79; 436-440, 1982. Carnot, P. and Deflandre, C: Sur l'activite hemopoietique des differents organes au cours de la regeneration du sang. Compt. Rend. 143; 432-435, 1906. Carsten, A.L: Tritium in the enviroment. Adv. Radiat. Biol. 8; 419-458, 1979. Casadevall, N., Vainchenker, W., Lacombe, C, Vinci, G., Chapman, J., Breton-Garius, J., Varet, B: Erythroid progenitors in polycythemia vera: Demonstration of their hypersensitivity to erythropoietin using serum free cultures. Blood 59; 447-451, 1982. Cashman, J., Henkelman, D., Humphries, R.K., Eaves, C.J., Eaves, A.C: Individual BFU-E in polycythemia vera produce bothe erythropoietin dependent and independent progeny. Blood 61; 876-884, 1983. Castor, L.N: Responses of protein synthesis and degradation in growth control of WI-38 cells. J. Cell Physiol. 92; 457-468, 1977. Castro-Malaspina, H., Gay, R.E., Safetans, S., Oettgea, B., Gay, S., Moore, M.A.S.: Phenotypic characterization of the adherent layer in long term mouse marrow culkture. Blood 58 (Suppl. 1); 107a, 1981. Chan,,H.S., Saunders, E.F., Freedman, M.H: Modulation of human hematopoiesis by prostaglandins and lithium. J. Lab. Clin. Med. 95; 125- 132, 1980. Chen, M.G., Schooley, J.C: A study of the clonal nature of spleen colonies using chromosome markers. Transplantation 6: 121, 1968. Chervenick, P.A., Ell i s , L.D., Pan, S.F., Lawson, A.L: Human leukemic cells: In vitro growth of colonies containing the Philadelphis (Ph^) chromosome. Science 174; 1134-1136, 1971. Cleaver, J.E: Frontiers of Biology: Thymidine Metabolism and Cell Kinetics. Vol. 6, Neuberger, A. and Tatum, E.L., (eds.), North Holland Publishing Company, Amsterdam, 1967. 90 Cleaver, J.E., Thomas, G.H., Burki, H.J: Biological damage from intranuclear tritium: DNA strand breaks and their repair. Science 177; 996-999, 1972. Cleaver, J.E: Induction of thioguanine and ouabain resistant mutants and single strand breaks in the DNA of Chinese hamster ovary cells by 3H-thymidine. Genetics 87; 129-138, 1977. Cochran, B.H., Rippel, A.C., Stiles, CD: Molecular cloning of gene sequences regulated by platelet derived growth factor. Cell 33; 939-947, 1983. — Cochran, B.H., Zullo, J., Verma, I, Stiles, CD: Expression of the c-fos gene and of an fos-related gene in stimulated by platelet derived growth factor. Science 226; 1080-1084, 1984. Collins, S. and Groudine, M: Amplification of endogenous myc-related DNA sequences in a human myeloid leukemia cell line. Nature 298; 679-681, 1982. Collins, S.J., Kubonishi, I., Miyoshi, I., Groudine, E.M: Altered transcription of the c-abl oncogene in K-562 and other chronic myelogenous leukemia cells. Science 225; 72-74, 1984. Cormach, D.H: Time-lapse characterization of erythrocytic colony forming cells in plasma culture. Exp. Hematol. 4; 319-327, 1976. Costa, M., Gerner, E.W., Russell, D.H: Ĝ  specific increase in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells. Biochem. Biophys. Acta 425; 246-255, 1976. Coulombel, L. , Eaves, A.C, Eaves, C J : Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62; 291-297, 1983. Coulombel, L., Kalousek, D., Eaves, C.J., Gupta, CM., Eaves, A.C: Long- term marrow cultures reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N.E.J.M. 308; 1493-1498, 1983. Croizat, H., Frindel, E., Tubina, M: Proliferative activity of the stem cells in the bone marrow of mice after single and multiply irradiations (total or partial body exposure). Int. J. Radiat. Biol. 18; 347-358, 1970. Cronkite, E.P., Harigaya, K., Garnett, H., Miller, M.E., Honikel, L., Shadduck, R.K: Production of colony-stimulating factor by murine bone marrow cell line derived from the dexter adherent layer and other properties of this cell line. In: Experimental Hematology Today, (Baum, S.J., Ledney, G.d., Thierfield, S., eds.), Basel:Karger, pp. 11-18, 1982. Cunningham, I., Gee, T., Dowling, M., Changanti, R., Bailey, R., Bowden, L., Turnbull, A., Knapper, W., Clarkon, B: Results of treatment of Phi-positive chronic myelogenous leukemia with an intensive treatment regimen (L-5 protocol). Blood 53; 375-395, 1979. Curry, J.L., and Trentin, J.J: Hemopoietic spleen colony studies in growth and differentiation. Dev. Biol. 15; 1967. 91 Dainiak, N., Davies, G. , Kalmanti, M., Lawler, J., Kulkarni, V: Platelet- derived growth factor promotes proliferation of erythropoietic progenitor cells in vitro. J. Clin. Invest. 71; 1206-1214, 1983. Dameshek, W: Some speculations on the myeloproliferative syndromes. Blood 6; 372-375, 1951. DeGowin, R.L. and Gibson, D.P: Prostaglandin mediated enhancement of erythroid colonies by marrow stromal cells (MSC). Exp. Hematol. 9; 274-280, 1981. DeKlein, A., Van Kessel, A.G., Grosveld, G., et al: A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature 300; 765-767, 1982. Dewey, W.C., Humphrey, R.M., Jones, B.A: Comparisions of tritiated thymidine, tritiated water, and cobalt-60 gamma rays in inducing chromosomal aberrations. Radiat. Res. 24; 214-227, 1965. Dewey, W.C., Sedita, B.A., Humphrey, R.M: Chromosomal aberration induced by tritiated thymidine during the S and G2 phase of Chinese hamster cells. Int. J. Radiat. Biol. 12; 597-606, 1967. Dexter, T.M. and Moore, M.A.S: In vitro duplication and "cure" of hemopoietic defects in genetically anaemic. Nature 269; 412-414, 1977. Dexter, T.M. and Shadduck, R.K: The regulation of hemapoiesis in long-term marrow cultures: I. The role of L-CSF. J. Cell Physiol. 102; 279-286, 1980. Dexter, T.M., Allen, T.D., Lajtha, L.G: Conditions controlling the proliferation of hemopoietic stem cells in vitro. J. Cell Physiol. 91; 335- 344, 1977a. ~~ Dexter, T.M., Wright, E.G., Krisza, F., Lajtha, L.G: Regulation of hemopoietic stem cell proliferation in long-term bone marrow cultures. Biomedicine 27; 344-349, 1977b. Dexter, T.M., Allen, T.D., Lajtha, L.G., Krizsa, F., Testa, N.G., Moore, M.A.S: In vitro analysis of self-renewal and commitment of hemopoietic stem cells. In: Differentiation of Normal and Neoplastic Hemopoietic Cells, Clarkson, B., Marks, P.A., T i l l , J.E., eds. Cold Spring Harbor Conferences on Cell Proliferation, New-York, Vol. 5, pp. 63-80, 1978. Dexter, T.M., Spooner, E., Toksoz, D., Lajtha, L.G: The role of cells and their products in the regulation of in vitro stem cell proliferation and granulocyte development. J. Supramol. Struct. 13; 513-524, 1980. Dexter, T.M., Testa, N.G., Allen, T.D., Rutherford, T., Scolnick, E: Molecular and cell biologic aspects of erythropoiesis in long-term bone marrow cultures. Blood 58; 699-707, 1981. Dexter, T.M., Simmons, P., Purnell, R.A., Spooner, E., Schofield, R: The regulation of hemopoietic cell development by the stromal cell enviroment and diffusible regulatory molecules. Prog. Clin. Biol. Res. 148; 13-33, 1984. 92 Dick, J.E., Magli, M.C., Huszar, D., Philips, R.A., Bernstein, A: Introduction of a selectable gene into primitive stem cells capable of long term reconstitution of the hemopoietic system of W/Wv mice. Cell 42; 71-79, 1985. Dippold, W.G., Joy, G., DeLeo, A.B., Khoury, G., Old, L.J: p53 transformation related protein: Detection by monoclonal antibody in mouse and human cells. Proc. Natl. Acad. Sci. U.S.A. 78; 1695-1699, 1981. Dorshkind, K. and Phillips, R.A: Characterization of early B lymphocyte prcursors present in long-term cultures. J. Immunol. 131; 1983. Douay, L., Barbu, V., Baillou, C, et al: The role of serum lipoproteins on the in vitro proliferative potential of human hematopoietic progenitors CFU- C and CFU-E. Exp. Hematol. 11; 499-505, 1983. Douer, D., Fabian, I., Cline, M.J.: Circulating pluripotent stem cells in patients with myeloproliferative disorders. Br. J. Hematol. 54; 373-381, 1983. Douer, D., Levin, A.M., Sparkes, R.S., Fabian, I., Sparkes, M., Cline, M.J., Koeffler, H.P: Chronic myelogenous leukemia: A pluripotent hematopoietic cell is involved in the malignant clone. Br. J. Hematol. 49; 615-619, 1981. Dube, I.D., Eaves, C.J., Kalousek, D.K., Eaves, A.C: A method for obtaining high quality chrmosome preparations from single hemopoietic colonies on a routine basis. Cancer Genet. Cytogenet. 4; 157-168, 1981. Dube, I.D., Gupta, CM., Kalousek, D.K., Eaves, CJ., Eaves, A.C: Cytogenetic studies of early myeloid progenitor compartments in Ph^-positive chronic myeloid leukemia (CML): I. Persistence of Ph^-negative committed progenitors that are suppressed from differentiating in vivo. Br. J. Haematol. 56; 633-644, 1984a. Dube, I.D., Arlin, Z.A., Kalousek, D.K., Eaves, CJ., Eaves, A.C: Nonclonal hemopoietic progenitor cells detected in long-term marrow cultures from a Turner synd rome mosiac with chronic myelogenous leukemia. Blood 64; 1284— 1287, 1984b. — Dulbecco, R. and Stoker, M.G.P: Conditions determining initiation of DNA synthesis in 3T3 cells. Proc. Natl. Acad. Sci. U.S.A 66; 204-210, 1970. Duplan, J.F., Feinendegen, L.E: Radiosensitivity of the colony forming cells of the mouse bone marrow. Proc. Soc. Exp. Med. 134; 319-321, 1970. Durham, A.C.H: The roles of small ions, especially calcium in virus disassembly, takeover, and transformation. Biomedicine 28; 307-317, 1978. Eaves, A.C, Bruce, W.R: Endotoxin induced sensitivity of hematopoietic stem cells to chemotherapeutic agents. Ser. Hematol. 2; 64, 1972. Eaves, A.C, Bruce, W.R: Altered sensitivity of hematopoietic stem cells to 5-fluorouracil (NSC-19893) following endotoxin (NSC-189681), cyclophosphamide (NSC-26271), or irradiation. Cancer Chemoth. Rep. 58; 813- 819, 1974. 93 Eaves, A.C. and Eaves, C.J: Abnormalities in the erythroid progenitor compartments in patients with chronic myelogenous leukemia (CML). Exp. Hematol. 7 (Suppl 5); 65-75, 1979. Eaves, A.C. and Eaves, C.J: In vitro studies of erythropoiesis in polycythemia vera. In: Current Concepts in Erythropoiesis, Dunn, C.D.R., ed. John Wiley and Sons Ltd., pp. 167-187, 1983. Eaves, A.C. and Eaves, C.J: Erythropoiesis in Culture. Clin. Hematol. 13; 371-391, 1984. Eaves, A.C, Henkelman, D.H., Eaves, CJ: Abnormal erythropoiesis in the myeloproliferative disorders: An analysis of underlying cellular and humoral mechanisms. Exp. Hematol. 8 (Suppl 8); 235-245, 1980. Eaves, A.C, Cashman, J., Coupland, R. , Eaves, C J : Erythropoietin- independence and altered proliferative status of early erythropoietic and granulopoietic progenitor cell populations in essential thromobocytosis. Blood 62 (Suppl 1); 169a, 1983b. Eaves, A.C, Cashman, J.D., Eaves, C J : Polycythemia vera: In vitro analysis of regulatory defects. In: Proceedings, Humoral and Cellular Regulation of Erythropoieis. Zanjani, E., Tavossoli, M., (eds.), Spectrum Publications Inc. Jamaica, New York, (in press). Eaves, C J . and Eaves, A.C: Erythropoietin dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood 52; 1196-1210, 1978. Eaves, C.J., Humphries, R.K., Eaves, A.C: In vitro characterization of erythroid precursor cells and the erythropoietic differentiation process. In: Cellular and Molecular Regulation of Hemoglobin Switching, Stamatoyannopoulos, C, Nienhuis, A.W., eds., pp. 251-278, Grune Stratton, New York, 1979a. Eaves, C.J., Humphries, R.K., Eaves, A.C: Marrow flask cultures: A system for examining early erythropoietic differentiation events. Blood Cells 5; 377-387, 1979b. Eaves, C.J., Coulombel, L., Eaves, A.C: Analysis of hemopoiesis in long- term human marrow cultures. In: Hemopoietic Stem Cells, Killmann, S.A., Cronkite, E.P., Muller-Berat, CN., eds. Munksgaard, Copenhagen, pp. 287- 298, 1983a. Eliason, J.F., Dexter, T.M., Testa, N.G: The regulation of hemopoiesis in long-term bone marrow cultures. III. The role of burst-forming activity. Exp. Hematol. 10; 444-450, 1982. Ell i s , J.T., Silver, T.T., Coleman, M., Geller, S.A: The bone marrow in polycythemia vera. Semin. Hematol. 12; 433-456, 1975. Eridani, S., Batten, E., Sawyer, B: Erythroid colony formation in primary thrombocythaemia: Evidence of hypersensitivity to erythropoietin. Br. J. Haematol. 55; 157-161, 1983. 94 Everett, N.B. and Perkin, W.D: Hemopoietic stem cell migration. In: Stem Cells of Renewing Cell Populations (Cairnie, A.B., Lala, P.K., Osmond, D.G., eds. Academic Press, New York, pp. 221-238, 1979. Fauser, A.A., Lohr, G.W: Recloned coloni es positive for T—cell associated antigens derived from hempoietic colonies (CFU-GEMM). Proc. Soc. Exp. Biol. Med. 170; 220-224, 1982. Fauser, A.A., Messner, M.A: Granuloerythropoietic colonies in human bone marrow, peripheral blood and cord blood. Blood 52: 1243-1248, 1978. Fauser, A.A., Messner, M.A: Identification of megakaryocytes, macrophages, and eosinophils in colonies of human bone marrow containing granulocytes and erythroblasts. Blood 53; 1023-1027, 1979. Fauser, A.A. and Messner, H.A: Pluripotent hemopoietic progenitors (CFU-GEMM) in polycythemia vera: Analysis of erythropoietin requirement and proliferative activity. Blood 58; 1224-1227, 1981. Fauser, A.A., Messner, H.A: Proliferative state of human pluripotent hemopoietic progenitors (CFU-GEMM) in normal individuals and under regenerative conditions after bone marrow transplantation. Blood 54; 1197- 1200, 1982. — Fauser, A.A., Kanz, L., Bross, K., Lohr, G.W: Identification of "pre-B" cells in multilineage hemopoietic colonies (CFU-GEMMT). Blood 62: 134a, 1983. ~~ Feher, I. and Gidali, J: Prostaglandin E 2 as stimulator of hemopoietic stem cell proliferation. Nature 247; 550-551, 1974. Fialkow, P.J., Gartler, S.M., Yoshida, A: Clonal origin of chronic myelocytic leukemia in man. Proc. Natl. Acad. Sci. U.S.A. 58; 1468-1471, 1967. — Fialkow, P.J., Jacobson, R.J., Papayannopoulou, T: Chronic myelocytic leukemia: Clonal evolution in a stem cell common to the granulocyte, erythrocyte, platelet, and monocyte/macrophage. Am. J. Med. 63; 125-130, 1977. Fialkow, P.J., Denman, A.N., Jacobson, R.J., Lowenthal, M.N: Chronic myelogenous leukemia: Origin of some lymphocytes from leukemia stem cells. J. Clin. Invest. 62; 815-823, 1978a. Fialkow, P.J., Denman, A.M., Singer, J., Jacobson, R.J., Lowenthal, M.N: Human myeloproliferative disorders: Clonal origin in pluripotent stem cells. In: Differentiation of Normal and Neoplastic Hematopoietic Cells. Cold Spring Harbor Laboratory, pp. 131-143, 1978b. Fialkow, P.J., Faguet, G.B., Jacobsen, R.J., Vaidya, K., Murphy, S: Evidence that essential thrombocythemia is a clonal disorder with origin in a multipotent stem cell. Blood 58; 916-919, 1981. Fowler, J.H., Wu, A.M., T i l l , J.E., McCulloch, E.A., Siminovitch, L: The cellular composition of hemopoietic colonies. J. Cell. Physiol. 69: 65-72, 1966. 95 Fox, C.F., Vale, R., Peterson, S.W., Das, M: Cell growth and division. In: Hormones and Cell Culture, Sato, G., Ross, R., Eds. Cold spring Harbor Laborator, pp. 143-158, 1979. Frindel, E., Croizat, H., Vassort, F: Stimulating factors liberated by treated bone marrow: in vitro effect on CFU kinetics. Exp. Hemat. 4; 56-61, 1976. Fung, M.C., Hapel, A.J., Ymer, S., et al: Molecular cloning of cDNA for murine IL-3. Nature 307; 233-237, 1984. Gale, R.P. and Canaani, E: An 8-kilobase abl RNA transcript in chronic myelogenous leukemia. Proc. Natl. Acad. Sci. USA 81; 5648-5652, 1984. Gartner, S. and Kaplan, H.S: Long-term culture of human bone marrow cells. Proc. Natl. Sci. U.S.A. 77; 4756-4759, 1980. Gazdar, A.F., Stull., H.B., Kilton, L., Bachrach, U: Increased ornthine decarboxylase activity in murine sarcoma virus infected cells. Nature 262; 696-698, 1976. Gewitz, A., Bruno, E., Elwell, J., Hoffman, R: In vitro studies of megakaryopoiesis in thrombocytotic disorders of man. Blood 61; 384-395, 1983. Gidali, J., Lajtha, L.G: Regulation of hemapoietic stem cell turnover in partially irradiated mice. Cell Tissue Kinet. 5; 147-157, 1972. Gilbert, H.S: Definition, clinical features and diagnosis of polycythemia vera. Clin. Haematol. 4; 263-290, 1975. Glasser, R.M. and Walker, R.J: Transitions amoung the myeloproliferative disorders. New Eng. J. Med. 304; 441-447, 1981. Goldberg, N.D., Haddox, M.K., Dunham, E: The cyclic nucleotides and cell growth. In: Control of Proliferation in Animal Cells. Clarkson, B., Baserga, R. (eds.) Cold Spring Harbor Laboratory, pp. 609-626, 1974. Golde, D.W., Rothman, B., Cline, M.J: Production of colony-stimulating factor by malignant leukocytes. Blood 43; 749-756, 1974. Golde, D.W., Burgaleta, C., Sparkes', R.S., Cline, M.J: The Philadelphia chromosome in human macrophages. Blood 49; 367-370, 1977. Goldman, J.M., Th'ng, K.H., Lowenthal, R.M: In vitro colony forming cells and colony stimulating factor in chronic granulocytic leukemia. Br. J. Cancer 30; 1-12, 1974. Goldman, J.M., Shiota, F., Th'ng K.H., Orchard, K.H: Circulating granulocyte and erythroid progenitor cells in chronic granulocytic leukemia. Br. J. Haematol. 46; 7-13, 1980. Goto, T., Nishikori, M., Arlin, Z., Adamson, J.W., Kempin, S.J., Clarkson, B., Fialkow, P.J: Growth characteristics of leukemic and normal hematopoietic cells in Ph^-positive chronic myelogenous leukemia and the effects of intensive treatment. Blood 59; 793-808, 1982. 96 Gough, N.M., Gough, J., Metcalf, D., et al: Molecular cloning of cDNA encoding a murine hematopoietic growth regulator, granulocyte-macrophage colony-stimulating factor. Nature 309; 763-767, 1984. Greenberg, P.L. and Mosney, S.A: Cytotoxic effects of interferon in vitro on granulocytic progenitor cells. Cancer Res. 37; 1794-1799, 1977. Greenberg, H.M., Newburger, P.E., Parker, L.M., Novak, T., Greenberger, J.S: Human granulocytes generated in continuous bone marrow are physiologically normal. Blood 58; 724-732, 1981. Greenberger, J.S: Sensitivity of corticosteroid dependent insulin resistant lipogenesis in marrow preadipocytes of obese-diabetic (db/db) mice. Nature 275; 752-754, 1978. Greenberger, J.S., Sakakeeny, M., Parker, L.M: In vitro proliferation of hemopoietic stem cells in long-term marrow cultures: Principles in mouse applied to man. Exp. Hematol. 7 (Suppl 5): 135-148, 1979. Gregory, C.J: Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: Studies of three erythropoietic colony responses in culture. J. Cell. Physiol. 89; 289-302, 1976. Gregory, C.J., Eaves, A.C: Human marrow cells capable of erythropoietic differentiation in vitro. Definition of three erythroid colony responses. Blood 49; 855-864, 1977. Gregory, C.J., Eaves, A.C: Three stages of erythropoietic progenitor cell differentiation distinguished by a number of physical and biological properties. Blood 51: 527-537, 1978a. Gregory, C.J. and Eaves, A.C: In vitro studies erythropoietic progenitor cell differentiation. In: Differentiation of Normal and Neoplastic Hemopoietic Cells, Clarkson, B., Marks, P.A., T i l l , J.E., eds. Cold Spring Harbor Conferences on Cell Proliferation, New York, Vol. 5 pp. 179-192, 1978b. Gregory, C.J. and Henkelman, R.M: Relationship between early hemopoietic progenitor cells determined by correlation analysis of their numbers in individual spleen colonies. In: Experimental Hematology Today, Baum, S.J. and Ledney, G.D. (Eds). Springer Verlag, New York, pg. 93, 1977. Gregory, C.J., McCulloch, E.A., T i l l , J.E: Erythropoietic progenitors capable of colony formation in culture: State of differentiation. J. Cell Physiol. 81; 411-420, 1973. Gregory, C.J., Tepperman, A.D., McCulloch, E.A., T i l l , J.E: Erythropoietic progenitors capable of colony formation in culture: Response of normal and genetically anemic W/Wv mice to manipulations of the erythron. J. Cell Physiol. 84; 1-12, 1974. Greulich, R.C: Deleterious influence of orally administered tritiated thymidine on the reproductive capacity of mice. Radiat. Res. 14; 83-99, 1967. 97 Griffin, J.C., Trantravahi, R., Canellos, G.P., Wisch, J.S., et al: T cell antigens in a patient with blast crisis of chronic myeloid leukemia. Blood 61; 640-645, 1983. Grobstein, C: Developmental role of intercellular matrix: Retrospective and prospectives. In: Extracellular Matrix Influences on Gene Expression, Slavkin, H.C. and Greulich, R.C, eds. Academic Press, New York, pp. 9-16, 1975. Groffen, J., Stephenson, J.R., Heisterkamp, N., de Klein, A., Bartram, CR., Grosveld, G: Philadelphia chromosomal breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell 36; 93-99, 1984. Gualtieri, R.J., Shadduck, R.K., Quesenberry, P.J: Characteristics of hemopoietic regulatory factors produced by long-term murine bone marrow cultures. Blood 60 (Suppl 1); 98A, 1982. Gualtieri, R.J., Shadduck, R.K., Baker, D.G., Quesenberry, P.J: Hematopoietic regulatory factors produced in long-term murine bone marrow cultures and the effect of in vitro irradiation. Blood 64; 516-525, 1984. Gurney, C.W: Pathogenesis of the polycythemias. In: Polycythemia: Theory and Management, Klein, H., ed. Charles C. Thomas, Ltd, Springfield, 111. pp 42, 1973. Hapel, A.J., Fung, M.C, Johnson, R.M., Young, I., Johnson, G., Metcalf, D: Biological properties of molecularly cloned and expressed murine interleukin-3. Blood 65; 1453-1459, 1985. Hara, H: Presence of cells in B-cell lineage in mixed (GEMM) colonies from murine marrow cells. Int. J. Cell Cloning 1; 171-181, 1983. Hara, H. Ogawa, M: Murine hemopoietic colonies in culture containing normoblast, macrophage and megakaryocytes. Am. J. Hematol. 4; 23-34, 1978. Hara, H., Kai, S., Fushimi, M., Taniwaki, S., Ifuku, H., Okamoto, T., Fujita, S., Noguchi, K., Kanamaru, A., Nagai, K., Inada, E: Pluripotent, erythropoietic, and granulocytic hemopoietic precursors in chronic granulocytic leukemia. Exp. Hematol. 9; 871-877, 1981. Harigaya, K., Cronkite, E.P., Miller, M.E., Shadduck, R.K: Murine bone marrow cell line producing colony stimulating factor. Proc. Natl. Acad. Sci. U.S.A. 78; 6963-6966, 1981. Heard, J.M., Fichelson, S., Varet, B: Role of colony-stimulating activity in murine long-term bone marrow cultures. Evidence for its production and consumption by the adherent layer. Blood 59; 761-767, 1982. Heby, 0., Marton, L.J., Zardij, L., Russell, D.H., Baserga, R: Changes in polyamine metabolism in WI-38 cells stimulated to proliferate. Exp. Cell Res. 90; 8-14, 1975. Heisterkamp, N., Stam, K., Groffen, J., de Klein, A., Grosveld. G: Structural organization of the bcr gene and its role in the Phi translocation. Nature 315; 758-761, 1985. 98 Hellman, S., Botnick, L.e., Hannon, E.c, Vigneulle, R.M: Proliferation capacity of murine hemopoietic stem cells. Proc. Natl. Acad. Sci. USA 75; 490-494, 1978. Hendry, J.H: The f number of primary transplanted colony-forming cells. Cell Tissue Kinet. 4; 217-223, 1971. Hernandez, P., Carnot, J., Cruz, C: Chronic myeloid leukemia blast crisis with T-cell features. Br. J. Hematol. 51; 175-182, 1982. Herrman, F, Kamischke, B., Kolecki, P., Ludwig, W.D., Sieber, G., Teichmann, H., Ruhl, H: Phi positive blast crisis of chronic myeloid leukemia exhibiting features characteristic of early T blasts. Scand. J. Haematol. 32; 411-417, 1984. Hibbin, J.A., McCarthy, D.M., Goldman, J.M: Antigenic expression and proliferative status of multilineage myeloid progenitor cells (CFU-GEMM) in normal individuals and patients with chronic granulocytic leukemia. Scand. J. Haematol. 31; 454-460, 1983. Hibben, J.A., Njoku, O.S., Matutes, E., Lewis, S.M., Goldman, J.M: Myeloid progenitor cells in the circulation of patients with myelofibrosis and other myeloproliferative disorders. Br. J. Haematol. 57; 495-503, 1984. Hocking, W.G. and Golde, D.W: Long-term human bone marrow cultures. Blood 56; 118-124, 1980. Hodgson, G.S. Bradley, T.R., Martin, R.F., Sumner, M., Fry, P: Recovery of proliferation hemapoietic progenitor cells after killing by hydroxyurea. Cell Tissue Kinet. 8; 51-60, 1975. Hoffman, R., Bruno, E., Gewirtz A: Kinetic analysis of megakaryocyte progenitor cells in myeloproliferative disorders. Clin. Res. 31; 482-493, 1983. Holley, R.N., Armour, R., Baldwin, J.H: Density dependent regulation of growth of BSC-1 cells in cell culture: Growth inhibitors formed by the cells. Proc. Natl. Acad. Sci. U.S.A. 75; 1864-1866, 1978. Holley, R.W: Control of growth of mammalian cells in cell culture. Nature 258; 487-490, 1975. Hori, T. and Nakai, S: Unusual dose-response of chromosome aberrations induced in human lymphocytes by very low dose exposure to tritium. Mutat. Res. 50; 101-110, 1978. Horland, A.A., Wolman, S.R., Murphy, M.T., Moore, M.A.S: Proliferation of erythroid colonies in semi-solid agar. Br. J. Haematol. 36; 495-499, 1977. Houba-Herin, N., Calberg-Bacq, CM., Piette, J., Van de Vorst, A: Mechanisms for dye mediated photodynamic action, singlet oxygen production, deoxyguanosine oxidation and phage inactivating effeciencies. Photochem. Photobiol. 36; 297-306, 1982. 99 Howard, A. and Pelc, S.R: Synthesis of deoxynucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heredity, (Suppl. 6); 261-273, 1953. Humphries, R.K., Eaves, A.C, Eaves, CJ: Characterization of a primitive erythropoietic progenitor found in mouse marrow before and after several weeks in culture. Blood 53; 746-763, 1979a. Humphries, R.K., Jacky P.B., Di l l , F.J., Eaves, A.C, Eaves, C J : CFU-S in individual erythroid colonies derived in vitro from adult mouse marrow. Nature 279; 718-720, 1979b. Humphries, R.K., Eaves, A.C, Eaves, CJ: Self-renewal of hemopoietic stem cells during mixed colony formation in vitro. Proc. Natl. Acad. Sci. U.S.A. 78; 3629-3633, 1981. Hung, C C , Ninan, T.A. , Petriccini, J.C: Extensive chromosome aberrations caused by ^-thymidine incorporation in a diploid monkey cell line DBS-FrhL- 2. In Vitro 11; 234-238, 1973. Iscove, N.N., Senn, J.S., T i l l , J.E., McCulloch, E.A: Colony formation by normal and leukemic human marrow cells in culture: Effect of conditioned medium from human leukocytes. Blood 37; 1-5, 1971. Iscove, N.N: The role of erythropoietin in regulation of population size and cell cycling of early and late erythroid precursors in mouse bone marrow. Cell Tissue Kinet. 10; 323-334, 1977. Islam, A., Catovsky, D., Galton, D.A.G: Histological study of bone marrow regeneration following chemotherapy for actute myeloid leukemia and chronic granulocytic leukemia in blast transformation. Br. J. Haematol. 45; 535- 540, 1980. Islam, A., Catovsky, D., Goldman, J.M., Galton, D.A: Studies on cellular interactions between stromal and hemopoietic stem cells in normal and leukemic bone marrow. Biblthca. Haematol. 50; 17-30, 1984. Itani, S., Hoshino, T: Mitogen responsive Philadelphia chromosome-positive T and B cells in the chronic phase of chronic myeloid leukemia. Proceedings of the 13th Internationsl Cancer Congress, Seattle, Washington, p. 399, 1982. Jacobs, K., Shoemaker, C, Rudersdorf, R., et al: Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313; 806-810, 1985. Jacobs, P. Greaves, M: Ph1 -positive T lymphoblastic transformation. Leuk. Res. 8: 737-739, 1984. Jacobson, L.O., Goldwasser, E., Fried, W., and Plzak, L: Role of the kidney in erythropoiesis. Nature 179; 633-634, 1957. Jacobson, R.J.. Salo, A., Fialkow, P.J: Agnogenic myeloid metaplasia: A clonal proliferation of hematopoietic stem cells with secondary myelofibrosis. Blood 51; 189-194, 1978. 100 Janossy, G., Greaves, M.F., Revesz, T., et al: Blast crisis of chronic myeloid leukemia (CML). II Cell surface marker analysis of lymphoid and myeloid cases. Br. J. Haematol. 34'; 179-192, 1976. Jannossy, G., Woodruff, R.K., Paxton, Greaves, M.F., Capellaro, D., Kirk, B., Innes, E.M., Eden, O.B., Lewis, C., Catovsky, D., Hoffbrand, A.V: Membrane marker and cell separation studies in Ph^-positive leukemia. Blood 51; 861-869, 1978. Johnson, G.R., Metcalf, D: Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin. Proc. Natl. Acad. Sci. U.S.A. 74: 3879-3882, 1977. Jones-Villeneuve, E. and Phillips, R.A: Potentials for lymphoid differentiation by cells from long-term cultures of bone marrow. Exp. Hematol. 8; 65-76, 1980. Juraskova, V., Tkadlecek, L., Character of primary and secondary colonies of hematopoiesis in the spleen of irradiated mice. Nature 206: 951-952, 1965, Kawasaki, E.S., Ladner, M.B., Wang, A.M., Arsdell, J.A., et al: Molecular cloning of a complementary DNA encoding human macrophage specific colony- stimulating factor (CSF-1). Science 230; 291-296, 1985. Kay, H.E., Lawler, S.D., Millard, R.E: The chromosomes in polycythemia vera. Br. J. Haematol. 12; 507-528, 1966. Kearney, L., Orchard, K.H., Hibbin, J., Goldman, J.M: T cell cytogenetics in CGL. Lancet 1; 858, 1982. Keating, A. and Singer, J.W: Further characterization of the in vitro hemopoietic microenviroment. Exp. Hematol. 11 (Suppl. 14); 144, 1983. Kelly, K., Cochran, B.H., Stiles, Cd., Leder, P: Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35; 603-610, 1983. Kerk, D.K., Henry, E.A., Eaves, A.C, Eaves, CJ: Two classes of primitive pluripotent hemopoietic progenitor cells: Separation by adherance. J. Cell Physiol. 125; 127-134, 1985. Klimpel, G.R., Fleishmann, W.R., Klimpel, K.D: Gamma interferon (IFN gamma) and IFN alpha/beta suppress murine myeloid colony formation (CFU-C) 2. Magnitude of suppression is dependent upon level of colony stimulating factor (CSF). J. Immunol. 129; 76-80, 1982. Konopka, J.B., Watanabe, S.M., Witte, 0.N: An alteration of the human c-abl protein in K562 leukemia cells unmasks associated tyrosine kinase activity. Cell 37; 1035-1042, 1984. Konopka, J.B., Watanabe, S.M., Singer, J.W., Collins, S.J., Witte, 0.N: Cell lines and clinical isolates derived from Phi-positive chronic myelogenous leukemia patients express c-abl proteins with a common structural alteration. Proc. Natl. Acad. Sci. USA 82; 1810-1814, 1985. 101 Korn, A.P., Henkelman, R.M., Ottenmeyer, F.P., T i l l , J.E: Investigations of a stochastic model of hemapoiesis. Exp. Hematol. 1; 362-375, 1973. Krantz, S.B., Jacobson, L.0: Erythropoietin and the Regulation of Erythropoiesis. Chicago, University of Chicago Press, 1970. Kurland, J.I., Hadden, J.W., Moore, M.A.S: Role of cyclic nucleotides in the proliferation of committed granulocyte-macrophage progenitor cells. Cancer Res. 37; 4534-4538, 1977. Kurland, J.I., Bockman, R.S., Broxmeyer, H.E., Moore, M.A.S: Limitation of excessive myelopoiesis by the extrinsic modulation of macrophage derived prostaglandin E. Science 199; 552-556, 1978. Kurnick, J.E., Robinson, W.A: Colony growth of human peripheral white blood cells in vitro. Blood 37; 136-141, 1971. Lacombe, C, Casadevall, N., Varet, B: Polycythemia vera: In vitro studies of circulation erythroid progenitors. Br. J. Haematol. 44; 189-199, 1980. Lajtha, L.G: On the concept of the cell cycle. J. Cell. Comp. Physiol. (Suppl. 1) 62; 143-145, 1963. Lambertson, R.H: Interdigitative coupling of presumptive hemopoietic stem cells to macrophages in endocloned marrow colonies. Blood 63; 1225-1229, 1984. Lanotte, M., Metcalf, D., Dexter, T.M: Production of monocyte/macrophage colony-stimulating factor by preadipocyte cell lines derived from murine bone marrow stroma. J. Cell Physiol. 112; 123-127, 1982. Leary, A.G., Ogawa, M., Strauss, L.C., Civin, C.I: Single cell origin of multilineage colonies in culture. Evidence that differentiation of multipotent progenitors and restriction of proliferative potential of monopotent progenitors are stochastic processess. J. Clin. Invest. 74; 2193-2197, 1984. Leary, A.G., Strauss, L.C., Civin, C.I., Ogawa, M: Disparate differentiation in hemopoietic colonies derived from human paired progenitors. Blood 66; 327-332, 1985. LeBien, J.W., Hozier, J., Minowada, J., Kersey, J: Origin of chronic myelocytic leukemia in a precursor of pre-B lymphocytes. N. Eng. J. Med. 301; 144-147, 1979. Lee, F., Yokota, T., Otsuka, T., Gemmell, L . , Larson, N., Lah, J. Arai, K., Rennick, D: Isolation of cDNA for a human granulocyte/macrophage colony- stimulating factor by functional expression in mammalian cells. Proc. Natl. Acad. Sci. 82; 4360-4364, 1985. Lee-Huang, S: Cloning and expression of human erythropoietin cDNA in E. coli. Proc. Natl. Acad. U.S.A. 81; 2708-2712, 1984. Lembach, K.I: Induction of human fibroblast proliferation by epidermal growth factor (EGF); enchancement by an EGF-binding arginine esterase and by 102 an EGF-binding arginine esterase and by ascorbate. Proc. Natl. Acad. Sci. USA 73; 183-187, 1976. Lepault, F., Fache, M.P., Frindel, E: Effect of adult thymectomy and splenectomy on pluripotent stem cells and progenitors of the myeloid and B- lymphoid lineages. Leuk. Res. 4; 663-670, 1980. Lepine, J.and Messner, H.A: Pluripotent hemopoietic progenitors (CFU-GEMM) in chronic myelogenous leukemia. Intern. J. Cell Cloning. 1; 1230-1239, 1983. Lewis, J.P., Trobaugh, F.E: Hematopoietic stem cells. Nature 204: 589-590, 1964. Lieberman, M.A., Glaser, L: Density-dependent regulation of cell growth: An example of a cell-cell recognition phenomenon. J. Memb. Biol. 63; 1-11, 1981. Lieberman, M.A., Raben, D., Glaser, L: Cell surface associated growth inhibitory proteins. Exp. Cell Res. 126; 413-419, 1981. Lim, B., Jamal, N., Tritchler, D., Messner, H.A: G6PD isoenzyme analysis of myeloid and lymphoid cells in human multilineage colonies. Blood 63: 1481- 1487, 1984a. Lim, B., Jamal, N., Messner, H.A: Flexible association of hemopoietic differentiation programs in multilineage colonies. J. Cell Physiol. 121; 291-297, 1984b. Liskay, R.M., Kornfeld,B., Fullerton, P., Evans, R: Protein synthesis and the presence or absence of a measurable Ĝ  in cultured C h i n e s e hamster cells. J. Cell. Physiol. 104; 461-467, 1980. Lobue, J. and Lobue, P.A: Control of cell proliferation. Transplant. Proc. 16; 341-348, 1984. Lord, B.I., Mori, K.J., Wright, E.G., Lajtha, L.G: An inhibitor of stem cell proliferation in normal bone marrow. Br. J. Hematol. 34; 441-445, 1976. Lord, B.I., Mori, K.J., Wright, E.G: A stimulator of stem cell proliferation in regenerating bone marrow. Biomedicine 27; 223-226, 1977. Lord, B.I., Wright, E.G., Mori, K.J: The role of proliferation inhibitors in the regulation of hemopoiesis. In: Stem Cells and Tissue Homeostasis, Lord, B.I., Potten, C.S., Cole, R., eds. Cambridge University Press, pp. 236, 1977. Lord, B.I. and Wright, E.G: Interaction of inhibitor and stimulator in the regulation of CFU-S proliferation. Leuk. Res. 6; 541-551, 1982. Lu, L.U., Broxmeyer, H.E., Meyers, P.A., et al: Association of cell cycle expression of la-like antigenic determinants on normal human multipotential (CFU-GEMM) and erythroid (BFU-E) progenitor cells with regulation in vitro by acidic isoferritins. Blood 61; 250-256, 1983. 103 Lu, L., Pelus, L.M., Broxmeyer, H.E: Modulation of the expression of HLA-DR (Ia) antigens and the proliferation of human erythroid (BFU-E) and multipotential (CFU-GEMM) progenitor cells by prostaglandin E. Exp. Hematol. 12; 741-748, 1984. Lyon, M.F: Gene action in the X-chromosome of the mouse. Nature 190; 372- 373, 1961. MacManus, J.P., Whitfield, J.F., Boynton, A.C, Rixon, R.H: Cyclic AMP and calcium as intracycle regulators in the control of cell proliferation. Adv. Cyclic. Nucleotide Res. 9; 485-491, 1978. Magli, M.C, Iscove, N.N. , Odartchenko, N: Transient nature of early hematopoietic spleen colonies. Nature 295; 527-529, 1982. Martin, P.J., Najfeld, V., Hansen, J.A., Penfold, G.K., Jacobson, R.J., Fialkow, P.J: Involvement of the B lymphoid system in chronic myelogenous leukemia. Nature 287; 49-57, 1980. Mauch, P., Greenberger, J.S., Botnick, L., Hannon, E., Hellman, S: Evidence for structered variation in self-renewal capacity within long-term bone marrow cultures. Proc. Natl. Acad. Sci. U.S.A. 77; 2927-2930, 1980. Mazur, E.M., Hoffman, R., Chasis, J., Marchesi, S., Bruno, E: Immunofluorescent identification of human megakaryocyte colonies using an antiplatelet glycoprotein antiserum. Blood 57; 277-281, 1981. McCaffrey, R., Smoler, D.F., Baltimore, D: Terminal deoxynucleotidyl transferase in a case of childhood acute lymphoblastic leukemia. Proc. Natl. Acad. Sci. U.S.A. 70; 521-525, 1973. McCaffrey, R., Harrison, T.A., Parkman, R., Baltimore, D: Terminal deoxynucleotidyl transferase activity in human leukemic cells and in normal thymocytes. N. Eng. J. Med. 292; 775-780, 1975. McCulloch, E.A; Les clones de cellules hematopoietiques in vivo. Rev. Franc. Etudes Clin, et Biol. 8: 15-19, 1963. McCulloch, E.A., Siminovitch, L., T i l l , J.e., russell, E.S., Bernstein, S.E: The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype Sl/Sl d. Blood 26; 399-410, 1965. McLeod D.L., Shreeve, M., Axelrad, A.A: Induction of megakaryocytic colonies with platelet formation in vitro. Nature 261; 492-494, 1976. McNeil, T.A. and Fleming, W.A: The relationship between serum interferon and an inhibitor of mouse hemopoietic colonies in vitro. Immunology 21; 761-766, 1971. Messner, H.A., Fauser, A.A., Lepine, J., Martin, M: Properties of human pluripotent hemopoietic progenitors. Blood Cells 6; 595-607, 1980. Messner, H.A., Izaguirre, C.A., Jamal, N: Identification of T-lymphocytes in human mixed hemopoietic colonies. Blood 58; 402-405, 1981. 104 Messner, H.A., Jamal, N., Izaquirre, C: the growth of large megakaryocyte colonies from human bone marrow. J. Cell Physiol. (Suppl 1); 45-51, 1982. Metcalf, D: Hemopoietic colonies: In vitro cloning of normal and leukemic cells. In: Recent Results in Cancer Research, Vol. 61, Springer-Verlag, Berlin, 1977. Metcalf, D: Regulation of hemopoiesis. Nouv. Rev. Fr. Hematol. Blood Cells 20; 521-533, 1979. Metcalf, D: The granulocyte/macrophage colony stimulating factors. Science 229; 6-22, 1985 Metcalf, D. and Johnson, G.R: Interactions between purified GM-CSF, purified erythropoietin and spleen conditioned medium on hemopoietic colony formation in vitro. J. Cell Physiol. 99; 159-174, 1974. Metcalf, D. and Johnson, G.R: Production by spleen and lymph node cells of conditioned medium with erythroid and other hemopoietic colony stimulating activity. J. Cell Physiol. 96; 31-42, 1978. Metcalf, D., Moore, M.A.S., Sheridan, J.W., Spitzer, G: Responsiveness of human granulocytic leukemia cells to colony stimulating factor. Blood 43; 847-859, 1974. — Metcalf, D., Macdonald, H.R., Odartchenko, N., Sordat, B: Growth of mouse megakaryocyte colonies in vitro. Proc. Nat. Acad. Sci. 72; 1744-1748, 1975. Metcalf, D., Johnson, G.R., Mandel, T.E: Colony formation in agar by multipotential hemopoietic cells. J. Cell. Physiol. 98; 401-420, 1979. Michalevicz, R., Francis, G.E., Price, G., Hoffbrand, A.V: The role of platelet derived growth factor on human pluripotent progenitor (CFU-GEMM) growth in vitro. Leuk. Res. 9; 399-405, 1985. Micklem, H.S., Ogden, D.A: Ageing of hematopoietic stem cell populations in the mouse. In: Stem Cells of Renewing Cell Populations, Cairnie, A.B., Lala, P.K., Osmond, D.G. (Eds). Academic Press, New York, pg. 331, 1976. Micklem, H.S., Anderson, N., Ross, E: Limitied potential of circulation hemapoietic stem cells. Nature 256; 41-43, 1975. Millard, R.E., Lawler, S.D., Kay, H.E., et al., Further observation on patients with a chromosomal abnormality associated with polycythemia vera. Br. J. Haematol. 14; 363-374, 1968. Minot, J.B., Buckman, T.E., Isaacs, R: Chronic myelogenous leukemia; age, incidence, duration, and benefit derived from irradiation. J.A.M.A. 82; 1489, 1924. Mladenovic, J. and Adamson, J.W: Characteristics of circulating erythroid colony-forming cells in normal and polycythemic man. Br. J. Haematol. 51; 377-384, 1982. 105 Moberg, C., Olofsson, T., Olsson, J: Granulopoiesis in chronic myeloid leukemia. I. In vitro cloning of blood and bone marrow cells in agar culture. Scand. J. Haematol. 12; 381-390, 1974. Moore, M.A.S., Metcalf, D: Cytogenetic analysis of human acute and chronic myeloid leukemic cells clone in agar culture. Int. J. Cancer 11; 143-152, 1973. Moore, M. and Robinson, W.A: Granulopoietic activity of urine and cells from patients with chronic granulocytic leukemia. Proc. Soc. Exp. Biol. Med. 146; 499-503, 1974. Moore, M.A.S., Williams, N., Metcalf, D: In vitro colony formation by normal and leukemia human hematopoietic cells: Characterization of the colony forming cells. J. Natl. Cancer Inst. 50; 603-623, 1973. Morley, A., Blake, S: An animal model of chronic aplastic marrow failure. I . Late marrow failure after busulphan. Blood 44; 49-57, 1974. Nakahata T., Ogawa, M: Clonal origin of murine hemopoietic colonies with apparent restriction to granulocyte-macrophage-megakaryocyte (GMM) differentiation. J. Cell Physiol. I l l : 239-246, 1982a. Nakahata, T., Ogawa, M: Identification in culture of a class of hemopoietic colony forming units with extensive capability to self-renew and generate multipotential hematopoietic colonies. Proc. Natl. Acad. Sci. U.S.A. 79: 3843-3847, 1982b. Nakahata, T., Ogawa, M: Hemopoietic colony-forming cells in umbilical cord blood with extensive capability to generate mono- and multipotential hemopoietic progenitors. J. Clin. Invest. 70: 1324-1328, 1982c. Nakahata, T., Gross, A.J., Ogawa, M: A stochastic model of self-renewal and committment to differentiation of the primitive hemopoietic stem cells in culture. J. Cell Physiol. 113; 455-458, 1982. Nakeff, A., Daniels-McQueen, S: In vitro colony assay for a new class of megakaryocyte precursor: Colony-forming unit megakaryocyte (CFU-M). Proc. Soc. Exp. Biol. Med. 151; 587-590, 1976. Nakeff, A., Dicke, K.A., Von Noord, M.J: Megakaryocytes in agar cultures of mouse bone marrow. Ser. Haemat. 8; 4-21, 1975. Natarajan, A.T. and Meyers, M. Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages. Hum. Genet. 52; 127- 132, 1979. National Council on Radiation Protection and Measurements: Tritium and other radionuclide labeled organic compounds incorporated in genetic material. Preport No. 63, Washington, D.C, 147pps. 1979. Natraj, CV. and Datta, P. Control of DNA synthesis in growing BALB/c 3T3 mouse cells by a fibroblast growth regulatory factor. Proc. Natl. Acad. Sci. U.S.A. 75; 6115-6119, 1978. 106 Neumann, H.A. and Fauser, A.A: Effect of interferon on pluripotent hemopoietic progenitors (CFU-GEMM) derived from human bone marrow. Exp. Hematol. 10; 587-590, 1982. Niskanen, E., Kallio, A., McCann, P.P., Baker, D.G: The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice. Blood 61; 740-745, 1983. Nissley, S.P., Passamoni, J., Short, P: Stimulation of DNA synthesis, cell multiplication and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA). J. Cell Physiol. 89; 393-402, 1976. Nitta, M., Kato, Y., Strife, A., Wachter, M., et al: Incidence of involvement of the B and T lymphocyte lineages in chronic myelogenous leukemia. Blood 66; 1053-1061, 1985. Nowell, P.C., Hungerford, D.A: A minute chromosome in human chronic granulocytic leukemia. Science, 132; 1497, 1960. Oliviera, G. and Oliviera, A: The mutagenic effect of tritiated uridine in Drosophila spermatocytes. Mutat. Res. 2; 381-389, 1965. Olofsson, T. and Olsson, I: Granulopoiesis in chronic myeloid leukemia. II. Serial cloning of blood and bone marrow cells in agar culture. Blood 48; 351-360, 1976. Palutke, M., Eisenberg, L., Nathan, L: Phi-positive T lymphoblastic transformation of chronic granulocytic leukemia in a lymph node. Lancet 2; 1053-1054, 1982. Paran, M. and Sachs, L: The continued requirement for inducer for development of macrophage and granulocyte colonies. J. Cell Physiol. 72; 247-253, 1968. Paran, M., Sachs, L: The single cell origin of normal granulocytic colonies in vitro. J. Cell. Physiol. 73; 91-92, 1969. Pardee, A.B: A restriction point for control of normal animal cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 71; 1286-1290, 1974. Pardee, A.B., Dubrow, R. , Hamlin, J.C, Kletzien, R.F: Animal cell cycle. Ann. Rev. Biochem. 47; 715-750, 1978. Parker, J.W., Metcalf, D: Production of colony-stimulating factor in mixed leukocyte cultures. Immunology 26; 1039-1044, 1974. Partenen, S., Ruutu, T., Vuopio, P: Hematopoietic progenitors in essential thrombocythaemia. Scand. J. Haematol. 30; 130-134, 1983. Pasten, I.H., Johnson, G.S., Anderson, W.B: role of cyclic nucleotides in growth control. Ann. Rev. Biochem. 44; 491-522, 1975. Peak, J.G., foote, C.S., Peak, M.J: Protection by DABC0 against inactivation of transforming DNA by near ultra-violet light: action spectra and implications for involvement of singlet oxygen. Photochem. Photobiol. 34; 45-49, 1981. 107 Pearson, M.G., Lawson, R.A., Golomb, H.M: Clinical significance of chromosome patterns in malignant diseases. In: Chromosomes and Cancer, Academic Press, New York, pp 311-331, 1983. Pelus, L.M., Broxmeyer, H.E., Kurland, J.I: Regulation of macrophage and granulocyte proliferation: Specificities of prostoglandins E and lactoferrin. J. Exp. Med. 15: 277-292, 1979. Pelus, J.M., Broxmeyer, Clarkson, B.D., Moore, M.A.S: Abnormal responsiveness of GM-committed colony-forming cells from patients with CML to inhibition by prostaglandin E. Cancer Res. AO; 2512-2515, 1980. Person, S., Snipes, W.S., Kradin, F: Mutation production from tritium decay: A local effect for [3H]2-adenosine and [3H]6-thymine decay. Mutat. Res. 3A; 327-335, 1976. Phillips, R.A., Bosma, M., Dorshkind, K: Reconstitution of immune deficient mice with cells from long term bone marrow cultures. In: Long Term Bone Marrow Cultures, Allan R. Liss, Inc., p 309, 198A. Piette, J., Lopez, M., Calberg-Bacq, CM., Van de Vorst, A: Mechanism for strand break induction in DNA-proflavine complexes exposed to visible light. Int. J. Radiat. Biol. AO; A27-A33, 1981. Pike, B.L., Robinson, W.A: Human bone marrow colony growth in agar-gel. J. Cell. Physiol. 76; 77-8A, 1970. Playfair, J.H.L., Cole, L.J: Quantitative studies on colony forming units in isogenic radiation chimeras. J. Cell. Comp. Physiol. 65; 7-19, 1965. Pledger, W.J., Stiles, CD., Antoniades, H.N., Scher, CD: Induction of DNA synthesis in BALB/c-3T3 cells by serum components: Reevaluation of the committment process. Proc. Natl. Acad. Sci. USA 7A; AA81-AA85, 1977. Ploemacher, R.E., Van't Hull, E., Van Soest, P.L: Studies of the HIM: Effects of acid mucopolysaccharides and dextran sulphate on erythroid and granuloid differentiation in vitro. Exp. Hematol. 6; 311-320, 1978. Pluznick, D.H., Sachs, L: The induction of normal "mast" cells by a substance in conditioned medium. Exp. Cell Res. A5; 553-563, 1966. Powell, J.S., Fialkow, P.J., Adamson, J.W: Polycythemia vera: Studies of hemopoiesis in continuous long-term culture of human marrow. J. Cell Physiol. (Suppl. 1); 79-85, 1982. Prchal, J.F. and Axelrad, J.W: Bone marrow responses in polycythemia vera. New Eng. J. Med. 290; 1382 (letter), 197A. Prchal, J.F., Axelrad, A.A., Crookston, J.H: Erythroid colony formation in plasma culture from cells of peripheral blood in myeloproliferative disorders. Blood AA; 912, 197A. Prchal, J.F., Adamson, J.W., Steinmann, L., Fialkow, P.J: Human erythroid colony formation in vitro: evidence for clonal origin. J. Cell. Physiol. 89, A89-A92, 1976. 108 Prchal, J.P., Throckmorton, D.W., Carroll, A.J., Fuson, E.W., Gams, R.A.. Prchal, J.F: A common progenitor for human myeloid and lymphoid cells. Nature 274: 590-591, 1978a. Prchal, J.F., Adamson, J.W., Murphy, S., Steinmann, L., Fialkow, P.J: Polycythemia vera: The in vitro response of normal and abnormal stem cell lines to erythropoietin. J. Clin. Invest. 61; 1044-1047, 1978b. Prescott, D.M: Control of the initiation of DNA synthesis in mammalian cells. Ann. N. Y. Acad. Sci. 397; 101-109, 1982. Rachmeler, M. and Pardee, A.B: Loss of viability and beta-galactosidase forming ability as a consequence of tritium decay in Escherichia coli. Biochim. Biophys. Acta 68; 62-67, 1963. Ralph, R.K: Cyclic AMP, calcium, and control of cell growth. F.E.B.S. 161; 1-8, 1983. Rao, P.N. and Johnson, R.T: Mammalian cell fusion: Studies on the regulation of DNA synthesis and mitosis. Nature 225: 159-164, 1970. Rapp, U.R., Cleveland, J.L., Brightman, K., Scott, A., Ihle, J.N: Abrogation of IL-3 and IL-2 dependence by recombinant murine retroviruses expressing v-myc oncogenes. Nature 317; 434-438, 1985. Rastrick, J.M.. Fitzgerald, P.H., Gunz F.W: Direct evidence for the presence of Phi chromosome in erythroid cells. Br. Med. J. 1; 96-98, 1968. Rencricca, N.J., Rizzoli, V., Howard, D., Duffy, P., Stohlman, F: Stem cell migration and proliferation during severe anemia. Blood 36; 764-771, 1970. Rennick, D.M., Lee, F.D., Yokota, T., Arai, K., Cantor, H., Nabel, G.J: A cloned MCGF cDNA encodes a multilineage hematopoietic growth factor; Multiple activities of interleukin-3. J. Immunol. 134; 910-914, 1985 Rickard, K.A., Brown, K.A., Wilkinson, T., Kronenberg, H: The colony- forming cell in the myeloproliferative disorders and aplastic anemia. Scand. J. Haematol. 22; 121-128, 1979. Roodman, G.D. and Zanjani, E.D: Endogenous erythroid colony-forming cells in fetal and newborn sheep. J. Lab. Clin. Med. 94; 699-713, 1979. Rosendaal, M., Hodgson, G.S., Bradley, T.R: Organization of hemopoietic stem cells: The generation age hypothesis. Cell Tissue Kinetics 12; 17-29, 1979. Ross, E.M. and Gilman, A.G: Biochemical properties of hormone sensitive adenylate cyclase. Ann. Rev. Biochem. 49; 533-564, 1980. Rossi, G.B., Migliaccio, A.R., Migliaccio, G., et al: In vitro interactions of PGE and cAMP with murine and human erythroid precursors. Blood 56; 74- 79, 1980. — Rossow, P.W., Riddle, V.G., Pardee, A.B: Synthesis of labile, serum dependent protein in early Ĝ  controls animal cell growth. Proc. Natl. Acad. Sci. U.S.A. 76; 4446-4450, 1979. 109 Rothstein, H: Regulation of the cell cycle by somatomedins. Int. Rev. Cytol. 78; 127-232, 1982. Rovera, G. and Baserga, R: Effect of nutritional changes on chromating template activity and nonhistone chromasomal protein synthesis. Exp. Cell Res. 78; 118-126, 1973. Rowley, J.D: A new consistent chrmosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and giemsa staining. Nature 243; 290-293, 1973. Rowley, J.D: The role of cytogenetics in hematology. Blood 8; 1-7, 1976. Rowley, J.D: Ph^-positive leukemia, including chronic myelogenous leukemia. In: Clinics in Hematology, Pennington, D.G., ed., Vol 9; Praeger Scientific, New York, pp. 55-86, 1980. Rubin, H: Nonspecific nature of the stimulus to DNA synthesis in cultures of chick embryo cells. Proc. Natl. Acad. Sci. U.S.A. 72; 1676-1680, 1975a. Rubin, H. Central role for magnesium in coordinate control of metabolism and growth in animal cells. Proc. Natl. Acad. Sci. U.S.A. 72; 3551-3555, 1975b. Sakurai, M., Hayata, I., Sandberg, A.A: Prognostic value of chromosomal findings in Ph^-positive chronic myelocytic leukemia. Cancer Res. 36; 313- 318, 1976. — Sandberg, A.A: The Chromosomes in Human Cancer and Leukemia. Elsvier/North Holland, New York, 1980 Saunders, G. and Pardee, A.B: Transport changes in synchronously growing CHO and L cells. J. Cell Physiol. 80; 267-272, 1972. Scher, CD., Shepard, R.D., Antoniades, H.N. , Stiles, CD: Platelet derived growth factor and the regulation of the mammalian fibroblast cell cycle. Biochim. Biophys. Acta 560; 217-241. 1979. Schofield, R: The relationship between the spleen colony forming cell and the hemopoietic stem cell. Blood Cells 4; 7-25, 1978. Schofield, R. and Lajtha, L.G: Effect of isopropyl methane sulphanate (IMS) on hemopoietic colony forming cells. Brit. J. Haemat. 25; 195-202, 1973. Schofield, R. , Lord, B.I., Kyffin, S., Gilbert, C.W: Self-maintenance capacity of CFU-S. J. Cell Physiol. 103; 355-362, 1980. Schrader, J.W. and Schrader, S: In vitro studies of lymphocyte differentiation. I. Long-term in vitro culture of cells giving rise to functional lymphocytes in irradiated mice. J. Exp. Med. 148; 823-828, 1978. Seeker-Walker, L.M., Hardy, J.D. Philadelphia chromosome in PHA-stimulated lymphocytes in acute leukemia. Lancet i ^ : 1301, 1975. 110 Senn, J.S., McCulloch, E;A., T i l l , J.E: Comparision of colony forming ability of normal and leukemic human marrow in cell culture. Lancet i ^ ; 597-598, 1967. Shabtai, F., Gafter, U., Weiss, S., Djaldetti, M., Halbrecht, I: New complex Phi translocation t(10;14;22) in bone marrow cells and in PHA- stimulated peripheral blood cultures in chronic myelocytic leukemia. J. Cancer Res. Clin. Oncol. 96; 287-292, 1980. Shadduck, R.K., Waheed, A., Greenberger, J.S., Dexter, T.M: Production of colony-stimulating factor in long-term bone marrow cultures. J. Cell Physiol. 114; 88-92, 1983. Shields, R. and Smith, J.A: Cells regulate their proliferation through alterations in transition probability. J. Cell Physiol. 91; 345-356, 1977. Shtivelman, E., Lifshitz, B., Gale, R.P., Canaani, E: Fused transcript of abl and bcr genes in chronic myelogenous leukemia. Nature 315; 550-554, 1985. Silverstein, M.N: The evolution into and the treatment of late stage polycythemia vera. Semin. Hematol. 13; 79-93, 1976. Silverstein, M.N: Diagnosis and treatment of polycythemia vera, agnogenic myeloid metaplasia, and primary thrombocythemia. In: Neoplastic Diseases of the Blood, (Wiernik, P.H., Canellos, G.P., Kyle, R.A., Sciffer, C.A., eds.), Churchill Livingston, New York, 1985. Siminovitch, L., McCulloch, E.A., T i l l , J.E: The distribution of colony forming cells amoung spleen colonies. J. Cell. Physiol. 62: 327-336, 1963. Siminovitch, L., T i l l , J.E., McCulloch, E.A: Decline in colony forming ability of marrow cells subjected to serial transplantation into irradiated mice. J. Cell Comp. Physiol. 64; 23-32, 1964. Singer, J.W., Fialkow, P.J., Dow, L.W., Ernst, C., Steinmann, L: Unicellular or multicellular origin of human granulocyte-macrophage colonies in vitro. Blood 54; 1395-1399, 1979a. Singer, J.W., Fialkow, P.J., Steinmann, L., et al: Chronic myeloid leukemia (CML): Failure to detect residual normal committed stem cells in vitro. Blood 53; 264-268, 1979b. Singer, J.W., Fialkow, P.J., Adamson, J.W., Steinmann, L., Ernst, C., Murphy, S., Kopecky, K.J: Polycythemia vera: Increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine. J. Clin. Invest. 64; 1320-1324, 1979c. Singer, J.W., Arlin, Z., Najveld, V., Adamson, J.W., Kempin, S.J., Clarkson, Fialkow, P.J: Restoration of nonclonal hematopoiesis in chronic myelogenous leukemia (CML) following a chemotherapy-induced loss of the Phi-chromosome. Blood 56; 356-360, 1980a. Singer, J.W., Adamson, J.W., Ernst, C, Lin, N., Steinmann, L., Murphy, S., Fialkow, P.J: Polycythemia vera: Physical separation of normal and I l l neoplastic committed granulocyte-macrophage progenitors. J. Clin. Invest. 66; 730-735, 1980b. Singer, J.W., Adamson, J.W., Arlin, Z.A., Kempin, S.J., Clarkson, B.D., Fialkow, P.J: Chronic myelogenous leukemia: In vitro studies of hemopoietic regulation in a patient undergoing intensive chemotherapy. J. Clin. Invest. 67; 1593-1598, 1981. Singh, J.P., Chaiken, M.A., Pledger, W.J., Scher, CD., Stiles, CD: Persistence of the mitogenic response to platelet-derived growth factor does not reflect a long-term interaction between the growth factor and the target cell. J. Cell Physiol. 96; 1457-1502, 1982. Smith, J.A. and Martin, L: Do cells cycle? Proc. Natl. Acad. Sci. U.S.A. 70; 1263-1267, 1973. Smith, J.A. and Martin, L: Regulation of cell proliferation. In: Cell Cycle Controls. Pailla, CM., Cameron, I.L., Zimmerman, A., eds. Academic Press, New York, pp. 43-60, 1974. Smith, J.C, Stiles, CD: Cytoplasmic transfer of the mitogenic response to platelet-derived growth factor. Proc. Natl. Acad. Sci. 78; 4363-4367, 1981. Sokal, J.E: Significance o f Ph1 -negative cells in Ph^-positive chronic granulocytic leukemia. Blood 56; 1072-1076, 1980. Sonia, S.I. and Sandburg, A.A: Chromosomes and causation of human cancer and leukemia: XXIX. Further studies of karyocytic progression in CML. Cancer 41; 153-163, 1978. Sorrell, J.M. and Weiss, L: Cell interactions between hemopoietic and stromal cells in the embryonic chich bone marrow. Anat. Rev. 197; 1-19, 1980. Spinks, J.W., Woods, R.J: Chapter 7: Water and Agueous Solutions. In: An Introduction to Radiation Chemistry, pp 247-359, John Wiley and Sons, New York, 1976. Spooner, E., Gallagher, J.T., Krizsa, F., Dexter, T.M: Regulation of hemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimulation of hemopoiesis by beta-D-xylosides. J. Cell Biol. 96; 510-514, 1983. Stanley, E.R. and Heard, P.M: Factors regulating macrophage growth and production. J. Biol. Chem. 252; 4305-4312, 1977. Stanley, E.R., Hansen, G., Woodcock, J., Metcalf, D: Colony stimulating factor and the regulation of granulopoiesis and macrophage production. Fed. Proc. 34; 2272-2279, 1975. Stanners, C.P, Adam, M.E., Harkins, J.C, Pollard, J.W: Transformed cells have lost control of ribosome number through their growth cycle. J. Cell. Physiol. 100; 127-138, 1979. 112 Stephenson, J.R., Axelrad, A.A., McLeod, D.L., Shreeve, M: Induction of colonies of hemoglobulin synthesizing cells by erythropoietin in vitro. Proc. Natl. Acad. Sci. U.S.A. 68; 1542-1546, 1971. Stiles, CD., Capone, G.T., Scher, CD., Antoniades, H.N., Van Wyk, J.J., Pledger, W.J: Dual control of cell growth by somatomedins and platelet- derived growth factor. Proc. Natl. Acad. Sci. USA 76; 1279-1283, 1979. Stoker, M. and Piggott, D: Shaking 3T3 cells: Further studies on diffusion boundary effects. Cell 3; 207-215, 1974. Strome, J.A., McLeod, P.L., Shreeve, M.M: Evidence for the clonal nature of erythropoietic bursts: Application of an in situ method for demonstrating centromere heterochromatin in plasma culture. Exp. Hemat. 6; 461-467, 1978. Suda, T., Suda, J., Ogawa, M: Single cell origin of mouse hemopoietic colonies expressing multiple lineages in variable combinations. Proc. Natl. Acad. Sci. U.S.A. 80; 6689-6693, 1983. Suda, J., Suda, T., Ogawa, M: Analysis of differentiation of mouse hemopoietic stem cells in culture by sequential replating of paired progenitors. Blood, 64; 393-399, 1984a. Suda, T., Suda, J., Ogawa, M: Disparate differentiation in mouse hemopoietic colonies derived from paired progenitors. Proc. Natl. Acad. Sci. U.S.A. 81; 2520-2524, 1984b. Taetle, R. and Mendelsohn, J: Modulation of normal and abnormal myeloid progenitor proliferation by cyclic nucleotides and PGÊ . Blood Cells 6; 701-718, 1980. Tavossoli, M. and Crosby, W.H: Transplantation of marrow to extramedullary sites. Science 161; 54-57, 1968. Taylor, J.H: Sister chromatid exchanges in tritium labelled chromosomes. Genetics 43; 515-529, 1958. Tchernia, F., Mielot, F., Coulombel, L., Mohandas, N: Characterization of circulating erythroid progenitor cells in human newborn blood. J. Lab. Clin. Med. 97; 322-331, 1981. Testa, J.R: Cytogenetic patterns in polycythemia vera. Can. J. Gen. Cytogen. 1; 207-215, 1980. Testa, N.G. and Dexter, T.M: Long term production of erythroid precursor cells (BFU-E) in bone marrow cultures. Differentiation 9; 193-195, 1977. Third International Workshop on Chromosomes in Leukemia; Introduction; Cancer Genet. Cytogenet. 4; 95-99, 1981 T i l l , J.E., McCulloch E.A: A direct measurement of the radiation sensitivity of normal mouse bone marrow. Radiat. Res. 14: 213-222, 1961. T i l l , J.E., McCulloch, E.A: Hemopoietic stem cell differentiation. Biochem. Biophys. Acta 605; 431-459, 1980. 113 T i l l , J.E., McCulloch, E.A., Siminovitch, L: A stochastic model of stem cell proliferation based on growth of spleen colony cells. Proc. Natl. Acad. Sci. U.S.A. 51; 29-36, 1964. Toksoz, D., Dexter, T.M., Lord, B.T., Wright, E.G., Lajtha, L.G: Blood 55; 931-936, 1980. Toogood, I.R., Dexter, T.M., Allen, T.D., Suda, T., Lajtha, L.G: The development of a liquid culture system for the growth of human bone marrow. Leuk. Res. 4; 449-464, 1980. Tough, I.M., Jacobs, P.A., Courtbrown, W.M: Cytogenetic studies on bone marrow in chronic myeloid leukemia. Lancet 1; 844-846, 1963. Trentin, J.J: Influences of hematopoietic organ stroma (hematopoietic inductive microenviroment) on stem cell differentiation. In: Regulation of Hematopoiesis. Gordon A.S., (Ed). Appleton-Century-Crofts, New York, page 161, 1970. Trentin, J.J: Determination of bone marrow stem cell differentiation by stromal hemopoietic inductive microenviroments (HIM). Am. J. Path. 65: 621- 627, 1971. Tushenski, R.J. and Stanley, E.R: The regulation of macrophage protein turnover by a colony-stimulating factor (CSF-1). J. Cell Physiol. 116; 67- 75, 1983. Upton, A.C: The biological effects of low-level ionizing radiation. Scientific American 246; 41-49, 1982. Vainchenker, W., Guichard, J., Breton-Gorius, J: growth of human megakaryocyte colonies in culture from fetal, neonatal, and adult peripheral blood cells: Ultrastructural analysis. Blood Cells 5; 25-31, 1979a. Vainchenker, W., Bouguet, J., Guichard, J., Breton-Gorius, J: Megakaryocyte colony formation from human bone marrow precursors. Blood 54; 940-945, 1979b. Vainchenker, W., Testa, U., Deschamps, J.F., Henri, A., et al: Clonal expression of the Tn antigen in erythroid and granulocytic colonies and its application to determination of the clonality of the human megakaryocyte colony assay. J. Clin. Invest. 69; 1081-1091, 1982. Van Zant, G. and Goldwasser, E. The simultaneous effects of erythropoietin and colony stimulating factor on bone marrow cells. Science 198; 733-735, 1977. Van Zant, G. and Goldwasser, E: Competition between erythropoietin and colony stimulating factor for target cells in mouse marrow. Blood 53; 946- 965, 1979. Vassort, F., Winterholer, M., Frindel, E., Tubania, M: Kinetic parameters of bone marrow stem cells using in vivo suicide by tritiated thymidine or by hydroxyurea. Blood 41; 789-796, 1973. 114 Vig, B.K., Kontras, S.B., Paddock, E.F: ^-thymidine induced chromosome aberrations in cultured human leukocytes. Cytogenetics 7; 189-195, 1968. Vig, B.K: Lack of relationship between chromosome aberrations induced by and localized incorporation of ^H-thymidine in human leukocytes. Radiat. Res. 59; 206, 1974. Wang, J.C. and Tobin, M.S: Erythropoietin independent erythroid progenitors in essential thrombocytosis. Blood 54; (Suppl. 1) 147a, 1979 Ward, H.P. and Block, M.H: The natural history of agnogenic myeloid metaplasia (AMM) and a critical evaluation of its relationship with the myeloproliferative syndrome. Medicine 50; 357-420, 1971. Wasserman, L.R: Polycythemia vera - its course and treatment: Relation to myeloid metaplasia and leukemia. Bull. N. Y. Acad. Med. 30; 343-375, 1954. Waterfield, M.D., Scrace, G.T., Whittle, N., et al: Platelet derived growth factor is structurally related to the putative transforming protein p28sis of simiam sarcoma virus. Nature 304; 35-39, 1983. Weinberg, R.S: Regulation of erythropoiesis in polycythemia vera and fetal sheep: Comparative in vitro and in vivo studies. Diss. Abstr. Int. (B) 38; 2069B 1977. ~~ Weiss, L: The hemapoietic microenvironment of bone marrow: an ultrastructural study of the interactions of blood cell, stroma, and blood vessels. In: Blood Cells and Vessel Walls: Functional Interactions. Elsevier/North Holland; Ciba Foundation Series 71, pp 3-19, 1980. Welsh, M.J., Dedman, J.R., Brinkly, B.R., Means, A.R: Tubulin and calmodulin: Effects of microtubule and microfilament inhibitors on localization in the mitotic apparatus. J. Cell Biol. 81; 624-634, 1979 Westin, H. and Bainton, D.F: Association of alkaline-phostphatase positive reticulum cells in bone marrow with granulocytic precursors. J. Exp. Med. 150; 919-937, 1979. Westin, J., Wahlstrom, J., Swolin, B: Chromosome studies in untreated polycythemia vera. Scand. J. Haematol. 17; 183-196, 1976. Whang, J., Frei, E., Tijio, J.H., Carbone, P.P., Brecher, G: The distribution of the Philadelphia chromosome in patients with chronic myelogenous leukemia. Blood 22; 664-673, 1963. Whang-Peng, J., Canellos, G.P., Carbone, P.P., Tijo, J.H: Clinical implications of cytogenetic variants in chronic myelocytic leukemia (CML). Blood 32; 755-766, 1968 Wharton, W., Gillespie, G.Y., Russell, S.W., Pledger, W.J: Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c-3T3 cells. J. Cell Physiol. 110; 93-100, 1982. Wheatley, D: Cell Growth and Division. E. Arnold Ltd., London, 57 pp, 1982. 115 Whitlock, C.A. and Witte, O.N: Long term cultures of B lymphocytes and their precursors from murine bone marrow. Proc. Natl. Acad. Sci. U.S.A. 79; 3608-3612, 1982. Whittenberger, B. and Glaser, L: Inhibition of DNA synthesis in cultures of 3T3 cells by isolated surface membranes. Proc. Natl. Acad. Sci. U.S.A. 74; 2251-2255, 1977. Whittenberger, B., Raben, D., Lieberman, M.A., Glaser, L: Inhibition of growth of 3T3 cells by extract of surface membranes. Proc. Natl. Acad. Sci. U.S.A. 75; 5457-5461, 1978. Whittenberger, B., Raben, D., Glaser, L: Regulation of the cell cycle of 3T3 cells in culture by a surface membrane enriched cell fraction. J. Supramol. Struct. 10; 307-327, 1979. Williams, N: Preferential inhibition of murine macrophage colony formation by prostaglandin E. Blood 53; 1089-1096, 1979. Williams, N. Burgess, A.W: The effect of mouse lung granulocyte-macrophage colony-stimulating factor and other colony-stimulating activities on the proliferation and differentiation of murine bone marrow cells in long-term cultures. J. Cell Physiol. 102; 287-295, 1980. Williams, N., Eger, R.R., Moore, M.A.S., Mendelsohn, N: Differentiation of mouse bone marrow precursor cells into neutrophil granulocytes by an activity separated from WEHI-3 cell conditioned medium. Differentiation 11_; 59-63, 1978a. Williams, N., Jackson, H., Sheridan, A.P., Murphy, M.J., Elste, A., Moore, M.A.S: Regulation of megakaryopoiesis in long term murine bone marrow cultures. Blood 51; 245-255, 1978b. Williams, N., Jackson, H., Meyers, P: Isolation of pluripotent hemopoietic stem cells and clonable precursor cells of erythrocytes, granulocytes, macrophages, and megakaryocytes from mouse bone marrow. Exp. Hematol. 7; 524-534, 1979. Wintrobe, M.M: Clinical Hematology. Lea and Febiger, Philadelphia, pp. 1565- 1584, 1981. Wolf, N.S: Dissecting the hemopoietic microenvironment: III Evidence for a positive short range stimulus for cellular proliferation. Cell Tissue Kinet. 11; 335-345, 1978. Wolf, N.S. and Trentin, J.J: Hemopoietic colony studies: V. Effects of hemopoietic organ stroma on differentiation of pluripotent stem cells. J. Exp. Med. 127; 205-214, 1968. Wong, G.G., Witek, J.S., Temple, P.A., et al: Human GM-CSF; Molecular cloning of the complementary DNA and purification of the natural and recombinent proteins. Science 228; 810-815, 1985. Worton, R.G., McCulloch, E.A., T i l l , J.E: Physical separation of hemopoietic stem cells differing in their capacity for self-renewal. J. Exp. Med. 130; 91-103, 1969. 1 1 6 Wright, E.G., Lord, B.I: Regulation of CFU-S proliferation by locally producted endogenous factors. Biomedicine 27; 215-218, 1977. Wright, E.G and Lord, B.I: Production of stem cell proliferation stimulators and inhibitors by hemopoietic cell suspensions. Biomedicine 28; 156-160, 1978. Wright, E.G. and Lord, B.I: Production of stem cell proliferation regulators by fractioned hemopoietic cell suspensions. Leuk. Res. 3; 15-22, 1979. Wu, A.M., T i l l , J.E., Siminovitch, L., McCulloch, E.A: A cytological study of the capacity for differentiation of normal hemopoietic colony forming cells. J. Cell. Physiol. 69: 177-184, 1967. Wurster-Hill, D.H. and Mclntyre, 0.R: Chromosome studies in polycythemia vera. Virchow Archiv. B. Cell Path. 29; 39-44, 1978. Wurster-Hill, D., Whang-Peng, J., Mclntyre, R., Hsu, L., et al: Cytogenetic studies in polycythemia vera. Semin. Hematol. 13; 13-29, 1976. Yanishevsky, R.M. and Prescott, D.M: Late S phase cells (Chinese hamster ovary) induce early S phase DNA labelling patterns in Ĝ  phase nuclei. Proc. Natl. Acad. Sci. U.S.A. 75; 3307-3311, 1978. Yanishevsky, R.M. and Stein, G.H: Ongoing DNA synthesis continues in young human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited. Exp. Cell Res. 126; 469-472, 1980. Yeh, J. and Fisher, H.W: A diffusible factor which sustains contact inhibition of replication. J. Cell Biol. 40; 382-388, 1969. Yen, A., Warrington, R., Pardee, A.B: Serum stimulated 3T3 cells undertake a histidinol sensitive process which Gj cells do not. Exp. Cell Res. 114; 458-462, 1978. Yuspa, S.H., Lichti, U., Ben, T., Patterson E., et al: Phorbol esters stimulate DNA synthesis and ornithine decarboxylase activity in mouse epidermal cell cultures. Nature 262; 402-404, 1976. Zanjani, E.D., Lulton, J.D., Hoffman, R., Wasserman, L.P: Erythoid colony formation by polycythemia vera bone marrow in vitro: Dependence on erythropoietin. J. Clin. Invest. 59; 841-848, 1977. Zanjani, E.D., Weinberg, R.S., Nomdedev, B., Kaplan, M.E., Wasserman, CR; In vitro simularities between erythroid precursors of fetal sheep and patients with polycythemia vera. In: In Vitro Aspects of Erythropoiesis, Murphy, M.J., ed., Springer Verlag, Berlin, pp. 118, 1978. Zech, L., Gahrston, C, Killander, d., Franzen, S., Hagland, U: Specific chromosomal aberrations in polycythemia vera. Blood 48; 687-688, 1976. Zeilig, C.E. and Goldberg, N.D: Cell cycle related changes of 3'5' cyclic GMP levels in Novikoff hepatoma cells. Proc. Natl. Acad. Sci. U.S.A. 74; 1052-1056, 1977. 117 Zuckerman, K.S. and Wicha, S: Extracellular matrix production by adherent cells of long term murine bone marrow cell cultures. Blood 61; 540-547, 1983. Zuckerman, K.s., Wicha, M.S., Goodwin, D.D., Patel, V.Rr., Mayo, L.A: Hematopoietic extracellular matrix production in murine long-term bone marrow cultures. Exp. Hematol. 11 (Suppl. 14); 124, 1983. 118 C H A P T E R I I MATERIALS AND METHODS 1) Marrow and Peripheral Blood Peparation Peripheral blood and marrow specimens were obtained with informed consent and collected in preservative free sterile heparin present at a final concentration of 50 U/ml and 100-400 U/ml respectively. Marrow buffy coat cells were separated from the majority of red cells using a two step procedure of light centrifugation followed by sedimentation at unit gravity for 10-20 minutes. The buffy coat rich plasma was then removed, washed twice in serum free Iscoves medium and resuspended in the same for plating. Peripheral blood was separated by layering a 10 ml aliquot over 15 ml of 1.077 gm/ml Ficoll-Hypaque (LSM-Bionetics, Kensington, Maryland) and spinning at 800g for 30 minutes. The light density mononuclear cell fraction at the plasma/Ficoll-Hypaque interface was then carefully removed and washed twice in 10 ml of serum free Iscove's medium and diluted to an appropriate concentration for plating. 2) Hemopoietic Colony Assays Erythropoietic colony forming units (CFU-E) and burst forming units (BFU-E) as well as granulopoietic (CFU-C) and pluripotent (CFU-G/E) progenitors were assayed in a previously standardized culture medium consisting of 0.8% methylcellulose in Iscove's medium, supplemented with 1% deionized bovine serum albumin (BSA), 30% fetal calf serum (FCS), 200 mM L- glutamine, and 10~̂ M 2-mercaptoethanol (Eaves and Eaves, 1978; Eaves et al, 1984). In addition, 5 U/dish human urinary erythropoietin (purified in this 119 laboratory to a specific activity of >100 U/mg) (Krystal et al, 1984) and an appropriate concentration of phytohemagglutin or agar stimulated human leukocyte conditioned medium (Coulombel et al, 1983) was added to achieve optimal growth by a l l primitive clonogenic cell types (Gregory and Eaves, 1978). A 1.1 ml aliquot of this methylcellulose assay mixture, containing an appropriate concentration of hemopoietic cells was placed in each 35 mm Greiner Petri dish. Routinely, normal marrow buffy coat cells were plated at a concentration of 2xl0-> cells per 1.1 ml culture. In normal peripheral blood assays, 4x10^ cells were plated per 1.1 ml culture. However, if the patient's i n i t i a l WBC count was elevated, additional dishes were set up at a lower concentration to ensure accurate colony counts. Adherent cells from long term bone marrow or peripheral blood cultures were routinely plated at a concentration of l x l C P cells per 1.1 ml culture. Nonadherent cells were also plated at this concentration, except in older cultures when the total number of nonadherent cells was insufficient and necessitated plating fewer cells. Cultures were incubated at 37°C in a strictly controlled 5% CO2 in air environment under conditions of high humidity. Each dish was scored on two different occasions. After 10 to 12 days of incubation small isolated clusters or pairs of clusters composed of 8 or more hemoglobin-containing cells were counted to determine the number of CFU-E plated. At this time, small bursts consisting of from 3 to 8 clusters of hemoglobinized cells were also scored to give mature BFU-E numbers. After an incubation period of 18-20 days a l l larger bursts, i.e. those containing 9 to 16 clusters, and those composed of more than 16 clusters as well as mixed colonies (CFU-G/E) were counted. In assays of fresh marrow and peripheral blood cells described in Chapter III, the term primitive BFU-E refers to high proliferative potential erythroid progenitors defined by their capacity to produce colonies containing 120 Marrow unit g r a v i t y aedlmented buf fy c e i l c a l l * Blood V F i c o l l H y p a q u e s e d i m e n t e d n o n o n u c l u r c a l l s w a s h e d and di luted 8 e m i - a o l i d n e t n y c e l l u l o a • Medium Including F C S . ISA and f - H E 1 h - i ^ ^ - - f S t imulants l i u k o c y t * c o n d i t i o n e d stadium ( L C M ) • r y t h r o p o i a t i n ( E p ) incubate 1 0 - 1 8 d a y s DAY 10 - 12 C F U - E ( 1 - 2 c l u s t e r s ) msture B F U - E (3 -8 c lustera ) DAY IS - 20 pr imi t ive B F U - E (>16 c luatera) C F U - G / E C F U - C ( 2 0 - 6 0 0 celts) pr imi t ive C F U - C ( > 6 0 0 c e l l s ) Figure 13. Schematic diagram discribing the methylcellulose assay system for the growth of hemopoietic cells. L 121 16 or more clusters. However, in the data presented in Chapters IV, V, and VI, the colony counts for a l l larger erythroid bursts were pooled, both for assays of nonadherent and adherent cells from long-term cultures as well as in assays of the fresh marrow and blood samples used to initiate these cultures. Thus, in these studies the term primitive BFU-E refers to clonogenic precursors capable of forming erythroid colonies containing 8 or more clusters. All granulocytic colonies were also scored at 18-20 days. These colonies were usually also subdivided into two catagories, those containing 20-500 cells and those containing more than 500 cells. Values for the latter yield counts for primitive CFU-C numbers. For assessment of progenitor numbers in primary or cultured cell suspensions, counts were averaged from 2-4 replicate 1.1 ml methylcellulose assay cultures. For assessment of cycling status counts were averaged from 6 replicates per treatment group. 3) Long Term Bone Marrow Cultures An aliquot of the untreated marrow aspirate specimen containing 2-2.5x10? nucleated marrow cells was placed in 8 ml of growth medium in a 60x15mm Falcon tissue culture dish. The growth medium was composed of a medium supplemented with inositol (40 mg/1), folic acid (10 mg/1), extra glutamine (400 mg/1), fetal calf serum (12.5%), horse serum (12.5%), 2-mercaptoethanol (10~^M), and hydrocortisone sodium succinate (10~^M). The cultures were incubated for 3 to 4 days at 37°C in an atmosphere of 5% CO2 in air. After this in i t i a l period of incubation a l l nonadherent cells were removed and layered over 1.077 gm/cc Ficoll-Hypaque to remove the red blood cells and mature granulocytes. The light density cells were washed in a medium supplemented with 2% FCS and returned to their original dishes A. iC /L. NON-ADHERENT ADHERENT Figure 14. Schematic representation of the 3-layer composition of a long- term culture after 2-3 weeks of incubation. From Eaves et a l , 1983. Used with permission. 123 The cultures were fed on a weekly basis by removal of half of the medium and half of the nonadherent cells. This was accomplished by pipetting 2-3 ml of medium from the dish, and then gently swirling the dish to ensure removal of a l l the nonadherent cells with the remaining 6-5 ml of the medium. The culture medium was placed in a tube, vortexed to distribute the cells evenly, and 4 ml of this suspension was returned to the culture dish, along with 4 ml of fresh culture medium. The remaining 4 ml of growth medium containing half of the nonadherent cells was centrifuged, the growth medium removed, and the cells washed once in 2% FCS, counted, and assayed in methylcellulose cultures If more than one culture was initiated from a single marrow specimen, the nonadherent cell fractions were pooled before washing. 4) Long Term Peripheral Blood Cultures Marrow adherent layer "feeders" were obtained by subculturing primary 2 to 3 week old confluent normal marrow adherent layers established in the growth medium described above, but without the addition of hydrocortisone and fed weekly with removal of a l l nonadherent cells and complete replacement of the growth medium. The primary marrow adherent layer was suspended by treatment with trypsin (see procedure below), washed and counted. Aliquots containing .5-1x10° cells were then placed in secondary culture dishes, and incubated at 37°C for 7 to 14 days until a confluent adherent layer was re- established. Just prior to use, these secondary adherent layers were irradiated with 15-20 gray (60co y-rays or 280 kvp X-rays at a dose rate of 240 centigrays/min) to completely eliminate residual hemopoiesis. Peripheral blood samples from CML patients were separated over Ficoll-Hypaque by centrifugation at 800 g for 30 minutes to obtain the mononuclear cell fraction. An aliquot containing 2.0-2.5x10̂  cells was then 124 added to the culture dishes containing the irradiated preestablished feeders in 8 ml of growth medium supplemented with hydrocortisone. These "reconstituted" blood cultures were subsequently handled as standard long-term marrow cultures. Since normal blood samples contained fewer WBC per ml, the establishment of control cultures necessitated the processing of a large quantity of blood. This lengthy, multistep procedure for the separation of normal blood was performed by Dr. Louis Gaboury. A unit of normal blood, containing approximently 500 ml, was aliquoted into 50 ml tubes and centrifuged at 2000 rpm for 5 minutes. The buffy coat cells were then removed, and 10 ml aliquots of this cell suspension were then layered over Ficoll-Hypaque. After centrifugation at 800 g for 30 minites the mononuclear cell fraction was removed and 100 U/ml of heparin was added to the cell preparation (to prevent subsequent clotting) which was then washed twice in 2% FCS and counted. The cell suspension was then diluted to a concentration of 5x10^ cells/ml and subjected to the following T cell depletion procedure. A 1% suspension of 5- 2-aminoethylisothiouronium bromide hydrobromide (AET) treated sheep red blood cells in 40% FCS was then added in equal volume to the cells. The tubes were incubated at 37°C for 5 minutes, centrifuged for 5 minutes at 150 g and then incubated at 4°C for 1 hour. After this period the supernatant from each tube was removed and the tubes centrifuged at 300 g for 10 minutes, the pellets resuspended and pooled for a second separation over Ficoll-Hypaque. The number of light density mononuclear cells recovered from the plasma/Ficoll- Hypaque interface after this step was approximately 3% of the original nucleated cell content and usually yielded sufficient cells for initiation of 2-3 long term cultures. These were subsequently maintained using the same protocol established for long-term marrow cultures and for reconstituted CML blood cultures. 125 In most experiments, with both CML patients and normal subjects, additional long term cultures were set up without pre-established feeders. These were initiated and handled in the same manner as the previous cultures except for designated dishes where a l l the nonadherent cells were returned to the cultures at each weekly half medium change. 5) Enzymatic Detachment of Adherent Cells The adherent layer of long term marrow or peripheral blood cells was removed enzymatically using either collagenase (bacterial type I, 200 U/mg of protein) or trypsin (.25% in solution containing 5% citrate, 10% KC1 and 1% glucose) following the procedure of Coloumbel et al (1983). The cultures were prepared for either procedure by removal of a l l the growth medium and nonadherent cells followed by a vigorous washing of the adherent layer with calcium- and magnesium-free Hank's balanced salt solution (HBSS-Ca-Mg). The additional detached cells were added to the nonadherent cell suspension. The collagenase solution was prepared just prior to use by dissolving the collagenase in HBSS-Ca-Mg to a final concentration of 0.13% and sterilizing by passage through a 22u Millipore filter. To detach the adherent cells, 8 ml of the collagenase solution was pipetted onto the adherent layer, 2 ml of FCS was added, and the culture dishes incubated undisturbed for 3 hours at 37°C in an atmosphere of 5% CO2 in air. When the cultures were removed from the incubator at the end of this period many cells were detached, and most of the remaining cells could be removed by gentle pipetting and washing. The cells were centrifuged at 300 g for 10 minutes to remove the collagenase solution and then washed twice in serum free Iscove's medium, carefully resuspended, counted, and diluted. 126 When trypsin was used, the cultures were prepared as for the collagenase procedure. Five ml of the enzyme solution was then placed in each of the culture dishes which were then incubated for 10 minutes at 37°C. At the end of this incubation period, 1 ml of FCS was added to stop further trypsin action, and a l l adherent cells could then be easily detached by gentle pipetting. Cells were then processed in the same manner as those harvested using the collagenase procedure. 6) The ^H-thymidine Cell Suicide Assay Each hemopoietic cell suspension was washed twice in serum free Iscove's medium prior to the ̂ H-thymidine cell suicide assay to eliminate contamination by endogenous unlabelled thymidine. The cell suspension was resuspended in the same pre-warmed medium (pH 7.2) and the cells incubated with (tube Tj>) or without (tube T c) 20 uCi/ml 3H-thymidine (25 Ci/mol) for 20 minutes at 37°C at a final cell concentration of 4x10^ (blood), 2x10^ (marrow), or 1x10^ (adherent or nonadherent cells from long-term cultures) in a volume of 1 ml. Ten ml of cold thymidine (400 ug/ml) in 2% FCS was then added and the cells washed twice in the same medium prior to resuspension in 2% FCS in Iscoves's medium and plating in methylcellulose. Suicide (or % k i l l ) values were calculated from colony counts according to the formula in Figure 15. Values less than one are shown in the Tables as zero. The concentration of -^-thymidine used in these experiments was selected on the basis of reported studies (Becker et al, 1965; Iscove, 1977) and confirmation of these results for the types of samples and reagents used in this study (see Chapter III). These showed that percent suicide values were the same whether cells were exposed to 20 or 100 uCi/ml. In addition, in order to establish that there was no nonspecific toxicity due to incubation in 127 3H-THYMIDINE SUICIDE ASSAY FOR HEMOPOIETIC PROGENITORS I 1 Hemopoietic cel l suspension I 1 Iscoves medium Iscoves medium with 20 j iCu/ml Yl-Tdr Incubated for 20 minutes at 3 7 ° C W a s h e d twice in 2% F C S with e x c e s s thymidine and plated Incubated for 2 - 3 weeks at 3 7 ° C « ^ • • » Viable cel ls form colonies Mc" M T % KILL Z X 100 Figure 15. A schematic diagram demonstrating the application of the ^H-thymidine c e l l suicide assay to the study of hemopoietic c e l l kinetics. 128 the ^H-thymidine solution, other aliquots of the same marrow suspension were exposed to each -^-thymidine concentration in the presence of excess cold thymidine (400 ygms). This procedure was followed upon receipt of each new vial of -^-thymidine used in these experiments. 7) Cytogenetic Methods Cytogenetic procedures were used in some of the studies of CML cultures to determine the presence or absence of the Pĥ  chromosome. Direct marrow metaphases were prepared by conventional methods (Tijo and Whang, 1962) using solid Giemsa staining or G banding (Seabright, 1971). In addition, metaphases were obtained from individual colonies from methylcellulose assays established with aliquots of the initial marrow specimen, or with the adherent or nonadherent cell fraction of long term cultures. This procedure was developed in the Terry Fox laboratory (Dube et al, 1981). Previous experience has shown that analyzable metaphases could be obtained by a careful selection of recognizable but immature colonies in which the largest number of dividing cells are present. To obtain such colonies, dishes were examined using an inverted microscopic after 8 or 9 days incubation and subsequently at daily intervals. When a culture was judged to be optimal, 0.1 ml of colcemid (1 yg/ml in HBSS) was carefully applied drop-wise over the methylcellulose surface of each 1.1 ml culture to arrest cell division at metaphase. A finely drawn out Pasteur pipette was used to remove individual single colonies which were each then transferred into a microtiter well containing 0.1 ml of .075M KCl. The cells in each colony were gently dispersed by pipetting and were left undisturbed in the microtiter wells for 15 to 20 minutes at 20°C. At the end of this time the entire contents of the microtiter well were transferred onto a microscope slide that had been coated 1-2 hours previously with a drop 129 of a 0.01% polylysine (w/v) solution. To spread the polylysine a coverslip was placed over the drop of polylysine until the slide was to be used. The coverslip was then removed, the slide rinsed with water, and gently blotted dry. Each colony was placed on the polylysine treated area of a single slide and then kept in a humid environment for 10 minutes. Excess hypotonic solution was removed with a cotton swab, and 0.5 ml of 3:1 methanol:acetic acid fixative gently dropped over the colony, followed by another 0.5 ml of the same fixative 30 seconds later. After 1 minute the slide was slowly air dried by passage over an open flame, then immersed in fresh fixative for 15 minutes, and air dried prior to staining. All cytogenetic preparations were made by an experienced cytogenetic technician, and the analyses overseen by Dr. D.K. Kalousek. 130 REFERENCES Becker, A.J., McCulloch, E.A., Siminovitch, L., T i l l , J.E: The effect of differing demands for blood cell production of DNA synthesis by hemopoietic colony forming cells of mice. Blood 26; 296-308, 1965. Coulombel, L., Eaves, A.C, Eaves, CJ: Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62; 291-297, 1983 Dube, I.D., Eaves, C.J., Kalousek, D.K., Eaves, A.C: A method for obtaining high quality chromosome preparations from single hemopoietic colonies on a routine basis. Cancer Genet. Cytogent. 4; 157-168, 1981. Eaves, C J . and Eaves, A.C: Erythroid progenitor cell numbers in human marrow - implications for regulation. Exp. Hematol. 7; 54-64, 1979. Eaves, C.J., Coulombel, L., Eaves, A.C: Analysis of hemopoiesis in long- term human marrow cultures. In: Hemopoietic Stem Cells, Killman, S.A., Cronkite, E.P., Muller-Berat, C.W., (eds), Munksgaard, Copenhagen, pp. 287- 302, 1983. Eaves, C.J., Krystal, C, Eaves, A.C: Erythropoietic Cells. Biblthca. Haematol. 48; 81-111, 1984. Gregory, C J . and Eaves, A.C: Three stages of erythropoietic progenitor cell differentiation distinquished by a number of physical and biological properties. Blood 51; 527-537, 1978. Iscove, N.N: The role of erythropoietin in regulation of population size and cell cycling of early and late erythroid precursors in mouse bone marrow. Cell Tissue Kinet. 10; 323-334, 1977. Krystal, C, Eaves, C.J., Eaves, A.C: CM Affi-Gel Blue chromatography of human urine: a simple one-step procedure for obtaining erythropoietin suitable for in vitro erythropoietic progenitor assays. Brit. J. Haematol. 58; 533-546, 1984. Seabright, M. A rapid banding technique for human chromosomes. Lancet 2; 971-972, 1971. Tijo, J.H. and Whang, J: Chromosome preparations of bone marrow cells without prior in vitro culture or in vivo colchicine administration. Stain Technol 37; 17-20, 1962. C H A P T E R I I I 131 ANALYSIS OF THE PROLIFERATIVE ACTIVITY OF HEMOPOIETIC PROGENITORS IN THE MYELOPROLIFERATIVE DISORDERS 1) INTRODUCTION The myeloproliferative disorders - PV, ET, and CML - are a group of closely related syndromes characterized by a clinically significant increase in the number of circulating mature blood cells. Considerable cytogenetic and isoenzyme evidence has been presented for the pluripotent stem cell origin of a l l of these diseases (Nowell and Hungerford, 1960; Fialkow et al, 1967, 1977, 1981; Adamson et al, 1976; Jacobson et al, 1978). A number of investigators have applied in vitro colony assay techniques to the study of the MPD in an effort to elucidate the mechanisms that permit the progeny of a single abnormal stem cell to completely dominate a l l of the mature myeloid cell compartments. In CML, assessment of the number of clonogenic precursors in marrow and blood samples has provided evidence that these compartments are also greatly expanded. In PV and ET, where the increase in numbers of mature blood cells in any lineage is much less pronounced (usually less than a 2-3 fold increase) the size of the clonogenic progenitor compartments also remains on average within normal limits (Gregory and Eaves, 1977). Comparison of the relative numbers of clonogenic progenitors committed to different pathways of differentiation has revealed no evidence for a significant shift in the committment behaviour of neoplastic pluripotent stem cells in any of these disorders (Eaves and Eaves, 1979; Adamson et al, 1980). 132 Demonstration of the clonal nature of a l l of these diseases, already apparent at the level of early progenitor cell types, focussed our interest on the assessment of progenitor cell cycle status as a possible mechanism contributing to clonal expansion of the circulating cell compartment in either the presence (CML) or absence (PV and ET) of a concommittant detectable increase in progenitor cell numbers. In view of the conflicting and incomplete cycling data reported by others ,for PV and CML progenitors (see Chapter I) and the lack, of any published cycling data for ET progenitors, a systematic analysis of this parameter was undertaken. In these experiments primitive and mature erythropoietic, granulopoietic, and, in some cases, pluripotent progenitors, from both marrow and blood were assessed. To evaluate the specificity of cycling changes measured in the MPD, samples from patients with secondary erythrocytosis and various other secondary perturbations of hemopoiesis were also evaluated. Since some of the marrow and blood samples did not reach the laboratory for processing until several hours after being obtained, additional control studies included an evaluation of the effect of storage on ice and at room temperature on progenitor number and cycling status, in addition to standard 3H-thymidine dose response measurements. 2) RESULTS A) Patients PV Sixteen PV patients were used in this study, ranging in age from 38 to 77 years. Cells from thirteen of the 16 patients were cultured at the time of diagnosis. In this group of untreated patients hemoglobin values ranged from 16.9-19.4 g/dl (5 women) and from 18.9-21.2 g/dl (8 men). Two other patients, both women, had been diagnosed 10 years earlier and had been 133 treated intermittently with hydroxyurea or myleran. Their hemoglobin values were 13.8 g/dl and 18/2 g/dl at the time of culture. One patient had been treated regularly by phlebotomy since the initial diagnosis had been made 5 years previously. Her hemoglobin was 15.3 g/dl at the time of culture. All PV patients studied had WBC values within normal limits or slightly elevated (range 7,500-16,300 cu/mm) and al l demonstrated the presence of Ep- independent erythroid colony-forming cells in their peripheral blood and marrow (Eaves and Eaves, 1983) SE Sixteen SE patients (13 men and 3 women) were used in this study. These ranged in age from 34-84 years. Cells for culture were in a l l cases obtained at the time of diagnosis. Hemoglobin values ranged from 16.4-20.1 g/dl (men) and from 15.7-18.8 g/dl (women). All SE patients had WBC within or close to normal limits (4,200-11,000 cu/mm). No Ep-independent erythroid colony-forming cells were detected in assays of blood and marrow from any of these patients. ET Eight female and 11 male (i.e. a total of 19) patients with thrombocytosis were used in this study. These ranged in age from 32-83 years. Cells from 17 of these were cultured at the time of presentation. One patient, diagnosed a year previously had been treated with anturan. Another had been diagnosed 6 months previously, but had not required treat- ment. The platelet counts for a l l of the patients ranged from 912,000- 2,632,000 cu/mm at time of culture. The WBC levels were normal or slightly elevated (range of 4,900-17,200 cu/mm). The hemoglobin values were normal (8.6-16.5 g/dl). Eight of 19 patients in this group had Ep-independent erythroid colony-forming cells present in their blood or marrow. CML All 20 CML patients (12 females and 8 males, ranging in age from 24-82 years) in this study were Phi-positive. Four of these patients had 134 been previously treated and in this group WBC values ranged from 16,000- 151,000 cu/mm. WBC values in the group of 16 remaining untreated patients ranged from 10,700-209,000 cu/mm. With the exception of the 4 treated patients, a l l members of this latter group were cultured within a year of diagnosis. Normals Normal peripheral blood was obtained from laboratory personnel and from the Red Cross. Normal bone marrow specimens were obtained from 3 allogenic bone marrow transplant donors and from 17 patients undergoing hematological assessment for a variety of disorders including lymphoma, anemia, leukocytosis, and ankylosing spondylitis but whose marrow showed no malignant infiltration or other abnormality. The hematological indices for these individuals were normal, with the exception of the two male subjects with anemia, who had hemoglobin values of 8.2 and 8.0 g/dl. Other patients Three other patients with perturbed hemopoiesis were included in this study. The first was a 26 year old female patient with a leukocytosis of unknown origin. She had a WBC count of 15,300 cu/mm, with a normal hemoglobin (12.1 g/dl) and platelet count (474,000 cu/mm). The se- ond patient in this group was a 12 year old boy with thalassemia major. He had a WBC of 4,100 cu/mm, a platelet count of 134,000 cu/mm and a hemoglobin of 9.1 g/dl. The third patient was a 59 year old male with thrombocytopenia. He had a platelet count of 30,000 cu/mm, a WBC of 4,200 cu/mm and a hemoglobin of 15.5 g/1. None of these 3 patients demonstrated the presence of Ep-independent erythroid colony-forming cells in their blood or marrow. B) 3H-thymidine Dose Response Curve Percent suicide values for erythroid and granulocytic progenitors from 6 normal marrows exposed to 0, 10, 20, and 100 uCi/ml of 3H-thymidine are shown in Figure 15. The results for actively cycling hemopoietic progenitors (i.e. CFU-E, CFU-C, and a l l BFU-E) indicated that 3H-thymidine 135 Primitive BFU-E s\T .^tf-a.--- — •••• - - • o . 10 20 100 0 10 20 100 [ 3H] - Thymidine Concentration (/xCi/ml) Figure 16. 3 H _ t n y m i Q i n e a o S e response curve for e ry thro id and g ranu locy t i c progeni tors from normal marrow. Each point represents the ar i thmet ic mean + 1 SEM of values obtained from 6 experiments. The s o l i d l i n e (c losed symbols) ind ica tes values for c e l l s incubated for 20 minutes in tubes conta in ing only the appropr iate concentrat ion of ^H-thymidine in a n u c l e o s i d e - f r e e nutr ient medium. The dotted l i n e (open symbols) re fe rs to the values obtained when a l iquots of the same c e l l suspension were incubated at each ^H-thymidine concentrat ion plus an excess (400 ygm/ml) of cold thymidine. 136 concentrations of 20 y Ci/ml gave maximum percent k i l l values with no change when the ^H-thymidine concentration was increased up to 100 yCi/ml. In contrast, the plating efficiency of primitive BFU-E (i.e. erythroid progenitors forming colonies consisting of more than 16 clusters) in the same marrow cell suspensions was not affected by exposure to any of the concentrations of 3 H -thymidine tested. The absence of toxicity in the ^H-thymidine solution, unrelated to its 3 H content, was also demonstrated in this series of experiments. In the presence of excess cold thymidine the percent k i l l at each concentration was reduced to insignificant levels indicating that cell death resulting from incubation in the 3n-thymidine solution in each case was attributable to the specific incorporation of the radioisotope into S-phase cells. C) Cell Cycle Status of Hemopoietic Progenitors From Marrow and Blood Differences in progenitor cell cycling behaviour between normal subjects and PV or CML patients were most readily apparent in the circulating cell compartments (Table 1). In normal subjects and patients with SE a l l classes of circulating hemopoietic precursors were found to be quiescent. In contrast, in patients with PV or CML, a significant elevation in the number of S-phase progenitors was readily evident. A similar alteration in the cycling behaviour of PV or CML marrow could also be seen at the level of the most primitive progenitor compartment of the erythroid and granulocytic lineages. In later compartments, most progenitors were found to be in S-phase even in normal subjects (Table 2). The results obtained for peripheral blood and marrow progenitors from a group of patients with elevated platelet counts are summarized in Tables 3 and 4. The alterations in cell cycle activity observed with the blood and marrow progenitors from CML and PV patients was not a consistent finding 137 TABLE 1 PERCENT KILL FOR PERIPHERAL BLOOD PROGENITORS FROM NORMAL INDIVIDUALS AND FROM PATIENTS WITH MYELOPROLIFERATIVE DISORDERS No. of BFU-E CFU-C CFU-GEMM Diagnosis Subjects (>2 clusters) Normal 9 0+1.2 0+1.9 0+2.0 SE 12 0+1.0 1.25+1.8 0+3.5 PV 14 33.2 + 2.7 26.4 + 3.4 46.7 + 4.9 CML 16 34.1 + 3.3 32.2 + 3.9 48.0 + 5.1 Values shown are the arithmetic means + 1 S.E.M. 138 TABLE 2 PERCENT KILL FOR MARROW PROGENITORS FROM NORMAL INDIVIDUALS AND PATIENTS WITH MYELOPROLIFERATIVE DISORDERS No. of Primitive BFU-E Primitive CFU-C CFU-GEMM Diagnosis Subjects (>16 clusters) (>500 cells) Normal 20 3.6 + 1.7 4.4 + 2.0 SE 15 2.5 + 2.2 4.9 + 2.9 PV 16 46.8 + 3.4 40.4 + 3.8 CML 13 40.4 + 3.6 44.2 + 7.0 Values shown are the arithmetic mean + 1 S.E.M. 139 TABLE 3 PERCENT KILL FOR PERIPHERAL BLOOD PROGENITORS FROM PATIENTS WITH ESSENTIAL THROMBOCYTOSIS Group1 Patient Ep BFU-E CFU-C CFU-GEMM No. Independence^ (>2 clusters) I 1 No 0 0 0 2 No 0 0 _* 3 No 3 5 - 4 No 3 0 - 5 No 0 3 0 6 No 0 0 0 7 No 1 5 0 8 No 0 0 - 9 No 0 9 - 10 No 0 0 0 11 No 2 10 11 II 1 Yes 30 30 _ 2 Yes 67 23 - 3 Yes 22 12 - 4 Yes 32 29 - 5 No 43 22 - 6 Yes 52 46 - 7 Yes 25 29 - 8 Yes 52 60 - 1 - Patients were divided into these two groups on the basis of the proliferative activity of their circulating progenitor cells. No clinical correlation was evident within each group. - Ep-independence was determined by the presence of detectable erythroid colonies in culture dishes to which no Ep was added (<.002 U Ep/ml). * - Insufficient counts for meaningful % k i l l calculations. 140 TABLE 4 PERCENT KILL FOR MARROW PROGENITORS FROM PATIENTS WITH ESSENTIAL THROMBOCYTOSIS Group1 Patient . Ep Primitive BFU-E Primitive CFU-C CFU-GEMM No. Independence^ (>16 clusters) (>500 cells) I 1 No 39 0 -* 2 No 10 8 3 No 0 10 4 No 5 5 No 0 6 0 6 No 0 7 No 6 0 8 No 0 8 9 No 11 6 10 No 18 II 1 Yes 75 8 - 2 Yes 47 16 3 Yes 28 22 4 No 53 5 Yes 46 41 39 9 Yes 36 15 1 - Patients were divided into these two groups on the basis of the proliferative activity of their circulating progenitor cells. No clinical correlation was evident within each group. 2 - Ep independence was determined by the presence of detectable erythroid colonies in culture dishes to which no Ep was added (<.002 U Ep/ml). 3 - Insufficient counts for meaningful % k i l l calculations. 141 for a l l patients studied. However, when these latter patients were divided into two groups on the basis of the proliferative activity of their circulating progenitor cells, it became apparent that many patients did not show an increase over normal values in the number of progenitors in S-phase in their peripheral blood or marrow. In addition, alterations in cycling behaviour, when present, were usually found in both the marrow and peripheral blood compartments alike. The only marked exception to this was patient no. 1 where cycling was apparent in the primitive BFU-E compartment in the marrow but not in the peripheral blood. As seen with the other MPD patients, such cycling abnormalities were not lineage specific, i.e., both erythroid and granulocytic progenitors were affected, regardless of whether the patient was diagnosed as having PV, CML, or ET. Another interesting association was revealed when erythroid progenitor cells from these patients were assessed for their ability to form recognizable, hemoglobin-containing colonies without the addition of Ep. The presence of such endogenous erythroid colony forming cells has previously been documented in ET patients (Eaves et al, 1983; Eridani et al, 1983; Partanum et al, 1983). As shown in Tables 3 and 4, in 7 out of 8 experiments with peripheral blood cells, and 5 out of 6 experiments with marrow cells, an increase in the number of hemopoietic progenitors in S-phase was found only in those patients in whom endogenous erythroid colony forming cells were also detected. In contrast to the MPD the proliferative activity of a l l classes of hemopoietic progenitors from the bone marrow or peripheral blood of 3 patients with various other forms of perturbed hemopoiesis (i.e. reactive leukocytosis, thalassemia major or thrombocytopenia) were indistinguishable from normal values. 142 TABLE 5 THE EFFECT OF VARYING TEMPERATURE AND TIME OF SPECIMEN STORAGE ON THE PERCENT KILL OF HEMOPOIETIC PROGENITORS DIAGNOSIS NORMAL ET CML PV Experiment Progenitor Fresh 8 hours Fresh 8 hours Fresh 8 hours Fresh 8 hours Class 0°C 22°C 0°C 22°C o°c 22°C 0°C Blood - 1 BFU-E 0 3 4 0 0 1 35 38 44 48 49 44 2 0 0 4 52 46 47 26 34 27 44 45 42 3 0 2 4 1 late* 1 0 1 0 0 12 22 25 32 37 32 41 2 CFU-C 0 3 0 46 52 . 45 47 49 36 37 49 49 3 5 0 0 1 earl** - _ _ 0 0 0 40 26 28 49 42 48 2 CFU-C 0 9 5 43 54 46 50 55 60 52 63 57 3 0 0 0 1 CFU-GEMM _ _ 0 _ 13 _ _ _ 54 45 56 2 - - - - - - 31 40 56 54 39 62 Marrow - 1 early 0 5 0 11 13 6 60 50 50 50 42 59 2 BFU-E 0 4 4 57 56 51 47 36 45 25 32 29 3 9 0 6 1 mature 39 56 47 57 52 46 14 23 24 36 37 45 2 BFU-E 37 35 33 •46 55 58 45 46 33 36 47 37 3 30 20 31 1 late 43 37 40 42 41 48 12 25 30 42 40 36 2 CFU-C 45 46 45 59 46 56 51 51 47 14 18 13 3 48 47 50 1 early _ _ _ 5 0 6 _ 32 50 22 2 CFU-C 6 0 0 48 _ _ _ _ _ _ 3 - - - 48 - mean of difference 1.0 .06 . •' •• 1.14 .77 2.77 1.07 2.1 t value .52n S .05n'S .4 n S .56" 5 .34 n s .84 n s .43 n S l .26' degrees of freedom 17 17 12 13 12 12 14 14 : late CFU-C - 20-500 cells/colony: : early BFU-E - >16 clusters/colony; ; early CFU-C - >500 cells/colony f : mature BFU-E - 3-8 clusters/colony not significant - no significant difference was seen in percent ki l l of hemopoietic progenitors between the fresh specimen aliquot processed immediately and the aliquots kept at 0 C and 22 C and processed 8 hours later as determined by the Students T test. 143 D) Time Course Experiments A number of experiments were undertaken to examine the effect of time and temperature on the proliferative activity of hemopoietic progenitors from the blood and bone marrow of MPD patients and normal subjects. These studies were necessary to demonstrate that the values shown in Tables 1-4 were not affected by variations in the interval between removing the cells from the patient and testing the cells for their sensitivity to ^H-thymidine. To evaluate the effect of storage time on -^-thymidine suicide values, freshly obtained MPD specimens were placed on ice or kept at room temperature for 8 hours before processing. In this series of experiments a l l normal and patient peripheral blood and bone marrow specimens were obtained locally and were processed within an hour of their removal from the subject to generate zero storage time values. For periods of up to 8 hours there was no detectable effect of storage on the absolute numbers or cycling status of the hemopoietic progenitors in any given sample (p>0.5) (Table 5). This was true whether the specimen was from a normal subject or a MPD patient. It would appear therefore, that the consistent differences observed here in the cell cycle activity of hemopoietic progenitors from patients with MPD as compared to normal subjects cannot be accounted for by differences in accrual procedures. 3) DISCUSSION The present studies confirm those of others indicating an increase in progenitor turnover with progression down the erythroid or granulocytic differentiation pathway. Pluripotent progenitors, capable of mixed colony formation in vitro are quiescent in normal marrow (Fauser and Messner, 1979) as are the majority of the most primitive erythroid progenitors (Eaves et 144 al, 1979). The present studies show that the most primitive granulopoietic progenitors, as identified by their high proliferative capacity are quiescent also. More mature progenitor types characterized by a lower proliferative potential normally show a significant proportion of their numbers in S-phase. Such differentiation stage-specific alterations in proliferative activity have also been noted in the mouse (Gregory and Eaves, 1978). The absence of S-phase progenitors in the circulation of normal individuals also confirms previous findings in man (Ogawa et al, 1977; Tebbi et al, 1976). The mechanisms that regulate the cycling status of progenitors in the marrow and peripheral blood are not known. Both negative signals leading to quiescence, and positive signals leading to activation may exist and some evidence for localized changes in such signals has been reported (Gidali and Lajtha, 1972; Lord, 1979). In this context the apparent abnormalities of cycling behaviour observed in the MPD might then be attributed to 1) an intrinsic block within the cells that prevents responsiveness to negative regulatory signals, and/or 2) the activation of a mechanism(s) of autostimulation within the cells that overrides an extrinsically derived negative signal. An increase in the suicide index after exposure to ^H-thymidine and hence an increase in the number of cells in S-phase, may not necessarily result in an expansion of intermediate compartments if the transit time through these is decreased. However the final result expected in both situations would be an increase in the number of terminally differentiating cells as is typical in the MPD. The application of clonal assay systems to the study of the MPD has demonstrated other common growth and in vitro progenitor differentiation 145 characteristics, such as the presence of Ep-independent erythroid progenitor cells (Prchal and Axelrad, 1974: Eaves and Eaves, 1978, 1979; Lacombe, 1980; Eaves et al, 1983) and increased numbers of light density CFU-C (Moore et al, 1973; Greenberg et al, 1976; Metcalf, 1977; Singer et al, 1980). The findings presented in this study indicate that abnormalities in progenitor proliferative behaviour may also be a common feature of primitive cells in different MPD clones. However, such alterations do not readily account for the preferential expansion of one mature cell type characteristic of each of the MPD, since the progenitor cycling changes observed were not lineage specific. Thus other anomalies in growth characteristics, such as altered production or sensitivity to regulatory factors resulting in a growth advantage of a particular cell line may act in tandem with alterations in cell cycle activity to produce the disease state. 146 REFERENCES Adamson, J.W., Fialkow, P.J., Murphy, S., Prchal, J.F., Steinmann, L: Polycythemia vera: Stem cell and probable clonal origin of the disease. N. Eng. J. Med. 295; 913-916, 1976 Adamson, J.W., Singer, J.W., Catalono, P., Murphy, S., Lin, N., Steinmann, L., Ernst, C., Fialkow, P.J: Polycythemia vera. Further in vitro studies of hemopoietic regulation. J. Clin. Invest. 66; 1363-1368, 1980. Eaves, C.J. and Eaves, A.C: Erythropoietin dose-response curves for three classes of erythroid progenitors in normal human marrow and in patients with polycythemia vera. Blood 52; 1196-1210, 1978. Eaves, A.C. and Eaves, C.J: Abnormalities in the erythroid progenitor compartments in patients with chronic myelogenous leukemia (CML). Exp. Hematol. 7 (Suppl 5); 65-75, 1979. Eaves, A.C. and Eaves, C.J: In vitro studies of erythropoiesis in polycythemia vera. In: Current Concepts in Erythropoiesis, Dunn, C.D.R., (ed.), John Wiley and Sons, Toronto, pp. 167-188, 1983. Eaves, C.J., Humphries, R.K., and Eaves, A.C: In vitro characterization of erythroid precursor cells and the erythropoietic differentiation process. In: Cellular and Molecular Regulation of Hemoglobin Switching. Stamatoyannopoulos, G., and Nienhuis, A.W., eds, Grune and Stratton, New York pp. 251-273, 1979. Eaves, A.C, Cashman, J., Coupland, R. , Eaves, C J : Erythropoietin- independence and altered proliferative status of early erythropoietic and granulopoietic progenitor cell populations in essential thromobocytosis. Blood 62 (Suppl 1); 169a, 1983. Eridani, S., Batten, E., Sawyer, B: Erythroid colony formation in primary thrombocythaemia: Evidence of hypersensitivity to erythropoietin. Br. J. Haematol. 55; 157-161, 1983. Fialkow, P.J., Gartler, S.M., Yoshida, A: Clonal origin of chronic myelocytic leukemia in man. Proc. Natl. Acad. Sci. U.S.A. 58; 1468-1471, 1967. Fialkow, P.J., Jacobson, R.J., Papayannopoulou, T: Chronic myelocytic leukemia: Clonal evolution in a stem cell common to the granulocyte, erythrocyte, platelet, and monocyte/macrophage. Am. J. Med. 63; 125-130, 1977. Fialkow, P.J., Faguet, G.B., Jacobsen, R.J., Vaidya, K., Murphy, S: Evidence that essential thrombocythemia is a clonal disorder with origin in a multipotent stem cell. Blood 58; 916-919, 1981. Gidali, J., Lajtha, L.G: Regulation of haemapoietic stem cell turnover in partially irradiated mice. Cell Tissue Kinet. 5; 147-157, 1972 147 Gregory, C.J., Eaves, A.C: Human marrow cells capable of erythropoietic differentiation in vitro. Definition of three erythroid colony responses. Blood 49; 855-864, 1977. Gregory, C.J., Eaves, A.C: Three stages of erythropoietic progenitor cell differentiation distinguished by a number of physical and biological properties. Blood 51; 527-537, 1978. Greenberg, P., Mara, B., Bax, I., et al: The myeloproliferative disorders; correlation between clinical evolution and alterations of granulopoiesis. Am. J. Med. 61; 878-891, 1976. Hossfeld, D.K: Additional chromosomal indication, for the unicellular origin of chronic myeloid leukemia. Z. Krebsforsch. 83; 269-273, 1975. Ogawa, M., Grush, O.C., O'Dell, R.F., Hara, H., MacEachern, M.D: Circulating erythropoietic precursors assessed in culture: Characterization in normal men and patients with hemoglobinopathies. Blood 54; 1081-1092, 1977. Jacobson, R.J.. Salo, A., Fialkow, P.J: Agnogenic myeloid metaplasia: A clonal proliferation of hematopoietic stem cells with secondary myelofibrosis. Blood 51; 189-194, 1978. Lacombe, C., Casadevall, N., Varet, B: Polycythemia vera: In vitro studies of circulation erythroid progenitors. Br. J. Haematol. 44; 189-199, 1980. Lord, B.I: Proliferation regulators in hemopoiesis. Clin. Hematol. 8; 435- 463, 1979. Metcalf, D: Hemopoietic colonies: In vitro cloning of normal and leukemic cells. In: Recent Results in Cancer Research, Vol. 61, Springer-Verlag, Berlin, 1977. Moore, M.A.S., Metcalf, D: Cytogenetic analysis of human acute and chronic myeloid leukemic cells clone in agar culture. Int. J. Cancer 11; 143-152, 1973. Nowell, P.C., Hungerford, D.A: A minute chromosome in human chronic granulocytic leukemia. Science, 132; 1497, 1960. Partenen, S., Ruutu, T., Vuopio, P: Hematopoietic progenitors in essential thrombocythaemia. Scand. J. Haematol. 30; 130-134, 1983. Prchal, J.F. and Axelrad, J.W: Bone marrow responses in polycythemia vera. New Eng. J. Med. 290; 1382 (letter), 1974. Singer, J.W., Adamson, J.W., Ernst, C., Lin, N., Steinmann, L., Murphy, S., Fialkow, P.J: Polycythemia vera: Physical separation of normal and neoplastic committed granulocyte-macrophage progenitors. J. Clin. Invest. 66; 730-735, 1980. Tebbi, K., Rubin, S., Cowan, D.H., McCulloch, E.A: A comparison of granulopoiesis in culture from blood and marrow cells of non-leukemic individuals and patients with acute leukemia. Blood 48; 235-241, 1976. 148 C H A P T E R I V REGULATED PROLIFERATION OF PRIMITIVE HEMOPOIETIC PROGENITOR CELLS IN LONG-TERM HUMAN MARROW CULTURES 1) INTRODUCTION Under normal conditions very few of the most pri m i t i v e hemopoietic progenitor c e l l s i n the marrow are a c t i v e l y c y c l i n g , although following various treatments this s i t u a t i o n may be altered (Becker et a l , 1965; Eaves et a l , 1979; Fauser and Messner, 1979). Data from a number of studies suggest that changes i n the c y c l i n g a c t i v i t y of primitive progenitors may be mediated at least i n part by mechanisms that operate l o c a l l y within the marrow and involve c e l l types that do not commonly c i r c u l a t e (McCulloch et a l , 1965; G i d a l i and Lajtha, 1972). These findings have provided considerable impetus for developing an i n v i t r o model where the same mechanism(s) that regulate primitive hemopoietic c e l l behavior i n vivo continue to operate. A system of this type could then be used to characterize the relevant c e l l types and analyze their mode of act i o n . Dexter and co-workers have obtained two l i n e s of evidence that the long-term mouse marrow culture system may represent such a model. They have found f i r s t , that the maintenance of pluripotent stem c e l l s (CFU-S) i n such cultures i s dependent on the presence of other c e l l s whose function i s adversely affected by the S l / S l d genotype (Dexter and Moore, 1977) and second, that the c y c l i n g a c t i v i t y of the stem c e l l population maintained i s regulated by feeding or simple physical disturbance of the culture m i l i e u (Toksoz et a l , 1980). 149 Recently conditions for supporting primitive human hemopoietic progenitors for periods of 8 weeks or more in what appear to be similar cultures have been described (Gartner and Kaplan, 1980; Greenberg et al, 1981; Coulombel et al, 1983). I therefore undertook to evaluate the cycling status of primitive progenitor cells in such cultures and to look for comparable changes in this parameter that might be related to changes in culture conditions subject to manipulation. 2) RESULTS A) Patients Fifteen marrow specimens were used in this study to establish a total of 82 long term cultures. The specimens were obtained from 2 normal bone marrow donors and from 13 individuals undergoing hematological assessment for a variety of disorders including lymphoma, anemia, leukocytosis, and ankylosing spondylitis but whose marrow showed no malignant infiltration or other abnormality. B) Cellularity and composition of the adherent and nonadherent fractions For each experiment the nonadherent fraction was assessed on a weekly basis for total cellularity and numbers of progenitors in standard methylcellulose cultures. Individual long term cultures from each experiment were sacrificed after varying periods of incubation and the adherent layer assessed in the same manner. The results are shown in Fig. 17. As noted by other investigators (Coulombel et al, 1983, 1984), the total number of nucleated cells in the nonadherent fraction declined rapidly during the first four weeks of incubation, to an average value of 2x10^ cells/dish. During the next several weeks there is a "plateau" phase where the nonadherent cell count per dish remained relatively constant despite weekly 150 J I 1 L J L 2 3 4 5 6 7 Weeks in Culture J L Weeks in Culture Figure 17. The cellularity and progenitor content of the adherent (solid line) and nonadherent (dotted line) fractions assessed at varying incubation times. Each point shown represents the geometric mean + SEM. Of the 15 experiments initiated, 5 were terminated at 3 weeks, 6 at 4 weeks, and 4 were maintained until 7 weeks and sacrificed for assessment at that time. The downward arrows indicate maximum mean values obtained i f one colony had been seen in any of the assay dishes scored in each individual experiment. 151 removal of half of the nonadherent cells. In contrast the number of nucleated cells per adherent fraction of the same experiments demonstrated very l i t t l e variation between 3 and 7 weeks. The values obtained on a weekly basis from the nonadherent fraction for CFU-E, BFU-E, and CFU-C declined rapidly during the first four weeks of culture. Subsequently, progenitor values remained fairly constant or decreased more slowly. The most rapid decline in progenitor numbers over time was seen in the CFU-E compartment, and these could not usually be detected after two weeks. BFU-E were present in the nonadherent fraction for up to 7 weeks, but at low levels. The initial decrease in CFU-C numbers was not as pronounced as that seen in the erythroid compartment, and from 4 to 7 weeks of culture CFU-C values remained at a higher level than observed for either CFU-E or BFU-E. Higher values for each progenitor class were obtained in the adherent fraction compared to the nonadherent fraction and these values remained relatively constant for up to 7 weeks. C) 3H-thymidine suicide assay The effect of exposing cells to high specific activity ^H-thymidine for 20 minutes was usually determined both for progenitors present in the original marrow used to initiate long-term cultures and for progenitors obtained from long-term marrow cultures after various periods of maintenance. Whenever possible nonadherent progenitors as well as those released by collagenase treatment of the adherent layer were evaluated. Total progenitor numbers in both fractions were similar to previously published values (Coulombel et al, 1984). As shown in Figure 18 and 19 assessment of primitive BFU-E and CFU-C starting populations showed these to include a low or undetectable S-phase component. Just prior to each weekly medium change, 1 5 2 Figure 18. Thymidine suicide values for primitive BFU-E present in normal long-term marrow cultures assessed at various times after feeding. Cells were exposed to ̂ H-thymidine immediately after removal from the cultures and then plated in methylcellulose as described in the text. Each value represents the arithmetric mean + 1 S.E.M. of values from 1 (no error bars) to 10 experiments (different marrows). 153 60 -o 5 " 5 5 o o o o o o ID IT) o o (0 CO CP Q) c "c o o o o 0 o 1 i • • 20 0 i 1 r — Non-adherent Fraction I -fh Adherent Fraction O • •D O o 60 - 405 20- ^—fh 4 7 W e e k s of Culture 8 Figure 19. Thymidine suicide values for CFU-C present i n normal long-term marrow cultures assessed at various times a f t e r feeding. C e l l s were exposed to -^-thymidine immediately a f t e r removal from the cultures and then plated i n methylcellulose as described i n the text. Each value represents the arithmetic mean + 1 S.E.M. of values from 1 (no error bars) to 10 experiments ( d i f f e r e n t marrows). 154 a similar lack of detectable S-phase cells in the primitive BFU-E and CFU-C compartments of the adherent layer, where the great majority of these progenitor types are located, was routinely evident. Then 2 to 3 days later, the proportion of primitive BFU-E and CFU-C in S-phase regularly increased (Figs 18 and 19 lower panels). This pattern was observed in a l l 10 experiments undertaken (different marrows), irrespective of differences in the total number of primitive progenitors present or the age of the culture. Occasionally sufficient numbers of nonadherent primitive BFU-E were available for cycling determinations also. In contrast to primitive BFU-E in the adherent layer, those found in the nonadherent fraction were always actively cycling (Fig 18, top panel). High proliferative potential CFU-C were not found in the nonadherent fraction after 2 weeks as reported previously (Coulombel et al, 1983). However, large numbers of low proliferative potential CFU-C were maintained in both the nonadherent and adherent fractions. Like the nonadherent primitive BFU-E, low proliferative potential CFU-C in both fractions remained continuously in cycle (Fig 19). As expected, the numbers of low proliferative potential BFU-E detected were too small to permit meaningful assessment of their proliferative state. Table 6 shows the results of experiments in which the effect of the physical manipulations involved in a routine medium change was tested independently. In these experiments the cultures were handled as before, except that a l l 8 ml of the old medium and a l l of the nonadherent cells were simply returned to the culture and no fresh medium was added. It can be seen that this was insufficient to activate the primitive progenitor cells present in the adherent layer into DNA synthesis. In some of these experiments the effect of removing half of the nonadherent cells (without changing the medium) or of adding fresh glutamine and fresh hydrocortisone (in the amount Table 6. Effect of Dif ferent Feeding Procedures on Thymidine Suicide Values {% K i l l ) of Pr imi t ive Hemopoietic Progenitors in the Adherent Layer of Normal Long-Term Human Marrow Cul tures* Expt. Age of Standard Medium Mock Medium No Medium Change Culture Change 2 days Change 2 days but Fresh Glut/HC When Fed previously+ previously* Added 2 Days Previously^ (wks) BFU-E* CFU-C* BFU-E* CFU-C* BFU-E* CFU-C* 1 3 59 60 2 3 44 55 3 3 56 36 4 4 46 36 5 7 35 46 6 7 39 _ 0** 8 0 0 0** 0 4 0** 4 5 7 11 6 9 16 16 0 0 _ •Values shown are for pr imi t ive BFU-E (>8 c lusters) and pr imi t ive CFU-C (>500 c e l l s ) . Negative values (**) ranging from -5 to -12% are shown as 0. Simultaneously recorded values for CFU-C (<500 ce l l s ) in the adherent layer ranged between 31% and 66% k i l l . + Hal f of the medium replaced with fresh medium and ha l f of the nonadherent c e l l s removed. ^A l l of the medium and nonadherent c e l l s were f i r s t removed as for a standard medium change, but both were then returned and no new medium was added. ^1.2 mg of glutamine and 40 ul of 10~4M hydrocortisone were added to cul tures that were otherwise not handled. 156 normally added at each medium change) was also examined. Neither of these treatments was found to be sufficient to trigger primitive adherent layer progenitors into S-phase (Table 6). 3) DISCUSSION The majority of clonogenic erythropoietic and granulopoietic progenitor cells present in human marrow are normally actively cycling although those characterized by a particularly high proliferative potential can be shown to represent a small, quiescent subset (Figs 18 and 19) (Eaves et al, 1979). In this respect they resemble other primitive hemopoietic progenitor cell types identified by their pluripotentiality (Fauser and Messner, 1979) or, in the mouse, a capacity for spleen colony formation in irradiated recipients (Becker et al, 1965). The implication of these findings is that common stage-specific (but not lineage specific) mechanisms may serve to control the proliferative behaviour of a l l of these early progenitor types. The data presented here support such a model. They demonstrate that a l l of the various erythropoietic and granulopoietic progenitor populations detected in long-term human marrow cultures are proliferatively active, but when subclassified according to their demonstrated proliferative potential show marked differences in the regulation of their cycling status. Specifically, low proliferative potential progenitors were found to cycle continuously regardless of their location, whereas in the adherent layer high proliferative potential progenitors of both erythropoietic and granulopoietic lineages underwent periodic activation and re-entry into a non-cycling state. This unique behaviour of committed but high proliferative potential progenitors is similar to that previously documented for spleen colony- forming cells in murine long-term cultures (Tokoz et al, 1980). However, 157 although the mechanisms involved appear to be reproduced in human marrow cultures also, in this system they are restricted to progenitor types in the adherent layer. This difference between human and murine cultures may be due to the more cohesive structure of the adherent layer typical of the former and its greater tendency to retain the most primitive hemopoietic cells within its framework. A similar explanation may underlie the failure of physical perturbations associated with feeding of human long-term cultures to reproduce the activating effect of adding new growth medium. How a balance between quiescence and activation of primitive hemopoietic cells is regulated is not yet resolved. Simple nutrient exhaustion seems unlikely to be a factor since low proliferative potential progenitors co- existing in the adherent layer or even primitive cells present in the non- adherent fraction proliferate continuously. A possible role of hydrocortisone or glutamine, two temperature-sensitive components of the medium has also been ruled out. Recent studies with peripheral blood progenitors cultured in the presence or absence of an irradiated marrow adherent layer show that progenitor proliferation occurs continuously in the absence of adherent marrow cells (see Chapter VI). In the presence of a feeder the pattern described for normal marrow cultures is reproduced (Eaves et al, 1985). Thus, a major part of the supportive function of cells in the adherent layer may lie not in an ability to activate primitive hemopoietic cells but in a function that forces primitive hemopoietic cells into a quiescent state and which itself can be regulated. Non-toxic, reversible negative regulators of primitive hemopoietic cells of murine origin have been described (Lord, 1979; Tokoz et al, 1980). The present studies suggest that analogous human factors exist and that they may be anticipated to act on early committed as well as pluripotent progenitors. 158 REFERENCES Becker, A.J., McCulloch, E.A., Siminovitch, L., T i l l , J.E: The effect of differing demands for blood cell production on DNA synthesis by haemopoietic colony-forming cells of mice. Blood 26:296-308, 1965. Coulombel, L., Eaves, A.C, Eaves, CJ: Enzymatic treatment of long-term human marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62:291-297, 1983. Coulombel, L., Eaves, C.J., Dube, I.D., Kalousek, D.K., Eaves, A.C; Variable persistence of leukemic progenitor cells in long-term CML and AML marrow cultures. In: Wright, D.C, Greenberger, J.S., (eds): Long-Term Bone Marrow Culture, New York, Alan R Liss, Inc, 1984, p 243. Dexter, T.M., Moore, M.A.S: In vitro duplication and "cure" of haemopoietic defects in genetically anemic mice. Nature 269:412-414, 1977. Eaves, CJ., Humphries, R.K., Eaves, A.C: In vitro characterization of erythroid precursor cells and the erythropoietic differentiation process, in Stamatoyannopoulos, G., Nienhuis, A.W., (eds): Cellular and Molecular Regulation of Hemoglobin Switching, New York, Grune and Stratton, 1979, p 251. Eaves, A.C, Cashman, J.D., Gaboury, L.A., Kalousek, D.K., Eaves, CJ; Abnormal cycling behaviour of CML progenitors in vivo reproduced in long-term culture. Clin. Res. 33(No. 2):338A, 1985 (abstr). Fauser, A.A., Messner, H.A: Proliferative state of human pluripotent hemopoietic progenitors (CFU-GEMM) in normal individuals and under regenerative conditions after bone marrow transplantation. Blood 54:1197- 1200, 1979. Gartner, S., Kaplan, H.S; Long-term culture of human bone marrow cells. Proc. Natl. Acad. Sci. USA 77:4756-4759, 1980. Gidali, J., Lajtha, L.G: Regulation of haemopoietic stem cell turnover in partially irradiated mice. Cell Tissue Kinet. 5:147-157, 1972. Greenberg, H.M., Newburger, P.E., Parker, L.M., Novak, T., Greenberger, J.S; Human granulocytes generated in continuous bone marrow culture are physiologically normal. Blood 58:724-732, 1981. Lord, B.I; Proliferation regulators in haemopoiesis. Clin. Hematol. 8:435- 463, 1979. McCulloch, E.A., Siminovitch, L., T i l l , J.E., Russell, E.S., Bernstein, S.E: The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype Sl/Sl d. Blood 26:399-410, 1965. ToksOz, D., Dexter, T.M., Lord, B.I., Wright, E.G., Lajtha, L.G: The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation. Blood 55:931-936, 1980. 159 C H A P T E R V UNREGULATED PROLIFERATION OF PRIMITIVE HEMOPOIETIC PROGENITORS IN LONG TERM POLYCYTHEMIA VERA MARROW CULTURES 1) INTRODUCTION Although several studies on the proliferative behaviour of hemopoietic progenitors in PV have now been reported, the possible role of increased cell cycle activity in the expansion of the abnormal clone in PV remains obscure. In Chapter III, results from a comprehensive study of progenitor cell cycle status in the MPD were presented and these clearly showed that alterations in primitive erythroid and granulocytic progenitor cell turnover are characteristic in these malignancies. Subsequent studies of normal progentior populations maintained in the long-term marrow culture system showed that reproducible fluctuations between primitive progenitor quiescence and cycling could be observed, and it was further demonstrated that these changes were subject to modifications to the composition of the growth medium. The establishment of an in vitro model where normal primitive cells could thus be manipulated to enter and exit from the cell cycle provided an opportunity to evaluate its applicability for detecting abnormal components of this control mechanism in the MPD. Earlier work, by another laboratory (Powell et al, 1982) had shown that standard long term cultures could be established with marrow from PV patients, and provided preliminary evidence that the neoplastic clone remained stable when maintained under these conditions. Initial experiments using Ep-independence as a marker for the abnormal clone confirmed that the culture conditions employed in our laboratory also did not result in selection for the normal clone in this disease, although the opposite finding had been documented for long-term cultures initiated with 1 6 0 CML marrow (Coulombel et al, 1983). Accordingly long-term cultures were established with marrow from a number of PV patients and cell cycle studies of the progenitors in both the adherent and nonadherent fractions of long- term cultures were preformed. 2) RESULTS A) Patients Cells from a l l 7 PV patients used in this study were cultured at time of diagnosis and therefore had not been previously treated. The hemoglobin values for the 3 women were 15.8, 16.7, and 20.6 g%. Corresponding values for the 4 men were 19.9, 20.0, 20.3, 20.5, and 20.6 g%. The patients ranged in age from 55 to 83 years. WBC values were within normal limits or slightly elevated (4,600-36,000 cu/mm). All patients demonstrated the presence of endogenous colony-forming cells in their blood and marrow (Eaves and Eaves, 1983). Data for many of the fresh speciments have also been included in the results reported in Chapter III. B) Cellularity and Composition of PV Long Term Cultures The total cellularity, as well as the CFU-E and CFU-C progenitor content of both the nonadherent and adherent cell fractions is shown in Figure 20. As in previous studies with normal cultures (see Chapter IV) the cellularity and composition of the nonadherent fraction in each experiment was determined on a weekly basis. For adherent layer assessment, individual long term cultures from each experiment were sacrificed 3,4, or 7 weeks after initiation. The general behaviour of marrow cells from PV patients in long term culture was similar to that of marrow cells from normal individuals (see Chapter IV). An initial steep decline in total nucleated cell and progenitor 161' W e e k s W e e k s W e e k s Figure 20. Total number of nucleated cells, erythroid colony forming cells and granulocyte progenitors in the adherent and nonadherent fractions of PV marrow cultures assayed at varying times after initiation. All cultures were established with marrow cells from newly diagnosed untreated patients. Each point represents the geometric mean + SEM. The downward arrows indicate maximum mean values obtained if one colony had been seen in any of the assay dishes scored in each individual experiment. 162 content of the nonadherent fraction occurred during the first 3-4 weeks of incubation. This was then followed by a "plateau phase" where these values remained fairly constant. The CFU-E compartment experienced the most rapid decline in progenitor number over time, with no, or very few CFU-E detectable in the nonadherent fraction after two weeks. The cellularity and composition of the adherent fraction in contrast to the nonadherent fraction, remained fairly constant throughout the duration of the experiment. C) Erythropoietin-Independence in Long-Term PV Cultures The presence of neoplastic erythroid progenitors capable of forming mature, hemoglobin-containing progeny in assay cultures that contained no added Ep was determined for both the adherent and nonadherent fractions of long term cultures. A similar assessment was also made on the i n i t i a l marrow specimen used to establish the cultures. The results are shown in Figure 21. The proportion of erythroid progenitors from each fraction that was capable of Ep-independent colony formation remained constant regardless of the age of the culture, and was comparable to the values obtained in the marrow sample used to establish the culture. It would therefore appear that the abnormal clone remained stable in long term cultures established from these PV marrow aspirates. D) ^H-Thymidine Suicide Assay of Hemopoietic Progenitors The proliferative activity of primitive and mature hemopoietic progenitors in the adherent fraction of 3, 4, and 7 week old cultures was determined. In the majority of experiments additional dishes were sacrificed 2 days later for similar progenitor cell cycling determinations. The results are shown in Table 7. Regardless of the age of the culture, a l l classes of hemopoietic progenitors appeared to be in active cell cycle. This finding contrasts with 163 Figure 21. Assessment of BFU-E number in the adherent and nonadherent fractions of PV long term cultures at varying periods of incubation. Each point represents the geometric mean + SEM. The solid line indicates the total number of BFU-E present in cultures where the proportion of erythroid burst forming cells capable of colony formation in the absence of added Ep was also assessed (dashed line). Seven experiments were initiated of which 4 were terminated by week 4. Only 1 of the 3 experiments carried until week 7 produced sufficient numbers of nucleated cells to permit assessment of the proportion of endogenous colony forming cells. The solid region represents the range of nonadherent BFU-E number per long term culture for a l l 7 experiments, as determined by assay in methylcellulose cultures containing Ep. Simularly, the total number of BFU-E and the proportion of endogenous BFU-E was determined for the adherent layer. Cultures from 3 experiments were assayed in this manner at week 3, from 4 experiments at week 4, and from 3 experiments at week 7. 164 TABLE 7 THYMIDINE SUICIDE (Z KILL) OF HEMOPOIETIC PROGENITORS IN PV LONG TERM MARROW CULTURES BFU -El CFU- Cell Age of Exp. Fraction Culture No. Early Late Early Late Adherent 3-4 weeks 1 55 57 40 2 32 47 30 61 3 69 - 48 55 4 50 54 53 54 5 38 47 37 57 6 58 47 57 55 7 50 66 42 67 3-4 weeks + 3 52 65 58 49 2 days 4 42 71 53 46 5 49 62 22 64 6 62 50 54 52 7 70 66 44 63 7 weeks 5 43 11 57 6 53 62 43 7 53 64 48 51 7 weeks + 7 51 34 66 59 2 days Non- 3-4 weeks 3 47 64 adherent 4 45 - - 52 7 52 - - 47 4 weeks + 7 56 62 2 days 1 Early - >8 clusters; Late - 3-8 clusters 2 Early - >500 cells; Late - 20-500 cells 165 the results obtained with normal cultures (see Chapter IV) where at week 3, 4, or 7, just prior to the weekly feeding, primitive BFU-E and CFU-C in the adherent layer were found to have a low or undetectable S-phase component. These normal early progenitors, however, could be triggered into active cell cycle by the weekly replacement of half of the culture medium. Such a pattern of regulated proliferation is not apparent in the cycling behaviour of primitive progenitors from long-term PV cultures. The majority of these cells were in S-phase irrespective of whether the cultures had been fed 7 days or 2 days previous to the ^-thymidine assay determinations. In 3 experiments, a sufficient number of non-adherent cells were obtained to permit assessment of the proliferative state of progenitors in this fraction. As reported in Chapter IV for normal marrow cultures quiescent primitive BFU-E and late CFU-C were not found in this fraction of the culture at any time. Primitive CFU-C were not detected in the nonadherent fraction after 2 weeks, and mature BFU-E were found in insufficient quantity to permit meaningful determinations of their cell cycle status. 3) DISCUSSION Studies on the proliferative behaviour of clonogenic progenitors in the marrow of normal individuals suggest that differentiation stage-specific mechanisms may regulate the cycling activity of primitive cell types. Subsequent studies suggested that conditions prevailing in the long-term marrow system may allow mechanisms to operate in vitro and hence be subjected to further analysis. In contrast, stage-specific alterations in progenitor proliferative activity are not observed in PV marrow where the majority of high proliferative potential progenitors of the erythroid and granulocytic 166 lineages appear to be actively turning over. In this series of experiments we have demonstrated that this unique pattern of continuous proliferative activity is also seen when PV cells are maintained in the long-term marrow culture system where, in normal controls, primitive cells become quiescent. The mechanism that permits primitive PV progenitors to appear to bypass or ignore negative regulatory signals is not known. The defect could be intrinsic to the cell, such as an abnormality in the cell membrane of neoplastic cells that prevents normal interactions with negative regulatory cell types, or an abnormality in gene expression that leads to an autocrine phenotype. However, from the data presented here for PV, it is not possible to exclude faulty negative signal production as an explanation for the results reported in this Chapter. 167 REFERENCES Coulombel, L., Kalousek, D., Eaves, C.J., Gupta, CM., Eaves, A.C: Long- term marrow cultures reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N.E.J.M. 308; 1493-1498, 1983. Eaves, A.C. and Eaves, C J : In vitro studies of erythropoiesis in polycythemia vera. In: Current Concepts in Erythropoiesis, Dunn, C.D.R., ed. John Wiley and Sons Ltd., pp. 167-187, 1983. Fauser, A.A. and Messner, H.A: Pluripotent hemopoietic progenitors (CFU-GEMM) in polycythemia vera: Analysis of erythropoietin requirement and proliferative activity. Blood 58; 1224-1227, 1981. Powell, J.S., Fialkow, P.J., Adamson, J.W: Polycythemia vera: Studies of hemopoiesis in continuous long-term culture of human marrow. J. Cell Physiol. (Suppl. 1); 79-85, 1982. Prchal, J.F., Erythroid progenitors in peripheral blood of normal and polycythemic subjects. J. Supramol. Struct. (Suppl. 2); 168-169, 1978. Singer, J.W., Fialkow, P.J., Adamson, J.W., Steinmann, L., Ernst, C, Murphy, S., Kopecky, K.J: Polycythemia vera: Increased expression of normal committed granulocytic stem cells in vitro after exposure of marrow to tritiated thymidine. J. Clin. Invest. 64; 1320-1324, 1979c. Zanjani, E.D., Weinberg, R.S., Nomdedev, B., Kaplan, M.E., Wasserman, C.R; In vitro simularities between erythroid precursors of fetal sheep and patients with polycythemia vera. In: In Vitro Aspects of Erythropoiesis, Murphy, M.J., ed., Springer Verlag, Berlin, pp. 118, 1978. 168 C H A P T E R VI UNRESPONSIVENESS OF PRIMITIVE CML PROGENITORS TO THE NEGATIVE REGULATORY EFFECT OF AN ADHERENT CELL TYPE IN NORMAL MARROW 1) INTRODUCTION A minimal requirement for analyzing the molecular basis of the abnormal proliferative behaviour of primitive Phi-positive cells would be a culture system where they could be maintained and their turnover shown to be indifferent to normal control. As demonstrated in Chapter IV, normal primitive committed and pluripotent myeloid progenitors undergo cyclic changes in their proliferative behaviour in response to changes of the growth medium. However, the long-term system has not been useful for the study of CML progenitor regulation since primitive Ph^-positive progenitor numbers rapidly decline when such cultures are initiated with CML marrow, even though conditions may be sufficient to support an initially undetectable but persisting normal progenitor population (Coulombel et al, 1983; Dube et al, 1984). In collaboration with others in the Terry Fox Laboratory, I therefore sought ways of modifying this type of culture system to achieve a higher yield of Ph1 -positive progenitors. Two approaches were evaluated. In the first, CML marrow cells were seeded onto pre-established adherent marrow "feeder" layers derived from normal long-term marrow cultures. In the second, CML peripheral blood (PBL) rather than marrow was used as a source of primitive Ph^-positive progenitor cells and these were then seeded onto irradiated normal marrow adherent layers. As controls, allogeneic, T cell depleted, light density PBL cells from normal donors were also cultured on similar adherent marrow feeder layers. In each experiment progenitor numbers 169 in both the non-adherent and adherent fractions were determined after varying intervals by cloning in methylcellulose. Cycling characteristics of these cells were measured using the -^-thymidine suicide technique and progenitor genotypes in CML cultures determined by cytogenetic analysis of plucked colonies as described in Chapter II. 2) RESULTS A) Patients Marrow and/or PBL from 5 untreated CML patients were used for co-culture studies. Additional co-culture studies were performed with PBL from 2 other CML patients. These had been previously treated but had elevated WBC counts at the time of study and PBL progenitor numbers were greatly elevated. Marrow was also obtained from a previously studied patient (Dube et al, 1984) 6 years postdiagnosis and on hydroxyurea at the time the present aspirate was taken. Normal PBL was obtained though the courtesy of the Canadian Red Cross. Normal marrow for the generation of adherent layers were from normal marrow transplant harvests or from lymphoma and Hodgkin's patients without marrow involvement. B) Culture of CML marrow on normal marrow adherent layers. The effect of seeding CML marrow aspirate specimens on irradiated normal marrow adherent layers established as described Chapter II was evaluated in 4 separate experiments using marrow cells from 4 different untreated CML patients. Results in a l l were similar. A representative experiment is shown in Figure 23. In general, the non-adherent fraction of cultures containing pre-established feeders was found to contain higher numbers of nucleated cells, BFU-E and CFU-C. Assessment of the adherent fraction required 170 sacrificing a whole culture and was therefore done less frequently (usually at 4 weeks and occasionally again at 7-8 weeks). Higher numbers of clonogenic progenitors were also found in the adherent fraction of CML marrow cultures established on feeders as compared to controls without feeders. Data for primitive progenitors is given in Table 8. Cytogenetic analysis of the large colonies produced from these progenitors showed that the maintenance of a significant population (>100 per culture) of primitive, i.e. high proliferative potential, Ph^-positive progenitors had not been achieved in any experiment. Where larger numbers of primitive progenitors were present, these proved to be Phi-negative. Thus, the use of feeders appeared to improve progenitor maintenance but only to a limited degree, and with l i t t l e selectivity for the Ph^-positive line. As a result, from none of these experiments were sufficient numbers of primitive Ph^-positive progenitors obtained after 4 weeks to allow their cycling behaviour to be evaluated. However, in a previous study (Dube et al, 1981) we had identified an anomalous patient whose long-term marrow cultures maintained Ph^-positive progenitor numbers in the range typical of cultures established from normal marrow. A marrow specimen obtained from this same patient 3 years later was used to initiate a second series of cultures. As shown in Table 9 extended maintenance of Ph1 -positive progenitors was again achieved. In anticipation that the numbers of those classified as primitive would be sufficient for cycling determinations, such measurements were also undertaken. As this proved to be the case these results are also presented in Table 9. It can be seen that primitive BFU-E and CFU-C in the adherent layer of these cultures did not become quiescent 7 days after a previous medium change as do their normal counterparts in control cultures (Chapter IV). gure 22. The adherent layer of a reconstituted CML long term blood culture 3 weeks after initiation (X160). 172 Figure 23. Comparison of total cell and progenitor content of a long-term CML marrow culture initiated with (solid symbols) or without (open symbols) a pre-established normal marrow feeder. Circles - data for the non-adherent fraction assessed weekly. Squares - data for the adherent layer assessed at week 4. 173 Table 8. Enhancing effect of feeders on primitive progenitor numbers maintained in long-term CML marrow cultures and assessed after 4 weeks Progenitor Experiment no. With feeders No.* Genotype (X Phi) Without feeders No.* Genotype (X Phi) Primitive BFU-E (>8 clusters) 1 2 3 4 25 1320 52 5 100 0 0 5 <26 3 6 0§ Primitive CFU-C (>500 cells) 1 2 3 4 <13 2260t 26 2 lOOt Ot lOOt 0 <5 1720t 3 1 Ot lOOt 30t * Values shown are the number of primitive progenitors detected per 4 week culture from adherent layer assays. Except in 2 cases, where 11 and 6 primitive BFU-E per nonadherent fraction, respectively, were measured, no primitive BFU-E or CFU-C were detectable in this fraction. Values for total progenitor numbers were higher but similarly distributed. t All types of CFU-C. } No data. § From cytogenetic analysis of colonies derived from mature BFU-E. 174 Table 9. Prim i t i v e hemopoietic progenitor numbers and t h e i r c y c l i n g status at week 4 i n long-term marrow cultures at week 4 ( i . e . 7 days a f t e r the routine 3 week medium change) from an anomalous CML patient whose Ph^- po s i t i v e c e l l s were maintained under these conditions. Normal Progenitor* Fraction assayed Number X K i l l X k i l l values* P r i m i t i v e BFU-E Nonadherent 946 46 3 8 + 7 (> 8 c l u s t e r s ) Adherent 280 35 0 + 3 Primi t i v e CFU-C Nonadherent 0 -§ (> 500 c e l l s ) Adherent 53 57 1 + 4 A l l colonies analyzed at this time were found to be Ph 1 - p o s i t i v e . +" X K i l l values + 1 SEM from Chapter IV for these progenitor classes found in 4 week old long-term cultures established from normal marrows and handled i n the same fashion. § No data. 175 C) Cultures of CML PBL on normal marrow adherent layers. Since the PBL of untreated high count CML patients contains highly elevated numbers of neoplastic progenitors of a l l types, we next tested whether Ph1 -positive progenitor maintenance might be more reproducibly achieved when these were seeded onto pre-established irradiated normal marrow feeders. Preliminary experiments by my supervisor and others (Eaves et al, 1984) had revealed that such cultures rapidly assumed the appearance of hemopoietically active long-term marrow cultures and >1000 clonogenic progenitors per culture could commonly be detected after 4 weeks (Figure 22). This suggested that cycling studies of Phi-positive progenitors would be feasible using this type of reconstructed culture. Accordingly, a number of experiments were set up for this purpose. Figure 24 shows a summary of the progenitor maintenance achieved in the 11 experiments from 7 different CML patients. Assessment of their cycling status was also undertaken and the results of these studies are given in Table 10 and 11. From Figure 24 it can be seen that clonogenic progenitor numbers were typically maintained at levels in excess of 1000 per culture for periods of 4 weeks or longer when feeders were present, the majority being located in the adherent (feeder layer containing) fraction. In the absence of pre-established feeders, no adherent layer formed and virtually a l l of the cells present were recovered in the non-adherent fraction. Total progenitor numbers in cultures without feeders were consistently lower than in cultures with feeders, although readily detectable numbers of progenitors usually persisted for several weeks. From the data shown in Tables 10 and 11 it can be seen that a l l types of progenitors in these CML PBL cultures were actively dividing regardless of their differentiation lineage, proliferative potential, location in the 176 Figure 24. Comparison of total cell and progenitor content of long-term CML PBL cultures initiated with (solid symbols) or without (open symbols) a pre-established normal marrow feeder. Circles - data for non-adherent fractions assessed weekly. Squares - data for adherent fractions (cultures with feeders only, <10̂  adherent cells and no adherent progenitors were detected in cultures without feeders). Triangles - data for cultures established without feeders, but from which no cells were removed at each weekly medium change until the culture was sacrificed. Values shown are the geometric means + 1 SEM from data of 11 experiments. 177 Table 10. Thymidine suicide measurements (X k i l l ) of nonadherent progenitors in CML blood cultures BFU-E* CFU-Ct Age of Exp. Feeders culture no. Primitive Mature Primitive Mature Present 3-4 weeks 3-4 weeks + 2 days 7 weeks 1 2 3 4 5 6 7 8 2 3 4 1 3 60 69 46 56 55 52 35 43 52 54 61 45 38 69 62 58 50 37 75 54 46 62 60 42 47 47 39 59 57 42 50 54 42 37 Absent 3-4 weeks 4 weeks + 2 days 1 2 3 4 9 10 1 4 43 44 28 51 58 43 46 33 51 45 52 57 63 45 47 51 44 55 58 51 42 55 7 weeks 55 * Primitive - >8 clusters; Mature - 3-8 clusters. + Primitive - >500 cells; Mature - 20-500 cells. + No data. 178 Table 11. Thymidine suicide measurements (% ki l l ) of adherent progenitors in CML blood cultures BFU-E* CFU-Ct Age of Exp. culture no. Primitive Mature Primitive Mature 3-4 weeks 3-4 weeks + 2 days 7 weeks 7 weeks + 2 days 1 39 50 46 59 2 50 39 44 41 3 32 51 - 42 4 49 63 65 45 5 48 46 43 49 6 54 42 64 56 7 58 56 70 57 8 44 52 53 49 9 44 44 52 53 10 75 69 73 62 11 36 44 59 42 2 52 42 51 52 3 51 55 - 42 4 41 49 65 45 5 48 49 58 52 6 52 50 50 53 7 49 - 46 42 1 36 57 _ 37 2 40 - - 43 3 42 _ 42 * Primitive + Primitive - >8 clusters; Mature - 3-8 clusters. - >500 cells; Mature - 20-500 cells. 179 adherent or non-adherent fraction, time of assessment after feeding the cultures, or presence or absence of a pre-established feeder. These findings corroborate those described above for the one conventional long-term CML marrow culture where Ph^-positive progenitor yields were sufficient to allow the cycling behaviour of neoplastic progenitors to be evaluated. However, these results contrast markedly with the findings for normal primitive progenitor types maintained in conventional long-term marrow cultures (Chapter IV). Colonies generated in assays of cells from both types of CML PBL cultures were also cytogenetically analyzed. The results are summarized in Table 12 These show that the majority of the progenitors in a l l cultures were Ph^-positive at the time cycling measurements were undertaken. D) Cultures of normal PBL on normal marrow adherent layers Because the difference in primitive Ph^-positive progenitor cell cycling noted in this study was revealed in a somewhat different system (i.e. CML PBL added to normal marrow adherent layers), a final series of experiments were undertaken to evaluate the cycling behaviour of normal peripheral PBL progenitors maintained under similar conditions. Weekly changes in the total number of nucleated cells and clonogenic erythroid and granulopoietic precursors detected in normal PBL cultures set up with and without feeders are shown in Figure 25. Results were very similar to those obtained with CML cells, although the ini t i a l inoculum of 2 x 10? light density T-depleted normal PBL cells contained 10-100 fewer progenitors, and progenitor numbers measured after 3-7 weeks were correspondingly lower. Nevertheless, adherent layer values, where the majority of the progenitors were again found, were sufficient to allow cycling data to be obtained. The results, shown in Table 13, reveal the same Table 12. Cytogenetic analysis of CML peripheral blood cultures with and without feeder l a y e r s * With feeders Without feeders Age of Culture Patient Ph+ Ph~ Ph+ Ph~ 1 38 1 1 7 2 87 0 5 0 3 21 0 21 0 4 6 8 45 11 5 35 0 8 0 6 33 0 25 0 7 26 0 9 0 1 15 0 1 2 2 14 0 _+ -3 - - - -4 - - - -5 21 0 - - 6 - - - -7 - — 0 1 * Values shown are t o t a l metaphases obtained from BFU-E and CFU-C colonies cultured i n methylcellulose assays. + No data. 181 W e e k s in C u l t u r e W e e k s in C u l t u r e W e e k s in C u l t u r e Figure 25. Comparison of total cell and progenitor content of long-term normal PBL cultures initiated with (solid symbols) or without (open triangles) a pre-established normal marrow feeder. Circles - data for non-adherent fractions of cultures with feeders assessed weekly. Squares - data for adherent fractions of cultures with feeders. Triangles - data for non-adherent fractions of cultures without feeders (no adherent fraction obtained), from which no cells were removed at each weekly medium change until the culture was sacrificed. Values shown are the geometric means + 1 SEM from data of 5 experiments. 182' Table 13. Thymidine suicide measurements (% kil l ) of hemopoietic progenitors from the adherent fraction of normal blood cultures BFU-E" Feeders Age of culture CFU-Ct Exp. no. Primitive Mature Primitive Mature 3-4 weeks 1 0 50 18 53 2 0 41 • 5 48 3 2 53 0 45 4a 0 40 0 42 4b 9 46 11 44 5 1 54 0 43 3-4 weeks + 1 41 72 59 57 2 days 2 47 50 48 46 3 54 60 50 51 4 49 33 40 43 5 56 59 53 42 7 weeks 1 0 43 0 38 2 4 33 -* 41 3-4 weeks 3 44 46 _ 54 4 57 50 62 53 5 56 70 52 48 3 weeks + 4 58 46 _ 59 2 days * Primitive - >8 clusters; Mature - 3-8 clusters, t Primitive - >500 cells; Mature - 20-500 cells. ^ No data. 183 pattern of alternating proliferation and quiescence, characteristic of primitive (high proliferative potential) but not mature (low proliferative potential) progenitor cell types located in the adherent layer. In cultures without feeders, the number of progenitors had generally decreased by 3 weeks to values below those measured in cultures with feeders, even when no cells were removed at each weekly medium change (Figure 25). However, at 3 weeks they were present in high enough numbers for cycling measurements to be performed. Interestingly, the results of these showed that in the absence of a feeder, a l l progenitor types were proliferating continuously, irrespective of the time since the previous medium change (Table 13). 3) DISCUSSION Currently, there is much interest in defining the mechanisms that regulate pluripotent hemopoietic stem cell turnover. In the murine system, conclusive evidence of direct acting soluble factors that support both self- renewal and differentiative divisions of pluripotent cells in vitro has now been obtained (Johnson et al, 1977; Metcalf et al, 1980; Metcalf and Nicola, 1983). Although similar studies with purified factors have yet to be performed with human cells, recent purification and gene cloning studies (Metcalf, 1985; Uelte et al, 1985; Kaushansky et al, 1985) indicate a strong analogy between these species. Thus it may be anticipated that soluble factors capable of activating or contributing to the direct activation of human pluripotent stem cells will be found. On the other hand, studies of the genetically determined Sl/Sl d anemic mouse indicate that primitive hemopoietic cell proliferation in vivo is dependent on close range interactions with cells that normally do not circulate and do not appear to be derived from hemopoietic progenitors (McCulloch et al, 1965). 184 Demonstration of localized effects of radiation on stem cell activation in partially shielded mice provide additional support for a non-humoral component to normal stem cell regulation (Gidali et al, 1972). More recently, evidence for both positive and negative regulation of primitive hemopoietic cell turnover in vitro has been obtained by time course studies of murine (Toksoz et al, 1980) and human long-term marrow cultures (Chapter IV). In the present study we have shown that after activation in culture primitive normal cells of PBL origin are rendered quiescent in the presence of an adherent marrow population, but not in its absence. This indicates that maintenance of primitive hemopoietic progenitor quiescence is a function of an adherent cell unique to the marrow and not present in, or derived from, cell types circulating in the PBL. The present studies have further revealed that primitive Ph1 -positive progenitors from CML patients are not sensitive to this negative control mechanism in vitro. The fact that CML cells remained in cycle despite their maintenance in higher numbers than in those typical of cultures initiated with normal blood, provides additional evidence that the arrest of normal primitive cell proliferation is not due simply to nutrient exhaustion (Chapter IV). In the CML experiments, the lack of cycling control would not appear to be explained by a faulty "stroma", since normal marrow adherent layers were used as feeders. It therefore seems likely that the abnormal behaviour observed is due to a change in the CML cells themselves. What the nature of this change may be is not clear, although two possibilities invite consideration. One is based on the observation that primitive CML progenitor cells may have altered cell surfaces (Baker et al, 1985). This could lead to a reduced ability to bind to surface determinants on adherent marrow cells that may be an essential aspect of the negative 185 regulation process. The second, not exclusive, possibility is that primitive CML progenitor cells may have autocrine or autostimulatory capabilities. Such a property might then allow these cells to override a negative signal or replace a transient positive signal, resulting in the phenotype observed. 186 REFERENCES Baker, M.A., Taub, R.N., Kanani, A., Brockhausen, I. & Hindenburg, A; Increased activity of a specific sialyl transferase in chronic myelogenous leukemia. Blood 66: 1068-1071, 1985. Coulombel, L., Kalousek, D.K., Eaves, C.J., Gupta, CM. & Eaves, A.C; Long- term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosome-positive chronic myelogenous leukemia. N. Engl. J. Med. 308: 1493-1498, 1983. Dube, I.D., Eaves, C.J., Kalousek, D.K. & Eaves, A.C; A method for obtaining high quality chromosome preparations from single hemopoietic colonies on a routine basis. Cancer Gen. Cytogen. 4: 157-168, 1981. Dube, I.D., Kalousek, D.K., Coulombel, L., Gupta, CM., Eaves, C.J., Eaves A.C; Cytogenetic studies of early myeloid progenitor compartments in Phi- positive chronic myeloid leukemia. II. Long-term culture reveals the persistence of Phi-negative progenitors in treated as well as newly diagnosed patients. Blood 63: 1172-1177, 1984. Eaves, A.C, Kalousek, D.K., Cashman, J.D. & Eaves CJ; Extended maintenance of proliferating Phi-chromosome positive (Phl+) progenitors following addition of CML peripheral blood but not marrow cells to pre-established normal marrow adherent layers. Blood 64 (Suppl 1): 188a, 1984. Gidali, J. & Lajtha, L.G; Regulation of haemopoietic stem cell turnover in partially irradiated mice. Cell Tissue Kinet. 5: 147-154, 1972. Johnson, G.R., Metcalf, D; Pure and mixed erythroid colony formation in vitro stimulated by spleen conditioned medium with no detectable erythropoietin. Proc. Natl. Acad. Sci. USA 74: 3879-3882, 1977. Kaushansky, K., O'Hara, P., Berkner, K., Segal, CM., Broudy, V., Hagen, F. & Adamson, J.W.; Genomic cloning and expression of human granulocyte/macrophage colony-stimulating factor (GM-CSF): Evidence for multilineage effect of the CSF. Blood 66 (Suppl 1): 153a, 1985. McCulloch, E.A., Siminovitch, L., T i l l , J.E., Russell, E.S. & Bernstein, S.E; The cellular basis of the genetically determined hemopoietic defect in anemic mice of genotype Sl/Sl d. Blood 26: 399-408, 1965. Metcalf, D., Johnson, G.R. & Burgess, A.W; Direct stimulation by purified GM-CSF of the proliferation of multipotent and erythroid precursor cells. Blood 55: 138-147, 1980. Metcalf, D., Nicola N.A; Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells. J. Cell Physiol. 116: 198-211, 1983. Metcalf, D; The granulocyte-macrophage colony stimulating factors. Cell 43: 5-6, 1985 Toksoz, D., Dexter, T.M., Lord, B.I., Wright, E.G. & Lajtha, L.G; The regulation of hemopoiesis in long-term bone marrow cultures. II. Stimulation and inhibition of stem cell proliferation. Blood 55: 931-937, 1980. Welte, K., Gabrilove, J., Platzer, E., Lu, L., Souza, L., Boone, T., Keever, C., Mertelsmann, R. & Moore, M.A.S; Biochemical and biological characterization of human hematopoietic pluripoietin. Blood 66 (Suppl 1): 165a, 1985. 188 CHAPTER VII CONCLUSIONS AND FUTURE DIRECTIONS Although clonal assay systems have permitted elucidation of a number of abnormalities characteristic of MPD progenitors, the contribution of these to the pathogenesis of the MPD is unclear. A major limitation of clonal assay systems is their inadequacy to measure pluripotent stem cell self-renewal and their failure to reproduce close range regulatory mechanisms believed to operate in vivo. By their very nature clonal assays minimize direct interactions between cells. Thus, some regulatory factors which may be important in vivo could be totally absent from clonal culture systems. A growing appreciation of the importance of hemopoietic:stromal cell interactions in in vivo hemopoiesis has led to increased interest in the application of the long term culture system in the study of normal and neoplastic hemopoiesis. Long term cultures provide a complex in vitro system in which hemopoietic progenitors of a l l myeloid cell lines can be maintained for periods in excess of 8 weeks. Such a culture system permits interactions between various hemopoietic and non-hemopoietic cell types that may closely approximate conditions occuring in vivo. Whether such accessory cell populations are an appropriate model of the intramedullary environment and whether they are able to produce factors that regulate normal stem cell turnover are areas of current investigation, addressed in part by the work described. The primary objective of this thesis was to characterize possible disease-related alterations in the cycling behaviour of normally quiescent hemopoietic cells in patients with various MPD and to develop an in vitro 189 model where the biology underlying such alterations might be analyzed further. I therefore first undertook a comprehensive study of the cycling characteristics of primitive and mature progenitors of the erythroid and granulocytic lineages, as well as pluripotent progenitors, in the blood and marrow of patients with PV, ET, and CML. These studies were then extended in vitro by appropriate application or modification of the long-term marrow culture system. Analysis of cell cycle control in MPD patients Peripheral blood cells from 9 normal individuals and from 12 SE, 14 PV, and 16 CML patients were examined for the presence of S-phase cells by short term exposure to ̂ H-thymidine. All hemopoietic progenitors from normal blood were found to be quiescent, regardless of cell lineage or stage of differentiation, while the majority of such progenitors from the peripheral blood of patients with CML or PV were in S-phase. Marrow cells from 20 normal individuals and from 15 SE, 16 PV, and 13 CML patients were subjected to the same procedure for analysis for their cell cycle activity. In normal marrow stage-specific increases in proliferative activity occur with progressive progenitor cell maturation in both lineages studied. In contrast this progression was absent in PV or CML patients, where a l l marrow progenitors including the most primitive erythropoietic and granulopoietic compartments were found to be in cycle. The majority of these are quiescent in normal marrow. Such alterations in cycling behaviour were also found to be typical of ET patients in whom erythroid progenitors capable of Ep- independent erythropoiesis could usually also be demonstrated. This series of experiments indicates that neoplastic hemopoietic progenitor cells from a l l classes of MPD patients are capable, in some manner, of bypassing normal extrinsic regulatory control. In order to 190 further characterize the cycling abnormalities observed in the MPD, experiments were undertaken to determine if such abnormalities are also characteristic of neoplastic progenitors produced in the long-term marrow culture system. To obtain control data, long term cultures were established with cells from normal individuals and the proliferative behaviour of progenitors present in these cultures 3-8 weeks later was examined Normal long term cultures A total of 82 long-term cultures were initiated with marrow from 15 normal individuals. Progenitors of large erythroid colonies (>8 erythroblast clusters) present in the nonadherent fraction, and progenitors of small granulocyte/macrophage colonies (<500 cells) present in both the nonadherent and adherent fractions were found to be actively cycling at a l l times examined. In contrast, progenitors of large granulocyte/macrophage colonies (>500 cells) and progenitors of large erythroid colonies (>8 erythroblast clusters), present in the adherent layer, consistently alternated between a quiescent state at the "time of each weekly medium change, and a proliferating state 2-3 days later. Additional experiments revealed that the activation of primitive progenitors in the adherent layer was not dependent on the addition of fresh glutamine or hydrocortisone, nor on the physical manipulations involved in changing the growth medium. These studies provide the first direct evidence that normal long-term human marrow cultures support the continued turnover of a variety of early hemopoietic progenitor cell types. In addition, they indicate that the proliferative activity of the most primitive of these progenitors is normally regulated in vitro by stage-specific cell-cell interactions, that are subject to extrinsic manipulation (i.e. feeding). I then proceeded to analyze the cycling behaviour of progenitors present in long-term MPD cultures. PV Long-Term Cultures 191 PV Long-Term Cultures Marrow cells from 7 PV patients were used to establish long-term cultures. The ability of these cultures to maintain the abnormal clone in vitro was examined using Ep-independence as a marker of neoplastic erythropoietic cells. The proportion of erythroid progenitors exhibiting this phenotype was found to remain unchanged in standard long-term cultures for periods of up to 7 weeks. The cell cycle status of hemopoietic progenitors in these cultures was also examined. All classes of hemopoietic progenitors were found to be in active cell cycle, regardless of the age of the culture, time after the weekly medium change, progenitor proliferative capacity, or progenitor location (adherent versus nonadherent fraction). Thus, the pattern of regulated proliferation observed in normal long-term marrow cultures was not reproduced in PV cultures. These differences suggest that neoplastic progenitors in PV do not respond to negative influences of regulatory adherent elements that suppress the proliferative activity of their normal counterparts. CML Long-Term Cultures Initial experiments with CML cells were concerned with achieving some maintenance of Phi-positive progenitors under long-term culture conditions. The effect of seeding CML cells on irradiated normal marrow adherent layers was evaluated with marrow specimens from 4 patients. Cytogenetic analysis and the assessment of progenitor numbers revealed that such modifications of the culture system resulted in a limited improvement in the maintenance of Phi progenitors. As a next step, peripheral blood cells from CML patients were layered over irradiated normal adherent layers. Eleven such experiments were undertaken, using cells from 7 CML patients. Under these conditions CML 192 PBL progenitors could be maintained for periods of 1-2 months. Progenitor numbers were found to be higher in the adherent layer than in the nonadherent fraction, and the number of Ph^-positive primitive progenitors of high proliferative potential present in the adherent layer was sufficient for determinations of their cycling status. Such measurements demonstrated that primitive CML progenitors cycle continuously regardless of the presence or absence of an adherent feeder layer. Similar results were obtained with marrow cells from one anomalous CML patient whose Ph1 -positive progenitors could be maintained in standard long-term marrow cultures. In contrast, primitive PBL normal progenitors, when cultured in the presence of a pre-established adherent marrow feeder layer were found to go in and out of cycle after each medium change. In the absence of a feeder layer they remained continuously in cycle. In both CML and normal PBL cultures, low proliferative potential progenitors were in constant cell cycle whether or not a feeder layer was present. This series of long-term culture experiments extends the observations obtained with the in vitro studies of PV marrow cells and demonstrates that in CML, as in PV, the abnormalities in cell cycle behaviour seen in vivo in the MPD can be reproduced in vitro. Conclusions Two significant findings emerge from this work: 1) Regulation of normal primitive progenitor proliferation in long-term cultures is determined by close range interactions with a cell type(s) derived from and unique to the marrow. These interactions may be directly between cells, or may involve short range regulatory factors. An important function of these adherent cells is to return and maintain primitive progenitors in a quiescent state. 193 2) Primitive progenitor cells in at least 3 of the MPD demonstrate a common abnormality in the control of primitive progenitor cell turnover in vivo. Studies with the long-term cultures indicate that such alterations in cycling behaviour may be due to an intrinsic fault in the neoplastic stem cells themselves, which permits them to overide or ignore negative regulation imposed by normal adherent marrow elements. Such insensitivity to negative regulation may be explained either by the activation within MPD progenitors of mechanisms for autostimulation and/or mechanisms that impair negative signal recognition or transduction. Future Directions Although data presented in this thesis supports the concept that alterations in cycling behaviour may be involved in the pathogenesis of the MPD, a number of questions remain unanswered. The absence of lineage specific alterations in cell cycle activity indicates that other mechanisms must be operative to produce the typical clinical presentation of PV, ET, or CML. The possibility that abnormalities in cycling behaviour and the expression of Ep-independence may have a common mechanistic basis remains to be explored. In addition, the nature of the lesion in the abnormal stem cell that permits its progeny to bypass normal regulatory controls has not yet been determined. Long term cultures form a complex in vitro system that has not been fully characterized. Regulatory influences, stimulatory or inhibitory, may have numerous sources. Further work, is needed to examine the components of the system, and to identify those involved in stem cell regulation. One possible approach is to isolate pure populations of adherent marrow cells and determine the ability of such cells to maintain and regulate hemopoiesis. Another approach is to undertake biochemical analysis of spent 1 9 4 growth medium from such cells to identify and characterize factors that may be released by them. Finally, one must consider the ultimate goal of a l l medical research - the application of new information to the treatment, and perhaps the cure, of disease. If abnormalities in cycling behaviour are a significant factor in the pathogenesis of the MPD, then elucidation of the underlying mechanism may eventually lead to the development of techniques by which hemopoietic cells in these diseases could be made responsive to normal regulatory influences. Johanne D. Cashman PUBLICATIONS 1. Cashman JD, Henkelman D, Eaves CJ & Eaves AC. Individual BFU-E in polycythemia vera produce erythropoietin dependent and independent progeny. Blood 61: 876-884, 1983. 2. Eaves C, Coulombel L, Dube I, Kalousek D, Cashman J & Eaves A. Behavior of human leukemic progenitor populations in long-term marrow culture. In: "Modern Trends in Human Leukemia VI", (eds Neth, Gallow, Greaves & Janko), Springer-Verlag, Heidelberg, pp 163-167, 1985. 3. Eaves C, Coulombel L, Dube I, Kalousek D, Cashman J & Eaves A. Maintenance of normal and abnormal hemopoietic cell populations in long- term marrow culture. In: "Hematopoietic Stem Cell Physiology", (eds. EP Cronkite, N Dainiak, RP McCaffrey, J Palek & PJ Quesenberry), Alan R Liss, Inc, New York, pp 403-413, 1985. 4. Cashman J, Eaves AC & Eaves CJ. Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66: 1002-1005, 1985. 5. Eaves C, Coulombel L, Dube I, Kalousek D, Cashman J & Eaves A. Behavior of human leukemic progenitor populations in long-term marrow culture. In: "Modern Trends in Human Leukemia VI", (eds. Neth, Gallo, Greaves & Janka), Springer-Verlag, Heidelberg, pp 163-167, 1985. 6. Eaves AC, Cashman JD & Eaves CJ. Polycythemia vera: In vitro analysis of regulatory defects. In: "Proceedings, Humoral and Cellular Regulation of Erythropoiesis", (eds. E Zanjani & M Tavassoli), Spectrum Publications, Inc, Jamaica, New York (in press). ABSTRACTS 1. Charbonneau J, Finkelstein S. A survey of the drug education needs among health professionals. Research on Drug Abuse Handbook, Department of Health and Welfare Publications, 1975. 2. Bressler BH & Charbonneau JD. Isometric contractile properties of amphibian skeletal muscle in temperature range 0° to 20°C. Biophys J 25: 11A, 1979. 3. Charbonneau JD & Bressler BH. Isometric contractile properties of amphibian skeletal muscle in solutions of decreased toxicity. Can Fed Biol Soc 22: 389, 1979. 4. Cashman J, Eaves C & Eaves A. In vitro studies of the erythropoietin requirements of erythroid progenitor cells in polycythemia vera (PV). Can Fed Biol Soc 24: 150, 1981. 5. Eaves AC, Cashman JD & Eaves CJ. In vitro studies of the erythropoietin requirements of erythroid progenitor cells in polycythemia vera (PV). Blood 58 (Suppl 1): 96a, 1981. Johanne D. Cashman 6. Eaves A, Cashman J, Henkelman D & Eaves C. Variable expression of Ep- independence by progeny of single BFU-E in polycythemia vera. Stem Cells 2: 359, 1982. 7. Eaves AC, Cashman J, Coupland R & Eaves CJ. Erythropoietin-independence and altered proliferative status of early erythropoietic and granulopoietic progenitor cell populations in essential thrombocytosis. Blood 62 (Suppl 1); 169a, 1983. 8. Cashman JD, Eaves AC & Eaves CJ. Cyclic variation in the proliferative status of primitive hemopoietic progenitor populations characteristic of the adherent layer of normal long-term human marrow cultures. Blood 64 (Suppl 1): 112a, 1984. 9. Eaves AC, Kalousek DK, Cashman JD & Eaves CJ. Extended maintenance of proliferating Phi chromosome positive (Phl+) progenitors following addition of CML peripheral blood but not marrow cells to pre-established normal marrow adherent layers. Blood 64 (Suppl 1): 188a, 1984. 10. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK & Eaves CJ. Abnormal cycling behaviour of CML progenitors in vivo reproduced in long-term culture. Clin Res 33: 338A, 1985.

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