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Genetic investigations of human hemopoiesis : studies of clonality and gene transfer to hemopoietic progenitors Hogge, Donna Eileen 1987

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GENETIC INVESTIGATIONS OF HUMAN HEMOPOIESIS: STUDIES OF CLONALITY AND GENE TRANSFER TO HEMOPOIETIC PROGENITORS by DONNA EILEEN HOGGE B .Med .Sc . , The U n i v e r s i t y of A l b e r t a , 1971 M.D. , The U n i v e r s i t y of A l b e r t a , 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Patho logy) We accept t h i s t hes i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA May 1987 © Donna Hogge, 1987 In p resen t i ng this thesis in part ial fu l f i lment of the requ i remen ts for an a d v a n c e d d e g r e e at the Univers i ty of Bri t ish C o l u m b i a , I agree that the Library shall m a k e it freely avai lable for re ference and s tudy. I fur ther agree that pe rm iss ion for ex tens ive c o p y i n g o f this thesis for scho lar ly p u r p o s e s may be gran ted by the h e a d of m y depa r tmen t o r by his o r her representa t ives . It is u n d e r s t o o d that c o p y i n g o r pub l i ca t ion of this thesis for f inanc ia l gain shall no t b e a l l o w e d w i thou t m y wr i t ten pe rm iss ion . D e p a r t m e n t of T h e Un ivers i ty of Brit ish C o l u m b i a 1956 M a i n M a l l V a n c o u v e r , C a n a d a V 6 T 1Y3 DE-6(3 /81) i i ABSTRACT In most neoplasms mal ignant change occurs i n a s i n g l e c e l l which then p r o l i f e r a t e s . My purpose was to exp lo re methods to study the c e l l that g i v e s r i s e to hemopoiet ic cancer and to i n v e s t i g a t e the a b n o r m a l i t i e s at a mo lecu la r l e v e l . Cy togene t i c a n a l y s i s of c e l l s from i n d i v i d u a l hemopoiet ic c o l o n i e s revea led that monosomy 7 syndrome, a hematologic d i s o r d e r of c h i l d h o o d , a r i s e s i n a p r i m i t i v e c e l l capable of d i f f e r e n t i a t i n g down both myelo id and e r y t h r o i d pathways. Long-term bone marrow c u l t u r e s (LTC) from p a t i e n t s w i th ch ron i c myelogenous leukemia (CML) favor the growth of P h i l a d e l p h i a chromosome (Ph) nega t i ve p rogen i t o r s wh ich , a l though c y t o g e n e t i c a l l y normal , cou ld have been par t of the mal ignant c lone at a stage p r i o r to the development of the Ph . LTC ' s were i n i t i a t e d w i th c e l l s from 2 women w i th CML who were heterozygous f o r 2 e l e c t r o p h o r e t i c a l l y d i s t i n c t g lucose-6-phosphate dehydrogenase (G6PD) enzyme v a r i a n t s . In one p a t i e n t , 2/11 p rogen i to rs were Ph-nega t i ve a f t e r 4 to 6 weeks i n LTC and 4/30 were nonc lona l by G6PD enzyme a n a l y s i s , i . e . the c o l o n i e s expressed the enzyme not found i n the mal ignant c l o n e . In t h i s c a s e , k a r y o t y p i c a l l y normal c e l l s were t r u l y normal . Nex t , gene t r a n s f e r to human hemopoiet ic c e l l s was demonstrated u s i n g recombinant r e t r o v i r u s c a r r y i n g the s e l e c t a b l e marker gene, n e o r . With the K562 human leukemic c e l l l i n e as ta rge ts up to 60% of i n f e c t e d c e l l s became G418 r e s i s t a n t ( G 4 1 8 r ) . Cloned popu la t ions of G418 r c e l l s showed unique pa t t e rns of r e t r o v i r a l i n t e g r a t i o n i n K562 DNA. When the ta rget c e l l s were p rogen i t o r s from normal marrow, CML blood or f e t a l l i v e r , the h ighes t f r equenc ies of G418 r granulocyte-macrophage or l a rge e r y t h r o i d c o l o n i e s was 16% and 5% r e s p e c t i v e l y . i i i Experiments infecting bone marrow cells in LTC with neor virus produced up to 2% G418r colonies after as long as 3 weeks in culture. Using v-src virus to infect LTC failed to perturb hemopoiesis, although infection of bone marrow-derived cells in these cultures was documented. In summary: 1. Unique populations of hemopoietic progenitors can be identified in culture using several genetic markers including chromosomes, G6PD analysis or gene transfer. 2. The feasibility of retroviral-mediated gene transfer for use on human hemopoietic cells has been demonstrated. i v TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS i x CHAPTER I INTRODUCTION 1) An Overview of Hemopoiesis 1 2) Markers of C l o n a l i t y 11 3) C l o n a l D iso rde rs of Hemopoiesis 17 4) Oncogenes 24 5) Gene T rans fe r 34 6) Present Ob jec t i ves 60 References 64 CHAPTER I I JUVENILE MONOSOMY 7 SYNDROME: EVIDENCE THAT THE DISEASE ORIGINATES IN A PLURIPOTENT HEMOPOIETIC STEM CELL 1) I n t r o d u c t i o n 75 2) P a t i e n t s 76 3) M a t e r i a l s and Methods 78 4) R e s u l t s 79 5) D i s c u s s i o n 84 References 87 CHAPTER I I I HEMOPOIETIC PROGENITORS THAT ARE NOT PART OF THE MALIGNANT CLONE REVEALED IN LONG-TERM MARROW CULTURES FROM A G6PD HETEROZYGOTE WITH CHRONIC MYELOGENOUS LEUKEMIA 1) I n t r o d u c t i o n 90 2) M a t e r i a l s and Methods 91 3) R e s u l t s 94 4) D i s c u s s i o n 97 References 100 CHAPTER IV GENE TRANSFER TO PRIMARY NORMAL AND MALIGNANT HUMAN HEMOPOIETIC PROGENITORS USING RECOMBINANT RETROVIRUSES 1) I n t r o d u c t i o n 102 2) M a t e r i a l s and Methods 103 3) R e s u l t s 109 4) D i s c u s s i o n 124 References 128 V CHAPTER V INFECTION OF HUMAN LONG-TERM CULTURES WITH RECOMBINANT RETROVIRUSES: PRELIMINARY EXPERIMENTS 1) I n t r o d u c t i o n 131 2) M a t e r i a l s and Methods 132 3) R e s u l t s 136 4) D i s c u s s i o n 148 References 153 CHAPTER VI SUMMARY AND CONCLUSIONS 156 APPENDIX I LIST OF ABBREVIATIONS 166 APPENDIX I I SELECTED CYTOGENETIC NOMENCLATURE 170 APPENDIX I I I METHOD FOR ESTABLISHMENT AND MAINTENANCE OF HUMAN 173 LONG-TERM CULTURES APPENDIX IV METHOD FOR HEMOPOIETIC COLONY ASSAYS IN 175 METHYLCELLULOSE CULTURE APPENDIX V METHOD FOR CYTOGENETIC ANALYSIS OF METAPHASES FROM 177 CELLS IN INDIVIDUAL HEMOPOIETIC COLONIES v i LIST OF TABLES Page TABLE I C l a s s i f i c a t i o n of the Acute Nonlymphoblastic 21 Leukemias TABLE I I De Novo Acute Nonlymphocytic Leukemia i n Adults 23 with S p e c i f i c Chromosome Defects TABLE I I I Oncogenes TABLE IV The Propor t i o n of Target C e l l s Showing Expression 38 of the Transferred Gene Following Various Gene Transfer Techniques TABLE V D i r e c t Cytogenetic Studies 77 TABLE VI Bone Marrow Culture - Progenitor Numbers 80 TABLE VII P e r i p h e r a l Blood Progenitor Numbers From P a t i e n t 1 81 TABLE V I I I Bone Marrow Culture - Hemopoietic Colonies 83 Cytogenetics TABLE IX Ra t i o of G6PD Isoenzyme Va r i a n t s i n Lysates of 95 P e r i p h e r a l Blood and Skin F i b r o b l a s t C e l l s TABLE X Hemopoietic Colony Cytogenetic and G6PD A n a l y s i s 96 TABLE XI T i t e r s of v-neo r From Producer C e l l Lines and pZipNeo 110 TABLE XII Maximum Frequency of G418 r 1° Hemopoietic Colonies 119 a f t e r C o - C u l t i v a t i o n with Various v-neo r Producer C e l l s TABLE X I I I Demonstration of v-neo r Production i n G418 r CFU-GM 122 by I n f e c t i o u s Center Assay TABLE XIV Propor t i o n of G418 Res i s t a n t Granulocyte-Macrophage 137 Colonies from Nonadherent C e l l s i n Human Long-Term Marrow Cultures Infected with Helper-Containing v-neo r TABLE XV v-neo r T i t e r s i n Medium From Infected Long-Term 139 Cultures (LTC) TABLE XVI I n f e c t i o u s Center Assays: G418 r CFU-GM From v-neo r 140 Infected Long-Term Marrow Cultures TABLE XVII Transforming V i r a l Assays From v-src Infected Human 143 C e l l s v i i LIST OF FIGURES Page FIGURE 1. O u t l i n e of Hemopoiesis as Defined by Colony Assays 5 of Stem C e l l s and Committed P r o g e n i t o r s . FIGURE 2 . C l o n a l O r i g i n of Human Hematopoiet ic C e l l s i n 12 Chronic Myelogenous Leukemia. FIGURE 3. Chromosomal Rearrangement i n CML. 18 FIGURE 4. Giemsa-Banded Human Chromosome Map w i t h 21 Oncogenes 30 (Do t ) , 6 C e l l u l a r Genes ( T r i a n g l e ) , and Breakpoin t s (Arrows) for Chromosomal Rearrangements Found i n Cancer. FIGURE 5. Chromosomal Rearrangement i n B u r k i t t ' s Lymphoma. 31 FIGURE 6. S t ruc tu re of Moloney Murine Leukemia V i r u s R e t r o v i r a l 41 P r o v i r u s DNA. FIGURE 7. R e t r o v i r a l L i f e C y c l e . 44 FIGURE 8. C r i t i c a l Steps i n the Synthes is of R e t r o v i r a l DNA. 46 FIGURE 9. C o n s t r u c t i o n of a Recombinant R e t r o v i r u s and 54 Genera t ion of V i r a l Stocks for Gene Transfer Exper iments . FIGURE 10. C o n s t r u c t i o n of a T~~ Amphotropic R e t r o v i r u s . 56 FIGURE 11. I n f e c t i o n of Human Hemopoietic C e l l s w i t h v - n e o r 106 FIGURE 12. G418 r K.562 Colon ies {% of Co lon ie s Grown wi thout 112 G418) a f t e r I n f e c t i o n of 1 0 5 K562 C e l l s w i t h Var ious Sources of v - n e o r . FIGURE 13. G418 r K562 Co lon ie s (X of Co lon ie s Grown without 114 G418) a f t e r I n f e c t i o n of 105 C e l l s w i t h v - n e o r from 3 ml Und i lu t ed Supernatant or a 60 mm Dish of Conf luen t , I r r a d i a t e d V i r a l Producer C e l l s of Var ious Types. FIGURE 14. Southern B l o t s of T o t a l C e l l u l a r DNA H y b r i d i z e d w i t h 115 a 32p_L a b e ]_ e cl neo r S p e c i f i c Bam H l - H i n d I I I Fragment from pRSV-neo. FIGURE 15. Frequency (%) of G418 r Primary Hemopoietic Co lon ie s 117 (G418 r C o l o n i e s / T o t a l Co lon ies without G418 x 100) a f t e r 24 Hours Exposure of 5 x 10^ C e l l s to a 60 mm Dish of I r r a d i a t e d , Confluent v - n e o r Producer C e l l L ines at Var ious V i r a l T i t e r s . v i i i FIGURE 16. Frequency (%) of G418 r Pr imary Hemopoiet ic P rogen i t o r s 120 (G418 r C o l o n i e s / T o t a l Co lon ies wi thout G418 x 100) a f t e r I n f e c t i o n of 5 x lO^ C e l l s by v - n e o r from a 60 mm Dish of I r r a d i a t e d Conf luent Producer C e l l s or 5 ml Und i lu ted Supernatant of Var ious Types. FIGURE 17. RNA Spot B l o t of T o t a l C e l l u l a r RNA from Pooled CML 123 Granulocyte-Macrophage Co lon ies or K562 C e l l s Hyb r i d i zed w i th a 32p_L a beled n e o r S p e c i f i c Bam HI-Hind I I I Fragment from pRSV-neo. FIGURE 18. T o t a l Nonadherent C e l l s per Long-Term Cu l t u re 144 (Pane l a) and Progen i to r Numbers per 10^ Nonadherent C e l l s (Panels b and c) i n Cu l t u res I n i t i a t e d w i th P e r i p h e r a l Blood C e l l s from a Pa t i en t w i th CML on Normal Bone Marrow F i b r o b l a s t Feeders . FIGURE 19. T o t a l Nonadherent C e l l s per Long-Term Bone Marrow C u l t u r e from a Pa t i en t w i th Mye lodysp las ia or Pre leukemia . 146 FIGURE 20. T o t a l Nonadherent C e l l s per Long-Term Bone Marrow C u l t u r e (Pane l a) or Granulocyte/Macrophage Colony-Forming C e l l s (CFU-C) per 1 0 5 Nonadherent C e l l s (Pane l b) from a Pa t i en t w i th Mye lodysp las ia or P re leukemia . 147 ACKNOWLEDGEMENTS I wish to express my sincere appreciation: To my supervisor, Dr. R.K. Humphries, and Dr. C.J. Eaves for their support and guidance throughout my research; To members of my advisory Committee, Drs. P.A. Baird, A.C. Eaves, D.K. Kalousek. and W.L. Dunn for their helpful suggestions; To Drs. L. Coulombel and K. Shannon for s c i e n t i f i c collaboration and help in obtaining patient samples suitable for these studies; To Gloria Shaw, Marjorie Hutchison, Dianne Reid, Darlene Nipius and Sheryle Taylor for expert technical assistance; To Susan Hayley and Michele Coulombe for s e c r e t a r i a l assistance during preparation of the manuscript; To the National Cancer Institute of Canada for f i n a n c i a l support. C H A P T E R I 1 INTRODUCTION 1) AN OVERVIEW OF HEMOPOIESIS The maintenance of hemopoiesis i s f e l t to be the result of the behavior of p r i m i t i v e hemopoietic stem c e l l s . These c e l l s r e t a i n c apacities for both self-renewal and d i f f e r e n t i a t i o n along a pathway committed to the development of a p a r t i c u l a r lineage of blood c e l l s . The property of self-renewal allows hemopoiesis to be maintained throughout the l i f e t i m e of an animal. This occurs although functional blood c e l l s , including granulocytes, monocytes, red blood c e l l s , p l a t e l e t s and most lymphocytes which are the ultimate products of stem c e l l commitment and d i f f e r e n t i a t i o n , are r e l a t i v e l y s h o r t - l i v e d . Current evidence favours the existence of a hierarchy among hemopoietic progenitors along each committed pathway. Each successive l e v e l exhibits reduced p r o l i f e r a t i v e and l i t t l e or no self-renewal capacities as i t d i f f e r e n t i a t e s toward functional blood c e l l s . The regulation of this complex process of hemopoiesis and the precise factors which determine stem c e l l commitment vs self-renewal are subjects of intense interest and in v e s t i g a t i o n but have not been completely elucidated. Major discoveries which have formed our current understanding of hemopoietic stem c e l l and growth factor physiology w i l l be described i n the following section. Hemopoietic progenitors are extremely rare c e l l types even i n blood-forming organs such as the bone marrow (less than one i n one thousand nucleated c e l l s ) . In addition, they cannot be i d e n t i f i e d morphologically. 2 These two f ac to r s have made i t necessary to use i n d i r e c t methods to s tudy stem c e l l behav io r . In 1961 T i l l and McCul loch (1) i n j e c t e d murine bone marrow c e l l s i n t o l e t h a l l y i r r a d i a t e d syngeneic mice. D i s c r e t e nodules of hemopoiet ic c e l l s formed on the r e c i p i e n t a n i m a l ' s sp leen 8 to 10 days a f t e r i n j e c t i o n . These nodules conta ined e i t h e r pure popula t ions or va r i ous combinat ions of e r y t h r o i d , g r a n u l o c y t i c , megakaryocytic or u n d i f f e r e n t i a t e d c e l l s ( 2 ) . Each of these nodules was shown to represent a c lone de r i ved from a s i n g l e p r i m i t i v e c e l l by i n j e c t i n g c e l l s w i t h s p e c i f i c s t a b l e chromosomal a b n o r m a l i t i e s i n t o i r r a d i a t e d , c y t o g e n e t i c a l l y normal r e c i p i e n t s . The abnormal karyotype was seen i n more than 95% of metaphase c e l l s i n some c o l o n i e s ( 3 ) . I f a suspension of c e l l s de r ived from a s i n g l e sp leen colony i s i n j e c t e d i n t o an i r r a d i a t e d mouse new spleen c o l o n i e s of approximate ly the same s i z e are seen to form ten to fourteen days l a t e r ( 4 ) . The format ion of these secondary sp leen c o l o n i e s i s evidence of the s e l f - r e n e w a l c a p a b i l i t y of the c e l l s from which the c o l o n i e s are d e r i v e d . (These c e l l s were c a l l e d CFU-S fo r sp leen colony forming u n i t s ) . I f c e l l s from a spleen colony c o n t a i n i n g a pure p o p u l a t i o n of c e l l s , such as g ranulocy tes or e r y t h r o c y t e s , were i n j e c t e d i n t o an i r r a d i a t e d r e c i p i e n t c o l o n i e s would form which conta ined s e v e r a l c e l l types ( 5 , 6 ) . Thus, one marrow c e l l , CFU-S, i s capable of forming a colony c o n t a i n i n g three myeloid c e l l types . Th i s same c e l l , i n a d d i t i o n to i t s a b i l i t y to p r o l i f e r a t e and d i f f e r e n t i a t e , can undergo s e l f - r e n e w a l . Fur the r s t u d i e s i n t o the stem c e l l h i e r a r chy have provided evidence fo r the ex i s t ence of a p l u r i p o t e n t stem c e l l capable of g i v i n g r i s e to lymphoid as w e l l as myeloid c e l l s ( 7 ) . More recent work us ing unique s i t e s of r e t r o v i r a l i n s e r t i o n i n t o hemopoiet ic c e l l DNA as markers of c l o n a l i t y have shown that the same stem c e l l can repopulate both thymus and bone marrow. The p r o l i f e r a t i v e c a p a c i t y of these c e l l s was cons ide rab le as a s i n g l e c lone was 3 of ten s u f f i c i e n t to repopulate an e n t i r e animal ( 8 , 9 ) . In human be ings , s tudy of g lucose 6 phosphate dehydrogenase (G6PD) heterozygotes w i t h c l o n a l d i s o r d e r s of hemopoiesis have a l so demonstrated a common c e l l of o r i g i n fo r lymphoid and myeloid c e l l s (10) . In v i t r o colony assays have been developed which permit the growth and development of p rogen i to r s which g ive r i s e to a l l types of mature blood c e l l s . Thus, r a re p r i m i t i v e c e l l s are. recognized by t h e i r d i f f e r e n t i a t e d progeny. C e l l s are suspended i n v i s c i d ( m e t h y l c e l l u l o s e ) or s e m i s o l i d (agar) media which p a r t i a l l y immobi l i zes the d i v i d i n g c e l l s so that the o f f s p r i n g of a s i n g l e p rogen i to r form a c l u s t e r or co lony . The concen t r a t i on of c e l l s p l a t e d i n each d i s h i s chosen so that c o l o n i e s are w ide ly d i spe r sed and can be recognized as separate e n t i t i e s and counted. The type of colony growth that i s favoured by a p a r t i c u l a r assay depends on the a d d i t i o n of va r i ous s t i m u l a t o r y f a c t o r s . Colony-forming u n i t s i n c u l t u r e (CFU-C) or g r a n u l o c y t e -macrophage colony forming u n i t s (CFU-GM) are descendants of CFU-S but are committed to granulocyte-macrophage l ineages and have l i m i t e d p r o l i f e r a t i v e c a p a c i t y ( 7 ) . E r y t h r o i d burs t forming u n i t s (BFU-E) are the p r i m i t i v e e r y t h r o i d counterpar t s of CFU-C and appear to be c l o s e l y r e l a t e d to CFU-S. BFU-E form l a r g e , hemoglobinized e r y t h r o i d c o l o n i e s composed of 3 or more c l u s t e r s of c e l l s i n the presence of h igh concen t ra t ions of e r y t h r o p o i e t i n . E r y t h r o i d colony forming u n i t s (CFU-E) form s m a l l c l u s t e r s of 8 to 32 e r y t h r o c y t e s and are l oca t ed l a t e i n e r y t h r o i d maturat ion (11) . Megakaryocyte colony forming u n i t s (CFU-Meg) have been desc r ibed which appear to be the megakaryocyte counterpar t s of CFU-C and BFU-E. In a d d i t i o n to p rogen i to r s of r e s t r i c t e d l i neage assays have been developed which i d e n t i f y p l u r i p o t e n t p r i m i t i v e c e l l s , (CFU-GEMM) which g ive r i s e to c o l o n i e s c o n t a i n i n g a mixed p o p u l a t i o n of g r anu locy t e s , e r y t h r o c y t e s , monocytes and megakaryocytes (12 ) . 4 In g e n e r a l , the more p r i m i t i v e p rogen i to rs g i ve r i s e to l a r g e r c o l o n i e s which r e q u i r e 2 to 3 weeks i n c u l t u r e and many c e l l d i v i s i o n s to produce t e r m i n a l l y d i f f e r e n t i a t e d b lood c e l l progeny. The more mature p r o g e n i t o r s , e . g . CFU-E, develop w i t h i n a week i n v i t r o and a f t e r on ly a few c e l l d i v i s i o n s produce s m a l l c o l o n i e s of mature c e l l s (F igu re 1 ) . The mechanism by which stem c e l l s are regu la ted and the way i n which cho i ce between s e l f - r e n e w a l and d i f f e r e n t i a t i o n i n t o one of the v a r i o u s types of committed p rogen i to rs i s made has not been e l u c i d a t e d . However, at l e a s t three models have been proposed* The f i r s t , known as the s t o c h a s t i c model proposed by T i l l et a l i n 1964 (13) ho lds that d i f f e r e n t i a t i o n a long any l i n e a g e i s a random event w i th a de f ined chance of o c c u r r i n g w i th each c e l l d i v i s i o n . Two other models s t a t e that stem c e l l de te rmina t ion i s dependent on e i t h e r the s p e c i f i c microenvironment sur round ing the c e l l s (14) or s o l u b l e humoral f a c t o r s (15 ) . S tud ies on the s e l f - r e n e w a l of hemopoiet ic stem c e l l s du r i ng mixed co lony format ion i n v i t r o suggest that the cho ice to s e l f -r e p l i c a t e or not i s determined by a mechanism that i s at l e a s t p a r t l y random and i n t r i n s i c to the c e l l i t s e l f (16 ) . Recent work has i d e n t i f i e d a unique type of co lony c o n s i s t i n g of p r i m i t i v e hemopoiet ic p rogen i to rs (17 ) . The study of these b l a s t c e l l c o l o n i e s has prov ided f u r t h e r ev idence f o r the s t o c h a s t i c model of s e l f - r e n e w a l and stem c e l l d i f f e r e n t i a t i o n . P a i r e d daughter c e l l s of u n i c e l l u l a r o r i g i n from b l a s t c e l l c o l o n i e s have been shown to generate c o l o n i e s of d i v e r s e mu l t i and o l i g o l i neage combina t ions . Each c e l l d i v i s i o n of p rogen i to rs may y i e l d p rogen i to rs w i th d i s s i m i l a r l i n e a g e p o t e n t i a l s . The l o s s of l i neage p o t e n t i a l does not appear to proceed i n a de f i ned sequence but i s a random p rocess . Va r ious substances have been found to be necessary to support the growth of hemopoiet ic c e l l s i n v i t r o and many of these may a l s o be important i n v i v o . A number of serum components are necessary f o r the growth of mammalian U L T I M A T E S T E M C E L L * Lymphopoiesis E R Y T H R O I D PROGENITORS Primitive BFU-E large erythroid colony or burst Mature BFU-E small erythroid colony or burst CFUE erythroid cluster selt-renewal M Y E L O I D S T E M C E L L (CFU-S, CFU-GEMM) mixed colony M E G A K A R Y O C Y T E P R O G E N I T O R S CFUM large megakaryocytic colony small megakaryocytic colony G R A N U L O C Y T E P R O G E N I T O R S CFU-C large granulocytic colony small granulocytic colony RED CELLS PLATELETS GRANULOCYTES 4 MACROPHAGES FIGURE 1. O u t l i n e of Hemopoiesis as Defined by Colony Assays of Stem C e l l s and Committed P r o g e n i t o r s . 6 c e l l s i n c u l t u r e . These i nc lude t race elements and n u t r i e n t s , va r i ous p r o t e i n s and endocrine hormones such as i n s u l i n , t h y r o i d hormones and s t e r o i d s . E r y t h r o p o i e t i n , another endocrine hormone, and c e r t a i n non-endocr ine growth f ac to r s are s p e c i f i c requirements for the growth of c e r t a i n hemopoiet ic c e l l s i n v i t r o (18) . The most convenient sources for en r iched concen t r a t ions of these l a t t e r p r o t e i n products a re ; a) medium cond i t i oned by the presence of white blood c e l l s that have been s t imu la t ed by agents such as phytohemagglu t in in , or b) cond i t ioned media from c e r t a i n immortal c e l l l i n e s . Leukocyte cond i t i oned medium, i n p a r t i c u l a r , conta ins a number of hemopoie t ic growth f a c t o r s s e v e r a l of which have been p u r i f i e d to homogeneity and the genes encoding t h e i r p r o t e i n sequence c loned . A b r i e f review of f a c t o r s f e l t to be important i n r e g u l a t i n g human hemopoiesis f o l l o w s . A. I n t e r l e u k i n 3 ( I L - 3 ) Both the murine and the human I L - 3 genes have been cloned and sequenced ( 1 9 , 2 0 ) . The p ro t e in s are g l y c o s y l a t e d and there i s r e l a t i v e l y l i t t l e homology between the mouse and human DNA sequences. The a c t i v i t i e s of I L - 3 on bone marrow c e l l s i n v i t r o i n c l u d e a pe rmiss ive r o l e for p r o l i f e r a t i o n of e a r l y m u l t i p o t e n t i a l p r o g e n i t o r s , i n c r e a s i n g the number of c e l l s capable of responding to e r y t h r o p o i e t i n , and promoting the growth and d i f f e r e n t i a t i o n of g ranu locy tes and macrophages and mast c e l l s . Thus, I L - 3 induces 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 of a r e l a t i v e l y e a r l y stem c e l l that can d i f f e r e n t i a t e to a v a r i e t y of c e l l types . B . Granulocyte-Macrophage S t i m u l a t i n g Fac tor (GM-CSF) Granulocyte/macrophage colony formation i n v i t r o r equ i r e s the c o n t i n u a l presence of a second g l y c o p r o t e i n produced by a c t i v a t e d T c e l l s and o ther c e l l 7 types , GM-CSF. Both the murine and human forms of t h i s p r o t e i n have been p u r i f i e d , c loned and sequenced (21 ,22 ) . The two molecules show s u b s t a n t i a l sequence homology at the n u c l e o t i d e (69%) and amino a c i d (54%) l e v e l s (23 ) . In s p i t e of t h i s s i m i l a r i t y , the murine and human GM-CSF's show no cross spec ies a c t i v i t y i n b i o l o g i c a l assays . The a v a i l a b i l i t y of recombinant human GM-CSF has a l lowed the i d e n t i f i c a t i o n of a c t i v i t i e s of t h i s f a c t o r not p r e v i o u s l y apprec ia ted (24) . I t s t imu la t e s the growth of BFU-E de r i ved e r y t h r o i d c o l o n i e s and mixed l i neage c o l o n i e s de r ived from CFU-GEMM as w e l l as granulocyte/macrophage c o l o n i e s . Human GM-CSF a l s o func t ions as a n e u t r o p h i l a c t i v a t o r as shown by i t s i n d u c t i o n of g ranu locy te superoxide p roduc t ion and i n h i b i t i o n of n e u t r o p h i l m o b i l i t y (25) . C. Granulocyte Colony S t i m u l a t i n g Fac tor (G-CSF) A t h i r d colony s t i m u l a t i n g f ac to r G-CSF s t i m u l a t e s the growth of n e u t r o p h i l c o l o n i e s and the t e rmina l d i f f e r e n t i a t i o n and f u n c t i o n of both normal and malignant g ranulocy tes i n v i t r o . L i k e GM-CSF, G-CSF i s ab le to support the growth and maturat ion of m u l t i l i n e a g e and e r y t h r o i d c o l o n i e s sugges t ing a c t i o n at m u l t i p l e l e v e l s of hemopoiet ic p rogen i to r c e l l d i f f e r e n t i a t i o n (26) . The human f a c t o r shows spec ies c r o s s - r e a c t i v i t y as i t w i l l s t i m u l a t e the growth of murine g ranu locy te c o l o n i e s . Both the murine and human forms of t h i s p r o t e i n have been p u r i f i e d and the genes c loned ( 2 7 , 2 8 ) . D. Macrophage Colony S t i m u l a t i n g Fac to r (M-CSF) A fou r th f a c t o r , M-CSF or CSF-1 , has a l s o been h i g h l y p u r i f i e d and the gene for the human f a c t o r c loned (29) . I t s t imu la t e s the growth and d i f f e r e n t i a t i o n , and func t ion of monocytes and macrophages and shows spec ies c ross r e a c t i v i t y . 8 E . E r y t h r o p o i e t i n A f i f t h f a c t o r which i s c r i t i c a l to i n v i t r o hemopoiet ic c e l l development i s e r y t h r o p o i e t i n . Th i s i s the only known hemopoiet ic growth f a c t o r which i s a t rue endocr ine hormone. I t i s produced by r e n a l tubu la r c e l l s i n response to t i s s u e hypoxia and l i k e other hemopoiet ic f ac to r s i s a g l y c o p r o t e i n (30 ) . The murine and human genes for e r y t h r o p o i e t i n have a l s o been c loned (31,32) and l a r g e q u a n t i t i e s of recombinant hormone are a v a i l a b l e . The predominant a c t i o n of e r y t h r o p o i e t i n i s to induce t e rmina l d i f f e r e n t i a t i o n of l a t e committed e r y t h r o i d p r o g e n i t o r s , no tab ly CFU-E (30) . F . I n t e r l e u k i n 2 ( I L - 2 ) Human I L - 2 t r i g g e r s the p r o l i f e r a t i o n of both mouse and human a c t i v a t e d T lymphocytes and may a l s o s t i m u l a t e B c e l l s v i a s p e c i f i c recep tors (33 ) . A l l o f the preceding 6 hemopoiet ic f ac to r s appear to act i n i t i a l l y by b i n d i n g to h igh a f f i n i t y c e l l surface receptors on respons ive c e l l s . The number of recep tors i s r e l a t i v e l y low on normal c e l l s ( l e s s than 1 0 0 0 / c e l l ) and the f ac to r s have t h e i r maximum a c t i v i t y at extremely low (pM) c o n c e n t r a t i o n s . The molecular mechanisms by which any of these molecules induce changes i n target c e l l s a f t e r receptor b i n d i n g i s unknown. The a v a i l a b i l i t y of l a r g e q u a n t i t i e s of recombinant hemopoiet ic growth f a c t o r s has a l lowed i n v i v o s tud i e s of t h e i r a c t i v i t y to be done. Recombinant e r y t h r o p o i e t i n has been shown to co r r ec t the anemia of ch ron ic r e n a l f a i l u r e i n human beings (34) . Human GM-CSF and G-CSF cause a dramatic r i s e i n g ranu locy te s i n cy topen ic monkeys (35 ,36) . GM-CSF causes a r i s e i n p l a t e l e t s and r e t i c u l o c y t e s as w e l l . Thus, i t appears that f ac to r s that have for some years been known to be e s s e n t i a l for i n v i t r o hemopoiet ic c e l l growth have important r o l e s i n v i v o as w e l l . 9 G. Hemopoiet ic Microenvironment Much of the r e g u l a t i o n of hemopoiesis i n v i v o probably occurs at a l o c a l l e v e l . Th i s i s suggested by the fact that hemopoiesis normal ly i s conf ined to the bone marrow i n adu l t humans and the bone marrow and sp leen i n adu l t roden t s . I t would seem l i k e l y that the s p e c i f i c composi t ion of these organs a l l o w s hemopoiesis to occur and the substances necessary to support hemopoiesis are not present i n other par ts of the body. The SlVsid mouse p rov ides a model i n which severe anemia and a decreased p roduc t ion i n a l l l i n e s of hemopoiet ic c e l l s i s the r e s u l t of a d e f e c t i v e hemopoiet ic microenvironment (37) . Much work has been done to study va r ious f a c t o r s of p o t e n t i a l importance i n the l o c a l c o n t r o l of hemopoiesis . Recen t ly c e l l s of the type present w i t h i n the bone marrow microenvironment have been shown to produce some of the growth f ac to r s to which hemopoiet ic p rogen i to r s respond. For example, a c t i v a t e d macrophages w i l l produce i n t e r l e u k i n 1 ( I L - 1 ) which then s t i m u l a t e s f i b r o b l a s t s and e n d o t h e l i a l c e l l s to make both GM and G-CSF (38 ) . Other substances such as tumor nec ros i s f ac to r w i l l a l s o induce hemopoiet ic growth f a c t o r p roduc t ion from mesenchymal c e l l types that are prominent w i t h i n the bone marrow microenvironment (39) . Other s t u d i e s have i n v e s t i g a t e d the p o t e n t i a l r o l e of the e x t r a c e l l u l a r mat r ix and d i r e c t c e l l u l a r i n t e r a c t i o n s i n r e g u l a t i n g hemopoiesis . The long term bone marrow c u l t u r e o r i g i n a l l y developed by Dexter (40) and modif ied for human s t u d i e s by Greenberger (41) and others (42) a l l ows one to i n v e s t i g a t e c e l l u l a r and s t r o m a l - c e l l u l a r i n t e r a c t i o n that take p lace i n the hemopoiet ic microenvironment . In t h i s system bone marrow c e l l s i n appropr i a t e n u t r i e n t medium are p laced i n a t i s s u e c u l t u r e d i s h . Over a per iod of s e v e r a l weeks a l a y e r of c e l l s adherent to the c u l t u r e d i s h forms which c o n s i s t s of f i b r o b l a s t s , e n d o t h e l i a l c e l l s , ad ipocytes and a v a r i e t y of hemopoiet ic c e l l s 10 i n c l u d i n g the ma jo r i ty of p r i m i t i v e p r o g e n i t o r s . The o r g a n i z a t i o n and compos i t ion of t h i s adherent l a y e r i n many ways resembles the bone marrow microenvironment i n v i v o . In the medium above t h i s adherent l a y e r i s a p o p u l a t i o n of nonadherent hemopoiet ic c e l l s which c o n s i s t s p r i m a r i l y of d i f f e r e n t i a t e d c e l l s but a l s o some p r o g e n i t o r s . C e l l s from t h i s nonadherent f r a c t i o n can be sampled p e r i o d i c a l l y and s tud ied for va r ious p r o p e r t i e s . Long term bone marrow c u l t u r e s e s t a b l i s h e d w i t h murine c e l l s w i l l ma in ta in hemopoiesis for 6 months or l onge r . C e l l s harvested from the nonadherent f r a c t i o n i n these c u l t u r e s are r i c h i n hemopoiet ic p rogen i to r s i n c l u d i n g CFU-S. Human long term marrow c u l t u r e s are l e s s s u c c e s s f u l i n that hemopoies is , a l though maintained for at l e a s t 10 weeks, d e c l i n e s s t e a d i l y as measured by both numbers of nonadherent c e l l s and p rogen i to r content w i t h i n both the adherent and nonadherent f r a c t i o n s . The composi t ion of the e x t r a c e l l u l a r ma t r ix i n the adherent l a y e r of long term c u l t u r e s has been ana lyzed and shown to con ta in va r ious types of c o l l a g e n , f i b r o n e c t i n , l a m i n i n and pro teoglycans as w e l l as other substances (43) . However, the r o l e of any of these substances i n suppor t ing hemopoiesis i s u n c l e a r . S i m i l a r l y , the r e l a t i v e importance of nonhemopoietic c e l l s i n the marrow environment i n hemopoiet ic r e g u l a t i o n i s unknown. In a d d i t i o n , the methods besides growth f a c t o r p roduc t ion by which these c e l l s may modulate hemopoiesis are s p e c u l a t i v e at t h i s t ime. Never the le s s , the long term marrow c u l t u r e p rov ides a convenient i n v i t r o system i n which to study va r ious aspects of hemopoiet ic r e g u l a t i o n . Thus, normal hemopoiesis i s a complex process which r equ i r e s an i n t r i c a t e network of r e g u l a t o r y c o n t r o l s . A number of i n v i t r o and i n v i v o model systems have been developed which have been used to e l u c i d a t e most of the cu r ren t knowledge i n t h i s a rea . 11 2) MARKERS OF CLONALITY Normal hemopoiesis i s p o l y c l o n a l . That i s , an organism con ta ins many hemopoiet ic stem c e l l s which through t h e i r p r o l i f e r a t i o n c o n t r i b u t e s to the poo l of d i f f e r e n t i a t e d blood c e l l s . Al though the u l t i m a t e p r o l i f e r a t i v e p o t e n t i a l of a s i n g l e stem c e l l i s unknown, i t has been demonstrated that a s i n g l e p l u r i p o t e n t c e l l i s capable of at l e a s t t empora r i ly r e p o p u l a t i n g the hemopoiet ic system of an i r r a d i a t e d mouse ( 8 , 9 ) . S i m i l a r i n fo rma t ion i s not a v a i l a b l e fo r human hemopoiesis . In con t ras t to the p o l y c l o n a l nature of normal hemopoiesis , hemopoiet ic neoplasms are almost always c l o n a l . The techniques which have been used to e s t a b l i s h these fac t s w i l l be d i scussed i n the f o l l o w i n g s e c t i o n . A . Chromosomal Markers R a d i a t i o n induced k a r y o t y p i c abno rma l i t i e s were used s e v e r a l decades ago to e s t a b l i s h the c l o n a l o r i g i n of spleen c o l o n i e s i n a host animal ( 3 ) . The d i s c o v e r y of unique chromosomal changes i n human mal ignancies was sugges t ive evidence of c l o n a l i t y i n d iseases such as CML (44) . F i n d i n g the same abnormal chromosome i n hemopoiet ic c e l l s of va r ious l ineages was the f i r s t i n d i c a t i o n that n e o p l a s t i c change i n CML o r i g i n a t e d i n a p l u r i p o t e n t hemopoiet ic c e l l (45) (F igu re 2 ) . The obse rva t ion of k a r y o t y p i c e v o l u t i o n i n acute phase CML and acute nonlymphoblas t ic leukemia (ANLL) where new chromosomal changes were t y p i c a l l y superimposed upon p r e v i o u s l y recognized abno rma l i t i e s as the d i sease progressed was fu r the r evidence that the cy togene t i c a b n o r m a l i t i e s represented unique c l o n a l markers (46 ,47 ) . Neve r the le s s , i t was r i g h t l y argued by some i n v e s t i g a t o r s that such k a r y o t y p i c a l t e r a t i o n s could be secondary changes o c c u r r i n g l a t e i n the e v o l u t i o n of a p a r t i c u l a r d isease and i n many c e l l s . In such a case chromosomal abnorma l i t i e s would not be r e l i a b l e i n d i c a t o r s of T CELL (? ) MEGAKARYOCYTE (+) FIGURE 2. Clonal Origin of Human Hematopoietic Cells in Chronic Myelogenous Leukemia. The schema is based on the results of isozyme and chromosome studies. A plus sign indicates that the cell is definitely involved in the leukemic clone, a question mark indicates that it is not clear whether the cell is involved in the leukemic clone. BFU-E denotes erythrocyte burst-forming unit; CFU-E, erythrocyte colony-forming unit; CFU-C, colony forming unit in culture; CFU-EO, eosinophil colony-forming unit; and CFU-MEG, megakaryocyte colony-forming unit. 13 c l o n a l i t y . In a d d i t i o n , there are many c o n d i t i o n s both benign and malignant i n which a n a l y s i s of c l o n a l i t y i s important but cy togene t i c markers are l a c k i n g . For these reasons i t was important to develop other techniques fo r i n v e s t i g a t i n g c l o n a l i t y . B . X-Chromosome I n a c t i v a t i o n Acco rd ing to the Lyon hypothes is (48) e a r l y i n embryogenesis one X chromosome i n each c e l l of a female organism becomes i n a c t i v a t e d . The genes on the a c t i v e X are the ones expressed i n a g iven c e l l and a l l the progeny of that c e l l . I f an organism i s heterozygous for a p a r t i c u l a r X - l i n k e d t r a i t some c e l l s w i l l express one phenotype for that t r a i t and other c e l l s the second phenotype. Because X chromosome i n a c t i v a t i o n occurs when the embryo i s composed of on ly a few c e l l s and because the de te rmina t ion of which X w i l l be i n a c t i v a t e d i s random the r a t i o of c e l l s which express each of the two phenotypes fo r a heterozygous t r a i t i s not always 50:50. In a m i n o r i t y of cases the r a t i o may be skewed towards one phenotype or another . The enzyme G6PD, which i s a component of the hexose monophosphate shunt, i s encoded on the X chromosome. The gene for t h i s enzyme i s h i g h l y polymorphic i n the human p o p u l a t i o n . Al though w e l l over 100 v a r i a n t s of G6PD have been d i s c o v e r e d , most of them are extremely r a r e . Some v a r i a n t s r e s u l t s i n a hemoly t i c anemia which i s exacerbated by o x i d a t i v e s t r e s s and others are c l i n i c a l l y s i l e n t . A number of these isoenzymes can be d i s t i n g u i s h e d by t h e i r e l e c t r o p h o r e t i c m o b i l i t y . These l a t t e r i n c l u d e G6PD-B which i s the most common, or "normal" isoenzyme i n human popula t ions and isoenzyme G6PD-A which i s a c l i n i c a l l y s i l e n t v a r i a n t found i n one t h i r d of the b lack popu la t ion i n the Uni ted Sta tes and pa r t s of A f r i c a . Another isoenzyme found i n one tenth of b l acks i s 14 G6PD-A - which has the same e l e c t r o p h o r e t i c m o b i l i t y as G6PD-A but causes a hemoly t i c anemia i n a f f ec t ed b lack males when they are exposed to o x i d a t i v e s t r e s s such as s u l f a or a n t i m a l a r i a l drugs (49) . One t h i r d of b l a c k women are heterozygous for G6PD-B and one of the A v a r i a n t s . Because of the phenomenon of X chromosome i n a c t i v a t i o n some of the c e l l s i n these women express on ly G6PD-B w h i l e some c e l l s express only G6PD-A. Because normal hemopoiesis i s p o l y c l o n a l both enzymes are expressed i n blood and bone marrow c e l l s of normal heterozygous i n d i v i d u a l s . S i m i l a r l y , both enzymes are seen i n s k i n f i b r o b l a s t s , or any other normal body t i s s u e . In c o n t r a s t , i f a neoplasm a r i s e s i n a s i n g l e c e l l one would expect that a l l the c e l l s i n that tumor would express a s i n g l e enzyme v a r i a n t i n G6PD heterozygotes w h i l e a neoplasm a r i s i n g i n many c e l l s would express both - isoenzymes (50) . G6PD a n a l y s i s has been used to demonstrate that the l a r g e ma jo r i t y of human mal ignancies are c l o n a l i n o r i g i n (50) . Th i s i nc ludes most hemopoie t ic neoplasms some of which t y p i c a l l y l a c k cy togene t i c markers, e . g . po lycy themia rubra vera (51) . In a d d i t i o n , c l o n a l i t y has been demonstrated i n some ins tances where malignancy was not o therwise apparent . For example, i n s e v e r a l cases of ANLL where c l i n i c a l l y normal hemopoiesis re turned a f t e r chemotherapy the p e r i p h e r a l blood c e l l s of the pa t i en t s who were G6PD heterozygotes cont inued to express only 1 isoenzyme, the same one that had been expressed by the leukemic b l a s t s (52) . Th i s f i n d i n g suggests e i t h e r that chemotherapy had merely re turned the pa t i en t from frank leukemia to a p re -leukemic malignant s t a t e or that normal hemopoiesis had regenerated from a s i n g l e p l u r i p o t e n t stem c e l l . G6PD a n a l y s i s of hemopoiet ic c e l l s i n a heterozygous p a t i e n t w i t h s i d e r o b l a s t i c anemia has been used to demonstrate the common o r i g i n of e r y t h r o c y t e s , g r anu locy te s , macrophages, p l a t e l e t s and T and B lymphocytes from a s i n g l e p l u r i p o t e n t c e l l (10 ) . Thus, G6PD a n a l y s i s 15 has provided cons ide rab l e i n s i g h t i n t o c l o n a l i t y i n both normal and malignant hemopoies is . However, because t h i s technique can on ly be used on the one t h i r d of black, women who are heterozygous for e l e c t r o p h o r e t i c a l l y d i s t i n c t isoenzymes of G6PD i t has l i m i t e d a p p l i c a b i l i t y . Recent developments i n molecu la r gene t i c s now a l l o w c l o n a l a n a l y s i s to be done on t i s s u e from women who are heterozygous for o ther , more common markers on the X-chromosome. C. HPRT, DNA M e t h y l a t i o n and RFLPs Genes on the i n a c t i v e X chromosome i n female c e l l s are more h e a v i l y methylated than those on the a c t i v e chromosome (53 ,54 ) . R e s t r i c t i o n endonucleases e x i s t which w i l l c leave demethylated but not methylated DNA at s p e c i f i c n u c l e o t i d e sequences (55) . Polymorphisms i n n u c l e o t i d e sequence e x i s t throughout the human genome and r e s u l t i n r e s t r i c t i o n endonuclease d i g e s t s of human DNA of ten producing fragments of d i f f e r e n t s i z e from otherwise normal genes (56) . For example, the gene for hypoxanthine phosphor ibosy l t r ans fe rase (HPRT) i s found on the X-chromosome. A r e s t r i c t i o n enzyme fragment l eng th polymorphism (RFLP) i n the HPRT gene can be de tec ted on 19% of X-chromosomes. Th i s a l lows d i s t i n c t i o n between the maternal and p a t e r n a l gene i n 19% of women. Fur ther d i g e s t i o n of DNA w i t h an enzyme which p r e f e r e n t i a l l y c leaves demethylated DNA w i l l a l l o w d e t e c t i o n of which of the two gene copies i s i n a c t i v e . I f DNA from a p o l y c l o n a l popu la t i on of c e l l s i s d iges t ed some of both the maternal and pa t e rna l X-chromosomes w i l l be i n a c t i v e , h e a v i l y methylated at the HPRT locus and w i l l not be cut by the second enzyme. The remainder of both copies of the gene w i l l be d i g e s t e d . T h i s means that on a Southern b l o t h y b r i d i z e d w i t h a probe for the HPRT gene both the fragments from the s i n g l e enzyme d iges t ( r ep re sen t ing methylated DNA r e s i s t a n t to the second enzyme) and new fragments generated by d i g e s t i o n of 16 demethylated DNA w i l l be present . In c o n t r a s t , i n DNA from a monoclonal p o p u l a t i o n of c e l l s i n which the same X chromosome i s i n a c t i v a t e d i n every c e l l , the DNA fragment(s) generated by the a c t i v e chromosome i n the s i n g l e d i g e s t w i l l be l o s t complete ly i n the double enzyme d iges t and new fragments generated w h i l e the fragment(s) from the s i n g l e d iges t of the i n a c t i v e chromosome w i l l remain. Diges t s of tumor c e l l DNA from women heterozygous fo r the RFLP i n the HPRT gene have been used to demonstrate c l o n a l i t y i n a number of s o l i d tumors (57) and ANLL (58) . In a d d i t i o n these techniques have shown that mature g ranu locy tes i n some p a t i e n t s w i t h ANLL i n r emis s ion are c l o n a l and may be par t of the leukemic popu la t ion (59) . The methods desc r ibed above have extended the use of the phenomenon of X-chromosome i n a c t i v a t i o n i n c l o n a l i t y s tud ie s to a l a r g e r p r o p o r t i o n of the p o p u l a t i o n than was p o s s i b l e w i t h G6PD a n a l y s i s . Fur ther developments ex tending the use of RFLPs to other genes on the X chromosome w i l l undoubtedly soon make i t p o s s i b l e to study a l l women i n t h i s f a s h i o n . D. "Marker G e n e s " - R e t r o v i r a l I n s e r t i o n In the l a s t s e v e r a l years i t has become p o s s i b l e to i n s e r t f o r e i g n genes i n t o mammalian chromosomes. The most e f f i c i e n t way to accompl ish t h i s and at present the on ly p r a c t i c a l method for hemopoiet ic c e l l s i s by r e t r o v i r a l i n f e c t i o n . R e t r o v i r a l genomes become incorpora ted i n t o c e l l u l a r DNA as par t of t h e i r l i f e c y c l e . I f the r e t r o v i r u s c a r r i e s an i d e n t i f i a b l e f o r e i g n gene t h i s can be l oca t ed i n the host c e l l by Southern b l o t t i n g . The s i t e of r e t r o v i r a l i n t e g r a t i o n w i t h i n the host c e l l DNA appears to be random. However, once the v i r u s i s i n t eg ra t ed w i t h i n the c e l l DNA a l l subsequent daughters of that c e l l i n h e r i t the r e t r o v i r a l gene. The presence of the r e t r o v i r a l - l i n k e d marker gene at a s p e c i f i c s i t e as i n d i c a t e d by r e s t r i c t i o n 17 enzyme d i g e s t s and Southern b l o t t i n g can be used to i d e n t i f y a monoclonal p o p u l a t i o n of c e l l s . R e t r o v i r a l - m e d i a t e d gene t r ans fe r has been used i n the a n a l y s i s of hemopoie t ic r e c o n s t i t u t i o n of l e t h a l l y i r r a d i a t e d mice ( 8 , 9 ) . In these experiments gene t r ans f e r techniques provide an oppor tun i ty to study the dynamics of hemopoiesis i n d e t a i l that has p r e v i o u s l y been i m p o s s i b l e . Many other exper imenta l and c l i n i c a l goa ls can be approached through the t r a n s f e r of new genes i n t o target c e l l s . The technology and i t s present and fu ture a p p l i c a t i o n s w i l l be d i scussed i n the f i n a l s e c t i o n of t h i s i n t r o d u c t i o n . 3) CLONAL DISORDERS OF HEMOPOIESIS The fac t that hemopoiet ic mal ignancies are c l o n a l , that i s , they o r i g i n a t e i n a s i n g l e c e l l was f i r s t demonstrated i n ch ron ic myelogenous leukemia (CML). Cytogene t ic a n a l y s i s of bone marrow c e l l s from p a t i e n t s w i t h t h i s d i sease revea led an abnormal chromosome 22 - the P h i l a d e l p h i a Chromosome (Ph) - which r e s u l t s from the t r a n s l o c a t i o n of gene t i c m a t e r i a l between the long arms of chromosomes 9 and 22 (59) (F igure 3 ) . Al though CML i s c h a r a c t e r i z e d p r i m a r i l y by increased members of p e r i p h e r a l blood g ranu locy te s and t h e i r p recursors other hemopoiet ic c e l l s i n c l u d i n g e r y t h r o b l a s t s , monocytes, e o s i n o p h i l s and B lymphocytes have been shown to have the Ph chromosome i n t h i s d isease (60-63) (F igure 2 ) . In a d d i t i o n , G6PD isoenzyme s t u d i e s of va r i ous hemopoiet ic c e l l s from pa t i en t s w i t h CML heterozygous fo r 2 e l e c t r o p h o r e t i c a l l y d i s t i n c t enzyme v a r i a n t s have shown that on ly one and the same isoenzyme i s expressed i n g ranu locy te s , monocytes, red c e l l s , p l a t e l e t s and some B lymphocytes (64 ,65 ) . These two l i n e s of evidence i n d i c a t e that CML i s a c l o n a l neoplasm a r i s i n g i n a p l u r i p o t e n t hemopoiet ic p r o g e n i t o r . Other hematologic d i s o r d e r s , i n c l u d i n g ANLL, polycythemia ve ra , i d i o p a t h i c 18 Chronic Mye logenous Leukemia Normal D e f e c t i v e Normal D e f e c t i v e FIGURE 3. Chromosomal Rearrangement i n CML. a b l and s i s are the c e l l u l a r protooncogenes c - a b l and c - s i s . bcr i s b reakpo in t c l u s t e r r e g i o n . • marks chromosomal l o c a t i o n of these genes. •> marks the s i t e of the b reakpo in ts i n the Ph t r a n s l o c a t i o n t ( 9 ; 2 2 ) ( q 3 4 ; q l l ) . 19 m y e l o f i b r o s i s , mye lodysp las ia and one case of a p l a s t i c anemia have been shown to be of c l o n a l o r i g i n us ing these techniques (66-68) . However, the c e l l u l a r compos i t ion of the abnormal c lone v a r i e s i n each d i s o r d e r and provides the bas i s for the c l i n i c a l d i a g n o s i s . For example, g ranulocy tes predominate i n CML w h i l e an e l eva ted red c e l l count c h a r a c t e r i z e s polycythemia v e r a . Neve r the l e s s , G6PD a n a l y s i s r evea l s the presence of at l e a s t the three l i n e a g e s of mye lopo ies i s i n the abnormal c lone of both d i s o r d e r s . As a c l o n a l hemopathy ANLL v a r i e s from the m y e l o p r o l i f e r a t i v e d i s o r d e r s such as CML and polycythemia vera i n two important ways. Us ing G6PD a n a l y s i s F i a l k o w et a l found that a l though some p a t i e n t s showed the u sua l pa t t e rn of t r i l i n e a g e m y e l o p o i e t i c d i f f e r e n t i a t i o n i n the abnormal c lone ; o thers showed g r a n u l o p o i e t i c but not e r y t h r o i d maturat ion (69) . These f i n d i n g s may i n d i c a t e that ANLL may begin e i t h e r i n p l u r i p o t e n t stem c e l l s or i n p rogen i to r s committed to g r a n u l o p o i e s i s . A l t e r n a t i v e l y , the t ransformat ion may occur i n p l u r i p o t e n t stem c e l l s which l o se the capac i ty for e r y t h r o p o i e s i s e i t h e r at the time of t rans format ion or dur ing c l o n a l e v o l u t i o n . The second d i s t i n c t i v e fea ture of ANLL c lones i s the presence w i t h i n them of a predominant p o p u l a t i o n of b l a s t c e l l s showing l i t t l e or no morpholog ica l evidence of d i f f e r e n t i a t i o n . B l a s t c e l l s appear du r ing the course of d iseases other than ANLL. In p a r t i c u l a r , the t e rmina l phase of CML i s accompanied by p r o l i f e r a t i o n of these u n d i f f e r e n t i a t e d c e l l s . Thus, the abnormal c lones i n t h i s and other hemopathies are g e n e t i c a l l y uns table and subject to p r o g r e s s i o n . The nature of the p r e c i s e gene t i c changes r e spons ib l e for the i n i t i a t i o n and p rog re s s ion of these or any other human malignancy are at present unknown. Neve r the l e s s , i t i s b e l i e v e d that gene t i c abno rma l i t i e s are c e n t r a l to malignant t r ans fo rma t ion . Some of the evidence for t h i s b e l i e f i s d i scussed below. 20 Theodor B o v e r i pub l i shed a t r e a t i s e on the o r i g i n of malignant tumors i n 1914 i n which he hypothes ized that malignant c e l l s have an abnormal chromosome c o n s t i t u t i o n and that these abnormal chromosomes cause the malignant growth (70 ) . Subsequent e v a l u a t i o n of metaphase c e l l s from human tumors i n the cy togene t i c e ra before chromosome banding techniques were a v a i l a b l e revea led many a b n o r m a l i t i e s i n cancer c e l l s . However, these appeared to be ext remely v a r i a b l e and were thought to most probably be random a b n o r m a l i t i e s of no pa thogenet ic importance. The Ph chromosome desc r ibed by Nowel l and Hungerford i n 1960 i n the leukemic c e l l s of 2 pa t i en t s w i t h CML was the f i r s t c o n s i s t e n t chromosomal abnormal i ty desc r ibed i n human cancer (44) . Subsequent a n a l y s i s of many hundreds of pa t i en t s w i t h CML have revealed the Ph abnormal i ty i n at l e a s t 90% of p a t i e n t s w i t h the d isease (71 ,72 ) . The a p p l i c a t i o n of chromosome banding techniques to metaphase c e l l s from pa t i en t s w i t h CML have revea led that the Ph abnormal i ty i s a c t u a l l y a r e c i p r o c a l t r a n s l o c a t i o n between the long arms chromosomes 9 and 22 and that the breakpoints i n these chromosomes are q u i t e s p e c i f i c : t ( 9 ; 2 2 ) ( q 3 4 ; q l l ) (59) (F igure 3 ) . O c c a s i o n a l l y more complex t r a n s l o c a t i o n s , i n v o l v i n g , fo r example, a t h i r d chromosome are obse rved - in CML c e l l s but d e t a i l e d a n a l y s i s t y p i c a l l y shows that the t r a n s l o c a t i o n of m a t e r i a l from the long arm of chromosome 9 to the long arm of 22 s t i l l takes p lace (71 ,72 ) . Ex t ens ive surveys of chromosomes abnorma l i t i e s i n a l l the common hema to log i ca l mal ignancies have now been accomplished (71) . A v a r i e t y of nonrandom changes have been descr ibed some of which appear to be a s s o c i a t e d w i t h a p a r t i c u l a r type of c l i n i c a l d i s o r d e r . For example, ANLL may be subgrouped acco rd ing to the F r e n c h - A m e r i c a n - B r i t i s h (FAB) c l a s s i f i c a t i o n i n t o a number of ca t egor i e s (Table I ) on the bas i s of morpholog ica l c r i t e r i a (73) . Up to 100% of p a t i e n t s w i th ANLL w i l l have cy togene t i c a b n o r m a l i t i e s i n t h e i r 21 TABLE I C l a s s i f i c a t i o n o f the A c u t e N o n l y m p h o b l a s t i c L e u k e m i a s M o r p h o l o g i c Types S u b t y p e s (FAB D e s i g n a t i o n ) M y e l o i d M y e l o m o n o c y t i c M o n o c y t i c E r y t h r o i d Mast c e l l M e g a k a r y o c y t e AML M y e l o b l a s t i c w i t h o u t m a t u r a t i o n ( M l ) M y e l o b l a s t i c w i t h m a t u r a t i o n (M2) H y p e r g r a n u l a r p r o m y e l o c y t e (M3) H y p o g r a n u l a r p r o m y e l o c y t e (M3 v a r i a n t ) AMML (M4) P o o r l y d i f f e r e n t i a t e d (M5^) AMoL D i f f e r e n t i a t e d ( M 5 B ) AMoL AEL (M6) A c u t e mast c e l l l e u k e m i a A c u t e m e g a k a r y o c y t i c l e u k e m i a A b b r e v i a t i o n s : F A B , F r e n c h - A m e r i c a n - B r i t i s h ; AML, a c u t e m y e l o b l a s t i c l e u k e m i a ; AMML, a c u t e m y e l o m o n o c y t i c l e u k e m i a ; AMoL, a c u t e m o n o c y t i c l e u k e m i a ; A E L , a c u t e e r y t h r o l e u k e m i a . 22 bone marrow metaphase c e l l s when they are examined by h igh r e s o l u t i o n banding techniques ( 7 4 , 7 5 ) . When these cy togene t i c changes are compared w i t h the FAB category of the corresponding leukemic c e l l s a number of s t r i k i n g c o r r e l a t i o n s are observed. A t (15;17) (q22;q21) i s v i r t u a l l y pathogenomic of acute p romye locy t i c leukemia , M3 (76) . An 8;21 t r a n s l o c a t i o n i s seen i n m y e l o b l a s t i c leukemia w i t h evidence of d i f f e r e n t i a t i o n , M2 (77) . Many o ther a s s o c i a t i o n s have been desc r ibed (Table I I ) some of which appear to have p r o g n o s t i c importance, i . e . i nv (17) i n myelomonocytic leukemia M4 w i t h d y s p l a s t i c e o s i n o p h i l s , i s a s soc ia t ed w i t h a good response to therapy and prolonged s u r v i v a l (78 ) . S i m i l a r s tud ie s of malignant c e l l s i n acute l ymphob la s t i c leukemia (ALL) (79) and nonHodgkin's lymphoma (80) have r evea led c h a r a c t e r i s t i c cy togene t i c abno rma l i t i e s i n va r i ous subgroups some of which appear to have p rognos t i c importance. Among s o l i d tumors d e t a i l e d k a r y o t y p i c i n f o r m a t i o n i s l e s s p l e n t i f u l . However, i t appears that most malignant tumors have an abnormal chromosome content (71) . The karyotype of malignant c e l l s t y p i c a l l y does not remain s t a b l e . A s tepwise rearrangement of the abnormal karyotype i s sometimes observed, o f t en as the c l i n i c a l i l l n e s s enters a more aggress ive phase. CML provides a convenient example. When t h i s d isease enters the phase of acute or b l a s t t r ans fo rmat ion the karyotype of bone marrow c e l l s shows changes i n a d d i t i o n to the Ph i s 80% of cases . The most common of these a d d i t i o n a l changes are an a d d i t i o n a l Ph, t r i somy of chromosome 8, and an isochromosome of the long arm of chromosome 17 (46) . Thus, cy togene t i c s tud i e s provided some of the f i r s t and most c o n v i n c i n g examples of gene t i c abno rma l i t i e s i n cancer c e l l s . However, because of the gross nature of these changes at the chromosomal l e v e l i t has not been p o s s i b l e to determine which genes are a l t e r e d e i t h e r i n t h e i r s t r u c t u r e or TABLE II De Novo Acute Nonlymphocytic Leukemia in Adults with Specific Chromosome Defects Frequency (X) FAB Chromosome Defects Median Survival Median Age of Category (month) Onset (year) 1 M1,M2,M4,M6 inv 3 or t(3;3) 8 44 1 M2 del 5q Few cases Few cases 3 M2,M1,M4,M5,M6 -7/del 7q 3 51 2 M2,M1,M4 t(6;9) Few cases 34 5-20 M2 t(8;21) 14+ 38 9 M2,M1,M4,M5,M6 +8 9 52 8 M1,M2 t(9;22) Few cases Few cases 9 M4,M5a,M2 t(V;ll) Few cases 34 6 M3 t(15;17) 19 31 9 M4,M2,M5b inv 16 15+ 49 14 Ml,M2,M4,H5a,M6 Complex defects 2.5 60 Abbreviations: FAB, French-American-British; V, variable. From Reference 72. 24 e x p r e s s i o n and func t ion by the documented a b n o r m a l i t i e s . Th i s has a l s o made i t d i f f i c u l t to prove that any cy togene t i c changes are of pa thogenet ic or pr imary importance i n the onset of malignant t r ans fo rma t ion . Recent developments i n molecular gene t i c s and recombinant DNA technology have made a n a l y s i s of s t r u c t u r a l chromosome rearrangements p o s s i b l e at the molecu la r l e v e l . The d i s c o v e r y of c e l l u l a r genes homologous to the t ransforming genes of oncogenic r e t r o v i r u s e s has i d e n t i f i e d a number of candidate genes on which to focus the search for tumor e t i o l o g y . 4) ONCOGENES I t has been known for a number of years that c e r t a i n RNA tumor v i r u s e s which cause tumors a f t e r shor t per iods of l a t ency i n animals do so because they con t a in gene t i c sequences that encode for t ransforming genes or oncogenes. A major s c i e n t i f i c d i scove ry of t h i s decade has been the i d e n t i f i c a t i o n of genes w i t h i n normal c e l l s that are h i g h l y homologous w i t h these oncogenes (81) . These genes have been h i g h l y conserved throughout e v o l u t i o n and thus are thought to encode p ro t e in s that are v i t a l to some face t of the c e l l u l a r l i f e c y c l e . I t appears that the r e t r o v i r u s has i nco rpo ra t ed a c e l l u l a r gene i n t o i t ' s genome as a consequence of i t s l i f e c y c l e which r e q u i r e s that the v i r u s i n t e g r a t e i n t o host c e l l u l a r DNA. The new gene becomes an oncogene or t ransforming gene i n the v i r u s as a r e s u l t of mutat ion to the gene that occurs at the time of i t s t r ansduc t ion i n t o the v i r u s or d u r i n g i t ' s subsequent r ep roduc t ive c y c l e s . Express ion of the gene may a l s o be a l t e r e d because of the presence nearby of r e t r o v i r a l promoters and enhancer sequences. Al though the p r e c i s e func t ion of most of these now more than 30 c e l l u l a r oncogenes i s unknown some genera l ca t egor i e s have begun to appear and c e r t a i n genes have been ass igned s p e c i f i c p r o t e i n products (82) (Table I I I ) . 25 TABLE III Oncogenes Name Or ig in Protein (Locat ion/Structure) Function abl bas Blym E1A e rb A e rb B ets 1,2 fes / fps fgr f ms t O S hst int 1,2 jun ma s met mil mo B myb c-Biyc L-myc N-myc P-myc R-myc neu H-ras K-ras N-ras raf r e l ros s i s s rc SV40-T trk yes Abelson virus (v) Murine sarcoma v B c e l l lymphoma Adenovi rus AEV AEV ALV E26 Fujinami SV Fel ine SV FBJ osteosarcoma v Human stomach ca M mammary tumor Avian SV Mouse sarcoma Human osteosarcoma AV-MH2 MSV-SD AMV AMV-MC2 9 Lung tumors Neuroblastomas Rhabdomyosarcoma Neuroblastoma Harvey SV Kirs ten SV Neuroblastoma MSV 3611 Turkey lymphoma v ASV UR2 Simian SV Rous SV SV40 virus Human colon ca ASV-Y73 cytoplasm/protein kinase (PK) ? ? nuclear prote in PK ? PK PK PK nuclear ? ? ? ? PK PK PK nuclear nuclear nuclear nuclear nuclear nuclear membrane/PK GTP binding GTP binding GTP binding PK ? PK PK PK nuclear PK PK ? s t ero id EOF r CSF-1 r receptor ceptor cepto r ? receptor ? s ignal transduction ? ? receptor PDGF-B ? ? ? ? 26 A number of the c e l l u l a r counterpar ts to v i r a l oncogenes encode p r o t e i n s that are l o c a l i z e d to the nuc leus , i n some cases b i n d i n g to DNA. These p r o t e i n s may have some r egu l a to ry func t ion i n c e l l d i v i s i o n . Th i s category i n c l u d e s the c-myb and the myc gene fami ly (82) . A second group of c e l l u l a r oncogenes appear to encode growth f a c t o r s or t h e i r r e c e p t o r s . C - s i s encodes a p r o t e i n h i g h l y homologous to the 3 cha in of p l a t e l e t - d e r i v e d growth f ac to r (PDGF) (83) . c -e rb-B i s homologous to the ep idermal growth f a c t o r receptor and v-fms to the M-CSF recep tor ( 8 4 , 8 5 ) . Other oncogenes have s t r u c t u r e s h i g h l y sugges t ive of the p o s s i b i l i t y that they, too may be growth f ac to r s or r ecep to r s , e . g . r o s , neu ( 8 6 , 8 7 ) . Sporn and Todaro have put forward a hypothes is that desc r ibes au toc r ine s t i m u l a t i o n of malignant c e l l s (88 ) . Accord ing to t h i s theory au toc r ine s t i m u l a t i o n occurs when a c e l l begins to produce a growth f ac to r for which i t a l r eady possesses a r ecep to r . In t h i s way a c e l l becomes independent of the need f o r exogenous s t i m u l a t o r y f a c t o r s . Examples of such behavior have been seen i n c e l l s transformed by V - s i s c o n t a i n i n g r e t r o v i r u s e s (89) . F i n a l l y , there are oncogenes that may func t ion w i t h i n the cytoplasm i n s i g n a l t r a n s m i s s i o n . For example, the ras gene fami ly bears many s t r u c t u r a l s i m i l a r i t i e s to the G p ro t e in s which are known to transduce s i g n a l s from v a r i o u s c e l l - s u r f a c e receptors to adeny lcyc lase (82) . Thus, the c e l l u l a r oncogenes for which such in fo rma t ion i s a v a i l a b l e appear to f u n c t i o n i n the c o n t r o l of c e l l growth and d i v i s i o n . I t seems s e l f -ev iden t that p e r t u r b a t i o n of such funct ions cou ld lead to malignant t r an s fo rma t ion . A f t e r the d i s cove ry of c e l l u l a r oncogenes i n normal c e l l s i n v e s t i g a t o r s began to look at malignant t i s s u e for abnorma l i t i e s i n v o l v i n g these genes or t h e i r p roduc t s . A number of t h e o r e t i c a l changes that could cause a normal 27 gene to become a cancer gene were hypothesized and examples of a l l o f these p o s s i b i l i t i e s have been found i n na ture . A . Gene A m p l i f i c a t i o n An increased number of copies of a gene could r e s u l t i n ove rp roduc t ion of an oncogene product . Members of the myc fami ly of oncogenes have been shown to be a m p l i f i e d i n the HL-60 c e l l l i n e , (c-myc) (90) me ta s t a t i c neuroblastoma (N-myc) (91) and s m a l l c e l l lung carcinoma (N-myc) (92) . In some cases dozens of copies of the gene are found i n tumor c e l l DNA and h igh l e v e l s of exp re s s ion of the gene are found at the RNA l e v e l . B . Po in t Mutat ions I t has been known for many years that a number of i n h e r i t e d human d i seases are caused by a s i n g l e n u c l e o t i d e s u b s t i t u t i o n i n a c r i t i c a l gene, e . g . s i c k l e c e l l anemia. I t would not be s u r p r i s i n g i f s i m i l a r changes i n oncogenes were important for malignant t r ans fo rmat ion . Members of the ras f a m i l y of oncogenes are of ten found to possess point mutations i n tumor c e l l DNA. These n u c l e o t i d e s u b s t i t u t i o n s seem to occur at p a r t i c u l a r s i t e s , e . g . codon 12, that are presumably important for ca rc inogenes i s (93 ) . N-ras and K - r a s are genes that have been found to have poin t mutat ion i n some samples of human leukemic c e l l DNA (94 ,95 ) . These mutated genes have been i d e n t i f i e d by t h e i r a b i l i t y to cause the formation of transformed f o c i of c e l l s when leukemic c e l l DNA i s t r ans fec ted i n t o murine f i b r o b l a s t c e l l l i n e s . Th i s suggests , but c e r t a i n l y does not prove, that the mutated ras genes are important for producing or ma in t a in ing the malignant phenotype i n the leukemic c e l l s from which they o r i g i n a t e d . 28 C. Gene Rearrangement Gross s t r u c t u r a l changes have been found to occur i n oncogenes i n many r e t r o v i r u s e s . For example, a l though the v -e rb -B oncogene i s h i g h l y homologous to the EGF recep tor gene i t has been seve re ly t runcated i n the e x t r a c e l l u l a r or l i g a n d - b i n d i n g r e g i o n . Th i s i s f e l t to r e s u l t i n a growth f a c t o r r ecep to r product which i s c o n s t i t u t i v e l y a c t i v e i n the absence of l i g a n d (84 ) . D. A l t e r e d Gene Express ion I t i s p o s s i b l e that a p o t e n t i a l oncogene could remain undamaged by a mu ta t i ona l event but that gene t i c rearrangements nearby could a f f e c t i t s exp re s s ion i n a s i g n i f i c a n t way. The av ian l e u k o s i s v i r u s causes lymphomas i n ch ickens a l though i t does not con t a in an oncogene. However, d e t a i l e d a n a l y s i s of tumor c e l l DNA from lymphomas induced by t h i s v i r u s r e v e a l that the v i r u s has i n t e g r a t e d near the c-myc oncogene i n the c e l l u l a r DNA. Th i s r e s u l t s i n o v e r - e x p r e s s i o n of the c-myc gene product , a c i rcumstance that may be important i n tumorigenesis (96) . E . I n t e r a c t i o n of More Than One Oncogene A number of oncogenes which e f f i c i e n t l y transform immor ta l i zed c e l l l i n e s t ransform primary c e l l s at extremely low frequency or not at a l l . However, when a second oncogene i s in t roduced i n t o the primary c e l l t r ans fo rmat ion occurs w i t h h igh e f f i c i e n c y . Th i s phenomenon of co -ope ra t ion between two oncogenes i n malignant t ransformat ion has been demonstrated most f r equen t ly fo r genes of the ras and myc f a m i l i e s . The target c e l l s have been v a r i o u s , i n c l u d i n g primary ra t embryo f i b r o b l a s t s and B lymphoblasts ( 9 7 , 9 8 ) . The a c t i v a t i o n of two t ransforming genes i n a s i n g l e tumor i s c o n s i s t e n t w i t h the o b s e r v a t i o n that most de novo tumors i n animals and man show prolonged l a t e n c y 29 and i n c r e a s i n g aggress iveness as the d isease progresses . Th i s suggests that m u l t i p l e gene t i c events are necessary to cause most ma l ignanc ie s . F . Oncogene Involvement i n Chromosomal Rearrangements There are now almost 40 d i f f e r e n t oncogenes i d e n t i f i e d e i t h e r by the i n v e s t i g a t i o n of t ransforming r e t r o v i r u s e s or the a n a l y s i s of tumor c e l l DNA i n v a r i o u s t r a n s f e c t i o n assays . Undoubtedly, many oncogenes remain to be d i s c o v e r e d . I t i s a formidable task to study human tumors for mutat ions i n a l l these p o t e n t i a l t ransforming genes so i n v e s t i g a t o r s have looked fo r c l ue s from other areas of gene t i c research on cancer c e l l s . Chromosomal a n a l y s i s of human malignant c e l l s has provided many va luab l e i n s i g h t s i n t o which of these many oncogenes might be perturbed i n a g iven tumor (71) . The c e l l u l a r oncogenes have i n most cases been mapped to s p e c i f i c regions on the human chromosomes (F igu re 4 ) . By comparing the l o c a t i o n of the oncogenes to the b reakpoin t s i n v o l v e d i n t r a n s l o c a t i o n s seen i n human mal ignancies i t has been p o s s i b l e to p r e d i c t that c e r t a i n genes would move from one chromosome to another as a consequence of the t r a n s l o c a t i o n . By c l o n i n g the reg ions of DNA around these chromosomal breakpoints and s tudy ing t h i s DNA fo r gene rearrangements i t has been p o s s i b l e to demonstrate s t r u c t u r a l or f u n c t i o n a l a l t e r a t i o n s i n both oncogenes and other c e l l u l a r genes i n s e v e r a l tumors. i ) B u r k i t t ' s Lymphoma C h a r a c t e r i s t i c cy togene t ic abno rma l i t i e s can be i d e n t i f i e d i n more than 80% of these tumors. In the major i ty of cases the karyotype of malignant c e l l s shows a t r a n s l o c a t i o n between the long arms of chromosomes 8 and 14. However, i n some cases chromosomes 8 and 2 or 8 and 22 are i n v o l v e d (99 ) . The breakpoin t on chromosome 8 i s at the s i t e where the c-myc gene has been 30 FIGURE 4 . Giemsa-banded human chromosome map w i t h 21 oncogenes ( d o t ) , 6 c e l l u l a r genes ( t r i a n g l e ) , and breakpoints (arrows) for chromosomal rearrangements found i n cancer . (Reference 72) . 31 Burkitt's Lymphoma translocation 8; 14= c-myc;lg rearrangement — T " 3 8 7« 1 M 3 i J 13, 13| 1 .2 :U .13 j 4 .21 .22[ .231 c-myc *C2T Ig-V - C J J c-myc Normal Defective 8 Normal Defective 14 FIGURE 5. Chromosomal Rearrangement i n B u r k i t t ' s Lymphoma. 32 mapped. On chromosome 14 the immunoglobulin heavy cha in gene i s at the breakpoin t whereas on chromosomes 2 and 22 i t i s the A and K l i g h t cha in immunoglobulin gene r e s p e c t i v e l y that i s at the breakpoint (F igu re 5 ) . C l o n i n g of the breakpoint i n the 8;14 t r a n s l o c a t i o n has revea led that the c-myc gene has been t r a n s l o c a t e d i n t o the Ig heavy cha in gene locus near the I g enhancer r e g i o n . I t has been shown that t h i s t r a n s l o c a t e d c-myc gene i s expressed i n the tumor c e l l s w h i l e the normal gene i s not (100) . In some B u r k i t t s Lymphoma tumor samples c-myc express ion i s unusua l ly h i g h . Thus, i t i s l i k e l y that the chromosomal t r a n s l o c a t i o n seen i n B u r k i t t ' s lymphoma, a B - c e l l mal ignancy, a l l ows a p o t e n t i a l t ransforming gene, c-myc, to be r egu la t ed by the I g enhancer which normal ly func t ions at h igh l e v e l s i n B c e l l s . The abnormal r e g u l a t i o n of c-myc i n these c e l l s may be c r i t i c a l to t h e i r malignant t r ans fo rma t ion . i i ) CML In t h i s d i s o r d e r the t (9 ;22) which i s seen i n more than 90% of p a t i e n t s r e s u l t s i n the t r a n s l o c a t i o n of the c - a b l oncogene from chromosome 9 i n t o a gene on chromosome 22 known as bcr (breakpoint c l u s t e r reg ion) (F igu re 3 ) . T h i s rearrangement causes abnormal s p l i c i n g of the two gene products so that a f u s i o n b c r - a b l RNA t r a n s c r i p t i s produced (101) . Th i s RNA i s t r a n s l a t e d i n t o an abnormal p r o t e i n w i t h a l t e r e d enzymatic a c t i v i t y , i . e . the normal c - a b l product , has l i t t l e or no de tec tab le t y r o s i n e p r o t e i n k inase a c t i v i t y whereas the b c r - a b l fus ion p r o t e i n has a very h igh and promiscuous l e v e l of t h i s a c t i v i t y (102 ,103) . Thus, the Ph t r a n s l o c a t i o n r e s u l t s i n a new rearranged or h y b r i d gene which u l t i m a t e l y y i e l d s a new and abnormal p r o t e i n . The abnormal b c r - a b l RNA spec ies has been seen i n c e l l s from p a t i e n t s w i t h CML who appeared 33 c y t o g e n e t i c a l l y normal (Ph negat ive CML) (104) and i n CML p a t i e n t s w i t h complex 3-way t r a n s l o c a t i o n s i n the karyotype of t h e i r malignant c e l l s (105) . Thus, t h i s rearrangement of the c - a b l oncogene appears to be h i g h l y c h a r a c t e r i s t i c and perhaps d i a g n o s t i c of CML. Cytogene t i c a b n o r m a l i t i e s have been used as c lues to begin i n v e s t i g a t i o n of h e r e d i t a r y carcinomas at the molecular l e v e l . He red i t a ry r e t inob la s toma i s o f ten accompanied by d e l e t i o n or rearrangements i n the long arm of chromosome 13 i n tumor c e l l metaphases (106) . Southern b l o t t i n g a n a l y s i s of tumor c e l l DNA h y b r i d i z e d w i t h probes to gene t i c sequences which map to t h i s r e g i o n of chromosome 13 have revea led the f o l l o w i n g f a c t s . I t appears that c h i l d r e n may i n h e r i t a mutat ion on one chromosome 13 and that tumors a r i s e when a somatic muta t ion occurs at the same s i t e on the p r e v i o u s l y normal chromosome 13. These mutations may be v i s i b l e as chromosomal abno rma l i t i e s or may r e q u i r e more s u b t l e molecular a n a l y s i s (107) . Recen t ly the p u t a t i v e " r e t i nob la s toma" gene has been cloned and i t remains to be seen what c h a r a c t e r i s t i c s i t may have i n common w i t h p r e v i o u s l y descr ibed oncogenes (108) . When i t was d i scovered that the c e l l u l a r homologues of s e v e r a l v i r a l oncogenes were growth f ac to r s or t h e i r receptors i t became n a t u r a l to suppose that o ther known growth f ac to r or receptor genes might act as oncogenes. As p r e v i o u s l y mentioned, a number of the hemopoiet ic growth f a c t o r gene have been c loned and recombinant f ac to r s produced. Us ing such reagents i t has been shown that a s i g n i f i c a n t m i n o r i t y of leukemic b l a s t c e l l s from p a t i e n t s w i t h ANLL both sec re te and respond to GM-CSF (109) . In a human leukemic c e l l l i n e the GM-CSF gene has been shown to be rearranged by Southern b l o t t i n g (110) . There fore , at l e a s t some leukemic c e l l s seem to be responding to a u t o c r i n e s t i m u l a t i o n and a hemopoiet ic growth f ac to r gene which has not been i d e n t i f i e d as an oncogene i n the usua l sense may be p l a y i n g an important r o l e i n malignant t r ans fo rma t ion . 34 5) GENE TRANSFER When s tudy ing e s t a b l i s h e d human mal ignancies the i n v e s t i g a t o r i s l o o k i n g at the r e s u l t of both gene t i c and nongenetic events which g i v e r i s e to a very compl ica ted a r ray of a b n o r m a l i t i e s . I t i s d i f f i c u l t to determine which changes are of primary importance and which are secondary to the n e o p l a s t i c p rocess . A more d i r e c t e d or s i m p l i f i e d approach to s tudy ing malignant t r ans fo rmat ion might be to use normal c e l l s as a target for i n t r o d u c t i o n of suspect cancer genes and study the r e s u l t s . S i m i l a r l y , the gene t i c c o n t r o l of many normal c e l l func t ions i s poor ly understood and could p o t e n t i a l l y be d i s s e c t e d i n t h i s way. The use of t r ans fe r r ed genes to mark and i d e n t i f y c e r t a i n c e l l popu la t ions has been d i scussed i n the previous s e c t i o n . A c l i n i c a l area i n which gene t r ans fe r may have a p p l i c a b i l i t y i s i n the therapy of i n h e r i t e d human d i so rde r s i n which a molecular gene t i c defec t has been i d e n t i f i e d (111) . In some cases the organ system i n which the d i sease i s p r i m a r i l y manifest would be a c c e s s i b l e to gene t i c man ipu l a t i on , e . g . the bone marrow i n va r ious hemoglobinopathies . However, because g l o b i n s y n t h e s i s and gene r e g u l a t i o n are unusua l ly compl icated and not complete ly unders tood, o ther d i seases are l i k e l y to be the f i r s t candidates for gene therapy. Those d i s o r d e r s which are caused by a mi s s ing enzyme or p r o t e i n whose l e v e l does not need to be regu la ted p r e c i s e l y are more approachable. Three d iseases which are undergoing a c t i v e i n v e s t i g a t i o n at present i nc lude the Lesch-Nyhan syndrome caused by the absence of hypoxanthine-guanine phosphor ibosy l t r ans fe rase (HPRT) and the two immune d e f i c i e n c y d i so rde r s caused by l a c k of pur ine nuc l eos ide phosphorylase (PNP) or adenosine deaminase (ADA). The i n t r o d u c t i o n of the normal gene for these f ac to r s i n t o pa t i en t bone marrow c e l l s may r e s u l t i n the produc t ion of on ly a f r a c t i o n of the normal enzyme l e v e l but t h i s may be s u f f i c i e n t to reverse many of the c l i n i c a l 35 a b n o r m a l i t i e s . On the other hand, a m i ld overp roduc t ion of these enzymes shou ld not be harmfu l to the c e l l s producing them; The above are but a few of the ins tances i n which an e f f i c i e n t means of t r a n s p o r t i n g s p e c i f i c gene t i c sequences i n t o s e l e c t e d ta rget c e l l s have p o t e n t i a l u s e f u l n e s s . Recent advances, to be d i scussed i n t h i s paper , make such man ipu la t ions p o s s i b l e now or i n the near f u t u r e . A. H i s t o r y of Gene T rans fe r - (112,113) T r a n s f e c t i o n exper iments began s e v e r a l decades ago w i th the a d d i t i o n o f metaphase chromosomes to c e l l suspens ions . The chromosomes were taken up r a t h e r i n e f f i c i e n t l y by the c e l l s and gave r i s e to uns tab le v a r i a n t s at low f requency . I n t ac t chromosomes r a r e l y s u r v i v e t h i s procedure and the r e c i p i e n t c e l l u s u a l l y ga ins a fragment of a donor chromosome which i s uns tab le because i t l a c k s a centromere. Only r a r e l y do s t a b l e l i n e s a r i s e a f t e r i n t e g r a t i o n of donor m a t e r i a l i n t o a r e c i p i e n t chromosome (112) . P u r i f i e d DNA i n suspens ion w i l l en ter euka ryo t i c c e l l s but w i th such low e f f i c i e n c y that the s u c c e s s f u l events cannot be e f f e c t i v e l y s t u d i e d . A g rea t t e c h n i c a l advance was made w i th the d i scove ry that when DNA i s p r e c i p i t a t e d w i t h ca l c ium phosphate (CaP04) before adding i t to c e l l s growing i n a monolayer, many more transformed f o c i appear than when CaPO^ i s absen t . A l though i t appears that the c e l l s a c t u a l l y phagocyt ize the DNA-CA++ g r a n u l e s , the mechanisms of p r e f e r e n t i a l uptake of CaP04 p r e c i p i t a t e d DNA by c e l l s i s u n c l e a r . Seve ra l s tages can be i d e n t i f i e d i n the t rans fo rmat ion p rocess . F i r s t , the CaPO/^-DNA p r e c i p i t a t e en te rs the cytoplasm of a l l or n e a r l y a l l of the c e l l s exposed. However, on ly 1-5% of c e l l s w i l l show the presence of DNA complexes i n the nuc leus . These c e l l s w i l l show t r a n s i e n t exp ress ion of the exogenous genes they have taken up. The frequency of t h i s t r a n s i e n t 36 e x p r e s s i o n i s 10 to 100 f o l d h igher than the f requency of s t a b l e gene e x p r e s s i o n which r equ i r es i n t e g r a t i o n of the f o r e i g n DNA i n t o a host chromosome. Even when DNA i s i n j e c t e d d i r e c t l y i n t o the nuc leus , a l though 50 to 100% of c e l l s show t r a n s i e n t gene e x p r e s s i o n , on ly 1 of 500-1000 show a s t a b l e gene t i c change (113) . The competence of the c e l l s to undergo t h i s permanent change i s the r e s u l t of a number of f a c t o r s , many of which are not unders tood. I t appears that c e l l s i n the e a r l y phase of DNA s y n t h e s i s i n the c e l l c y c l e are the most l i k e l y to be t ransformed, perhaps because enzymes i n v o l v e d i n DNA metabol ism such as DNA polymerase, are most a c t i v e at t h i s t ime (114) . The DNA s t a b l y in t roduced i n t o euka ryo t i c c e l l s by CaPO^ c o -p r e c i p i t a t i o n has been found to be i n t eg ra ted i n t o a s i n g l e random s i t e i n a host chromosome u s u a l l y i n a l a rge concatamer of many u n i t s l i n k e d together and c o n t a i n i n g up to 1000 k i l o b a s e s . Al though v i r t u a l l y any c loned DNA segment can be in t roduced i n t o adherent t i s s u e c u l t u r e c e l l s us i ng t h i s techn ique , the e f f i c i e n c y of gene t r a n s f e r i s never g r e a t e r than 1:10^ even i n the most competent c e l l s . The technique a l s o has a r e l a t i v e , though not a b s o l u t e , requirement f o r the ta rge ts to be adherent c e l l s ra the r than f l o a t i n g i n suspens ion . Th is l i m i t s the a p p l i c a b i l i t y of the technique to a r e l a t i v e l y s e l e c t group of c e l l t ypes . Recen t l y c e r t a i n groups of i n v e s t i g a t o r s have had success us i ng the technique of p r o t o p l a s t f u s i o n f o r gene t r a n s f e r (115,116) . P r o t o p l a s t s are de r i ved by t r e a t i n g b a c t e r i a con ta i n i ng the p lasmid DNA of i n t e r e s t w i t h lysozyme. The p r o t o p l a s t s are then fused to c e l l s i n the presence of po l ye thy lene g l y c o l . Reported e f f i c i e n c i e s of s t a b l e gene t r a n s f e r range around 3 x 10~3. Al though t h i s appears be t t e r than that ach ieved w i th CaPO^-DNA p r e c i p i t a t e s , the success comes from a very sma l l number of l a b o r a t o r i e s . In the hands of most i n v e s t i g a t o r s , p r o t o p l a s t f u s i o n i s very 37 t o x i c to many c e l l t ypes , once aga in l i m i t i n g the a p p l i c a b i l i t y of the techn ique . A t h i r d technique of DNA mediated gene t r a n s f e r uses e l e c t r o p o r a t i o n (117) . C e l l s are incubated i n suspension w i th the DNA of i n t e r e s t and then sub jec ted to an e l e c t r i c cur ren t of 2000-4000 v o l t s which opens c e l l u l a r membrane pores and a l l ows the DNA to enter the c e l l s ; The e f f i c i e n c y of t r a n s f e r r i n g s e l e c t a b l e genes i n t o lymphocytes or f i b r o b l a s t c e l l l i n e s has been repor ted at up to 3 x 1 0 - ^ . With t h i s technique the number of cop ies o f the genes t r a n s f e r r e d i s lower (1 to 15 per c e l l ) than i s u s u a l l y seen w i t h t r a d i t i o n a l CaPO^-DNA p r e c i p i t a t e s which may be an advantage f o r some exper iments . The range of c e l l types s u c c e s s f u l l y t r ans fec ted w i t h technique may a l s o be broader than w i th CaP04 p r e c i p i t a t e s , a l though repo r t s of s u c c e s s f u l e l e c t r o p o r a t i o n are s t i l l r e l a t i v e l y sca rce i n the l i t e r a t u r e . With any of the three gene t r a n s f e r techniques d i scussed so f a r ; CaP04~ DNA p r e c i p i t a t e s , p r o t o p l a s t f u s i o n or e l e c t r o p o r a t i o n , the f requency of gene t r a n s f e r i s s t i l l q u i t e low - too low to be r e l i a b l y s u c c e s s f u l when the ta rge t c e l l of i n t e r e s t i s ra re i n the t o t a l p o p u l a t i o n , e . g . the hemopoie t ic stem c e l l i n the bone marrow (Table I V ) . DNA can be i n j e c t e d d i r e c t l y i n t o the c e l l nuc leus w i th an e f f i c i e n c y of s t a b l e t rans fo rma t ion of about 1 x 10~3. The t e c h n i c a l demands of t h i s m i c r o i n j e c t i o n procedure are c o n s i d e r a b l e , making i t u n s u i t a b l e f o r the u s u a l gene t r a n s f e r exper iments . However, i t has been d i scovered t ha t , when DNA i s i n j e c t e d i n t o the pronucleus of a f e r t i l i z e d zygote , the f requency of s t a b l e i n t e g r a t i o n and exp ress ion i s v a s t l y inc reased over that ach ieved by i n j e c t i n g genes i n t o somat ic c e l l n u c l e i (118,119) . A f t e r a s u c c e s s f u l m i c r o i n j e c t i o n , from 1 to 25 cop ies of the gene are l oca ted at a s i n g l e chromosomal s i t e . In the order of 5% of s u c c e s s f u l l y i n j e c t e d zygotes r e s u l t i n v i a b l e o f f s p r i n g 38 TABLE IV The P r o p o r t i o n of Target C e l l s Showing Exp ress ion of the T rans fe r red Gene Fo l l ow ing Var ious Gene T rans fe r Techniques Technique T rans ien t Gene Exp ress ion E f f i c i e n c y of S t a b l e Exp ress ion CaPO^-DNA p r e c i p i t a t e 1-5% 1-10 x I O " 4 P r o t o p l a s t f u s i o n 3 x 10~3 E l e c t r o p o r a t i o n 3 x 10"^ M i c r o i n j e c t i o n 60-70% 5% R e t r o v i r u s -100% 39 when p laced i n a pseudopregnant mother. The fo r e ign DNA i n these mice i s then i n the germ l i n e , and thus w i l l be present i n a l l c e l l s of the new organism and t r ansmi t t ed to o f f s p r i n g as Mendelian t r a i t s . The c r e a t i o n of such " t r ansgen i c " animals has been used' to study t i s s u e s p e c i f i c r e g u l a t i o n of the immunoglobulin and i n s u l i n genes among others and to cure ?3 tha lassemia i n a murine model (120) . The l i m i t a t i o n s of the m i c r o i n j e c t i o n technique i n c l u d e i t s cons ide rab l e t e c h n i c a l demands and i t s s u c c e s s f u l use p r i m a r i l y on ly i n germ l i n e c e l l s . B . V i r a l Vec to rs E f f o r t s to harness the i n f e c t i v e c y c l e of v i r u s e s for gene t r a n s f e r have i n v o l v e d many c l a s se s of v i r u s e s . DNA v i r u s e s have shown promise fo r gene t r a n s f e r . Recombinant adeno or SV40 v i r u s e s (121) , DNA packaged pseudo SV40 (122) or polyoma v i r i o n s (123) and recombinant pa rvov i rus (124) a l l have been used w i t h some degree of success . A c o n d i t i o n a l l y n o n r e p l i c a t i n g a d e n o v i r a l vec to r has been developed that w i l l e f f i c i e n t l y i n f e c t animal and human c e l l s w i t h on ly one or a few copies of the recombinant v i r u s i n t e g r a t e d i n t o the host genome (121) . However, evidence of s t a b l e gene express ion i n pr imary c e l l s has not been for thcoming. Another DNA v i r u s , the bovine pap i l l oma v i r u s , which r e p l i c a t e s extrachromosomally may be u se fu l for m a i n t a i n i n g genes i n c e l l s i n an un in tegra ted s t a t e (125) , but experiments to show i t s usefu lness i n ta rge ts such as hemopoiet ic c e l l s , have yet to be r epo r t ed . SV40 i s of i n t e r e s t because of i t s broad range of spec ies and t i s s u e i n f e c t i v i t y , i t s w e l l - c h a r a c t e r i z e d genome and i t s genera l l a c k of p a t h o g e n i c i t y . A recombinant SV40 v i r u s has been used to c a r r y the ch loramphenico l a c e t y l t r a n s f e r a s e (CAT) gene i n t o hemopoiet ic c e l l l i n e s and pr imary bone marrow c e l l s and achieve t r a n s i e n t gene e x p r e s s i o n . However, 40 assays designed to demonstrate s t a b l e gene t r ans fe r and express ion w i t h s i m i l a r vec to r s were unsuccess fu l i n primary p rogen i to r s (126) . A method of gene t r ans fe r that shows great promise at present i s the use of r e t r o v i r a l vec to r s (111,127) . Genes can be t r ans fe r r ed as a consequence of the normal l i f e c y c l e of these v i r u s e s . The genes to be t ranspor ted can be those s e l e c t e d by the i n v e s t i g a t o r and i n s e r t e d i n t o the app rop r i a t e v e c t o r u s i n g s tandard recombinant DNA technology. The e f f i c i e n c y of gene t r a n s f e r should approach 100% and the procedure i s nontoxic to e u k a r y o t i c c e l l s . A r e l a t i v e l y s m a l l number of gene copies w i l l i n t eg ra t e i n host DNA, i n most cases on ly one. Because of these fea tu res , r e t r o v i r u s e s appear to have the most immediate promise fo r gene t r a n s f e r . In order to op t imize t h e i r use, i t i s necessary to understand d e t a i l s of t h e i r s t r u c t u r e and l i f e c y c l e which w i l l be d i s cus sed i n the f o l l o w i n g s e c t i o n s . C. R e t r o v i r a l S t ruc tu re (128,129) Dur ing i n f e c t i o n by r e t r o v i r u s e s , double-s t randed v i r a l DNA i s generated from an RNA template and c o v a l e n t l y j o i n e d to host chromosomes. The RNA genomes of a l l r e t r o v i r u s e s con ta in 3 coding regions that p a r t i c i p a t e i n r e p l i c a t i o n (F igure 6 ) . The gag reg ion codes for v i r a l core p r o t e i n s , p o l fo r the enzyme reverse t r a n s c r i p t a s e (RNA-di rec ted DNA polymerase) , and env encoding sequences for syn thes i s of envelope g l y c o p r o t e i n s . Each end of the v i r u s RNA conta ins a repeated sequence R and segments c h a r a c t e r i s t i c of the 3 ' (U3) or 5 ' (U5) end of the v i r u s . The U3 and U5 regions con t a in sequences necessary fo r r e t r o v i r a l i n t e g r a t i o n i n t o host DNA, i n i t i a t i o n of v i r a l t r a n s c r i p t i o n of the v i r a l genome, p o l y a d e n y l a t i o n of RNA t r a n s c r i p t s and a promoter which enhances t r a n s c r i p t i o n . A 150 bp sequence 5 ' to the gag r e g i o n 41 LTR U3 TT E P I U5h I — I 1 H 1- T LTR A U3 I R US FIGURE 6. Structure of Moloney Murine Leukemia Virus R e t r o v i r a l Provirus DNA. Abbrev iat ions: E , enhancer; P, promoter; I , i n i t i a t i o n (Cap) s i t e for v i r a l RNA synthesis; r~ , r e p l i c a t i o n i n i t i a t i o n s i t e for minus DNA strand; D, donor s p l i c e s i t e ; V packaging sequence; A, major acceptor s p l i c e s i t e ; r + , r e p l i c a t i o n i n i t i a t i o n s i t e for plus DNA strand; T, terminal (poly-A addi t ion) s i t e for v i r a l RNA synthes is ; LTR, long terminal repeat; U3, R, and U5 are port ions of the LTR; gag, v i r a l core prote ins ; pol RNA-dependent DNA polymerase (reverse t r a n s c r i p t a s e ) ; and env, envelope prote ins . 42 has a l s o been i d e n t i f i e d which i s e s s e n t i a l fo r packaging of v i r a l components i n t o completed v i r a l p a r t i c l e s . W i t h i n each v i r a l core are 2 i d e n t i c a l subuni t s of RNA genome, as desc r ibed above, each 5 to 9 Kb i n l e n g t h . The reason fo r t h i s gene t i c redundancy i s unknown and unique among animal v i r u s e s to the RNA tumor v i r u s e s . Al though the above represents the e s s e n t i a l components of a r e p l i c a t i o n competent r e t r o v i r u s , i n the course of i t s l i f e c y c l e a v i r u s may be a l t e r e d by mu ta t iona l events that occur w i t h i n the host c e l l genome or r ecombina t i ona l events among v i r u s e s themselves. Examples would i nc lude the endogenous r e t r o v i r u s e s present i n many animal s p e c i e s , most of which have l o s t the a b i l i t y to r e p l i c a t e except under s p e c i a l c i rcumstances , or the a c u t e l y t ransforming r e t r o v i r u s e s which appear to have acqui red a c e l l u l a r "oncogene" at some po in t s i n t h e i r h i s t o r y . D. Host Range of R e t r o v i r u s e s (130) One fea ture of r e t r o v i r u s e s which makes them a t t r a c t i v e fo r gene t r a n s f e r i s t h e i r wide host range. There are v i r u s e s which w i l l i n f e c t most p o t e n t i a l e u k a r y o t i c hosts and most f u n c t i o n a l t i s s u e s . However, each v i r u s does have a s p e c i f i c host requirement which i s determined by s e v e r a l f a c t o r s . Most i m p o r t a n t l y , there are receptor r e s t r i c t i o n s which are determined by the v i r a l env sequence. Al though the v i r u s adsorbs n o n s p e c i f i c a l l y to the c e l l sur face by a process which can be enhanced by p o l y c a t i o n s such a polybrene which decrease c e l l surface charge, the v i r u s must b ind to a s p e c i f i c recep tor i f i t i s to enter the c e l l and r e p l i c a t e . Most c e l l s possess 1 to 5 x 10^ such receptors on t h e i r su r face . While these s t r u c t u r e s presumably do not e x i s t s o l e l y for the convenience of the r e t r o v i r u s , t h e i r " n o n v i r a l " or usua l func t ion i s unknown. In v i v o s tud i e s have shown some 43 t i s s u e s p e c i f i c i t y e x i s t s for r e t r o v i r a l r ecep to r s . For example, recombinant thymic lymphoma v i r u s e s react w i t h receptors that are r e s t r i c t e d to a subset of thymocytes. R e t r o v i r u s e s have been c l a s s i f i e d accord ing to t h e i r r ecep to r s p e c i f i c i t y . C-type murine r e t r o v i r u s e s v i r u s e s are ca t ego r i zed as f o l l o w s : i ) e c o t r o p i c - r e p l i c a t i n g only i n mouse c e l l s . i i ) x eno t rop i c - r e p l i c a t i n g poor ly or not at a l l i n most mouse c e l l s , but i n f e c t i n g a wide range of other spec ies i n c l u d i n g r a t , mink, human and q u a i l . i i i ) amphotropic - r e p l i c a t i n g i n both mouse and other mammalian c e l l s , i n c l u d i n g human. Each of the above types of v i r u s recognizes a unique c e l l sur face r e c e p t o r . There a l s o e x i s t s a category of v i r u s known as p o l y t r o p i c from which the env product w i l l recognize more than one r ecep to r , fo r example, both the e c o t r o p i c and the xeno t rop ic r ecep to r . Recombinant v i r u s e s have been cons t ruc ted i n which an e c o t r o p i c v i r u s has i t s env sequence rep laced by a x e n o t r o p i c or amphotropic env. When t h i s i s done, the new v i r u s acqu i res the host range of the v i r u s from which the new env came. Such manipu la t ions a l l o w the i n f e c t i v i t y of a g iven v i r u s to be manipulated at w i l l . How the i n t e r a c t i o n between v i r u s and c e l l receptor f a c i l i t a t e s v i r a l r e p l i c a t i o n i s not known, a l though l i k e l y candidates i n c l u d e a i d i n g v i r a l p e n e t r a t i o n of the c e l l or v i r a l uncoa t ing . E . R e t r o v i r a l R e p l i c a t i o n (128,129) Once the v i r u s enters the c e l l i t begins to reproduce i t s e l f (F igu re 7 ) . The key event i n r e t r o v i r a l r e p l i c a t i o n occurs when the v i r a l enzyme reverse t r a n s c r i p t a s e conver ts s i n g l e - s t r a n d e d v i r a l RNA i n t o double-s t randed DNA. 44 Binding and FIGURE 7. R e t r o v i r a l L i f e C y c l e . Adso rp t ion of v i r u s to the c e l l and b i n d i n g to a c e l l sur face receptor i s fo l lowed by the en t ry of s i n g l e -stranded RNA genome i n t o the cytoplasm. There i t i s reverse t r a n s c r i b e d i n t o a double stranded complementary DNA molecule . Th i s DNA as a c i r c u l a r molecule i s t ranspor ted to the nucleus where i t i n t e g r a t e s i n t o a host chromosome. The i n t eg ra t ed p r o v i r u s serves as a template for syn thes i s of the subgenomic mRNA's encoding the gag, p o l and env p ro t e in s as w e l l as f u l l - l e n g t h t r a n s c r i p t s of the v i r a l genome. These components are then packaged and shed from the c e l l as i n f e c t i o u s v i r u s . (From Reference 126). 45 The v i r a l genome c o n s i s t s of dimers of 2 i d e n t i c a l s t rands of RNA j o i n e d at the 5 ' ends and w i t h a complex secondary s t r u c t u r e . Dur ing the course of r e p l i c a t i o n , a p o r t i o n of the v i r a l RNA i s d u p l i c a t e d to form the i d e n t i c a l LTRs ( long t e r m i n a l repeats) found at the 5' and 3' end of p r o v i r a l DNA. The LTR u n i t represents a fus ion of sequences from the 3 ' end of v i r a l RNA (U3) , the R (repeated) sequence and sequences from the 5 ' end of v i r a l RNA (U5) i n the order 5 ' - U 3 - R - U 5 - 3 ' and i s terminated by shor t sequences forming i n v e r t e d and of ten imperfect repea ts . On comparing the s t r u c t u r e of v i r a l RNA and DNA, i t becomes c l e a r that DNA syn thes i s r equ i r e s the t r ans fe r of DNA s t rand twice between templates du r ing reverse t r a n s c r i p t i o n (F igure 8 ) . L i n e a r duplex v i r a l DNA appears i n the c e l l cytoplasm w i t h i n a few hours of i n f e c t i o n . From there i s must migrate to the nucleus where a second un in teg ra ted form of v i r a l DNA i s formed; c o v a l e n t l y c losed c i r c u l a r DNA c o n t a i n i n g 1 or 2 copies of the LTR. I t i s now known that the format ion of the c i r c u l a r DNA spec ies w i th the two LTRs i s necessary for p r o v i r a l i n t e g r a t i o n . The j u n c t i o n between the two LTRs i n the c i r c u l a r DNA forms a s o - c a l l e d "a t t " s i t e which i s necessary for i n t e g r a t i o n (131) . Al though we do not understand the enzymatic mechanisms i n v o l v e d , r e t r o v i r a l i n t e g r a t i o n i n t o host DNA i s a h i g h l y ordered process i n f l u e n c e d by sequences w i t h i n the v i r a l LTR. A r e l a t i v e l y s m a l l number (1-20) copies of p r o v i r a l DNA are i n t e g r a t e d per c e l l . Th i s may be a s m a l l percentage of the v i r a l DNA a c t u a l l y syn thes ized i n the c e l l . The s i t e of i n t e g r a t i o n appears to be random i n that no s p e c i f i c host n u c l e o t i d e sequence has been i d e n t i f i e d that favors i t nor has r e s t r i c t i o n enzyme mapping l oca t ed gross gene t i c sequences that are l i k e l y to surround the s i t e of v i r a l DNA e n t r y . In c o n t r a s t , the v i r a l n u c l e o t i d e s which j o i n to c e l l u l a r DNA are r i g i d l y determined. In a l l pub l i shed cases, e x a c t l y 2 base p a i r s are mi s s ing from the 5 ' end of the 5 ' LTR and from the 3 ' end of the 3' LTR. 46 WP U3 R T A ' R U5 HPB ^ B - - . - , , . ' W M ^ W W V W A . H P U3 R < A> (U5)HPB < // ^ j —J r HPB M P U3 R ' U6 + H P B ' < 1 i H—I 1 U3 R U5 H P B f-+* H4-* C M H Ht-> FIGURE 8. C r i t i c a l Steps i n the Synthes is of R e t r o v i r a l DNA. The p r iming events for (-) and (+) stands of v i r a l DNA (A and C) and the two t r ans fe r s of nascent s t rands between templates (B and D) , p i c t u r e d on an expanded s c a l e , l ead to p roduc t ion of a l i n e a r duplex w i t h two copies of the LTR u n i t (E and F; reduced s c a l e ) . RNA i s shown as wavy l i n e s , DNA as s t r a i g h t l i n e s w i t h arrows denot ing d i r e c t i o n of s y n t h e s i s ; the 5 ' end of (-) s t r and DNA i s i n d i c a t e d by a f i l l e d c i r c l e , the 5 ' end of (+) s t r and DNA by an open c i r c l e . ( - )PB i s the t r ans fe r RNA primer b i n d i n g s i t e , (+)P i s the p u t a t i v e (+) s t rand primer sequence. The shor t v e r t i c a l l i n e s des ignate the boundaries of U3,R, and U5 i n a l l pane l s . (A) A nascent (-) s t rand copy of R-U5 at the extreme 5 ' end of i t s template . (B) A f t e r removal of the 5 ' end of the primary template , the nascent (-) s t rand has base-pa i red w i t h the R sequence at the 3 ' terminus of the same or a companion RNA subuni t and i s extended a long i t s secondary template of v i r a l RNA. (C) Synthes i s of (+) s t r and DNA commences at a pr iming s i t e at the boundary of U3, and (+) s t r and i s extended through a p o r t i o n of the t r ans f e r RNA sequence o r i g i n a l l y bound to v i r a l RNA; the (-) s t r and c o n c u r r e n t l y e longates toward the 5 ' end of v i r a l RNA, i n t o or beyond the ( - )PB s i t e . (D) The nascent (-) s t rand i n (C) has base-pa i red w i t h the (+) s t r a n d , w i t h complementary sequences from the ( - )PB r e g i o n . The (+) s t rand i s then extended on i t s second template, (-) s t r and DNA, and the (-) s t rand i s extended by displacement syn thes i s a long i t s t h i r d template , (+) s t rand DNA. (E) The events i n (D) are shown at a reduced s c a l e to i n d i c a t e the use of e i t h e r one RNA subuni t ( l e f t ) or two ( r i g h t ) du r ing DNA s y n t h e s i s . (F) Complete ex tens ion of both (+) and (-) s t rands has produced a l i n e a r duplex terminated w i t h L T R ' s . (From Reference 127). 47 Once the p r o v i r u s has s u c c e s s f u l l y i n t eg ra t ed i t must be expressed to complete the v i r a l l i f e c y c l e . Host c e l l f ac to r s are r e s p o n s i b l e for p roduc t ion of v i r a l RNA, maintenance of the DNA template and s y n t h e s i s and p roces s ing of v i r a l p r o t e i n s . The v i r a l LTR conta ins sequences which i n i t i a t e t r a n s c r i p t i o n and a promoter which i s found i n the U3 r e g i o n . Sequences are a l s o found i n the LTR for p o l y a d e n y l a t i o n and t e rmina t ion of the RNA t r a n s c r i p t s . Many molecules of v i r a l RNA are produced per c e l l . Some v i r a l or RNA molecules code for the e n t i r e genome w h i l e others code on ly fo r 3 ' segments. The RNA molecules may be c leaved i n t o i n d i v i d u a l p r o t e i n coding sequences before p r o t e i n t r a n s l a t i o n or the whole RNA molecule may be processed and the p r o t e i n product c leaved i n t o components l a t e r . 0.1% to 1% of t o t a l c e l l u l a r RNA i n a p r o d u c t i v e l y i n f e c t e d c e l l i s v i r a l - s p e c i f i c . Among mRNA molecules 5 to 10% may be of v i r a l o r i g i n , s e v e r a l thousand cop ies per c e l l . V i r a l p ro t e in s are syn thes ized on c e l l u l a r r ibosomes, modi f ied by g l y c o s y l a t i o n and/or phosphory la t ion , then c leaved and assembled a long w i t h the RNA genome, i n t o v i r a l p a r t i c l e s . The v i r u s e s then e x i t the c e l l by budding from plasma membranes and mature upon re lease (F igure 7 ) . There are many ways i n which the r e t r o v i r u s and the host c e l l can a f f e c t each o t h e r ' s behav io r . A f t e r v i r a l pene t r a t ion some of the f i r s t ins tances are seen at the time of p r o v i r a l i n t e g r a t i o n . R e t r o v i r u s e s may act as mutagens i f they i n t e g r a t e w i t h i n a c e l l u l a r gene and des t roy i t s f u n c t i o n , e . g . the transformed phenotype of some Rous sarcoma v i r u s (RSV) has been reve r t ed to normal by s u p e r i n f e c t i o n of the c e l l s w i th a nontransforming murine leukemia v i r u s which was shown to have in t eg ra t ed w i t h i n the RSV genome (128) . The 3 ' v i r a l LTR may act as a promoter for a c e l l u l a r gene that l i e s nearby. An example of the l a t t e r would be the av ian l e u k o s i s v i r u s which 48 acqu i r e s t ransforming a b i l i t y when i t i n t eg ra t e s near the c e l l u l a r "myc" gene (96 ) . Other a c u t e l y t ransforming r e t r o v i r u s e s appear to have been c rea ted by recombina t ion between the p r o v i r u s and c e l l u l a r DNA a l l o w i n g the v i r u s to a c q u i r e a c e l l u l a r sequence, an oncogene which i s r e spons ib l e fo r the t rans forming a c t i v i t y of the v i r u s (132) . There are now at l e a s t 30 examples of t h i s phenomenon. A r e l a t e d t h e o r e t i c a l , but as yet undemonstrated phenomenon, would be the t r a n s p o s i t i o n of a c e l l u l a r gene from one l o c a t i o n to another w i t h i n the genome much as t ransposable elements move gene t i c m a t e r i a l i n lower organisms. F i n a l l y , the v i r u s i t s e l f may be a l t e r e d a f t e r the process of i n t e g r a t i o n . As components of c e l l chromosomes, the p r o v i r u s i s subjec t to the same muta t iona l r i s k s and r e g u l a t o r y i n f luences as host DNA. I f any th ing , p r o v i r i o n s appear to be more l a b i l e than host chromosomes. S tud ies of c loned v i r a l transformed l i n e s have a l lowed the d i s c o v e r y of d e l e t i o n s , po in t muta t ion , d u p l i c a t i o n s of coding sequences, nonsense mutat ions and i n s e r t i o n a l mutations w i t h i n p r o v i r a l DNA and a f f e c t i n g v i r a l e x p r e s s i o n . Dur ing t r a n s c r i p t i o n , host c e l l f ac to r s a l s o operate to modify the e f f i c i e n c y of v i r a l RNA produc t ion (129) . The f ac to r s which cause the same p r o v i r u s to produce d i f f e r e n t amounts of RNA i n d i f f e r e n t c e l l s are p o o r l y understood but may i n c l u d e the nature of f l a n k i n g c e l l u l a r DNA sequences ( c i s f a c t o r s ) , the me thy la t ion s t a t e of p r o v i r a l DNA, and the host chromosome s t r u c t u r e i n the r eg ion of v i r a l i n t e g r a t i o n which could a f f e c t the 2° s t r u c t u r e of p r o v i r a l DNA. Regardless of the reasons for v a r i a t i o n s i n v i r a l RNA e x p r e s s i o n , there are now many ins tances where i t has been shown to vary by s e v e r a l logs (128) . For example: i ) s t r a i n s of the Rous sarcoma v i r u s which w i l l s u c c e s s f u l l y i n f e c t mammalian, as w e l l as av ian c e l l s , show 2-3 logs l e s s v i r a l RNA i n the mammalian t a r g e t s . 49 i i ) d i f f e r e n t v i r a l l y - i n f e c t e d clones of the same c e l l type containing the same type of v i r a l DNA w i l l show differences i n the number of v i r a l RNA molecules produced per c e l l from 0 to several thousands. i i i ) d i f f e r e n t proviruses of the same type within the same c e l l can show 1 to 2 log differences i n expression. iv) over time, a sing l e provirus within a c e l l may show changes i n v i r a l expression as shown by the spontaneous reversion to normal from a transformed phenotype of many v i r a l l y transformed c e l l l i n e s . This occurs without any observable change in the p r o v i r a l genome and i s caused by presumed epigenetic mechanisms. v) transacting factors, such as hormones, may also interact with the provirus, e.g. steroids interact with sequence i n the U3 region of the mouse mammary tumor virus LTR increasing v i r a l t r a n s c r i p t i o n and leading to the malignant transformation of c e l l s caused by the v i r u s . The general condition of the infected c e l l s also a f f e c t s the l e v e l of v i r a l expression. In p a r t i c u l a r , c e l l s a c t i v e l y i n cycle, p a r t i c u l a r l y i n the DNA synthesis phase, support the synthesis of v i r a l DNA whereas noncycling c e l l s do not. C e l l u l a r DNA synthesis i s necessary for p r o v i r a l i n t e g r a t i o n and v i r a l r e p l i c a t i o n and i s more e f f i c i e n t i n c y c l i n g c e l l s . In many c e l l s , one round of mitosis a f t e r the acute v i r a l i n f e c t i o n may be necessary to i n i t i a t e v irus production. Terminally d i f f e r e n t i a t e d c e l l s are not able to produce v i r a l DNA, at least partly because they are no longer able to divide themselves. Although i t i s clear that the v i r a l l i f e cycle proceeds most e f f i c i e n t l y i n c y c l i n g c e l l u l a r hosts, the requirement may not be absolute i n a l l circumstances. It has been shown that infected c e l l s which have been maintained i n a stationary state by serum starvation and which are not 50 producing v i r u s w i l l begin to show v i r a l r e p l i c a t i o n when the c e l l s are re fed serum and enter c e l l c y c l e . In s p i t e of the many f a c t o r s , both known and unknown as o u t l i n e d above, that w i l l a f f e c t the success of gene t r ans fe r mediated by r e t r o v i r a l v e c t o r s , the concept i s s t i l l more encouraging for many types of work than any other technique a v a i l a b l e at present . The p o t e n t i a l advantages of r e t r o v i r u s e s i n c l u d e (111) : i ) An e f f i c i e n c y of c e l l i n f e c t i o n approaching 100%. Th i s means that not on ly a l a rge number of c e l l s r ece ive f o r e i g n genes but that the o c c a s i o n a l ra re target i n a l a rge popu la t ion of l e s s i n t e r e s t i n g c e l l s , fo r example the hemopoiet ic stem c e l l among d i f f e r e n t i a t i n g bone marrow c e l l s , may be i n f e c t e d at h igh frequency. i i ) R e t r o v i r a l DNA g e n e r a l l y i n t eg ra t e s i n t o host c e l l chromosomes as a s i n g l e copy. This i s i n con t ras t to other methods of gene t r a n s f e r where the DNA u s u a l l y e x i s t s i n the r e c i p i e n t c e l l as a concatamer of as many as s e v e r a l hundred copies i n t eg ra t ed at a s i n g l e s i t e . In some s i t u a t i o n s t h i s may be important for gene f u n c t i o n or e x p r e s s i o n . i i i ) The s t r u c t u r e of the i n t eg ra t ed p r o v i r a l DNA i s maintained i n most i n f e c t e d c e l l s . Al though recombinat ion events do occur among r e t r o v i r u s e s , t h i s i s a r e l a t i v e l y rare event compared to the p o s s i b i l i t y of d e l e t i o n or rearrangement of the t r a n s f e r r e d DNA i n chemica l or p h y s i c a l techniques . i v ) R e t r o v i r a l i n f e c t i o n i s non tox ic to the target c e l l s . Again except ions occur ( e . g . Human immunodeficiency v i r u s appears t o x i c to many lymphocytes) but there are a v a i l a b l e many r e t r o v i r a l vec to r s that do not harm the i n f e c t e d c e l l s or a l t e r t h e i r growth i n any measurable way. 51 v) There are a v a i l a b l e a l a r g e number of p o t e n t i a l r e t r o v i r a l v e c t o r s w i t h many d i f f e r e n t host and t i s s u e type ranges such that there should be v i r t u a l l y no l i m i t to the target c e l l employed. v i ) The r e t r o v i r u s provides i t s own promoter for gene expres s ion i n i t s LTR. Th i s should a l l o w many fo r e ign genes to be expressed i n the host c e l l wi thout fu r the r man ipu l a t i on . For example, the neomycin r e s i s t a n c e gene (neo r ) can be expressed i n mouse bone marrow c e l l s from the r e t r o v i r a l LTR (8 ,133) . There a l s o e x i s t r e t r o v i r u s e s w i t h enhancers that can be manipulated by chemicals or hormones, such as the mouse mammary tumor v i r u s . Use of vec to r s based on such a v i r u s would a l l o w f l e x i b i l i t y and some c o n t r o l over how the host c e l l expressed the t r ans fe r r ed genes. The s u c c e s s f u l express ion of genes c a r r i e d by r e t r o v i r u s e s i s an important con t ras t to genes in t roduced by p h y s i c a l or chemical techniques where "normal" express ion i s the excep t ion ra ther than the r u l e . To c o r r e c t t h i s problem, the exogenous DNA i s of ten l i n k e d to a f o r e i g n promoter before use i n experiments i n v o l v i n g techniques such a m i c r o i n j e c t i o n (119) . However, on ly a handful of known genomic promoters have been s u c c e s s f u l i n t h i s s i t u a t i o n , e . g . m e t a l l o t h i o n e i n , immunoglobulin, t r a n s f e r r i n , e l a s t a s e . Many more such promoters f a i l to func t ion a f t e r DNA-mediated gene t r a n s f e r perhaps due to methy la t ion or other unknown mechanisms. Thus, the a b i l i t y of the r e t r o v i r u s to enhance i t s own express ion may prove to be one of i t s most a t t r a c t i v e f ea tu res . There are a number of t h e o r e t i c a l and r e a l disadvantages to r e t r o v i r a l gene d e l i v e r y systems (111) . 52 i ) There i s an i n t r i n s i c l i m i t to the s i z e of the DNA which can be i n s e r t e d i n t o these v e c t o r s . The Moloney murine leukemia v i r u s (MoMuLV) must not be l a r g e r than 9 to 12 kb i n order to be packaged. Two to 3 Kb are necessary for e s s e n t i a l f u n c t i o n l e a v i n g 6 to 9 Kb a v a i l a b l e for i n s e r t s . i i ) The DNA to be t r ans fe r r ed must be a c loned fragment w h i l e whole c e l l u l a r DNA can be used i n p h y s i c a l techniques such as ca l c ium phosphate t r a n s f e c t i o n . i i i ) The use of t r a d i t i o n a l r e t r o v i r a l vec to r s r e s u l t s i n a p roduc t i ve i n f e c t i o n of ta rget c e l l s which can spread to other c e l l s beyond the i n t e n t i o n of the i n i t i a l experiment. Th i s problem appears to have been overcome by newer cons t ruc ted vec to r s to be desc r ibed below. i v ) R e t r o v i r u s e s appear to have a s t rong p ropens i ty for d e l e t i n g sequences dur ing v i r u s r e p l i c a t i o n . For example, the murine sarcoma v i r u s (MSV) DHFR-NEO vec to r which produces neo r e x p r e s s i o n i n mice has l o s t a p o r t i o n of i t s DHFR gene dur ing p roduc t ion of the v i r a l p a r t i c l e s (134) . C e l l r e p l i c a t i o n a l s o appears to be necessary for v i r a l i n t e g r a t i o n . Th i s would make i t imposs ib l e to i n f e c t n o n d i v i d i n g c e l l s such as b r a i n c e l l s . v) The randomness of r e t r o v i r a l i n t e g r a t i o n may prove to be a d isadvantage . I t would be important for some experiments to d i r e c t the vec to r to a s p e c i f i c chromosomal s i t e . At present t h i s appears to be a formidable task i n mammalian c e l l s . v i ) Al though the r e t r o v i r a l LTR appears to func t ion i n most c e l l l i n e s as an e f f i c i e n t promoter i n primary c e l l s , p a r t i c u l a r l y those which are p r i m i t i v e or u n d i f f e r e n t i a t e d such as found i n embryos and 53 perhaps hemopoiet ic stem c e l l s , i t appears to operate l e s s w e l l ( 8 , 9 , 1 3 5 ) . Th i s may mean that exogenous promoters w i l l have to be l i n k e d to the gene to be transformed by the r e t r o v i r u s i n order to achieve adequate gene exp re s s ion . F . C o n s t r u c t i o n of R e t r o v i r a l Vec tors (111,127) (F igure 9) The p r o v i r a l DNA for the des i r ed r e t r o v i r a l v e c t o r , commonly e i t h e r the Moloney murine leukemia v i r u s (MoMuLV) or murine sarcoma v i r u s (MSV), i s i n s e r t e d i n t o a convenient b a c t e r i a l plasmid to make i t p o s s i b l e to produce i t i n l a r g e q u a n t i t i e s . The v i r a l s t r u c t u r a l genes can then be rep laced w i t h the exogenous genes of choice by standard recombinant DNA techniques . Th i s v e c t o r i s now incompetent to r e p l i c a t e because i t l a c k s the a b i l i t y to produce a f u l l complement of v i r a l p r o t e i n s . The vec to r cons t ruc t i s used to t r ans f ec t c e l l s such as NIH-3T3 c e l l s by ca lc ium phosphate c o - p r e c i p i t a t i o n . I f such c e l l s a l r eady con t a in packaging or he lper v i r u s p r o v i r a l DNA, appropr i a t e v i r a l p r o t e i n s are a v a i l a b l e to package the recombinant v i r u s . The recombinant v i r u s i s produced and buds o f f the c e l l s i n t o the medium. E i t h e r the medium or the v i r a l producer c e l l s can be used to i n f e c t target c e l l s . However, i f an i n t a c t he lper v i r u s such as the Moloney murine leukemia v i r u s (MoMuLV) i s used, he lpe r v i r u s as w e l l as recombinant v i r u s i s produced and the s u c c e s s f u l l y i n f e c t e d target c e l l s themselves now become i n f e c t i o u s . The continuous p roduc t ion of v i r u s , . b o t h he lper and recombinant, by i n f e c t e d ta rge t c e l l s w i l l be a disadvantage for many experiments i n c l u d i n g any attempt at gene t r ans f e r to human be ings . Two groups of i n v e s t i g a t o r s have developed r e t r o v i r a l packaging mutants that produce a d e f e c t i v e he lpe r r e t r o v i r u s (136,137) . A s i t e 5 ' of the r e t r o v i r a l gag gene termed the T r e g i o n has been found necessary for v i r a l packaging i n both the Rous sarcoma 54 PRODUCTION OF RETROVIRUSES FOR G E N E T R A N S F E R st«p ^Const ruc t Recombinant Virus in a Plasmid BUILDING BLOCKS 'Packaging Cell*' provide structural protein FIGURE 9. C o n s t r u c t i o n of a Recombinant R e t r o v i r u s and Genera t ion of V i r a l Stocks for Gene Transfer Exper iments . Step 1: V i r a l r egu l a to ry sequences present p r i m a r i l y i n the long t e r m i n a l repeat (LTR) of the r e t r o v i r a l genome and the gene of i n t e r e s t to be t r ans fe r r ed (neo r ) are i n s e r t e d i n t o a b a c t e r i a l p lasmid vec to r to a l l o w l a rge q u a n t i t i e s of the recombinant molecule to be generated i n b a c t e r i a l hos t s . Step 2: Plasmid DNA i s t r ans fec ted i n t o a packaging c e l l l i n e and i n t e g r a t e s i n t o the host c e l l DNA. The recombinant p r o v i r a l DNA provides the template for v i r a l RNA s y n t h e s i s . The packaging c e l l l i n e con ta ins v i r a l DNA sequences that code for v i r a l s t r u c t u r a l p r o t e i n s and a l l o w the recombinant v i r a l DNA to be packaged and bud from the c e l l s . I l l u s t r a t e d i s a "helper d e f e c t i v e " packaging c e l l i n which the he lper p r o v i r a l DNA l a c k s sequences necessary fo r he lper v i r u s r e p l i c a t i o n (see t e x t ) . Only recombinant " n e o r " v i r u s i s produced by t h i s c e l l . 55 v i r u s and MoMuLV (F igu re 10) . A d e l e t i o n of 350 bp fragment encompassing t h i s r e g i o n between the 5 ' LTR and the gag codon r e s u l t s i n a recombinant v i r u s which w i l l p rovide v i r a l p ro t e in s for another d e f e c t i v e recombinant v i r u s coding for exogenous genes without r e p l i c a t i n g i t s e l f . The p r o v i r a l DNA of the Y d e f i c i e n t vec to r i s i n s e r t e d i n t o NIH-3T3 c e l l s . P r o v i r a l DNA c o n t a i n i n g the exogenous gene of i n t e r e s t i s i n s e r t e d i n t o the same c e l l s by c a l c i u m phosphate t r a n s f e c t i o n . V i r a l p ro t e in s produced by the Y d e f i c i e n t p r o v i r u s are used to s u c c e s s f u l l y package the other recombinant v i r u s , which con ta ins the i n t a c t Y r e g i o n , and which then buds o f f the c e l l s and can be used to i n f e c t ta rge t c e l l s . The i n f e c t i n g v i r u s cannot r e p l i c a t e i n the t a rge t because i t l a c k s genes coding for v i r a l p r o t e i n s . The v i r u s thus ac t s as a gene d e l i v e r y system ra the r than an i n f e c t i o u s agent. U n f o r t u n a t e l y , i n p r a c t i c e when such Y-he lper v i r u s e s are used s u f f i c i e n t recombinat ion occurs between he lper and recombinant v i r a l genomes such that he lpe r v i r u s i s generated w i t h unacceptably h igh frequency i n many cases . To overcome t h i s problem fu r the r d e l e t i o n s have been made i n the he lper v i r u s genome p r i m a r i l y i n the LTRs. Th i s s t r a t egy appears to make recombinat ion events and the genera t ion of he lper v i r u s h i g h l y u n l i k e l y i f not imposs ib l e (138) . I t would be important to develop packaging d e f e c t i v e v i r u s e s of broader host range than the MoMuLV for experiments u s ing human c e l l s . Two groups of i n v e s t i g a t o r s have accomplished t h i s by r e p l a c i n g the env gene i n the Y d e f i c i e n t MoMuLV w i t h the env from an amphotropic r e t r o v i r u s (137,139) (F igu re 10) . There i s concern, p a r t i c u l a r l y among those hoping to use r e t r o v i r u s e s for gene therapy i n humans, that the v i r a l LTRs may act as abnormal promoters for c e l l u l a r genes w i t h p o t e n t i a l d e l e t e r i o u s r e s u l t s fo r the target c e l l s . CONSTRUCTION OF AN AMPHOTROPIC PACKAGING-DEFECTIVE VIRUS E c o t r o p i c MoMLV Amphotropic V i rus Amphotropic MoMLV Amphotropic Packaging-D e f e c t i v e V i r u s DCLt-TED FIGURE 10. Cons t ruc t ion of a ? " Amphotropic R e t r o v i r u s . A b b r e v i a t i o n s : MoMLV, Moloney murine leukemia v i r u s ; LTR, long t e rmina l repeat ; SD, s p l i c e donor s i t e for generat ion of env MRNA; V, packaging s i g n a l ; gag, v i r a l core p ro t e in s ; p o l , reverse t r a n s c r i p t a s e ; SA, s p l i c e acceptor fo r genera t ion of env mRNA; env, envelope p r o t e i n s . The env reg ion of the MoMLV i s rep laced w i t h the env of the amphotropic v i r u s to produce an amphotropic MoMLV. The packaging s i n g n a l i s then de le ted to g ive an amphotropic v i r u s that w i l l provide p ro te ins to package a recombinant v i r u s but cannot package i t s e l f . 57 S i t u a t i o n s e x i s t i n animal systems where, fo r example, the av i an l e u k o s i s v i r u s causes inc reased express ion of the c e l l u l a r myc gene w i t h r e s u l t i n g malignant t r ans format ion of the i n f e c t e d c e l l s (96) . For t h i s reason some i n v e s t i g a t o r s are deve lop ing r e t r o v i r a l vec to r s w i t h " c r i p p l e d " LTRs which enable the v i r u s to i n t e g r a t e i n t o host DNA, but which cannot promote or enhance gene expres s ion (140) . With these vec to r s i t w i l l be necessary to p rov ided an exogenous promoter and enhancer sequence to see exp res s ion of the p r o v i r a l DNA. G. S tudies on R e t r o v i r a l - M e d i a t e d Gene Transfe r to Hemopoietic C e l l s i ) Murine Studies Seve ra l i n v e s t i g a t o r s have now shown that i t i s p o s s i b l e to i n t roduce f o r e i g n genes i n t o murine bone marrow stem c e l l s and committed p rogen i to r s u s i n g recombinant r e t r o v i r u s e s . The f i r s t repor t of the s u c c e s s f u l use of r e t r o v i r u s e s to t r ans fe r genes i n t o hemopoiet ic c e l l s demonstrated G418 r e s i s t a n t granulocyte-macrophage colony format ion a f t e r exposure of mouse bone marrow c e l l s to v i r u s c o n t a i n i n g the gene for neomycin r e s i s t a n c e ( n e o r ) (133) . Subsequently, s e v e r a l groups have repor ted h igh e f f i c i e n c i e s of gene t r a n s f e r to more p r i m i t i v e hemopoiet ic c e l l s i n c l u d i n g CFU-S ( 8 , 9 , 1 3 4 , 1 4 1 ) . S u c c e s s f u l l y i n f e c t e d c e l l s have a l s o r e c o n s t i t u t e d W/Wv mice or normal i r r a d i a t e d r e c i p i e n t s ( 8 , 9 ) . Gene t r ans fe r e f f i c i e n c i e s to CFU-S approaching 100% have been obta ined us ing h igh t i t e r v i r u s (141,142) . Var ious f a c t o r s may be important to a l l o w such h igh e f f i c i e n c i e s . These i n c l u d e , c o - c u l t i v a t i o n of ta rge t w i t h v i r a l producer c e l l s , pre- treatment of mice w i t h 5 f l u o r o u r a c i l and the a d d i t i o n of growth f ac to r s such as I L - 3 dur ing the i n f e c t i o n p e r i o d . These l a t t e r two manipula t ions presumably act by i n c r e a s i n g the p r o p o r t i o n of ta rge t c e l l s i n the a c t i v e part of the c e l l c y c l e . 58 As mentioned i n a previous s e c t i o n , r e t r o v i r a l marking of hemopoie t ic stem c e l l s has been used to analyze pa t te rns of bone marrow r e p o p u l a t i o n and p r o g e n i t o r d i f f e r e n t i a t i o n i n v i v o . The s i t e of r e t r o v i r a l i n t e g r a t i o n i n t a rge t c e l l DNA i s random and provides a unique molecular marker for that c e l l and i t s progeny. In fec ted bone marrow c e l l s have been i n j e c t e d i n t o l e t h a l l y -i r r a d i a t e d r e c i p i e n t s . In some cases the pa t t e rn of r e t r o v i r a l i n t e g r a t i o n i n t o sp l een , bone marrow and thymus DNA from r e c o n s t i t u t e d mice i n d i c a t e s that a s i n g l e c lone can sometimes repopulate a l l three t i s s u e s ( 8 , 9 ) . Thus, a p l u r i p o t e n t stem c e l l of both myeloid and lymphoid p o t e n t i a l has been i n f e c t e d and t h i s c e l l has an enormous p r o l i f e r a t i v e c a p a c i t y . In the same experiment p rogen i to r s of more r e s t r i c t e d p o t e n t i a l were i d e n t i f i e d that populated o n l y the thymus or the bone marrow. Long term s tud i e s have been performed on such r e c o n s t i t u t e d animals and success ive waves of p r o l i f e r a t i o n have been i d e n t i f i e d from progen i to r s of d i f f e r e n t c l o n a l i t y (142) . That i s , a c lone that has repopula ted bone marrow may disappear and be rep laced by c e l l s of d i f f e r e n t c l o n a l i t y on ly to reappear at a l a t e r t ime. The fac t that very few stem c e l l c lones may be a c t i v e at a g iven time i n murine t r ansp lan t r e c i p i e n t s and the evidence for c l o n a l success ion of these c e l l s may be r e l evan t to human marrow t r a n s p l a n t a t i o n and hemopoiesis . In con t ra s t to the above s tud i e s where gene t r ans fe r was c l e a r l y h i g h l y e f f i c i e n t experiments designed to show express ion of the f o r e i g n gene i n pr imary hemopoiet ic c e l l s have been much l e s s s u c c e s s f u l . Most pub l i shed r e s u l t s have used vec to r s i n which promoter func t ion for the t r a n s f e r r e d gene would be provided by the r e t r o v i r a l LTR or SV40 v i r a l sequences. P rogen i t o r s d e r i v e d from CFU-S known to ca r ry the neo r have g iven r i s e to c o l o n i e s on ly 10% of which show G418 r e s i s t a n c e ( 8 , 9 ) . S i m i l a r d i f f i c u l t i e s have been encountered w i t h express ion of the human adenosine deaminase (hADA) gene 59 (143) . These vec to r s which r e s u l t i n poor express ion from CFU-S appear to f u n c t i o n w e l l i n e s t a b l i s h e d c e l l l i n e s or more d i f f e r e n t i a t e d p r o g e n i t o r s such as CFU-GM. Mechanisms may be a c t i v e i n p r i m i t i v e p l u r i p o t e n t c e l l s which r e s u l t i n i n a c t i v a t i o n of c e r t a i n promoters such as that found i n the r e t r o v i r a l LTR. Vec to rs which r e l y on d i f f e r e n t promoters for gene exp re s s ion are c u r r e n t l y being tes ted i n a number of l a b o r a t o r i e s . P r e l i m i n a r y evidence i n d i c a t e s that the herpes s implex thymidine k inase promoter or m y e l o p r o l i f e r a t i v e sarcoma v i r u s may a l l o w improved gene expres s ion i n p r i m i t i v e hemopoiet ic c e l l s (135,144) . P r e l i m i n a r y s tud i e s e x p l o r i n g the p o t e n t i a l r o l e of genes such as oncogenes or growth f ac to r s i n malignant t rans format ion of hemopoiet ic c e l l s have been conducted by a number of workers u s ing r e t r o v i r a l - m e d i a t e d gene t r a n s f e r to murine c e l l s . Lang et a l (145) i n f e c t e d a growth factor-dependent hemopoiet ic c e l l l i n e w i t h a v i r u s c o n t a i n i n g the mGM-CSF gene. I n f ec t ed c e l l s not on ly l o s t t h e i r dependence on exogenous growth f a c t o r but became leukemogenic when i n j e c t e d i n t o i r r a d i a t e d mice sugges t ing that escape from "normal" growth c o n t r o l i n these hemopoiet ic c e l l s was r e l a t e d to t h e i r malignant p o t e n t i a l . In somewhat analogous s tud i e s Wi t t e et a l (98) i n f e c t e d l ong term c u l t u r e s of murine c e l l s designed to favor the growth of B c e l l s w i t h v i r u s e s c o n t a i n i n g v i r a l oncogenes; H- ra s , v-myc, or both i n combina t ion . Growth of p r i m i t i v e B c e l l s was g r e a t l y enhanced i n the c u l t u r e s i n f e c t e d w i t h both v i r u s e s and the i n f e c t e d B c e l l s caused B - c e l l lymphomas i n mice . In t h i s study i t appears that the t r ans fe r r ed genes were ca r c inogen i c fo r murine B c e l l s . In a d d i t i o n to the i n t r i n s i c i n t e r e s t of the s tud i e s themselves, r e s u l t s such as those desc r ibed above provide a bas i s for the b e l i e f that the technique of r e t r o v i r a l - m e d i a t e d gene t r ans fe r i s a u s e f u l t o o l for d i s s e c t i o n of the gene t i c events i n v o l v e d i n malignant t r ans fo rma t ion . 60 i i ) Human Studies R e t r o v i r u s e s have a l s o been used for gene t r ans fe r to human c e l l s . Al though the a v a i l a b l e data i s l e s s ex tens ive than for murine t a rge t s i t has been p o s s i b l e to demonstrate c o r r e c t i o n s of hypoxanthine phosphor ibosy l t r ans fe rase (HPRT) d e f i c i e n c y i n human f i b r o b l a s t s (146) and B lymphoblas ts (147) a f t e r i n f e c t i o n w i t h an HPRT-conta in ing v i r u s . S i m i l a r r e s u l t s have been repor ted for experiments i n which a v i r u s c o n t a i n i n g the hADA gene was used to i n f e c t human ADA d e f i c i e n t B and T lymphocytes (143,148) . Evidence of sus t a ined r e t r o v i r a l i n f e c t i o n i n human long term bone marrow c u l t u r e s w i t h v i r u s c o n t a i n i n g oncogenes or the human HPRT gene has a l s o been repor ted (149 ,150) . However, q u a n t i t a t i v e s tud i e s of the l e v e l of gene t r a n s f e r or exp res s ion i n hemopoiet ic p rogen i to r s i n these c u l t u r e s was not determined. Hock and M i l l e r have r e c e n t l y pub l i shed more p r e c i s e data demonstra t ing r e t r o v i r a l - m e d i a t e d gene t r ans fe r to primary human marrow p rogen i to r s u s i n g r e t r o v i r u s e s c a r r y i n g the neo r or mutant d i h y d r o f o l a t e reductase gene (151) . In summary, gene t r ans fe r v i a r e t r o v i r u s e s i s not on ly f e a s i b l e but very e f f i c i e n t u s ing murine hemopoiet ic t a r g e t s . The a v a i l a b l e data on human hemopoiet ic c e l l s i s very l i m i t e d but a l s o i n d i c a t e s the f e a s i b i l i t y of the technique . Problems of o b t a i n i n g h igh l e v e l s of express ion of the t r a n s f e r r e d gene appear to be cons ide rab le when p r i m i t i v e primary c e l l s such as hemopoie t ic stem c e l l s are the t a r g e t s . Never the le s s , r e t r o v i r a l - m e d i a t e d gene t r a n s f e r i s an e x c i t i n g technique for use both i n the gene t i c l a b e l l i n g of c e l l s and i n the study of gene r e g u l a t i o n . 6) PRESENT OBJECTIVES Ma l ignanc i e s of the hemopoiet ic system present c l i n i c a l l y as the d i s o r d e r e d p roduc t ion of blood c e l l s . Al though the malignant c e l l s may 61 d i f f e r e n t i a t e along several lineages, i n most, i f not a l l , of these diseases, the e s s e n t i a l malignant event(s) occurs i n a single c e l l which then p r o l i f e r a t e s , i . e . hemopoietic malignancies are clonal i n o r i g i n . The hypothesis behind my research i s that genetic change i s fundamental to the development of malignancy. The purpose of this research was to explore methods to study not only the c e l l i n which the changes occur that give r i s e to hemopoietic cancer but also to investigate the s p e c i f i c abnormalities at a molecular l e v e l . In the f i r s t phase of my research, I used cytogenetic analysis of c e l l s from i n d i v i d u a l hemopoietic colonies of various lineages to demonstrate that, i n a myelodysplastic disorder of childhood associated with monosomy of chromosome 7, the malignancy arose i n a pluripotent stem c e l l capable of d i f f e r e n t i a t i o n down both myeloid and erythroid pathways. Although this i s a r e l a t i v e l y unusual p e d i a t r i c disorder, i t i s c l i n i c a l l y and cyto g e n e t i c a l l y very s i m i l a r to the increasingly common myelodysplasias of adulthood. Both diseases t y p i c a l l y terminate i n refractory acute leukemia. Thus, i t i s l i k e l y that myelodysplasic disorders originate i n a very primitive hemopoietic progenitor which i n i t i a l l y retains some of i t s a b i l i t y to d i f f e r e n t i a t e into functional c e l l s . However with time, progressive changes occur and a part of the clone loses this capacity and i s seen as leukemic b l a s t s . In the second part of my research, I took a d i f f e r e n t approach to study the c l o n a l evolution of malignant c e l l s . In this case, using the disorder CML and G6PD isoenzyme analysis, I investigated the p o s s i b i l i t y that there might be cytogenetically normal hemopoietic progenitors that would, nevertheless, be part of the malignant clone. Long-term bone marrow cultures from patients with CML are known to favour the growth of Ph-negative progenitors. Such cultures were i n i t i a t e d with c e l l s from 2 women who were heterozygous for 2 62 G6PD enzyme v a r i a n t s . In one of these pa t i en t s a . p r o p o r t i o n of hemopoie t ic p r o g e n i t o r s was found to be Ph-negat ive a f t e r 4 to 6 weeks i n long- te rm c u l t u r e . A s i m i l a r p r o p o r t i o n of p rogen i to r s were found to be n o n - c l o n a l by G6PD enzyme a n a l y s i s , i . e . some c o l o n i e s expressed the enzyme not found i n the malignant c l o n e . In t h i s case k a r y o t y p i c a l l y normal c e l l s appear to be t r u l y normal and a Ph-negat ive c l o n a l popu la t ion has not been i d e n t i f i e d . Al though i t i s c l e a r l y p o s s i b l e to i d e n t i f y the G6PD enzyme produced by i n d i v i d u a l hemopoiet ic c o l o n i e s , the technique i s l i m i t e d i n i t s a p p l i c a b i l i t y to the 1/3 of b l a c k women who are heterozygous for two e l e c t r o p h o r e t i c a l l y d i s t i n c t enzyme types . Exper imenta l approaches which would be v a l i d for a l a r g e r p r o p o r t i o n of c l i n i c a l samples are necessary before such s tud i e s can be c a r r i e d out i n a more comprehensive f a s h i o n . The t h i r d phase of my research i nvo lved deve lop ing the technique of r e t r o v i r a l - m e d i a t e d gene t r ans fe r to move f o r e i g n genes i n t o human hemopoie t ic p r o g e n i t o r s . The purpose of t h i s was t w o - f o l d . F i r s t l y , marking the DNA of hemopoie t ic p rogen i to r s w i th a f o r e ign gene would a l l o w them and t h e i r progeny to be fo l lowed through the processes of 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 . V a r i o u s c l o n a l popu la t ions of marked c e l l s could be i d e n t i f i e d by the unique s i t e of i n t e g r a t i o n of the r e t r o v i r a l genome i n t o c e l l u l a r DNA.. Secondly , genes of p o t e n t i a l i n t e r e s t i n the study of hemopoiesis , e . g . growth f a c t o r s or oncogenes, cou ld be t r ans fe r r ed i n t o target c e l l s to a l l o w the study of gene f u n c t i o n or r e g u l a t i o n . I have demonstrated the f e a s i b i l i t y of r e t r o v i r a l - m e d i a t e d gene t r ans f e r to human hemopoiet ic c e l l s u s ing recombinant v i r u s c a r r y i n g the s e l e c t a b l e marker gene, n e o r , which confer r e s i s t a n c e to the neomycin analogue G418 on s u c c e s s f u l l y i n f e c t e d c e l l s . These experiments e s t a b l i s h e d the f e a s i b i l i t y of r e t r o v i r a l - m e d i a t e d gene t r a n s f e r to human hemopoiet ic c e l l s . Fur ther experiments u s ing bone marrow 63 c e l l s i n long- te rm c u l t u r e as a target and the n e o r v i r u s as i n f e c t i n g agent were performed w i t h the hope of showing s u c c e s s f u l and s t a b l e gene t r a n s f e r over a pe r iod of s e v e r a l months. These were a l s o p r e l i m i n a r y experiments to those i n which we p lan to use v i r u s e s c o n t a i n i n g genes more pe r t i nen t to the s tudy of hemopoiet ic r e g u l a t i o n , e . g . growth f ac to r s or oncogenes. In summary, the experiments descr ibed i n t h i s t he s i s w i l l show tha t : i ) Hemopoietic p rogen i to r s can be s tud ied i n c u l t u r e u s ing s e v e r a l gene t i c markers. Cytogene t ic a n a l y s i s can be used i n p a t i e n t s w i t h a hemopoiet ic malignancy c h a r a c t e r i z e d by an abnormal ka ryo type . Where chromosomal markers are l a c k i n g other techniques such as G6PD isoenzyme a n a l y s i s or gene t r ans fe r may provide ways to i d e n t i f y unique popula t ions of c e l l s . i i ) R e t r o v i r a l - m e d i a t e d gene t r ans fe r may provide ways to study the r e g u l a t i o n of normal and malignant hemopoiesis at the molecu la r l e v e l u s ing i n v i t r o model system such as long- term bone marrow c u l t u r e . 64 REFERENCES 1. T i l l J E , McCul loch EA. A d i r e c t measurement of the r a d i a t i o n s e n s i t i v i t y of normal mouse bone marrow c e l l s . Radia t Res 14: 213, 1961. 2 . Fowler J H , Wu AM, T i l l J E , McCul loch EA, S i m i n o v i t c h L . The c e l l u l a r composi t ion of hemopoiet ic sp leen c o l o n i e s . J C e l l P h y s i o l 69: 65, 1967. 3. Becker A J , McCul loch EA, T i l l J E . 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Nature 320: 275, 1986. 75 C H A P T E R I I JUVENILE MONOSOMY 7 SYNDROME: EVIDENCE THAT THE DISEASE ORIGINATES IN A PLURIPOTENT HEMOPOIETIC STEM CELL 1) INTRODUCTION The a s s o c i a t i o n of ch ron ic m y e l o p r o l i f e r a t i v e d i s o r d e r s or r e f r a c t o r y cy topenias w i t h a bone marrow karyotype showing monosomy of chromosome 7 i s well-documented i n the p e d i a t r i c l i t e r a t u r e ( 1 - 6 ) . The bone marrow i s t y p i c a l l y h y p e r c e l l u l a r w i t h d i so rde red maturat ion i n one or more c e l l l i n e s . Al though t h i s d isease may present as a t y p i c a l mye lodysp l a s t i c d i s o r d e r (5 ,6 ) i t o f ten has fea tures more commonly a s soc i a t ed w i t h the m y e l o p r o l i f e r a t i v e syndromes, such as hepatosplenomegaly and an e leva ted whi te blood c e l l count w i t h a l e f t - s h i f t e d d i f f e r e n t i a l and e o s i n o p h i l i a ( 1 , 3 , 4 ) . The p r e sen t i ng fea tures may vary somewhat, but the u l t i m a t e prognosis i s p r e d i c t a b l y poor and the ma jo r i t y of p a t i e n t s enter a t e rmina l phase of r e f r a c t o r y acute nonlymphoblas t ic leukemia (ANLL) w i t h i n a few yea r s . The c e l l i n which malignant change o r i g i n a t e s i n t h i s d i sease has not been e l u c i d a t e d . Some p a t i e n t s show only l e u k o c y t o s i s and l e f t - s h i f t e d p e r i p h e r a l blood granu locy tes i n a s s o c i a t i o n w i t h marrow myeloid h y p e r p l a s i a , sugges t ing an o r i g i n i n c e l l s committed to the granulocyte-macrophage pathway. In o ther c h i l d r e n d y s e r y t h r o p o i e s i s , r e t i c u l o c y t o p e n i a and severe anemia have been observed du r ing the preleukemic p e r i o d . This could r e f l e c t involvement of c e l l s of the e r y t h r o i d l ineage but might a l so be exp la ined by secondary e f f e c t s . D i r e c t i n v e s t i g a 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 76 malignant c e l l s i n c h i l d r e n w i t h bone marrow monosomy 7 and mye lodysp la s i a was made p o s s i b l e i n t h i s study by the combined use of hemopoiet ic p rogen i to r assays and hemopoiet ic colony c y t o g e n e t i c s . 2) PATIENTS A . Case 1 An 18 month o l d boy presented w i t h b r u i s i n g , hepatosplenomegaly and abnormal blood counts . H i s Hb was 8.9 gm/d l , p l a t e l e t s 3 6 , 0 0 0 / u l , wbc 4 9 , 0 0 0 / u l ( n e u t r o p h i l s 42%, metamyelocytes 4%, myelocytes 2%, promyelocytes 0.5%, monocytes 10%, b l a s t s 3%). His bone marrow was h y p e r c e l l u l a r w i t h reduced megakaryocytes and an abnormal karyotype i n c l u d i n g c e l l s showing monosomy of chromosome 7 (See Resu l t s and Table V ) . The pa t i en t was t r ea ted w i t h m u l t i p l e chemotherapeutic agents without b e n e f i t . He developed p r o g r e s s i v e organomegaly, pancytopenia and an i n c r e a s i n g p r o p o r t i o n of myelo id b l a s t s i n marrow and b lood . A d iagnos i s of ANLL was made 17 months a f t e r p r e s e n t a t i o n and the pa t i en t d ied 3 months l a t e r . B . Case 2 A 5 month o l d boy presented w i th hepatosplenomegaly, anemia and thrombocytopenia. H i s Hb was 4.9 gm/d l , p l a t e l e t s 3 9 , 0 0 0 / y l , wbc 1 0 , 4 0 0 / y l w i t h 14% c i r c u l a t i n g nuclea ted red blood c e l l s but no other b l a s t s . The bone marrow was h y p e r c e l l u l a r w i t h an M:E r a t i o of 1:3, d y s e r y t h r o p o i e s i s and reduced megakaryocytes. Bone marrow cy togene t i c a n a l y s i s showed the presence of c e l l s w i t h monosomy 7 (See Resu l t s and Table V ) . The pa t i en t was i n i t i a l l y t r ea t ed w i t h c o r t i c o s t e r o i d s . He underwent splenectomy at age 8 months for r e f r a c t o r y thrombocytopenia. A l l o g e n e i c bone marrow t r a n s p l a n t a t i o n was performed at age 9 1/2 months a f t e r c o n d i t i o n i n g w i th cyclophosphamide, TABLE V D i rec t Cytogenet ic Stud ies P a t i e n t T issue Stud ied Time of Sample Karyotype Number of Analyzed Metaphases 1 blood lymphocyte at d iagnos is 46,XY 50 bone marrow at d iagnos is 46 ,XY /45 ,XY , -7 /47 ,XY ,+8 9 /8 /4 bone marrow 17 months pos t -d iagnos is - onset of ANLL 45 ,XY , -7 25 2 bone marrow at d iagnos is 4 6 , X Y / 4 5 , X Y , - 7 NA* blood lymphocyte 3 months pos t -d iagnos is 46,XY 50 s p l e n i c a s p i r a t e 3 months pos t -d iagnos is 45 ,XY , -7 50 bone marrow 1 month post marrow t ransp lan t 4 6 , X Y / 4 5 , X Y , - 7 98/3 bone marrow 4 months post t ransp lan t 4 5 , X Y , - 7 / 4 6 , X Y 20/3 * NA = not a v a i l a b l e . 78 busu l fan and c y t o s i n e a r a b i n o s i d e . The donor marrow was from the p a t i e n t ' s mother and had been deple ted of a l l o r e a c t i v e T lymphocytes w i t h soy bean l e c t i n a g g l u t i n a t i o n and sheep e ry th rocy te r o s e t t i n g ( 7 ) . Al though he became h e m a t o l o g i c a l l y normal , bone marrow cy togene t i c s f o l l o w i n g t r a n s p l a n t a t i o n showed autologous r e c o n s t i t u t i o n . The pa t i en t remains a l i v e at age 10 months w i t h i n c r e a s i n g marrow dys func t ion and at the time of w r i t i n g had j u s t r e c e i v e d a second marrow a l l o g r a f t . 3) MATERIALS AND METHODS A . Hemopoietic Colony Assays E r y t h r o p o i e t i c (CFU-E and BFU-E) and g r a n u l o p o i e t i c (CFU-GM) p r o g e n i t o r s from bone marrow buffy coat or F i co l l -Hypaque - sepa ra t ed p e r i p h e r a l b lood mononuclear c e l l s were assayed i n m e t h y l c e l l u l o s e and scored as p r e v i o u s l y desc r ibed ( 8 , 9 , Appendix I V ) . A f i n a l concen t r a t ion of 10% l eukocy te cond i t i oned medium was added to a l l c u l t u r e s . Smal l and l a r g e e r y t h r o i d colony growth was evaluated i n c u l t u r e s both w i t h and wi thout human u r i n a r y e r y t h r o p o i e t i n (3 U / m l , 1000 U/mg, and <0.002 U / m l , r e s p e c t i v e l y ) . C e l l s were p l a t e d at s e v e r a l concen t ra t ions to prevent deve lop ing c o l o n i e s from o v e r l a p p i n g one another . C o n t r o l va lues were determined u s i n g s i m i l a r reagents and c r i t e r i a as repor ted p r e v i o u s l y ( 9 ) . B . Cy togene t i c S tudies Bone marrow was processed for d i r e c t cy togene t i c a n a l y s i s by s tandard techniques (10) and c e l l s harvested both immediately and a f t e r 24 hours i n l i q u i d c u l t u r e . To study the karyotype of i n d i v i d u a l hemopoiet ic c o l o n i e s l a r g e , w e l l - i s o l a t e d e r y t h r o i d or granulocyte/macrophage c o l o n i e s were p lucked and processed i n d i v i d u a l l y as p r e v i o u s l y desc r ibed (11, Appendix V ) . A 79 minimum of 2 G-banded metaphases were analyzed per co lony . Pools of s m a l l e r c o l o n i e s of the same l i neage were a l s o ana lyzed . Karyotypes were e s t a b l i s h e d by Giemsa banding (12) . 4) RESULTS A . P r o g e n i t o r Assays Hemopoietic p rogen i to r assays were performed on bone marrow c e l l s from both p a t i e n t s s e v e r a l times dur ing t h e i r c l i n i c a l course . P e r i p h e r a l b lood c e l l s from pa t i en t 1 were a l s o ana lyzed . Represen ta t ive colony counts from marrow samples obta ined s h o r t l y a f t e r d i agnos i s are shown i n Table V I . Q u a l i t a t i v e a b n o r m a l i t i e s i n colony growth were s t r i k i n g i n both p a t i e n t s ' c u l t u r e s a l though the a c t u a l y i e l d of e r y t h r o i d c o l o n i e s per 10^ marrow c e l l s d i d not vary s i g n i f i c a n t l y from normal . In both cases , 5 to 10% of CFU-E and BFU-E of ten generated v i s u a l l y de tec tab le hemoglobinized c o l o n i e s i n the absence of e r y t h r o p o i e t i n , and even those c o l o n i e s forming i n the presence of normal ly op t ima l concen t ra t ions of e r y t h r o p o i e t i n showed poor development and ma tu ra t ion . G r a n u l o p o i e t i c c o l o n i e s were increased i n frequency i n p a t i e n t l ' s marrow c u l t u r e s . Some of these became macroscopic, c o n t a i n i n g s e v e r a l thousand c e l l s . When such c o l o n i e s were i n d i v i d u a l l y plucked and c e l l morphology examined on Wright-Giemsa s t a ined cy tocen t r i f uge p r e p a r a t i o n s , the ma jo r i t y of c e l l s appeared to be monocytes or macrophages. CFU-GM i n p a t i e n t 2 c u l t u r e s were normal or reduced i n frequency and generated on ly s m a l l c o l o n i e s . P e r i p h e r a l b lood from pa t i en t 1 was c o n s i s t e n t l y found to con t a in e l eva ted numbers of p rogen i to r s of a l l c l a s se s (Table V I I ) . The frequency of CFU-E, BFU-E and CFU-GM i n blood g r a d u a l l y increased throughout the p a t i e n t ' s course so that by 12 months a f t e r d i agnos i s the p r o p o r t i o n of p rogen i to r s i n 80 TABLE VI Bone Marrow C u l t u r e - P rogen i to r Numbers* P a t i e n t CFU-E BFU-E CFU-GM Remarks 1 105 125 808 - e r y t h r o i d c o l o n i e s s m a l l , poor l y hemog lob in ized , 5-7% e r y t h r o p o i e t i n independent - many very l a r g e macrophage c o l o n i e s 2 48 224 180 - e r y t h r o i d c o l o n i e s s m a l l , poo r l y hemog lob in ized , 7-10% e r y t h r o p o i e t i n independent - repeat c u l t u r e showed no normal g r a n u l o c y t e / macrophage c o l o n i e s Normal Range (99%) (10-299) (4-336) (7-156) * Co lon ies per 10^ marrow buf fy coat c e l l s . 81 TABLE VI I P e r i p h e r a l Blood P rogen i t o r Numbers From P a t i e n t 1* Time S ince D iagnos i s (months) CFU-E BFU-E % Abnormal** CFU-GM Blood wbc /u l 3 0 752 0 4317 8000 9 1830 18800 36 166375 21000 12 22856 68572 0 348572 26300 Normal Range (99%) (1-345) (21-841) 0 (2-290) (6000-17500) * Co lon ies per ml of b l ood . * * % E r y t h r o i d c o l o n i e s grown wi thout added e r y t h r o p o i e t i n . 82 the blood was almost 2% of the t o t a l wbc count . These e x h i b i t e d the same spectrum of q u a l i t a t i v e abno rma l i t i e s of e r y t h r o i d and g r a n u l o p o i e t i c co lony growth as were seen i n the marrow c u l t u r e s . B . Cy togene t i c S tudies D i r e c t cy togene t i c s tud i e s of bone marrow c e l l s i n i t i a l l y showed more than one karyotype i n both p a t i e n t s : 4 5 , X Y , - 7 / 4 7 , X Y , + 8 / 4 6 , X Y i n pa t i en t 1 and 4 5 , X Y , - 7 / 4 6 , X Y i n pa t i en t 2 (Table V ) . In pa t i en t 1, the p r o p o r t i o n of bone marrow c e l l s w i t h the 4 5 , X Y , - 7 karyotype subsequently i nc reased , r each ing 100% i n s e v e r a l samples i n which at l e a s t 25 metaphases were analyzed by 17 months a f t e r d i a g n o s i s . Th i s co inc ided w i t h c l i n i c a l p rogress ion to acute l eukemia . In p a t i e n t 2, the 4 5 , X Y , - 7 karyotype was present i n a l l 50 metaphases from sp leen c e l l s obta ined 3 months a f t e r d i a g n o s i s . Wright-Giemsa s t a i n of the s p l e n i c a s p i r a t e processed for cy togene t i c s revea led predominantly e r y t h r o i d c e l l s , some of which showed d y s e r y t h r o p o i e s i s . A f t e r bone marrow t r a n s p l a n t a t i o n the pa t i en t showed cy togene t i c evidence of autologous recovery w i t h 3 of 101 c e l l s c o n t a i n i n g the 4 5 , X Y , - 7 karyotype and the p r o p o r t i o n of metaphases w i t h t h i s karyotype subsequently increased (Table V ) . Table V I I I shows the r e s u l t s of cy togene t i c analyses of hemopoiet ic c o l o n i e s . The ma jo r i ty of i n d i v i d u a l and pooled e r y t h r o i d c o l o n i e s from p a t i e n t l ' s c u l t u r e s showed the 4 5 , X Y , - 7 karyotype . One BFU-E was 47 ,XY,+8 . No c y t o g e n e t i c a l l y normal BFU-E were de tec ted . CFU-GM showed a s i m i l a r p i c t u r e , some be longing to the -7 c lone , some to the +8 clone and some showing a normal ka ryo type . In pa t i en t 2 ' s c u l t u r e s the major i ty of e r y t h r o i d c o l o n i e s , i n d i v i d u a l and pooled , were 4 5 , X Y , - 7 , even at times when metaphases from d i r e c t marrow samples were l a r g e l y normal . Attempts to ob t a in c y t o g e n e t i c data from the s m a l l granulocyte/macrophage c o l o n i e s produced i n p a t i e n t 2 ' s assays were unsucces s fu l . TABLE VIII Bone Marrow Culture - Hemopoietic Colony Cytogenetics Patient Karyotype BFU-E CFU-GM 1 45,XY,-7 47,XY,+8 46, XY 2 45,XY,-7 46, XY 9 (7)* 3 (12) 1 3 (20) 0 1 (9) 22 (48) 4 (16) * Number of individual colonies (number of metaphases from pools of c e l l s from similar colonies). 84 5) DISCUSSION Preleukemia d i s o r d e r s are uncommon' i n c h i l d r e n . However, they have many s i m i l a r i t i e s to the i n c r e a s i n g l y prevalent forms of adul t m y e l o d y s p l a s i a . In p a r t i c u l a r , k a r y o t y p i c abnorma l i t i e s are frequent i n the adu l t d i s o r d e r and d e l e t i o n s of 7q or monosomy 7 are among the most common cy togene t i c changes observed (13 ,14 ) . These changes confer a p a r t i c u l a r l y poor prognosis as a l a r g e p r o p o r t i o n of p a t i e n t s w i t h them u l t i m a t e l y develop r e f r a c t o r y ANLL. M y e l o d y s p l a s i a and a b n o r m a l i t i e s of chromosome 7 of ten f o l l o w treatment of a pr imary malignancy w i t h c y t o t o x i c chemotherapy, no tab ly a l k y l a t i n g agents , and these p a t i e n t s , too, fare badly (15-17) . In c h i l d r e n w i t h monosomy 7 and marrow d y s f u n c t i o n , whether the p re sen ta t ion has features more c o n s i s t e n t w i t h a m y e l o d y s p l a s t i c or m y e l o p r o l i f e r a t i v e na ture , the prognosis i s s i m i l a r l y poor ( 1 - 6 ) . Re f rac to ry cytopenias develop, become p r o g r e s s i v e l y severe and t y p i c a l l y evolve i n t o ANLL which i s unresponsive to conven t iona l a n t i - l e u k e m i c therapy. The fac t that hemopoiet ic mal ignancies may a r i s e i n a p l u r i p o t e n t hemopoiet ic p rogen i to r was f i r s t demonstrated i n ch ron ic myelogenous leukemia (CML) by cy togene t i c and glucose 6 phosphate dehydrogenase (G6PD) isoenzyme s t u d i e s (18 ,19 ) . In t h i s d i s o r d e r the malignant stem c e l l g i ve s r i s e to d i f f e r e n t i a t e d progeny that func t ion as r e l a t i v e l y normal blood c e l l s . However, the even tua l emergence of the b l a s t phase of CML i n more than 80% of p a t i e n t s i s remin iscen t of the e v o l u t i o n of mye lodysp las ia to ANLL. G6PD a n a l y s i s has a l s o been used to demonstrate involvement of e r y t h r o c y t e s , p l a t e l e t s , g ranu locy tes and lymphocytes i n the malignant c lone of two adu l t cases of mye lodysp la s i a (20 ,21) . Recen t ly a s i n g l e case of t h e r a p y - l i n k e d mye lodysp la s i a and monosomy 7 i n a c h i l d has been desc r ibed w i t h k a r y o t y p i c changes i n both BFU-E and CFU-GM (17) p r o v i d i n g fu r the r evidence for p l u r i p o t e n t p rogen i to r involvement i n preleukemic s t a t e s . 85 In t h i s repor t e r y t h r o i d p rogen i to r s that are c y t o g e n e t i c a l l y marked and d i f f e r e n t i a t e abnormally have been detected i n two c h i l d r e n w i t h dyshemopoiesis a s soc i a t ed w i t h monosomy of chromosome 7. In one case morphologic and k a r y o t y p i c changes i n c o l o n i e s de r ived from g r a n u l o p o i e t i c p r o g e n i t o r s were a l s o demonstrable. E ry th ropo ie t in - independen t e r y t h r o i d co lony growth was prominent i n both p a t i e n t s ' c u l t u r e s . Al though t h i s phenomenon was f i r s t desc r ibed i n polycythemia vera (22) , i t has subsequent ly been revea led i n o ther m y e l o p r o l i f e r a t i v e d i so rde r s (23) and e ry thro leukemias (24) , and appears to be s p e c i f i c a l l y a s soc ia t ed w i t h c l o n a l d i s o r d e r s of hemopoies is . The l a r g e increase i n p rogen i to r content of the p e r i p h e r a l b lood i n p a t i e n t 1 i s remin iscen t of s i m i l a r changes t y p i c a l of CML and o ther a d u l t m y e l o p r o l i f e r a t i v e d i s o r d e r s (25) . Var ious growth a b n o r m a l i t i e s have been desc r ibed i n c u l t u r e s of hemopoiet ic c e l l s from pa t i en t s w i t h m y e l o d y s p l a s i a . They i n c l u d e , reduced g r a n u l o c y t i c and e r y t h r o i d colony format ion (26-29) and inc reased s m a l l c l u s t e r formation (27-29) . Our second p a t i e n t ' s c u l t u r e s showed a decreased CFU-GM content i n l a t e r c u l t u r e s s i m i l a r to what was desc r ibed i n the above s t u d i e s . Thus, both p a t i e n t s ' p rogen i to r s e x h i b i t e d abnormal growth i n c u l t u r e cons i s t en t w i th the malignant nature of t h e i r hemopoiet ic c e l l s . Monosomy 7 was demonstrated i n many of the c y t o g e n e t i c a l l y analyzed e r y t h r o i d and g r a n u l o p o i e t i c c o l o n i e s v e r i f y i n g the p r o l i f e r a t i o n of n e o p l a s t i c hemopoiet ic p rogen i to r s i n these c u l t u r e s . Mal ignant change i n t h i s d i sease must therefore i n v o l v e p r i m i t i v e hemopoiet ic c e l l s capable of e r y t h r o i d and, i n at l e a s t some cases , myeloid d i f f e r e n t i a t i o n . S tud ies of blood and bone marrow c e l l s from pa t i en t s w i t h de novo acute leukemia a l s o suggest that some of the c e l l s w i t h i n the malignant c lone r e t a i n the a b i l i t y to d i f f e r e n t i a t e a long one or more l ineages under c e r t a i n c o n d i t i o n s both i n v i t r o and i n v i v o (30-32) . Thus, hemopoiet ic p rogen i to r s 86 and, i n p a r t i c u l a r the p l u r i p o t e n t stem c e l l appear to be common t a rge t s fo r events that g i v e r i s e to a v a r i e t y of malignant d i so rde r s i n c l u d i n g m y e l o d y s p l a s i a . 87 REFERENCES 1. Teasdale JM, Worth A J , Corey MJ. 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N Eng l J Med 290: 1382, 1974. 23. Eaves AC, Eaves C J . A b n o r m a l i t i e s i n the e r y t h r o i d p rogen i to r compartments i n pa t i en t s w i th ch ron ic myelogenous leukemia (CML). Exp Hematol 7: 65, 1979/ 24. Anderson NF, Beckman B, B e l t r a n G, F i s c h e r JW, S t u c k l y WJ. E ry th ropo ie t in - independen t e r y t h r o i d colony formation i n p a t i e n t s w i t h e ry th ro leukemia (M6) and r e l a t e d d i s o r d e r s . Br J Haematol 52: 311, 1982. 25. Goldman JM, Th'Ng KH, Lowenthal RM. In v i t r o colony forming c e l l s and colony s t i m u l a t i n g f ac to r i n ch ron ic g r a n u l o c y t i c leukemia . Br J Cancer 30: 1, 1974. 26. Chui DHK, C l a r k e B J . Abnormal e r y t h r o i d p rogen i to r c e l l s i n human pre leukemia . Blood 60: 362, 1982. 27. M i l n e r GR, Tes ta NG, Geary CG, Dexter TM, Mulda l S, Maclver J E , L a j t h a LG. Bone marrow c u l t u r e s tud ie s i n r e f r a c t o r y cy topen ia and "smothering leukaemia" . Br J Haematol 35: 251, 1977. 28. Greenberg PL. The smolder ing myeloid leukemic s t a t e s : C l i n i c a l and b i o l o g i c f ea tu res . Blood 61: 1035, 1983. 89 29. Pedersen-Bjergaard J , P h i l i p P, Mortensen BT, E s b o l l J , Jensen G, Panduro J , Thomsen M. Acute nonlymphocytic leukemia, pre leukemia , and acute m y e l o p r o l i f e r a t i v e syndrome secondary to treatment of o ther malignant d i s ea se s . C l i n i c a l and cy togene t i c c h a r a c t e r i s t i c s and r e s u l t s of i n v i t r o c u l t u r e of bone marrow and HLA t y p i n g . Blood 57: 712, 1981* 30. Re id MM, Tant ravah i R, G r i e r HE, O'Toole S, M i l l e r BA, L i p t o n JM, Weins t e in H J , Nathan DG. De tec t i on of l eukemia - r e l a t ed karyotypes i n granulocyte/macrophage c o l o n i e s from a pa t i en t w i t h acute myelomonocytic leukemia . N Eng l J Med 308: 1324, 1983. 31. Mar ie J P , I z a g u i r r e CA, C i v i n C I , M i r r o J , McCul loch EA. G r a n u l o p o i e t i c d i f f e r e n t i a t i o n i n AML b l a s t s i n c u l t u r e . Blood 58: 670, 1981. 32. Fearon ER, P h i l i p BA, Burke J , S c h i f f e r CA, Zehnbauer BA, V o g e l s t e i n B . D i f f e r e n t i a t i o n of leukemia c e l l s to polymorphonuclear l eukocy tes i n p a t i e n t s w i t h acute nonlymphocytic leukemia . N Engl J Med 315: 15, 1986. C H A P T E R I I I 90 HEMOPOIETIC PROGENITORS THAT ARE NOT PART OF THE MALIGNANT CLONE REVEALED IN LONG-TERM MARROW CULTURE FROM A G6PD HETEROZYGOTE WITH CHRONIC MYELOGENOUS LEUKEMIA 1) INTRODUCTION Chron ic myelogenous leukemia (CML) i s a c l o n a l neoplasm i n which malignant t rans format ion occurs i n a p r i m i t i v e hemopoiet ic c e l l w i t h the p o t e n t i a l to d i f f e r e n t i a t e a long both myeloid and lymphoid l i neages ( 1 , 2 ) . I n most p a t i e n t s 100% of bone marrow metaphases w i l l show a cy togene t i c marker, the P h i l a d e l p h i a chromosome (Ph) ( 3 , 4 ) . When marrow or blood c e l l s from CML p a t i e n t s are c u l t u r e d i n s e m i - s o l i d media u s u a l l y a l l of the e r y t h r o i d , g r a n u l o c y t i c and m u l t i - l i n e a g e c o l o n i e s generated are a l s o P h - p o s i t i v e , and these f i n d i n g s are not a l t e r e d a f t e r conven t iona l chemotherapy ( 5 , 6 ) . However, s t u d i e s of pa t i en t s t rea ted w i t h h igh dose chemotherapy (7-9) or a i n t e r f e r o n (10) have demonstrated the reappearance of Ph-nega t ive , and i n two cases , nonc lona l hemopoiesis . From q u a n t i t a t i v e s tud ies of p rogen i to r numbers and genotypes i n CML p a t i e n t s , i t has been shown that f a i l u r e to detec t Ph-nega t ive c e l l s i n untreated or l e s s a g g r e s s i v e l y managed p a t i e n t s i s due to two f a c t o r s : d i l u t i o n of p r i m i t i v e Ph-negat ive p rogen i to r c e l l s by e a r l y expansion at the stem c e l l l e v e l of the P h - p o s i t i v e c lone as w e l l as accompanying suppress ion of Ph-negat ive p rogen i to r c e l l d i f f e r e n t i a t i o n ( 6 , 1 1 ) . More recent s t ud i e s us ing the long- term marrow c u l t u r e system have shown that Ph-negat ive p rogen i to r s can of ten be revea led i n c u l t u r e s d e r i v e d 91 from r e c e n t l y diagnosed p a t i e n t s , i n c l u d i n g cases where these k a r y o t y p i c a l l y normal c e l l s were not i n i t i a l l y demonstrable (11-13) . Al though the Ph-nega t ive p rogen i to r s appearing i n long- term CML marrow c u l t u r e s are k a r y o t y p i c a l l y and p h e n o t y p i c a l l y normal, i t i s not c e r t a i n that they represent t r u l y normal c e l l s . An a l t e r n a t i v e p o s s i b i l i t y i s that they may belong to the n e o p l a s t i c c lone but d e r i v e from a c e l l at a stage p r i o r to development of the Ph t r a n s l o c a t i o n ( 2 ) . In a previous study of a p a t i e n t mosaic fo r the karyotypes 46,XX and 46,X w i t h CML, i t was shown that the Ph-negat ive hemopoiet ic p rogen i to r s detected a f t e r long- term c u l t u r e of marrow were not part of the malignant c lone (14) . In the present study I have extended t h i s obse rva t ion to a second CML pa t i en t us ing glucose-6-phosphate dehydrogenase (G6PD) a n a l y s i s to d i s t i n g u i s h c l o n a l and normal p o p u l a t i o n s . 2) MATERIALS AND METHODS Twelve b l ack women w i t h CML were i d e n t i f i e d and s k i n b i o p s i e s used as a source of f i b r o b l a s t s for G6PD a n a l y s i s . S i x of these p a t i e n t s were found to be heterozygous for two e l e c t r o p h o r e t i c a l l y d i s t i n g u i s h a b l e G6PD isoenzymes. Of the s i x p a t i e n t s ; one was l o s t to f o l l o w - u p , two had marrow a s p i r a t e s of i n s u f f i c i e n t q u a n t i t y for e s t a b l i s h i n g long- term c u l t u r e s because of p rev ious therapy or m y e l o f i b r o s i s and a four th pa t i en t refused marrow a s p i r a t i o n . The remaining 2 p a t i e n t s are the cases i n t h i s r e p o r t . A. Case Reports P a t i e n t 1. P a t i e n t 1 was a 45 y e a r - o l d woman from West A f r i c a . The d i a g n o s i s of CML was made i n March, 1985 when she was d i scovered to have massive splenomegaly and an abnormal p e r i p h e r a l blood p i c t u r e (whi te c e l l s 490,000/mm3 w i t h 30% immature myeloid forms i n c l u d i n g promyelocytes , 92 myelocytes and metamyelocytes, 3% b a s o p h i l s , hemoglobin 5.4 gm/d l , p l a t e l e t s 800,000/mm3). At that time cy togene t i c a n a l y s i s showed the s tandard Ph t r a n s l o c a t i o n : t (9 ;22 ) ( q 3 4 ; q l l ) i n a l l 30 marrow metaphases examined. The p a t i e n t was t r ea ted w i t h o r a l hydroxyurea for 10 days before a second bone marrow a s p i r a t i o n was performed, a p o r t i o n of which was used for the s t u d i e s repor ted here . At t h i s time her white blood c e l l count was 10,500/mm3, hemoglobin 7.8 gm/d l , p l a t e l e t s 500,000/mm 3 . P a t i e n t 2. P a t i e n t 2 was an 11-year o l d g i r l from Guadeloupe who presented i n August , 1983 w i t h a p e r i p h e r a l blood white c e l l count of 105,000/mm3 and a p l a t e l e t count of 1,000,000/mm 3 . She was observed wi thout treatment u n t i l August , 1984. P h y s i c a l examinat ion at that time showed hepatosplenomegaly and her p e r i p h e r a l blood white c e l l count was 185,000/mm3 w i t h 38% n e u t r o p h i l s , 3% e o s i n o p h i l s , 1% b a s o p h i l s , 25% myelocytes , 19% metamyelocytes, 8% promyelocytes , 4% b l a s t s , 2% lymphocytes. Her hemoglobin was 10 gm/dl and her p l a t e l e t count was g rea te r than 1,000,000/mm 3 . A bone marrow examinat ion revea led myeloid h y p e r p l a s i a at a l l l e v e l s of matura t ion and inc reased megakaryocytes. A l l 25 marrow metaphase c e l l s examined showed the s tandard Ph t r a n s l o c a t i o n : t ( 9 ; 2 2 ) ( q 3 4 ; q l l ) . She was then t rea ted w i t h hydroxyurea and a l l o p u r i n o l i n t e r m i t t e n t l y u n t i l November 1984 when the marrow sample used fo r the s tud i e s repor ted here was taken. At t h i s time the p e r i p h e r a l b lood whi te c e l l count was 58,300/mm 3 . B . C u l t u r e s S k i n samples were obta ined by punch biopsy and placed i n s t e r i l e medium. Bone marrow a s p i r a t e and blood c e l l s were c o l l e c t e d i n hepa r in . A l l samples were p laced on i c e from the time of c o l l e c t i o n i n P a r i s u n t i l a r r i v a l i n Vancouver 18 to 24 hours l a t e r . 93 S k i n samples were minced w i t h s c i s s o r s and a l lowed to adhere to t i s s u e c u l t u r e d i shes before f l o o d i n g the d ishes w i t h a medium plus 20% f e t a l c a l f serum. A f t e r three to four weeks conf luent f i b r o b l a s t s were harves ted by t r y p s i n i z a t i o n fo r G6PD a n a l y s i s . P e r i p h e r a l b lood mononuclear c e l l s were i s o l a t e d by F i c o l l - H y p a q u e s e p a r a t i o n . Bone marrow buffy coat c e l l s were used to i n i t i a t e long- term c u l t u r e s as p r e v i o u s l y desc r ibed (12, Appendix I I I ) . An a l i q u o t was p l a t ed i n m e t h y l c e l l u l o s e c u l t u r e s fo r assessment of e r y t h r o i d and g r a n u l o c y t i c c o l o n y -forming c e l l numbers, karyotype and G6PD a n a l y s i s . Long-term c u l t u r e s were mainta ined by weekly removal of h a l f of the growth medium and nonadherent c e l l s and replacement of h a l f of the growth medium. A f t e r 4 to 6 weeks adherent l a y e r s were suspended us ing co l lagenase and the c e l l s p l a t ed i n m e t h y l c e l l u l o s e at 10^ c e l l s / m l for assessment of the number and types of p rogen i to r s present (15, Appendix I V ) . C. G6PD A n a l y s i s Lysa tes were made from c e l l s p e l l e t e d by c e n t r i f u g a t i o n by repeated f r e e z i n g on dry i c e and thawing. Samples were then a p p l i e d to c e l l u l o s e ace ta te membranes. I n d i v i d u a l c o l o n i e s were plucked from m e t h y l c e l l u l o s e d i r e c t l y onto c e l l u l o s e ace ta te membranes before f r eeze / thawing . Samples were then subjec ted to e l e c t r o p h o r e s i s i n Supre-Heme buffer pH 8.4 (Helena L a b o r a t o r i e s , Beaumont, Texas) for 40 minutes at 375 v o l t s . The membranes were s t a i n e d immediately w i t h G6PD isoenzyme reagent (Helena L a b o r a t o r i e s ) and the p o s i t i o n and r e l a t i v e i n t e n s i t y of the enzyme bands es t imated v i s u a l l y and compared to e s t a b l i s h e d s tandards . 94 D. Cy togene t i c S tudies Cytogene t i c a n a l y s i s was performed on i n d i v i d u a l c o l o n i e s and pools of c o l o n i e s of the same l i neage plucked from m e t h y l c e l l u l o s e assays as p r e v i o u s l y de sc r i bed (16, Appendix V ) . Karyotypes were e s t a b l i s h e d a f t e r Giemsa banding (17) . 3) RESULTS G6PD a n a l y s i s of s k i n f i b r o b l a s t s (Table IX) showed the p a t i e n t to be heterozygous for enzyme v a r i a n t s A and B at a r a t i o of 50:50. Whole b lood l y s a t e s from pa t i en t 1 were 100% G6PD-B. Blood from pa t i en t 2 was separated i n t o red and whi te c e l l f r a c t i o n s and l y s a t e s of both were found to be 100% G6PD-A. Cy togene t i c a n a l y s i s of c o l o n i e s removed i n d i v i d u a l l y from m e t h y l c e l l u l o s e assays of the i n i t i a l marrow sample showed a l l 10 c o l o n i e s examined from pa t i en t 1 and a l l 12 c o l o n i e s from pa t i en t 2 to be P h - p o s i t i v e (Table X ) . G6PD a n a l y s i s of c o l o n i e s from the same assays showed 26/26 c o l o n i e s from pa t i en t 1 assays to be G6PD-B and 22/22 c o l o n i e s from p a t i e n t 2 assays to be G6PD-A. S i m i l a r r e s u l t s were obta ined for c o l o n i e s produced i n s imultaneous assays of p e r i p h e r a l blood p rogen i to r s (data not shown). For both p a t i e n t s the number of g r a n u l o p o i e t i c p rogen i to r s present i n the nonadherent f r a c t i o n of long- term marrow c u l t u r e s was c o n s i s t e n t l y low from the second week of c u l t u r e onward, and no e r y t h r o i d p rogen i to r s were de t ec t ab l e i n the non-adherent f r a c t i o n s a f t e r t h i s t ime. Adherent l a y e r s were harves ted a f t e r 4 weeks for pa t i en t l ' s c u l t u r e s , and a f t e r 4 and 6 weeks of c u l t u r e for pa t i en t 2. The major i ty of the g ranu locy te colony p rogen i to r s present at these times were i n the adherent l a y e r , as has been found p r e v i o u s l y (12,13) and hence cy togene t i c and G6PD data were obta ined on ly from these assays . An o c c a s i o n a l e r y t h r o i d colony was produced i n assays of the 4 TABLE IX Ratio of G6PD Isoenzyme Variants in Lysates of Peripheral Blood and Skin Fibroblast Cells Blood Fibroblast Patient A : B A : B 1 rbc* 0 : 100 50 : 50 (whole blood) 2 rbc 100 : 0 50 : 50 wbct 100 : 0 * rbc - red blood cells | wbc - white blood cells 96 TABLE X Hemopoiet ic Colony Cy togenet ic and G6PD A n a l y s i s P a t i e n t I n i t i a l Karyotype Marrow G6PD Long Term C u l t u r e Karyotype G6PD 1 BFU-E* 6 - Ph§ 18 - B 3 - Ph 0 - NU 0 - A 0 - N CFU-Cj A - Ph 8 - B 9 - Ph 0 - N 0 - A 0 - N Poo led -C^ 21 - Ph 0 - N 2 BFU-E 10 - Ph 0 - N 9 - A 0 - B CFU-C 2 - Ph 13 - A 9 - Ph 26 - A 0 - N 0 - B 2 - N 4 - B * BFU-E = l a r g e e r y t h r o i d c o l o n i e s . + CFU-C = granulocyte/macrophage c o l o n i e s . ^ Poo led-C = a poo l of many sma l l g ranu locy te c o l o n i e s . § Ph = 4 6 , X X , t ( 9 ; 2 2 ) ( q 3 4 ; q l l ) . 1 N = 46 ,XX. 97 week adherent l a y e r c e l l s from pa t i en t l ' s c u l t u r e s . No e r y t h r o i d c o l o n i e s were de tec ted i n the assays of the 4 and 6 week adherent l a y e r c e l l s of p a t i e n t 2 ' s c u l t u r e s . From the assays of pa t i en t l ' s long- term c u l t u r e s , 3 l a r g e e r y t h r o i d c o l o n i e s , 9 l a r g e g r a n u l o c y t i c c o l o n i e s and a pool of many sma l l e r g r a n u l o c y t i c c o l o n i e s were examined c y t o g e n e t i c a l l y . A l l were found to c o n t a i n on ly P h - p o s i t i v e metaphases (Table X ) . From the assays of pa t i en t 2 ' s long- term c u l t u r e s , 11 i n d i v i d u a l g r a n u l o c y t i c c o l o n i e s were examined c y t o g e n e t i c a l l y . Nine were P h - p o s i t i v e . Two were Ph-nega t ive . Another 30 i n d i v i d u a l g r a n u l o c y t i c c o l o n i e s were examined for t h e i r G6PD isoenzyme type. Twenty-s ix conta ined G6PD v a r i a n t A , the isoenzyme a s soc i a t ed w i t h the malignant CML c l o n e . Four conta ined G6PD v a r i a n t B . 4) DISCUSSION Prev ious s t u d i e s have shown that i n most cases , P h - p o s i t i v e hemopoiesis i s not maintained i n long- term c u l t u r e s i n i t i a t e d w i th marrow c e l l s from p a t i e n t s w i t h P h - p o s i t i v e CML. In c o n t r a s t , chromosomally normal p r o g e n i t o r s , even i f i n i t i a l l y unde tec tab le , appear to be maintained w i t h k i n e t i c s s i m i l a r to those t y p i c a l of long- term c u l t u r e s i n i t i a t e d w i th normal marrow ( 1 2 , 1 4 ) . However, whether or not Ph-negat ive p rogen i to r s are detected i n long- te rm CML c u l t u r e s depends on the extent of d i l u t i o n of the Ph-negat ive stem c e l l p o p u l a t i o n . I f the content of Ph-negat ive stem c e l l s i n the i n i t i a l marrow inoculum i s s u f f i c i e n t l y s m a l l , then i t i s u n l i k e l y that t h e i r progeny would be de tec ted i n s p i t e of a r a p i d d e c l i n e i n P h - p o s i t i v e c e l l s . Examples of such a pa t t e rn have been observed p r e v i o u s l y and are l i k e l y exp lana t ions fo r the absence of de tec tab le Ph-negat ive p rogen i to r s i n c u l t u r e s e s t a b l i s h e d w i t h 98 marrow from p a t i e n t 1, and the r e l a t i v e l y low numbers of Ph-nega t i ve p rogen i t o r s de tec ted i n c u l t u r e s e s t a b l i s h e d w i th marrow from p a t i e n t 2 . Both p a t i e n t s had h igh whi te blood c e l l counts at the time of d i a g n o s i s , and hence probab ly a l s o had a g r e a t l y expanded poo l of P h - p o s i t i v e p rogen i to rs (11 ) . Ph -nega t i ve p rogen i to rs are a l s o l e s s commonly detec ted i n long- te rm c u l t u r e s i n i t i a t e d w i th c e l l s from t rea ted p a t i e n t s , and aga in the present f i n d i n g s are c o n s i s t e n t w i th that o b s e r v a t i o n . Never the less G6PD a n a l y s i s c l e a r l y showed that at l e a s t some of the p rogen i t o r s from the pa t i en t 2 ' s c u l t u r e s were not par t of the mal ignant c lone as they expressed the G6PD enzyme v a r i a n t not found i n the CML c e l l s . These da ta con f i rm prev ious f i n d i n g s w i th a pa t i en t mosaic f o r karyotypes 46,XX and 46,X w i t h CML. Ph-negat i ve p rogen i to rs detec ted i n 4 to 6 week o l d long- te rm marrow c u l t u r e s were demonstrated to not be par t of the CML c lone by c y t o g e n e t i c a n a l y s i s i n t h i s case (14) . These r e s u l t s do not e l i m i n a t e the p o s s i b i l i t y of a Ph-negat i ve s tep i n the pathogenesis of CML, nor even that c l o n a l Ph-nega t i ve c e l l s were present i n low numbers i n the c u l t u r e s examined he re . However, at l e a s t f o r the 2 p a t i e n t s thus f a r examined, both the 4 6 , X X / 4 6 , X mosaic and pa t i en t 2 i n the present s tudy , the da ta suggest that the m a j o r i t y , i f not a l l , Ph-negat ive c e l l s are not par t of the mal ignant c l o n e . Conv inc ing ev idence of a c y t o g e n e t i c a l l y normal but c l o n a l popu la t i on i n G6PD he terozygotes w i l l depend on ana lyses of p a t i e n t s or c u l t u r e s where nonc lona l elements are e i t h e r absent or suppressed. Such a s i t u a t i o n has been r e c e n t l y deomonstrated i n a pa t i en t w i th acute myelogenous leukemia (AML) i n r e m i s s i o n (18 ) . I t has been shown long- term c u l t u r e may favor the maintenance of p h e n o t y p i c a l l y normal p rogen i to rs i n AML as w e l l as CML (19) . I t would be of i n t e r e s t to determine i f these apparen t l y normal p rogen i to rs s e l e c t e d f o r 99 long- te rm c u l t u r e are c l o n a l and p o s s i b l y "premalignant" or t r u l y normal i n d i seases such as AML. The unequivoca l demonstrat ion of normal hemopoiet ic p recursors i n p a t i e n t s w i t h CML and the enrichment for these p rogen i to r s i n long- te rm marrow c u l t u r e s o f f e r s p o s s i b i l i t i e s that may be e x p l o i t e d c l i n i c a l l y . S e l e c t i v e enrichment of normal p rogen i to r s i n CML marrow by i n v i t r o treatment w i t h 4 hydroxypercyclophosphamide has a l s o r e c e n t l y been repor ted (20) . Fu r the r development of i n v i t r o techniques, e i t h e r s epa ra t e ly or i n combina t ion , may e v e n t u a l l y p rov ide a method of h a r v e s t i n g normal autologous hemopoiet ic stem c e l l s fo r bone marrow t r a n s p l a n t a t i o n i n s i t u a t i o n s where an a l l o g e n e i c t r ansp lan t i s not p o s s i b l e . 100 REFERENCES 1. F i a lkow P J , Jacobson R J , Papayannopoulou T. Chronic m y e l o c y t i c l eukemia : C l o n a l o r i g i n i n a stem c e l l common to the g r anu locy t e , e r y t h r o c y t e , p l a t e l e t and monocyte/macrophage. Am J Med 63: 125, 1977. 2 . F i a l k o w P J , M a r t i n P J , N a j f e l d V, Penfold GK, Jacobson R J , Hansen J A . Evidence for a m u l t i s t e p pathogenesis of ch ron ic myelogenous l eukemia . Blood 58: 158, 1981. 3. Tough IM, Jacobs PA, Brown WMC, B a i k i e AG, W i l l i a m s o n ERD. Cytogene t i c s t u d i e s on bone-marrow i n ch ron ic myeloid leukaemia. Lancet 1: 844, 1963. 4 . Whang J , F r e i E I I I , T j i o JH , Carbone PP, Brecher G. The d i s t r i b u t i o n of the P h i l a d e l p h i a chromosome i n pa t i en t s w i t h ch ron ic myelogenous leukemia . Blood 22: 664, 1963. 5. Moore MAS, M e t c a l f D. Cytogene t ic a n a l y s i s of human acute and c h r o n i c myelo id leukemic c e l l s cloned i n agar c u l t u r e . In t J Cancer 11: 143, 1973. 6. Dube ID, Gupta CM, Kalousek DK, Eaves C J , Eaves AC. Cytogene t i c s t u d i e s of e a r l y myeloid p rogen i to r compartments i n P h ^ - p o s i t i v e ch ron i c myelo id leukaemia (CML). Br J Haematol 56: 633, 1984. 7. Goto T, N i s h i k o r i M, A r l i n A, Gee T, Kempin S, Burchenal J , S t r i f e A , Wisn iewski D, Lambek C, L i t t l e C, Jhanwar S, Chaganti R, C l a r k s o n B . Growth C h a r a c t e r i s t i c s of leukemic and normal hematopoie t ic c e l l s i n Ph+ ch ron i c myelogenous leukemia and e f f e c t s of i n t e n s i v e t reatment. Blood 59: 793, 1982. 8. Chaganti RSK, B a i l e y RB, Jhanwar SC, A r l i n ZA, C la rkson BC. Chron ic myelogenous leukemia i n the monosomic c e l l l i n e of a f e r t i l e Turner syndrome mosaic ( 4 5 , X 4 6 , X X ) . Cancer Genet Cytogenet 5: 215, 1982. 9. S inger JW, A r l i n ZA, N a j f e l d V, Adamson JW, Kempin S J , C la rkson BD, F i a lkow P J . R e s t o r a t i o n of nonc lona l hematopoiesis i n ch ron i c myelogenous leukemia (CML) f o l l o w i n g a chemotherapy-induced l o s s of the P h 1 chromosome. Blood 56: 356, 1980. 10. Ta lpaz M, Gutterman J . Leukocyte i n t e r f e r o n . I n : " C o n t r o l of Mye lo id P r o l i f e r a t i o n i n Chronic Myelogenous Leukemia. The B i o l o g y of the I n t e r f e r o n System " , DeNayer E , Schel lekens H (eds) , E l s e v i e r , New York , pp 493, 1983. 11. Kalousek DK, Eaves C J , Eaves AC. I n - v i t r o cy togene t i c s t u d i e s of haemopoiet ic ma l ignanc ies . Cancer Surv 3: 439, 1984. 12. Coulombel L , Kalousek DK, Eaves C J , Gupta CM, Eaves AC. Long-term marrow c u l t u r e r evea l s chromosomally normal hematopoiet ic p rogen i to r c e l l s i n p a t i e n t s w i t h P h i l a d e l p h i a chromosome-posit ive ch ron ic myelogenous leukemia . N Eng l J Med 308: 1493, 1983. 101 13. Dube ID, Kalousek DK, Coulombel L , Gupta CM, Eaves C J , Eaves AC. Cy togene t i c s tud i e s of e a r l y myeloid p rogen i to r compartments i n Ph -p o s i t i v e ch ron i c myeloid leukemia. I I . Long-term c u l t u r e r evea l s the p e r s i s t e n c e of Ph-negat ive p rogen i to r s i n t rea ted as w e l l as newly diagnosed p a t i e n t s . Blood 63: 1172, 1984. 14. Dube ID, A r l i n ZA, Kalousek DK, Eaves C J , Eaves AC. Nonc lona l hemopoiet ic p rogen i to r c e l l s detected i n long- term marrow c u l t u r e s from a Turner syndrome mosaic w i th ch ron ic myeloid leukemia . Blood 64: 1284, 1984. 15. Coulombel L , Eaves AC, Eaves C J . Enzymatic treatment of long- te rm human marrow c u l t u r e s r evea l s the p r e f e r e n t i a l l o c a t i o n of p r i m i t i v e hemopoiet ic p rogen i to r s i n the adherent l a y e r . Blood 62: 291, 1983. 16. Dube ID, Eaves C J , Kalousek DK, Eaves AC. A method for o b t a i n i n g h igh q u a l i t y chromosome prepara t ions from s i n g l e hemopoiet ic c o l o n i e s on a r o u t i n e b a s i s . Cancer Genet Cytogenet 4: 157, 1981. 17. Seabr ight M. A r a p i d banding technique for human chromosomes. Lancet 12: 971, 1971. 18. Jacobson R J , Temple MJ, Singer JW, Raskind W, Powe l l J , F i a l k o w P J . A c l o n a l complete r emis s ion i n a pa t i en t w i t h acute nonlymphocytic leukemia o r i g i n a t i n g i n a mul t ipo ten t stem c e l l . New Engl J Med 310: 1513, 1984. 19. Coulombel L , Eaves C, Kalousek D, Gupta C, Eaves A. Long-term marrow c u l t u r e of c e l l s from pa t i en t s w i t h acute myelogenous leukemia . J C l i n Inves t 75: 961, 1985. 20. D e g l i a n t o n i G, Mangoni L , R i z z o l i V . In v i t r o r e s t o r a t i o n of p o l y c l o n a l hematopoiesis i n a ch ron ic myelogenous leukemia a f t e r i n v i t r o treatment w i t h 4-hydroperoxycyclophosphamide. Blood 65: 753, 1985. 102 C H A P T E R IV GENE TRANSFER TO PRIMARY NORMAL AND MALIGNANT HUMAN HEMOPOIETIC PROGENITORS USING RECOMBINANT RETROVIRUSES 1) INTRODUCTION The d e l i v e r y of exogenous gene t i c m a t e r i a l i n t o va r ious ta rge t c e l l s i s important fo r many exper imenta l and c l i n i c a l g o a l s . Recombinant r e t r o v i r u s e s p rov ide an a t t r a c t i v e v e h i c l e for gene t r ans fe r for a number of reasons . These i n c l u d e the h igh e f f i c i e n c y w i t h which they are ab le to enter c e l l s and i n t e g r a t e i n t o host DNA, t h e i r wide target c e l l range and t h e i r l a c k of t o x i c i t y ( 1 ) . Mammalian bone marrow i s a convenient source of p r i m i t i v e c e l l s w i t h h igh p r o l i f e r a t i v e and s e l f - r e n e w a l c apac i t y on which to tes t the f e a s i b i l i t y of u s ing a gene t r ans fe r technique to primary c e l l s . A number of workers have a l ready shown that i t i s p o s s i b l e to in t roduce a v a r i e t y of f o r e i g n genes i n t o the bone marrow stem c e l l s of mice u s ing recombinant r e t r o v i r u s e s ( 2 - 7 ) . These authors have shown a h igh e f f i c i e n c y of gene t r a n s f e r and the t r ans f e r r ed gene has remained s t a b l y i n t eg ra t ed through s e r i a l marrow t r a n s p l a n t s . Al though some express ion of the t r a n s f e r r e d genes has been demonstrated, d i f f i c u l t i e s i n o b t a i n i n g s a t i s f a c t o r y l e v e l s of the products of c e r t a i n genes such as human adenosine deaminase have been encountered ( 7 ) . In a d d i t i o n , the long- term s t a b i l i t y of gene expres s ion has been quest ioned ( 5 ) . Neve r the le s s , i t i s c l e a r that r e t r o v i r u s e s are capable of d e l i v e r i n g f u n c t i o n a l genes i n t o murine hemopoiet ic p r o g e n i t o r s . 103 R e t r o v i r u s e s have a l s o been used for gene t r ans fe r to human c e l l s , a l though the a v a i l a b l e data i s l e s s ex tens ive than for murine t a rge t s ( 7 - 1 2 ) . The enzyme d e f i c i e n c y i n hypoxanthine phosphor ibosy l t r ans fe rase (HPRT) nega t ive human f i b r o b l a s t s and B lymphoblasts has been co r r ec t ed by i n f e c t i o n w i t h recombinant v i r u s c o n t a i n i n g the HPRT gene ( 8 , 9 ) . S i m i l a r r e s u l t s have been repor ted for experiments i n which a v i r u s c o n t a i n i n g the human adenosine deaminase (ADA) gene was used to i n f e c t human ADA d e f i c i e n t B lymphocytes ( 7 ) . Some data e x i s t s for c e l l s of the hemopoiet ic system from experiments i n which long- te rm human marrow c u l t u r e s were i n f e c t e d w i th v i r u s c o n t a i n i n g oncogenes or the human HPRT gene (10 ,11 ) . V i r a l r e p l i c a t i o n i n the hemopoiet ic c e l l s i n these c u l t u r e s was documented but the extent to which the gene of i n t e r e s t had been s u c c e s s f u l l y t r a n s f e r r e d and expressed by p r i m i t i v e blood c e l l p rogen i to r s was not s t u d i e d . Recen t ly , more d i r e c t q u a n t i t a t i v e data has been pub l i shed by Hock, and M i l l e r (12) demonstrat ing r e t r o v i r a l - m e d i a t e d gene t r a n s f e r to pr imary human marrow progen i to r s w i t h recombinant v i r u s e s c a r r y i n g the n e o r or the DHFR* gene. The present study was undertaken to i n v e s t i g a t e and i d e n t i f y v a r i a b l e s that may i n f l u e n c e the frequency of s u c c e s s f u l gene t r ans f e r to human hemopoie t ic p r o g e n i t o r s . A number of the a v a i l a b l e v i r a l packaging l i n e s and r e t r o v i r a l vec to r s have been evaluated us ing a spectrum of target c e l l types i n c l u d i n g an e s t a b l i s h e d c e l l l i n e as w e l l as f resh p rogen i to r s of both normal and leukemic o r i g i n . 2) MATERIALS AND METHODS A. C e l l s and C u l t u r e Cond i t ions C e l l l i n e s were c u l t u r e d i n Dulbecco ' s modif ied Eagle medium wi th h igh g lucose (4 .5 g/1) and 10% heat i n a c t i v a t e d c a l f serum ( f o r YAM or Y2 c e l l s ) or 104 10% f e t a l c a l f serum for a l l other c e l l types i n 5% CO2 atmosphere at 37°C. The amphotropic r e t r o v i r u s packaging l i n e s , YAM, PA12 and PA317, and the e c o t r o p i c packaging l i n e , Y2, have been p r e v i o u s l y desc r ibed (13-16) . Pr imary human c e l l s were obtained e i t h e r from consent ing a d u l t s (CML p e r i p h e r a l blood or normal bone marrow) or from second t r i m e s t e r abo r t i ons ( f e t a l l i v e r c e l l s ) a f t e r approval of the C l i n i c a l Screening Committee fo r Research I n v o l v i n g Human Subjects of the U n i v e r s i t y of B r i t i s h Columbia . Blood and bone marrow c e l l s were passed over a p e r c o l l d e n s i t y g rad ien t ( d e n s i t y 1.063) and l i g h t d e n s i t y c e l l s c o l l e c t e d and used i n subsequent exper iments . F e t a l l i v e r was minced w i t h s c i s s o r s , and incubated fo r 3 hours at 37°C i n a medium w i t h 20% f e t a l c a l f serum and co l lagenase 1 mg/ml (Sigma Chemical C o . , S t . L o u i s , MO). The c e l l s were then passed through a s u c c e s s i o n of needles of decreas ing gauge, washed and used for the experiments d e s c r i b e d . Pr imary human c e l l s were grown i n a medium supplemented w i t h 20% f e t a l c a l f serum and 10% aga r - s t imu la t ed leukocyte cond i t i oned medium w h i l e i n suspension c u l t u r e and i n m e t h y l c e l l u l o s e assays as p r e v i o u s l y desc r ibed (17, Appendix IV) for assessment of hemopoiet ic p r o g e n i t o r s . B . V i r u s P roduc t ion and Assay The gene ra l s t r a t egy for genera t ing h igh t i t e r r e t r o v i r a l producer c e l l l i n e s was as o u t l i n e d by M i l l e r et a l (18) . The r e t r o v i r u s packaging l i n e , Y2, was p l a t ed at 5 x 10^ c e l l s per 60 mm d i s h on day 1. On day 2, 10 yg of v i r a l p lasmid DNA (pSVXZipNeo or pN2) (17,19) was t r ans fec ted i n t o the c e l l s by ca l c ium phosphate c o - p r e c i p i t a t i o n . These plasmids are both MoMuLV-based vec to r s which code for the gene for neomycin phosphotransferase ( n e o r ) . A f t e r 24 hours , the medium was changed and on day 3 the medium which conta ined n e o r v i r u s ( v - n e o r ) produced by the Y2 c e l l s was removed, cen t r i fuged at 3000 rpm x 105 5 minutes to remove c e l l s and deb r i s and a l i q u o t s used to i n f e c t the amphotropic packaging l i n e s . YAM, PA12 or PA317 c e l l s had been p l a t e d at 10^ c e l l s per 60 mm t i s s u e c u l t u r e d i s h the previous day. They were incubated w i t h medium from the t r ans fec ted Y2 c e l l s c o n t a i n i n g 8 ug/ml polybrene fo r 2 hours at 37°C. Then f resh medium was added f o l l o w e d , i n 48 hours , by t r y p s i n i z a t i o n , d i l u t i o n 1:10 and s e l e c t i o n for v - n e o r - p r o d u c i n g c lones i n medium c o n t a i n i n g the neomycin analogue G418 at 1 mg/ml (Gibco L a b o r a t o r i e s , Chagr in F a l l s , O h i o ) . G418 was d i s s o l v e d i n d i s t i l l e d water and added to growth medium to achieve the des i r ed f i n a l concen t r a t ion i n t o t a l mg/ml ( the e f f e c t i v e drug concen t r a t i on was approximate ly 50% of that va lue fo r the 2 l o t s of G418 used) . Co lon ie s were i s o l a t e d by c l o n i n g r i n g s , expanded and examined fo r v - n e o r t i t e r on 3T3 c e l l s and for amphotropic he lper v i r u s u s i n g the S + L ~ assay (15) . The sarcoma v i r u s - c o n t a i n i n g non-producer ( S + L ~ ) c e l l s were cat CCC-81 c e l l s and the background c e l l s on which the r e l ea se of t ransforming v i r u s was scored were ra t NRK c e l l s . For amphotropic he lpe r v i r u s assay, 10^ CCC-81 c e l l s were p l a t ed per 60 mm d i s h on day 1. On day 2 the medium was a s p i r a t e d and replaced w i t h tes t medium c o n t a i n i n g v i r u s and 4 ug/ml po lybrene . On day 3 the medium was rep laced w i th 4 ml medium c o n t a i n i n g 2 x 10^ NRK c e l l s per d i s h . 4 to 5 days l a t e r , f o c i r e s u l t i n g from rescue of the sarcoma v i r u s present i n the CCC-81 c e l l s were counted. E c o t r o p i c v i r u s e s do not score i n t h i s assay because CCC-81 cat c e l l s are r e s i s t a n t to i n f e c t i o n by e c o t r o p i c v i r u s e s . E c o t r o p i c he lper v i r u s was assayed by secondary v - n e o r t i t e r s on 3T3 c e l l s . That i s , medium was removed from the o r i g i n a l 3T3 v i r a l assays and reassayed on new 3T3 c e l l s for v - n e o r . C. V i r a l I n f e c t i o n The genera l p r o t o c o l for v i r a l i n f e c t i o n i s i l l u s t r a t e d i n F igu re 11. 106 INFECTION OF HUMAN HEMOPOIETIC CELLS WITH V-NEOr LTR neor + X-rays LTR 1 pN2 or pZip neo retroviral packaging cell line I viral producer lines (tfAM, PA12 or PA317) K562 or normal marrow or fetal liver or CML blood + I 24h at 37°C suspension culture x48h I P l^ 1r ft I' 't 1 r: methylcellulose assay + G418 2mg/ml FIGURE 11. I n f e c t i o n of Human Hemopoietic C e l l s w i t h v - n e o r . V i r a l producer l i n e s were produced by t r a n s f e c t i n g n e o r p lasmid DNA i n t o v i r a l packaging l i n e s . I r r a d i a t e d producer c e l l s were used to i n f e c t human target c e l l s (K562, normal marrow, f e t a l l i v e r or CML blood c e l l s ) . Target c e l l s were incubated on v i r a l producer c e l l s fo r 24 hr ( i n some experiments the i n f e c t i o n was done w i t h v i r a l c u l t u r e supernates for 2 hr at 37°C) and then placed i n suspension c u l t u r e for 48 hr to a l l o w i n t e g r a t i o n and express ion of the v i r a l genome. Target c e l l s were then p laced i n m e t h y l c e l l u l o s e assay w i t h or without G418 2 mg/ml and c o l o n i e s scored 14 to 18 days l a t e r . 107 The K562 human leukemic c e l l l i n e or primary c e l l s were i n f e c t e d w i t h v - n e o r by e i t h e r c o - c u l t i v a t i o n w i t h amphotropic v i r a l producer c e l l s which had r e c e i v e d 1500R i r r a d i a t i o n or i ncuba t i on i n supernate from v i r a l producer c e l l s w i t h 8 ug/ml polybrene for va r ious per iods of t ime. A f t e r the i n f e c t i o n p e r i o d they were maintained i n suspension c u l t u r e for 24 to 48 hours before p l a t i n g i n m e t h y l c e l l u l o s e assay w i t h or without G418. C o n t r o l c u l t u r e s that were not exposed to v i r u s were grown i n suspension c u l t u r e and p l a t e d w i t h or wi thout G418 at the same time as the i n f e c t e d c e l l s . Co lon ie s were scored a f t e r p l a t i n g i n m e t h y l c e l l u l o s e on day 5 to 7 for K562 and day 10-14 fo r g ranu locy te macrophage c o l o n i e s (from CFU-GM) and day 18-21 for l a r g e e r y t h r o i d c o l o n i e s (from BFU-E) for primary p rogen i to r assays . C o l o n i e s were not scored unless they conta ined at l e a s t 30 c e l l s and, i n the case of BFU-E, had at l e a s t 3 c l u s t e r s and were c l e a r l y hemoglobinized . The i n f e c t i o u s center assay was done by p l u c k i n g i n d i v i d u a l G418 r e s i s t a n t (G418 r ) granulocyte-macrophage c o l o n i e s from m e t h y l c e l l u l o s e assay and p l a c i n g the d i spe r sed c e l l s from one colony i n a 2 cm^ t i s s u e c u l t u r e w e l l c o n t a i n i n g 10^ N1H-3T3 c e l l s i n medium w i t h 8 ug/ml polybrene . A f t e r ove rn igh t i n c u b a t i o n , the medium was rep laced w i t h f resh medium c o n t a i n i n g G418 1 mg/ml. Seven days l a t e r the assay was scored for the presence of G418 r 3T3 c e l l s . C e l l and v i r a l manipula t ions and c u l t u r e s were performed under L e v e l C containment f o l l o w i n g Med ica l Research C o u n c i l of Canada g u i d e l i n e s fo r h a n d l i n g r e t r o v i r u s e s and human samples. D. DNA and RNA Studies High molecular weight DNA was harvested from expanded c lones of K562 c e l l s and pooled hemopoiet ic c o l o n i e s by c e l l l y s i s w i t h 0.2% SDS and 108 p ro te inase K d i g e s t i o n overn ight at 37°C fo l lowed by phenol and ch loroform e x t r a c t i o n s . Southern b l o t t i n g of K562 and primary hemopoiet ic co lony DNA to n i t r o c e l l u l o s e f i l t e r s was performed by d i g e s t i o n of 10 pg of t o t a l c e l l u l a r DNA w i t h the app rop r i a t e r e s t r i c t i o n endonuclease f o l l o w i n g the manufac turer ' s recommendations. A f t e r agarose g e l e l e c t r o p h o r e s i s the DNA was denatured by soak ing the g e l i n 0.5 M NaOH, 1.5 M NaCl 2 x 30 minutes fo l lowed by 1 M N a C l , 0 .5 M T r i s H C l pH 7.A 2 x 30 minutes to n e u t r a l i z e . DNA was t r a n s f e r r e d to n i t r o c e l l u l o s e by Southern b l o t t i n g w i t h 6 x SSC (0 .9 M N a C l , 0.09 M N a 3 c i t r a t e ) overn ight and the f i l t e r s baked i n a vacuum oven for 2 hours at 80°C (20 ) . In order to detec t n e o r sequences, a 2.3 kb n e o r s p e c i f i c Bam H I , Hind I I I fragment i s o l a t e d from RSV-neo plasmid DNA was 3 2 p _ i a b e l e d by n i c k t r a n s l a t i o n (Bethesda Research L a b o r a t o r i e s , Ga i the r sbe rg , MD, k i t ) f o r use as a probe. F i l t e r s were p r e h y b r i d i z e d , h y b r i d i z e d i n 3 x SSC, A x Denhardts ' s o l u t i o n (.08% F i c o l l , .08% BSA, .08% p o l y v i n y l p y r o l i d i n e ) and s o n i c a t e d , denatured salmon sperm DNA 100 ug/ml at 68°C for 8 hours and h y b r i d i z e d under the same c o n d i t i o n s w i t h 3.3 x 10^ cpm/ml of , heat-denatured 32p_]_ a D e].]. e rj probe ( s p e c i f i c a c t i v i t y 5 x 10? cpm/ug) for 20 hours . H y b r i d i z e d f i l t e r s were washed fo r 30 minutes each time i n 0.1 x SSC, 0.1% SDS, 0.1% sodium pyrophosphate at 55°C x 3 and 65°C x 1. T o t a l c e l l u l a r RNA was harvested by the method of Meinkoth and Wahl (21) from K562 or from primary hemopoiet ic c o l o n i e s that had been i n d i v i d u a l l y p lucked and pooled from m e t h y l c e l l u l o s e assays . 1-5 x 10^ c e l l s were resuspended i n A5 y l i c e c o l d 10 mM T r i s - H C l (pH 7 . 0 ) , 1 mM EDTA, wi th . 10 mM vanadyl nuc l eos ide as a r ibonuc lease i n h i b i t o r . C e l l s were l y s e d by a d d i t i o n of 5 y l 5% Nonidet PA0, i ncuba t i on on i c e 5 minutes, a d d i t i o n of a fu r the r 5 y l 5% Nonidet PA0 and a fu r the r 5 minutes on i c e . The mixture was spun at 109 1 5 , 0 0 0 g f o r 2 . 5 m i n u t e s t o remove n u c l e i and t h e n e x t r a c t e d once w i t h p h e n o l and once w i t h c h l o r o f o r m . 50 y l o f s u p e r n a t a n t was added t o 30 y l 20 x SSC (3 M N a C l , 0 . 3 M N a 3 c i t r a t e , pH 7 . 0 ) and 20 y l 37% f o r m a l d e h y d e . The m i x t u r e was i n c u b a t e d a t 60°C f o r 15 m i n u t e s to d e n a t u r e RNA. S e r i a l d i l u t i o n s were p e r f o r m e d i n 15 x SSC and t he samp les a p p l i e d to n i t r o c e l l u l o s e f i l t e r s t h r o u g h a s p o t b l o t vacuum m a n i f o l d ( S c h l e i c h e r and S c h u e l l , I n c . , K e e n , N H ) . The f i l t e r s were d r i e d and t h e n baked i n a vacuum oven a t 80°C f o r 1 h o u r . The s p o t b l o t f i l t e r s were p r e h y b r i d i z e d and h y b r i d i z e d a t 42°C i n 50% d e i o n i z e d f o r m a m i d e , 5 x D e n h a r d t s ' s o l u t i o n ( 0 . 1 % F i c o l l , 0 . 1% BSA, 0 .1% p o l y v i n y l p y r o l i d i n e ) , 0 .1% SDS, 100 y g / m l h e a t - d e n a t u r e d s a l m o n sperm DNA and 5 x SSC ( 0 . 7 5 M N a C l , 0 . 0 7 5 M N a 3 c i t r a t e , pH 7 . 0 ) . P r e h y b r i d i z a t i o n was done f o r 8 h o u r s f o l l o w e d by h y b r i d i z a t i o n o v e r n i g h t w i t h 2 x 10^ cpm/ml o f 32p_ l a b e l e d , n i c k t r a n s l a t e d p robe as f o r S o u t h e r n b l o t s . Spot b l o t f i l t e r s were washed a t room t e m p e r a t u r e , 4 x 15 m i n u t e s i n 2 x S S C , 0 .1% SDS and 2 x 5 m i n u t e s i n 0 . 1 x S S C , 0 .1% SDS. F i l t e r s were a u t o - r a d i o g r a p h e d a t - 7 0 ° C w i t h t he use o f i n t e n s i f y i n g s c r e e n s ( 2 0 ) . 3 . RESULTS A . V i r a l P r o d u c e r C e l l s Many a t t e m p t s were made to o b t a i n t he h i g h e s t p o s s i b l e v - n e o r t i t e r f r o m v a r i o u s c o m b i n a t i o n s o f p a c k a g i n g l i n e s and e i t h e r the Z i p N e o o r t he N2 v e c t o r ( T a b l e X I ) . A l a r g e number o f c l o n e s were t e s t e d f o r b o t h r e c o m b i n a n t V - n e o r t i t e r s and h e l p e r v i r u s . The n e o r t i t e r f rom t h e s e c l o n e s v a r i e d o v e r a r a n g e o f 2 l o g s . F o r the e c o t r o p i c p a c k a g i n g l i n e s , Y 2 , the h i g h e s t V - n e o r t i t e r amongst 40 c l o n e s was 7 x 10^ c f u / m l u s i n g t he Z i p N e o p l a s m i d . A t l e a s t h a l f o f t h e Y2 c l o n e s were p o s i t i v e f o r h e l p e r v i r u s as j u d g e d by s e c o n d a r y 110 TABLE XI T i t e r s of v - n e o r From Producer C e l l L i nes and pZipNeo Type # Clones x T i t e r Range Tested ( c fu /m l ) Y2 e c o t r o p i c + / -he lper 40 1.3 x 1 0 5 <1 x 1 0 3 - 7 x 105 PA12 amphotropic + he lpe r 21 3.1 x 1 0 4 <1 x 1 0 3 - 3 x 105 YAM amphotropic + he lpe r - he lpe r 29 32 2 x 1 0 3 <1 x 1 0 2 - 3 x 1 0 4 T i t e r s of v - n e o r From pN2 Type V i r a l T i t e r ( c fu /m l ) PA12 amphotropic + he lper PA317 amphotropic - he lpe r 4 x 1 0 6 5 x 1 0 5 I l l i n f e c t i o n of 3T3 c e l l s by supernatant from primary v i r a l assays . For the combinat ion of YAM c e l l s and pZipNeo, the h ighes t t i t e r of h e l p e r - f r e e v i r u s ob ta ined a f t e r s c reen ing 61 clones was 3 x 10^ c f u / m l . With PA12 and pZipNeo, s e v e r a l of the 21 c lones s tud i ed produced approximate ly 10^ c fu /ml but these were found to produce he lper v i r u s as w e l l . With the combinat ion of PA12 and the N2 v e c t o r , h igher t i t e r s of v - n e o r (4 x 10^ c fu /ml ) were ob ta ined , but these a l s o conta ined he lper v i r u s . The h ighes t t i t e r s of h e l p e r - f r e e v - n e o r (5 x 1 0 5 c fu /ml ) were obta ined from the PA317 packaging c e l l l i n e c o n t a i n i n g the N2 p r o v i r a l DNA. B. K562 Experiments To op t imize c o n d i t i o n s for h igh frequency r e t r o v i r a l i n f e c t i o n of human hemopoiet ic p r o g e n i t o r s , we used K562 c e l l s as a convenient model for experiments i n which a number of v a r i a b l e s were e x p l o r e d . F igu re 12a shows the f requencies of G418 r K562 c o l o n i e s obta ined a f t e r exposure o f 1 0 5 K562 c e l l s to v a r i o u s numbers of i r r a d i a t e d v i r a l producer c e l l s of d i f f e r e n t types p l a t e d i n a 60 mm d i s h . The G418 concen t r a t i on of 1 mg/ml comple te ly i n h i b i t e d a l l colony growth i n c o n t r o l p l a t e s . The t rans format ion e f f i c i e n c y inc reased as the number of producer c e l l s increased from 10^ to 10-". Beyond t h i s l e v e l , the producer c e l l s began to approach confluence and the frequency appears to p l a t eau at 1% fo r PA12/ZipNeo, 40% for PA317/N2 and 60% for PA12/N2. The h igher the t i t e r of v - n e o r produced by the c e l l s , the h igher was the frequency of G418 r K562 transformants ob ta ined . F igu re 12b i s a graph of s i m i l a r data for i n f e c t i o n of 10^ K562 c e l l s a f t e r i n c u b a t i o n w i t h 3 ml of va r ious d i l u t i o n s of supernatant from c u l t u r e s of v - n e o r producer c e l l s . For the PA12/ZipNeo c lone producing v i r u s at 1 x 10^ c f u / m l , there was a c l e a r c o r r e l a t i o n between d i l u t i o n of v i r a l 0.01 106 Number of Irradiated Viral Producer Cells 1:103 1:X)2 1=10 neat Dilution Viral Supernatant FIGURE 12. G418 r K562 co lon ie s (X of co lon i e s grown without G418) a f t e r i n f e c t i o n of 10^ K562 c e l l s wi th va r ious sources of v - n e o r : PA12/N2 c e l l l i n e wi th t i t e r 4 x 1 0 6 c f u / m l , + he lpe r v i r u s ; PA317/N2 c e l l l i n e wi th t i t e r 5 x 1 0 5 c f u / m l , h e l p e r - f r e e PA12/ZipNeo c e l l l i n e w i t h t i t e r 1 x 1 0 5 c f u / m l , + he lpe r v i r u s ; a) c o - c u l t i v a t i o n for 24 hours w i t h va r ious numbers of i r r a d i a t e d v -neo r producer c e l l s . b) incuba t ion x 2 hours w i t h 3 ml of v - n e o r c o n t a i n i n g supernatant at va r ious d i l u t i o n s . 113 supernatant and frequency of G418 r K562 c e l l s , to a maximum of 0.25%. For h ighe r t i t e r v i r u s the frequency appeared to p la teau at 1:10 d i l u t i o n s and d e c l i n e d somewhat w i t h und i l u t ed supernatant . Maximum G418 r K562 c e l l f requencies were 20% fo r PA317/N2 supernatant and 60% for PA12/N2 superna tant . F igu re 13 shows the frequency of G418 r K562 c o l o n i e s obta ined by i n f e c t i o n w i t h u n d i l u t e d v - n e o r - c o n t a i n i n g medium or c o - c u l t i v a t i o n w i t h conf luen t c e l l s of va r ious types . The c e l l l i n e s s e l e c t e d were those producing the h ighes t v - n e o r t i t e r for the va r ious packaging l i n e / n e o r p lasmid combina t ions . At v i r a l t i t e r s <10^ c fu /ml the maximum frequency of G418 r K562 c e l l s was approximate ly 2% a f t e r c o - c u l t i v a t i o n w i t h producer c e l l s . Frequencies obta ined a f t e r i n f e c t i o n w i t h supernatant from the same c e l l l i n e s was at l e a s t 4 - f o l d lower than that obtained by c o - c u l t i v a t i o n . At the h ighes t t i t e r , 4 x 10^ c f u / m l , the d i f f e r e n c e i n e f f i c i e n c y between c o -c u l t i v a t i o n and supernatant d isappeared. A number of other v a r i a b l e s that were i n v e s t i g a t e d d i d not appear to a f f e c t the frequency of K.562 c e l l t ransformat ion to G418 r . These i n c l u d e d ex tend ing the d u r a t i o n of c o - c u l t i v a t i o n from 6 to 48 hours , or ex tending the d u r a t i o n o f exposure to supernatant from 2 to 48 hours . The n e o r gene was demonstrated i n G418 r K.562 c e l l DNA by expanding i n d i v i d u a l c o l o n i e s i n l i q u i d c u l t u r e and e x t r a c t i n g c e l l u l a r DNA fo r Southern b l o t s . F igu re 14a demonstrates the 4 kb neo r s p e c i f i c Xba l fragment found i n 4 G418 r K562 c o l o n i e s but not found i n c o n t r o l K562 DNA. F igu re 14b shows DNA from the same 4 G418 r K562 c o l o n i e s and 2 un infec ted c o n t r o l K562 c o l o n i e s cut w i t h enzymes Bam HI ( lanes 1-6) or EcoRI ( lanes 7-12) which cut on ly once w i t h i n the n e o r p r o v i r a l DNA. The neo r s p e c i f i c fragments demonstrated i n the G418 r c o l o n i e s ( lane 1-4 and 7-10) are s i n g l e and unique to each colony i n d i c a t i n g one random s i t e of i n t e g r a t i o n for the p r o v i r u s i n the c e l l u l a r DNA from each K562 co lony . 100 <M <o in oo O 10 >» o c 01 cr E E 1 x ra 2 0.1 cell lines/ plasmid ^AM/Z ipneo PA12/Zipneo PA317/N2 PA12/N2 helper virus — + — + viral titer (cfu/ml) 2x10* 1x10s 5x10s 4x10° FIGURE 13. G418 r K.562 c o l o n i e s (Z of c o l o n i e s grown without G418) a f t e r i n f e c t i o n of 1 0 5 c e l l s w i t h v - n e o r from 3 ml u n d i l u t e d supernatant or a 60 mm d i s h of con f luen t , i r r a d i a t e d v i r a l producer c e l l s of va r ious types . supernatant c o - c u l t i v a t i o n 115 FIGURE 14. Southern b l o t s of t o t a l c e l l u l a r DNA h y b r i d i z e d w i t h a 3 2 P - l a b e l e d neo r s p e c i f i c Bam H l - H i n d I I I fragment from pRSV-neo. a) K562 DNA from cloned G418 r c e l l s i n f e c t e d w i t h YAM/ZipNeo v i r a l s tocks ( lanes 1-4) or un infec ted ( lane 5) and d i g e s t e d wi th X b a l . * 4kb neo r s p e c i f i c fragment i n i n f e c t e d c e l l s . b) DNA from the same G418 r K562 c lones as i n (a) ( lanes 1-4 and 7-10) and un infec ted clones (Lanes 5 ,6 ,11 ,12) d iges t ed w i t h Bam HI ( lanes 1-6), or EcoRI ( lanes 7-12) . These enzymes cut once w i t h i n p r o v i r a l DNA and once w i t h i n c e l l u l a r DNA to generate a s i n g l e neo r s p e c i f i c fragment of unique s i z e i n each of the G418 r c l ones . c) DNA from pooled primary hemopoiet ic c o l o n i e s e i t h e r i n f e c t e d w i t h PA12/N2 v i r a l s tocks ( lanes 2 ,3 ,5 ) or un in fec ted ( lanes 1,6,7) and d iges ted w i t h EcoRI. The expected 1.5 kb n e o r s p e c i f i c fragment i s seen i n lane 2 ( c e l l s i n f e c t e d w i t h v i r a l supernatant but not s e l e c t e d i n G418), lane 3 (G418 r c e l l s a f t e r i n f e c t i o n w i th superna tan t ) , and lane 5 ( c e l l s i n f e c t e d by c o - c u l t i v a t i o n w i th producer c e l l s but not s e l e c t e d i n G418). Lanes 4 shows no neo r s p e c i f i c h y b r i d i z a t i o n from c e l l s exposed to supernatant from PA317/N2 producer c e l l s but not s e l e c t e d i n G418. 116 C. I n f e c t i o n s of Primary Hemopoietic P rogen i to r s A t o t a l of A normal marrow, 6 f e t a l l i v e r and 6 GML blood samples were i n f e c t e d w i t h v - n e o r from a v a r i e t y of packaging c e l l l i n e / n e o r p lasmid combina t ions . The i n f e c t i o n procedure d i d not appear to be t o x i c to the t a rge t c e l l s as shown by a l a c k of any e f f ec t on c e l l recovery or p l a t i n g e f f i c i e n c y . (Values for i n f e c t e d c e l l s were 80 to 100% of c o n t r o l v a l u e s ) . A GA18 c o n c e n t r a t i o n of 2 mg/ml ( e f f e c t i v e drug concen t r a t i on 1 mg/ml) comple te ly i n h i b i t e d a l l colony growth i n assays of un in fec ted pr imary p r o g e n i t o r s . No d i f f e r e n c e i n GA18 s e n s i t i v i t y between the v a r i o u s ta rge t c e l l sources or between p rogen i to r s of d i f f e r e n t l ineages ( i . e . BFU-E and CFU-GM) could be demonstrated. F igu re 15a shows the frequency of GA18 r CFU-GM seen a f t e r i n f e c t i o n by v - n e o r producer c e l l s at va r ious v i r a l t i t e r s . Each poin t represents a d i f f e r e n t experiment combining target c e l l s of a c e r t a i n type w i t h a s p e c i f i c v i r a l producer c e l l l i n e . Al though there was a c o r r e l a t i o n between v i r a l t i t e r and frequency of G A 1 8 r , cons ide rab le v a r i a t i o n i n t r ans format ion e f f i c i e n c y at any g iven t i t e r was a l s o found; for example, from 2.A to 15.7% GA18 r CFU-GM at a t i t e r of A x l O 6 c f u / m l . There was a l s o a low (0.0A5 to 0 .A%), but r e p r o d u c i b l e inc idence of GA18 r c o l o n i e s a f t e r i n f e c t i o n w i t h c e l l s producing v i r a l t i t e r s as low as 1 0 3 c f u / m l . F igu re 15b shows s i m i l a r data for BFU-E. I n f e c t i o n s w i t h c e l l s producing a v - n e o r t i t e r of 1 x 1 0 3 c f u / m l , y i e l d e d a frequency of l a r g e GA18 r e r y t h r o i d c o l o n i e s of l e s s than 1% w h i l e f requencies up to 5.6% were seen a f t e r i n f e c t i o n s done w i t h c e l l l i n e s producing h igher t i t e r s . However, the c o r r e l a t i o n of GA18 r frequency w i t h t i t e r was l e s s c l e a r and the v a r i a t i o n i n GA18 r frequency at any g iven t i t e r was s i m i l a r to that seen for CFU-GM. From these data (F igures 15a & 15b), i t appears that BFU-E may be l e s s r e a d i l y 100 o o 10 o o LU ki-rn eg O b o c a> D a a> 10 3 10" 10 5 10 6 10' 0.01 10 J Viral Titer of Confluent Viral Producers Cells (CFU/ml) 10" 10 s 10" 10 7 Viral Titer of Confluent producer Cells (CFU/ml) FIGURE 1 5 . F r e q u e n c y (X) o f G 4 1 8 r p r i m a r y h e m o p o i e t i c c o l o n i e s ( G 4 1 8 r c o l o n i e s / t o t a l c o l o n i e s w i t h o u t G418 x 100) a f t e r 24 h o u r s e x p o s u r e o f 5 x 10^ c e l l s to a 60 mm d i s h o f i r r a d i a t e d , c o n f l u e n t v - n e o r p r o d u c e r c e l l l i n e s a t v a r i o u s v i r a l t i t e r s . 0 no rma l bone marrow, O CML b l o o d , A f e t a l l i v e r a ) g r a n u l o c y t e , macrophage c o l o n i e s ; CFU-GM b) l a r g e e r y t h r o i d c o l o n i e s ; BFU-E C o r r e l a t i o n ( r ) o f l o g ^ n f r e q u e n c y G 4 1 8 r c o l o n i e s v s . l o g ^ v i r a l t i t e r i s 0 .71 (p<.001) f o r f i g u r e a and 0 . 6 4 (p< .01) f o r f i g u r e b. 118 t ransformed by v - n e o r than CFU-GM. A l though t h i s may be t r u e , i t i s p o s s i b l e that the apparent d i f f e r e n c e i s the r e s u l t of exper imenta l v a r i a b l e s which p a r t i c u l a r l y a f f e c t e r y t h r o i d co lony growth. For example, i t was found that i n g e n e r a l , G418 r e r y t h r o i d c o l o n i e s were not as l a rge or as red as those ob ta ined from i n f e c t e d BFU-E p la ted i n the absence of G418. I t i s p o s s i b l e that i m p u r i t i e s i n the G418 a f f e c t the growth of e r y t h r o i d c o l o n i e s c o n t a i n i n g the n e o r gene. A l t e r n a t i v e l y , the n e o r gene may be expressed at a lower l e v e l i n e r y t h r o i d c e l l s as they d i f f e r e n t i a t e . The re fo re , i t i s l i k e l y that the f requenc ies recorded here f o r G418 r BFU-E represent minimum e s t i m a t e s . The maximum f requenc ies of G418 r p rogen i to rs from these exper iments a re summarized i n Tab le X I I . The h ighes t f requency was u s u a l l y ob ta ined a f t e r i n f e c t i o n w i th the c e l l l i n e producing the h ighest v i r a l t i t e r , PA12/N2 c e l l s p roduc ing v - n e o r at 4 x 10^ c f u /m l i n a d d i t i o n to he lpe r v i r u s . The a b i l i t y of v i r a l supernatant , as compared to c o - c u l t i v a t i o n w i t h producer c e l l s of v a r i o u s types , to t ransform pr imary p rogen i to rs to G418 r i s compared i n F igu re 16. At a t i t e r of 6 x 1 0 3 c f u /m l produced by YAM/ZipNeo c e l l s , supernatant was unable to produce a s i g n i f i c a n t i nc idence of G418 r c o l o n i e s wh i l e c o - c u l t i v a t i o n y i e l d e d a frequency of approx imate ly 0.1%. At t i t e r s of 5 x 10^ or 4 x 10^ c f u / m l , supernatant was e f f e c t i v e at t rans fo rm ing both BFU-E and CFU-GM. However, the frequency was s t i l l 2 to 50 f o l d h ighe r u s i n g c o - c u l t i v a t i o n . Neve r t he l ess , the .frequency of G418 r c o l o n i e s was h ighe r a f t e r i n f e c t i o n w i th medium at h igh t i t e r s (10.6% fo r CFU-GM at 4 x 10^ c f u / m l ) than w i th c o - c u l t i v a t i o n at low t i t e r s (0.13% CFU-GM at 6 x 1 0 3 c f u / m l ) . The f requenc ies of t rans fo rmat ion f o r pr imary p rogen i to rs were not as h igh as those ob ta ined fo r K562 c e l l s . To t r y and i nc rease the e f f i c i e n c y , t a rge t c e l l s were a l lowed to remain on v i r a l producer c e l l s f o r as l ong as 7 days wi thout any i nc rease i n the e f f i c i e n c y of t r ans fo rma t i on . TABLE XII Maximum Frequency of G418 r 1° Hemopoietic Co lon ies a f t e r Co -Cu l t i va t i on wi th Various v - n e o r Producer C e l l s Target C e l l s V i r a l Producer C e l l s v -neo r T i t e r (c fu/ml) G418 r Co lon ies /Co lon ies Without G418 (Z) CFU-GM + Vi rus C o n t r o l * * BFU-E + V i r u s C o n t r o l * * Normal Marrow F e t a l L i v e r CML Blood YAM/ZipNeo PA317/N2 PA12/N2* YAM/ZipNeo PA317/N2 PA12/N2* TAM/ZipNeo PA317/N2 PA12/N2* 3 x 10 4 5 x 10 5 4 x 10 6 3 x 10 4 5 x 10 5 4 x 10 6 6 x 10 3 5 x 10 5 4 x 10 6 26/2775 (0.94) 0/2970 78/1860 (4.2) 0/630 127/1800 (7.1) 0/630 24/2960 (0.81) 0/3996 0/150 2/158 (1.3) 13/414 (3.1) 0/150 48/9900 (0.48) 0/6600 66/3120 (2.1) 0/3888 656/4170 (15.7) 0/16920 85/3930 (2.2) 0/4050 101/4260 (2.4) 0/4050 54/3333 (1.6) 0/18468 1/105 (1.0) 0/120 2/690 (0.31) 0/120 18/14320 (0.13) 0/9523 194/3480 (5.6) 0/6480 1672/31650 (5.3) 0/81180 * + Helper v i r us * * Con t ro l = C e l l s from the same sample plated i n methy lce l lu lose + G418 without exposure to v - n e o r producer c e l l s . 120 (0 o c 0> cn o co o c CT 0) 10 0.1 0.01 J I cell lines/plasmid ^AM/Zip neo ^AM/Zipneo PA 317/N2 PA12/N2 viral titer (cfu/ml) 6*103 3X104 5x10s 4xi06(+helper) FIGURE 16. Frequency (X) of G418 r pr imary hemopoiet ic p rogen i t o r s (G418 c o l o n i e s / t o t a l c o l o n i e s without G418 x 100) a f t e r i n f e c t i o n of 5 x l O 6 c e l l s by v - n e o r from a 60 mm d i s h of i r r a d i a t e d con f luen t producer c e l l s or 5 ml u n d i l u t e d supernatant of va r i ous types . A l l i n f e c t i o n s were done w i th CML p rogen i to rs except at t i t e r of 3 x 10 4 c fu /ml where the ta rge t was f e t a l l i v e r c e l l s . BFU-E CFU-GM super-natant conf1uent producer cel ls super- confluent natant producer eel 1 s 121 The a b i l i t y o f G418 r CFU-GM that had been i n f e c t e d w i th h e l p e r - c o n t a i n i n g v - n e o r to i n f e c t 3T3 c e l l s was tes ted by i n f e c t i o u s center assay. In two exper iments , 5 of 22 and 24 of 24 CFU-GM transformed 3T3 c e l l s to G418 r e s i s t a n c e (Table X I I I ) . The produc t ion of G418 r 3T3 i n t h i s assay r e q u i r e s that the i n f e c t i n g c e l l s be producing v - n e o r . To do so they, themselves, must have been i n f e c t e d by both v - n e o r and he lper v i r u s , which may occur r e l a t i v e l y i n f r e q u e n t l y . Th i s would account for l e s s than 100% of G418 v CFU-GM being p o s i t i v e i n the i n f e c t i o u s center assay. F igu re 14c i s a Southern b l o t of DNA harvested from pooled hemopoiet ic c o l o n i e s grown from CML blood c e l l s that were e i t h e r un in fec ted or exposed to v i r a l s tocks c o n t a i n i n g v - n e o r . A f t e r d i g e s t i o n w i th EcoRI the n e o r s p e c i f i c probe i d e n t i f i e d the expected 1.5 kb neo r s p e c i f i c fragment i n G418 r c e l l s ( l ane 3 ) . A l e s s in tense s i g n a l i s seen i n lane 2 where the same c e l l s were exposed to medium c o n t a i n i n g v - n e o r but not s e l e c t e d i n G418 i n d i c a t i n g that some of the c e l l s d i d not con ta in the neo r gene, (8% of the CFU-GM i n t h i s sample were G 4 1 8 r ) . In lane 5 the DNA from c e l l s exposed to v i r a l producer c e l l s but not s e l e c t e d i n G418 shows a s t rong s i g n a l . In colony assays 15.7% of the CFU-GM i n t h i s sample were G418 r . The s t r eng th of the h y b r i d i z a t i o n s i g n a l i n lane 5 as compared to lane 3 where an equ iva len t amount of DNA was h y b r i d i z e d i n d i c a t e s that at l e a s t 15.7% of the c e l l s i n t h i s sample con ta ined the n e o r gene. DNA from c e l l s from the same sample that were not exposed to v i r u s , i n lanes 1, 6 and 7 show no s i g n a l . Expres s ion of the neo r gene by v - n e o r i n f e c t e d , pooled primary p rogen i to r s or K562 c e l l s , was shown by RNA spot b l o t (F igu re 17) . T o t a l c e l l u l a r RNA was h y b r i d i z e d w i t h a neo r s p e c i f i c probe. Un in fec t ed , c o n t r o l c e l l s show no evidence of neo r h y b r i d i z a t i o n , wh i l e the same number of i n f e c t e d c e l l s show a s t rong s i g n a l . The s i g n a l i s s t ronges t fo r c e l l s that TABLE X I I I Demonstrat ion of v - n e o r P roduc t ion i n G418 r CFU-GM by I n f e c t i o u s Center Assay Experiment + ve CFU-GM/Total Tested 1 2 5/22 24/24 123 pZipneo Control- CFU-GM PA12/N2 I PA317/N2 a-lb-Control -PA12/N2-PA317/N2-i K562 4 1 Number of cells X 105 FIGURE 17. RNA spot b l o t of t o t a l c e l l u l a r RNA from pooled CML granulocyte-macrophage c o l o n i e s or K562 c e l l s h y b r i d i z e d w i t h a 3 2 p _ i a b e i e c j n eo r s p e c i f i c Bam HI-Hind I I I fragment from pRsv-neo. C o n t r o l c e l l s were not exposed to v - n e o r or G418. C e l l s exposed to e i t h e r PA12/N2 or PA317/N2 v - n e o r producer c e l l s were e i t h e r s e l ec t ed i n G418 (b) or unse lec ted (a) i n the case of CFU-GM. I n s u f f i c i e n t c e l l s were a v a i l a b l e to a l l o w samples for the PA317/N2, 4 x 10^ c e l l s column. Therefore , these two spots are b lank . A l l i n f e c t e d K562 c e l l s were s e l e c t e d i n G418. pZipNeo DNA serves as a p o s i t i v e c o n t r o l i n the top lane (2 and 0.5 pg DNA from l e f t to r i g h t ) . 124 were s e l e c t e d i n G418 ( lanes b, CFU-GM and a l l the i n f e c t e d K562 c e l l s ) but i s a l s o seen i n c e l l s that were i n f e c t e d but not s e l e c t e d ( lanes a, CFU-GM). 3) DISCUSSION These data demonstrate e f f i c i e n t t r ans fe r and express ion of the n e o r gene to both the K562 human leukemic c e l l l i n e and a v a r i e t y of normal and malignant pr imary human hemopoiet ic p rogen i to r s us ing recombinant r e t r o v i r u s e s . A number of v a r i a b l e s were i n v e s t i g a t e d to t r y to achieve the h ighes t p o s s i b l e e f f i c i e n c i e s of gene t r a n s f e r . C o - c u l t i v a t i o n w i t h v i r a l producer c e l l s r a the r than i n f e c t i o n w i t h v i r a l supernatant and use of h igh t i t e r v i r u s were both i d e n t i f i e d as important parameters, but v i r a l t i t e r c l e a r l y had the g rea t e s t i n f l u e n c e on the frequency of t ransformat ion to G418 r e s i s t a n c e . Cons ide rab le e f f o r t was devoted to s tudy ing v i r a l producer c e l l s generated by v a r i o u s packaging l i n e / n e o r - c o n t a i n i n g plasmid combinat ions . The cho ice of these v a r i a b l e s c l e a r l y had a major impact on the t i t e r of both v - n e o r and he lpe r v i r u s . We were unable to generate h igh t i t e r v - n e o r from the PA12 packaging l i n e that was not a s soc i a t ed w i t h he lper v i r u s . The use of the pN2 vec to r r a the r than ZipNeo r a i s e d the v - n e o r t i t e r but d i d not e l i m i n a t e the problem of he lpe r v i r u s . Al though the PA12 l i n e has been cons t ruc ted to generate h e l p e r - f r e e recombinant v i r u s , i t appears that s u f f i c i e n t g e n e t i c recombinat ion occurs between the d e f e c t i v e packaging and recombinant n e o r v i r a l sequences to f r equen t ly generate s i g n i f i c a n t he lper v i r u s t i t e r s w i t h the vec to r used i n t h i s s tudy. He lpe r - f r ee v - n e o r generated by the YAM packaging l i n e c o n t a i n i n g pZipNeo DNA, was always of r e l a t i v e l y low t i t e r (<10^ c f u / m l ) . The newer genera t ion of r e t r o v i r a l packaging l i n e s , such as the PA317 developed by M i l l e r and But t imore (16) , may so lve the problem of o b t a i n i n g h igh t i t e r recombinant v i r u s without a s soc i a t ed h e l p e r . 125 Al though both ZipNeo and N2 con ta in the same n e o r gene under the c o n t r o l of the promoter i n a Moloney leukemia v i r u s LTR the two vec to r s are not i d e n t i c a l i n s t r u c t u r e ( 6 , 2 0 ) . Al though the d i f f e r ences appear s u b t l e they seem to a f f e c t the v i r a l t i t e r s obta ined from producer c e l l s and might a l s o cause d i f f e r e n t l e v e l s of express ion of the t r ans f e r r ed f o r e i g n gene i n i n f e c t e d hemopoiet ic c e l l s ; For t h i s reason we e l e c t e d to t e s t v i r u s d e r i v e d from both the N2 and ZipNeo vec to r s on our target c e l l s . Al though our da ta are not d e f i n i t i v e , some of our r e s u l t s ( e . g . F igure 13 where K562 were the ta rge t c e l l s and a 5 f o l d increase i n v i r a l t i t e r lead to a 20 f o l d i nc rease i n frequency of G418 r c o l o n i e s ) may be exp la ined i f neo r was expressed more e f f i c i e n t l y i n c e l l s i n f e c t e d w i t h v i r u s de r ived from N2 ra the r than ZipNeo. In experiments done w i t h h e l p e r - f r e e v i r u s , from a producer l i n e d e r i v e d from PA317 c e l l s and the N2 p lasmid , we were s u c c e s s f u l i n demonstra t ing gene t r a n s f e r at l e v e l s on ly s l i g h t l y lower than those achieved w i t h he lpe r c o n t a i n i n g v i r u s of s i g n i f i c a n t l y h igher v - n e o r t i t e r . No obvious e f f e c t of he lpe r v i r u s on the frequency of gene t r ans fe r was de tec ted . In a d d i t i o n to normal bone marrow, we evaluated two a l t e r n a t i v e sources of pr imary human p rogen i to r s for r e t r o v i r a l i n f e c t i o n s . These were f e t a l l i v e r and CML b l o o d . In both cases, a h igh p r o p o r t i o n of the p r i m i t i v e p r o g e n i t o r s i n these t i s s u e s are i n the a c t i v e part of the c e l l c y c l e as compared to t h e i r counterpar t s i n normal marrow which are l a r g e l y qu iescent ( 2 2 , 2 3 ) . C e l l c y c l i n g s t a tus has been suggested as an important v a r i a b l e a f f e c t i n g the success of r e t r o v i r a l i n f e c t i o n and i n t e g r a t i o n i n t o host DNA (24 ) . Al though some of our h ighes t f requencies of G418 r c o l o n i e s were seen i n the CML blood c u l t u r e s , there was s u f f i c i e n t v a r i a b i l i t y from experiment to experiment that no o v e r a l l d i f f e r e n c e was ev iden t . However, there was c l e a r l y no dramat ic improvement i n the frequency of G418 r p rogen i to r s i n experiments 126 u s i n g e i t h e r of the primary human ta rge ts that were presumably c y c l i n g o p t i m a l l y . Al though encouraging, the frequency of gene t r ans fe r demonstrated to pr imary p rogen i to r s by these data i s s t i l l much lower than the t h e o r e t i c a l 100% that should be ob ta inab le w i t h r e t r o v i r u s e s ( 1 ) . Our f requencies are reasonably c l o s e to those repor ted by Hock and M i l l e r fo r gene t r a n s f e r to pr imary normal human hemopoiet ic p rogen i to r s from bone marrow (12) , but are much lower than the 75 to 100% frequency repor ted by E g l i t i s et a l fo r murine CFU-S ( 6 ) . However, i t i s notable that the l a t t e r authors and others have found the l e v e l of express ion of G418 r e s i s t a n c e or neo r gene product to be h i g h l y v a r i a b l e among d i f f e r e n t CFU-S c o n t a i n i n g the n e o r gene ( 4 , 5 , 6 ) . The f requencies of G418 r e s i s t a n c e i n murine CFU-GM i n f e c t e d and s e l e c t e d i n v i t r o have been approximate ly 10 to 30% (4 ,5) which i s a l s o lower than one might have p r e d i c t e d and cons i s t en t w i t h our r e s u l t s on human p r o g e n i t o r s . A l a r g e number of techniques e x i s t fo r t r a n s p o r t i n g gene t i c m a t e r i a l i n t o c e l l s . A number of these have been a p p l i e d to primary hemopoiet ic p r o g e n i t o r s i n c l u d i n g ca l c ium phosphate c o - p r e c i p i t a t i o n and e l e c t r o p o r a t i o n (25-27) . Al though i n i t i a l r e s u l t s u s ing the former technique to confer methotrexate r e s i s t a n c e on murine bone marrow c e l l s appeared p romis ing , s i m i l a r r e s u l t s have not been for thcoming for other genes or for human c e l l s . E l e c t r o p o r a t i o n i s a r e l a t i v e l y new technique that has not been wide ly a p p l i e d to gene t r a n s f e r i n t o primary c e l l s . I n i t i a l r e s u l t s w i t h human bone marrow p r o g e n i t o r s demonstrate low l e v e l s of t r ans fe r and express ion of the x a n t h i n e -guanine phosphor ibosy l t rans fe rase gene i n CFU-GM (27) . The f u l l p o t e n t i a l of t h i s technique remains to be e x p l o r e d . At the present time the bu lk of evidence would i n d i c a t e that the h ighes t e f f i c i e n c i e s of gene t r ans f e r are those mediated by r e t r o v i r u s e s . 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Human marrow c e l l s capable of e r y t h r o p o i e t i c d i f f e r e n t i a t i o n i n v i t r o : D e f i n i t i o n of three e r y t h r o i d colony responses . Blood 49: 855, 1977. 18. M i l l e r AD, Trauber DR, But t imore C. Fac tors i n v o l v e d i n the p roduc t ion of he lpe r v i r u s - f r e e r e t r o v i r u s v e c t o r s . Somatic C e l l Mol Genet 12: 175, 1986. 19. Cepko CL, Roberts BE, M u l l i g a n RC. C o n s t r u c t i o n and a p p l i c a t i o n s of a h i g h l y t r a n s m i s s i b l e murine r e t r o v i r u s s h u t t l e v e c t o r . C e l l 37: 1053, 1984. 20. M a n i a t i s T, F r i t s h EF, Sambrook J . Molecu la r c l o n i n g : A l a b o r a t o r y ' manual. Co ld Spr ing Harbor, NY, Cold Spr ing Harbor Labora to ry , pp 383, 1982. 21. Meinkoth J , Wahl G. H y b r i d i z a t i o n of n u c l e i c ac ids immobi l i zed on s o l i d suppor t s . Ana l Biochem 138: 267, 1984. 22. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves C J . Unregulated p r o l i f e r a t i o n of p r i m i t i v e ch ron ic myeloid leukemia p rogen i to r s i n the presence of normal adherent c e l l s . Proc N a t l Acad S c i USA 83: 5306, 1986. 23. Pesch le C, M i g l i a c c i o AR, M i g l i a c c i o G, C i c c a r i e l l o R, L e t t i e r i F , Q u a t t r i n S, Russo G, Mastroberadino G. I d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of three c l a s se s of e r y t h r o i d p rogen i to r s i n human f e t a l l i v e r . Blood 58: 565, 1981. 24. Varmus H, Swanstrom R. R e p l i c a t i o n of r e t r o v i r u s e s . I n : "RNA Tumor V i r u s e s , 2nd E d i t i o n " , Weiss R, Te ich N , Varmus H, C o f f i n J ( eds ) , Co ld Spr ing Harbor Labora to ry , Cold Spr ing Harbor, New York , pp 467, 1984. 25. C l i n e MJ, Stang H, Mercola K, Morse L , Ruprecht R, Browne J , S a l s e r W. Gene Transfe r i n i n t a c t an imals . Nature 284: 422, 1980. 26. Car r F , Medina WD, Dube S, B e r t i n o JR. Genet ic t rans format ion of murine bone marrow c e l l s to methotrexate r e s i s t a n c e . Blood 62: 180, 1983. 130 27. Toneguzzo F , K e a t i n g A . S tab le express ion of s e l e c t a b l e genes in t roduced i n t o human hematopoie t ic stem c e l l s by e l e c t r i c f i e l d mediated DNA t r a n s f e r . Proc N a t l Acad S c i USA ( i n p r e s s ) . 28. Anderson WF. Prospects for human gene therapy. Science 226: 401, 1984. 131 C H A P T E R V INFECTION OF HUMAN LONG-TERM CULTURES WITH RECOMBINANT RETROVIRUSES: PRELIMINARY EXPERIMENTS 1) INTRODUCTION The long- te rm bone marrow c u l t u r e system as developed by Dexter et a l (1) fo r murine c e l l s and modif ied by Greenberger (2) and others (3) fo r human c e l l s p rov ides an oppor tun i ty to study hemopoiesis i n v i t r o over a pe r iod of s e v e r a l months. The c e l l u l a r i n t e r a c t i o n s and r e g u l a t i o n that takes p lace i n these c u l t u r e s appear to mimic i n many ways events that occur i n v i v o i n the bone marrow microenvironment. One a p p l i c a t i o n of human long- te rm marrow c u l t u r e s to the study of hemopoiesis i s as a system i n which to i n v e s t i g a t e techniques i n v i t r o a l though t h e i r use i n v i v o i s not yet p o s s i b l e . For example, r e t r o v i r a l - m e d i a t e d gene t r ans fe r to hemopoiet ic p rogen i to r s has p o t e n t i a l fo r use i n the therapy of i n h e r i t e d gene t i c d i s o r d e r s ( 4 ) . However, p r i o r to i n v i v o experiments i n humans, the use of these v i r a l vec to r s r e q u i r e ex t ens ive t e s t i n g , not on ly i n an imals , but i n i n v i t r o human systems. Such systems should assess the frequency and s t a b i l i t y of gene t r ans fe r and the l e v e l s of express ion of the t r ans fe r r ed gene. The long- term marrow c u l t u r e p rov ides the oppor tun i ty to assess v i r a l l y - i n f e c t e d hemopoiet ic c e l l s over time fo r these f ea tu res . In a d d i t i o n , experiments designed to study the importance of a p a r t i c u l a r gene i n normal or abnormal hemopoiesis can be performed us ing r e t r o v i r a l - m e d i a t e d gene t r ans fe r to hemopoiet ic c e l l s i n long- te rm c u l t u r e s . Candidate genes i nc lude the va r ious oncogenes. 132 A number of i n v e s t i g a t o r s have i n f e c t e d murine and human long- te rm marrow c u l t u r e s w i t h a c u t e l y t ransforming r e t r o v i r u s e s c o n t a i n i n g oncogenes (5-8). Among the most i n t e r e s t i n g r e s u l t s are those repor ted by Dexter et a l i n which v i r u s c o n t a i n i n g the v - s r c oncogene was used to i n f e c t murine c u l t u r e s ( 7 ) . I n f ec t ed c u l t u r e s showed increased p rogen i to r numbers i n the non-adherent c e l l f r a c t i o n and sp leen colony forming c e l l s (CFU-S) from these c u l t u r e s showed inc reased a b i l i t y to s e r i a l l y repopulate the hemopoiet ic system of i r r a d i a t e d r e c i p i e n t an ima l s . A number of immorta l , non-leukemogenic f a c t o r dependent c e l l l i n e s were de r i ved from these c u l t u r e s ( 9 ) . Al though i t i s not c l e a r what r o l e the v - s r c oncogene i s p l a y i n g i n the e t i o l o g y of these phenomena, these events are c l e a r l y of i n t e r e s t to those i n v e s t i g a t i n g the r e g u l a t i o n of hemopoiesis p a r t i c u l a r l y i f they can be demonstrated to occur i n human systems. R e t r o v i r a l - m e d i a t e d t r ans fe r of the n e o r gene has r e c e n t l y been demonstrated to human hemopoiet ic c e l l s i n shor t - t e rm m e t h y l c e l l u l o s e assays (10 ) . The experiments desc r ibed i n t h i s paper desc r ibe attempts to extend that exper ience by u s ing human hemopoiet ic c e l l s i n long- term c u l t u r e as a ta rge t for t r ans fe r of the n e o r gene. Experiments i n which r e t r o v i r u s c o n t a i n i n g the v - s r c oncogene were used as the i n f e c t i n g vec to r are a l s o d e s c r i b e d . 2) MATERIALS AND METHODS A . C e l l s and C u l t u r e Cond i t i ons C e l l l i n e s were c u l t u r e d i n Dulbecco ' s modif ied Eagle medium w i t h h igh g lucose (4 .5 g /1) and 10% heat i n a c t i v a t e d c a l f serum ( fo r Y2 c e l l s ) or 10% f e t a l c a l f serum for a l l o ther c e l l types i n 5% C0£ atmosphere at 37°C. The amphotropic r e t r o v i r u s packaging l i n e , PA12, the e c o t r o p i c packaging l i n e , Y2, 133 and the 2-1-292 c e l l l i n e producing amphotropic v - s r c - c o n t a i n i n g r e t r o v i r u s have been p r e v i o u s l y desc r ibed (11 ,12 ,13 ) . Pr imary human c e l l s were obta ined e i t h e r from consent ing a d u l t s (CML p e r i p h e r a l b lood , preleukemic bone marrow, or s k i n f i b r o b l a s t s ) or from second t r i m e s t e r abor t ions ( f e t a l l i v e r c e l l s ) a f t e r approval of the C l i n i c a l Screening Committee for Research I n v o l v i n g Human Subjects of the U n i v e r s i t y of B r i t i s h Columbia . Blood and some bone marrow c e l l s were passed over a P e r c o l l d e n s i t y g rad ien t ( d e n s i t y 1.063) and l i g h t d e n s i t y c e l l s c o l l e c t e d and used i n subsequent exper iments . F e t a l l i v e r was minced w i t h s c i s s o r s , and incubated fo r 3 hours at 37°C i n a medium w i t h 20% f e t a l c a l f serum and co l l agenase 1 mg/ml (Sigma Chemical C o . , S t . L o u i s , MO). The c e l l s were then passed through a success ion of needles of decreas ing gauge, washed and used fo r the experiments d e s c r i b e d . S k i n samples were obta ined by 3 mm punch b iopsy , minced w i t h s c i s s o r s and a l lowed to adhere to p l a s t i c d i s h e s . A f t e r growth i n a medium w i t h 20% f e t a l c a l f serum for 3 weeks, f i b r o b l a s t s were t r y p s i n i z e d and s p l i t i n t o s e v e r a l d ishes for the experiments desc r ibed below. Human long- te rm marrow c u l t u r e s and m e t h y l c e l l u l o s e assays for hemopoiet ic p rogen i to r s were e s t a b l i s h e d w i t h marrow buffy coat c e l l s as p r e v i o u s l y desc r ibed (14 ,15 , Appendices I I I , I V ) . Recons t i t u t ed long- te rm c u l t u r e s were e s t a b l i s h e d us ing bone marrow f i b r o b l a s t feeders and p e r i p h e r a l b lood or marrow P e r c o l l - s e p a r a t e d l i g h t d e n s i t y c e l l s . The bone marrow f i b r o b l a s t feeders were produced by p l a c i n g marrow buffy coat c e l l s i n t o t i s s u e c u l t u r e d i shes i n a medium wi th 20% f e t a l c a l f serum. A f t e r 3 weeks, w i t h weekly t o t a l medium changes, a conf luent adherent l a y e r comprised p r i m a r i l y of sp ind le -shaped f i b r o b l a s t - l i k e c e l l s was present . Th i s was t r y p s i n i z e d and s p l i t i n t o s e v e r a l d ishes before use. Confluent bone marrow f i b r o b l a s t feeders i n 60 mm t i s s u e c u l t u r e d ishes were i n o c u l a t e d w i t h 134 approx imate ly 2 x 10^ l i g h t d e n s i t y marrow or blood c e l l s i n 8 ml long- te rm c u l t u r e medium to c rea te a r e c o n s t i t u t e d long- term c u l t u r e . B . V i r u s P roduc t ion and Assay The gene ra l s t r a t egy for genera t ing h igh t i t e r r e t r o v i r a l producer c e l l l i n e s was as o u t l i n e d by M i l l e r et a l (16 ) . The r e t r o v i r u s packaging l i n e Y2 was p l a t e d at 5 x 10^ c e l l s per 60 mm d i s h on day 1. On day 2, 10 ug of v i r a l p lasmid DNA (pN2) (17) was t rans fec ted i n t o the c e l l s by ca l c ium phosphate c o -p r e c i p i t a t i o n . Th i s plasmid i s a MoMuLV-based vec to r which codes fo r the gene fo r neomycin phosphotransferase ( n e o r ) . A f t e r 24 hours , the medium was changed and on day 3 the medium which conta ined n e o r v i r u s ( v - n e o r ) produced by the Y2 c e l l s was removed, cen t r i fuged at 3000 rpm x 5 minutes to remove c e l l s and d e b r i s and a l i q u o t s used to i n f e c t the amphotropic packaging l i n e . PA12 c e l l s had been p la t ed at 10^ c e l l s per 60 mm t i s s u e c u l t u r e d i s h the p rev ious day. They were incubated w i t h medium from the t r ans fec ted Y2 c e l l s c o n t a i n i n g 8 ug/ml polybrene for 2 hours at 37°C. Then f resh medium was added, fo l lowed i n 48 hours by t r y p s i n i z a t i o n , d i l u t i o n 1:10 and s e l e c t i o n fo r v - n e o r - p r o d u c i n g c lones i n medium c o n t a i n i n g the neomycin analogue G418 at 1 mg/ml (Gibco L a b o r a t o r i e s , Chagr in F a l l s , OH). G418 was d i s s o l v e d i n d i s t i l l e d water and added to growth medium to achieve the d e s i r e d f i n a l c o n c e n t r a t i o n i n t o t a l mg/ml ( the e f f e c t i v e drug concen t r a t i on was approx imate ly 50% of that value for the 2 l o t s of G418 used) . Co lon i e s were i s o l a t e d by c l o n i n g r i n g s , expanded and examined for v - n e o r t i t e r on 3T3 c e l l s and fo r amphotropic he lper v i r u s us ing the S + L~ assay (16) . The 2-1-292 c e l l l i n e producing amphotropic v - s r c - c o n t a i n i n g v i r u s was a g i f t from Dr . S. Anderson. The t i t e r of t h i s v i r u s was approximate ly 5 x 10^ f f u / m l of medium cond i t ioned by producer c e l l s assayed for the a b i l i t y to induce transformed f o c i on 3T3 c e l l s . 135 The i n f e c t i o u s center assay was done by p l u c k i n g i n d i v i d u a l G418 r e s i s t a n t (G418 r ) granulocyte-macrophage c o l o n i e s from m e t h y l c e l l u l o s e assay and p l a c i n g the d i spe r sed c e l l s from one colony i n a 2 cm 2 t i s s u e c u l t u r e w e l l c o n t a i n i n g 1 0 4 NIH-3T3 c e l l s i n medium w i t h 8 yg /ml polybrene . A f t e r ove rn igh t i n c u b a t i o n , the medium was rep laced w i t h f resh medium c o n t a i n i n g G418 1 mg/ml. Seven days l a t e r , the assay was scored for the presence of G418 r 3T3 c e l l s . Medium from long- term c u l t u r e s was assayed for the presence of v - n e o r or v - s r c on 3T3 c e l l s . C. V i r a l I n f e c t i o n C e l l s were i n f e c t e d w i t h v - n e o r by e i t h e r c o - c u l t i v a t i o n overn igh t w i t h amphotropic v i r a l producer c e l l s which had r ece ived 1500R i r r a d i a t i o n or i n c u b a t i o n i n supernate from v i r a l producer c e l l s w i t h 8 yg /ml polybrene fo r 2 hours . A f t e r the i n f e c t i o n p e r i o d , c e l l s were washed o f f the feeders or p e l l e t e d from the supernate . An a l i q u o t of the i n f e c t e d or c o n t r o l c e l l s were w i t h e l d from long- te rm c u l t u r e and assessed i n shor t - t e rm m e t h y l c e l l u l o s e assays w i t h or wi thout G418 at 2 mg/ml. (This concen t r a t ion of G418 comple te ly i n h i b i t e d colony growth i n un infec ted c u l t u r e s . ) The remaining c e l l s were resuspended i n long- term c u l t u r e medium and placed i n t i s s u e c u l t u r e d i s h e s , fo r marrow buffy coat c e l l s , or on p r e - e s t a b l i s h e d bone marrow feeders for l i g h t d e n s i t y blood or marrow mononuclear c e l l s . Non-adherent or adherent c e l l s were removed from the long- term c u l t u r e s at va r i ous i n t e r v a l s and p l a t e d i n m e t h y l c e l l u l o s e assay w i th or without G418. C o n t r o l c u l t u r e s were not exposed to v i r u s but were grown i n suspension c u l t u r e i n medium w i t h 8 yg /ml polybrene du r ing the i n f e c t i o n per iod and placed i n long- term c u l t u r e and m e t h y l c e l l u l o s e assays at the same time as the i n f e c t e d c e l l s . C o l o n i e s were scored a f t e r p l a t i n g i n m e t h y l c e l l u l o s e on day 10-14 for g ranu locy te 136 macrophage c o l o n i e s (from CFU-GM) and day 18-21 for l a rge e r y t h r o i d c o l o n i e s (from BFU-E) fo r primary p rogen i to r assays . Co lon ie s were not scored un less they conta ined at l e a s t 30 c e l l s and, i n the case of BFU-E, had at l e a s t 3 c l u s t e r s and were c l e a r l y hemoglobinized. C e l l s were i n f e c t e d w i t h v - s r c c o n t a i n i n g v i r a l supernate i n the same manner as the v - n e o r i n f e c t i o n s . Bone marrow f i b r o b l a s t s which had been i n f e c t e d w i t h and were producing v - s r c v i r u s were used to i n f e c t l i g h t d e n s i t y hemopoiet ic c e l l s i n s e v e r a l experiments i n which they were used as the permanent feeder or adherent l a y e r for long- term c u l t u r e s . C e l l and v i r a l manipula t ions and c u l t u r e s were performed under L e v e l C containment f o l l o w i n g Med ica l Research C o u n c i l of Canada g u i d e l i n e s fo r h a n d l i n g r e t r o v i r u s e s and human samples. 3) RESULTS A. v - n e o r i n Long-Term Cu l tu r e Three normal bone marrow samples were i n f e c t e d w i t h v i r u s from an amphotropic r e t r o v i r a l producer c e l l l i n e producing v - n e o r at 4 x 10^ c f u / m l w i t h he lpe r v i r u s . Two samples were i n f e c t e d as marrow buffy coats w i t h c e l l -f ree v i r a l supernatant . The t h i r d sample was processed to recover l i g h t d e n s i t y c e l l s which were i n f e c t e d by c o - c u l t i v a t i o n w i t h i r r a d i a t e d v i r a l producer c e l l s fo r 24 hours and then placed on p r e - e s t a b l i s h e d bone marrow f i b r o b l a s t feeder l a y e r s . Ne i the r i n f e c t i o n p r o t o c o l appeared to be t o x i c to the ta rge t c e l l s as the number of non-adherent and adherent c e l l s and the number of p rogen i to r s i n the i n f e c t e d long- term c u l t u r e s was the same as i n the u n i n f e c t e d , c o n t r o l c u l t u r e s . 137 Table XIV shows the p r o p o r t i o n of G418 r granulocyte-macrophage c o l o n i e s that were recovered from i n f e c t e d long- term c u l t u r e s a f t e r v a r i o u s pe r iods of time i n c u l t u r e . Bone marrow c e l l s i n f e c t e d by v - n e o r - c o n t a i n i n g supernate had a low but d e f i n i t e inc idence of G418 r c o l o n i e s . The frequency of G418 r c o l o n i e s remained roughly s t a b l e over t ime. When l i g h t d e n s i t y bone marrow c e l l s were i n f e c t e d by c o - c u l t i v a t i o n w i t h v i r a l producer c e l l s , the frequency of G418 r c o l o n i e s was higher and a l s o appeared to remain s t a b l e i n c u l t u r e , a l though the pe r iod of obse rva t ion was s h o r t . S ince the v i r u s used to do these i n f e c t i o n s conta ined he lpe r v i r u s , i t was expected that a p roduc t ive v i r a l i n f e c t i o n would be produced i n the t a rge t c e l l s . Table XV shows the v - n e o r t i t e r s present i n the medium of i n f e c t e d long- te rm c u l t u r e s at va r ious time p o i n t s . I n d i v i d u a l G418 r granulocyte-macrophage c o l o n i e s were p lucked from m e t h y l c e l l u l o s e assays of the f i r s t two experiments and assayed fo r the p roduc t ion of v - n e o r i n i n f e c t i o u s centre assays . Table XVI shows the r e s u l t s of these assays and i n d i c a t e s that at l e a s t some of these c o l o n i e s had been i n f e c t e d by both v - n e o r and he lper v i r u s . Some G418 r c o l o n i e s may have been i n f e c t e d by v - n e o r wi thout he lper which would account for the nega t ive c o l o n i e s i n these exper iments . B. v - s r c i n Long-Term C u l t u r e The above experiments i n d i c a t e that i t i s p o s s i b l e to i n f e c t human hemopoiet ic p rogen i to r i n long- term c u l t u r e w i t h recombinant r e t r o v i r u s and ma in t a in a cons tan t , a l b e i t low, l e v e l of transformed c e l l s . Based on t h i s i n f o r m a t i o n and data from our own, and other l a b o r a t o r i e s on the a b i l i t y of v - s r c c o n t a i n i n g r e t r o v i r u s e s to per turb hemopoiesis i n murine long- te rm c u l t u r e s ( 7 , 9 , 1 8 ) , a s e r i e s of experiments were performed to assess the e f f e c t s of t h i s v i r u s on human hemopoiesis . 138 TABLE XIV P r o p o r t i o n of G418 R e s i s t a n t Granulocyte-Macrophage Co lon ies From Nonadherent C e l l s i n Human Long-Term Marrow C u l t u r e s In fec ted w i th He lpe r -Con ta in i ng v - n e o r r Marrow Source Sample of V i r u s Time of A n a l y s i s (day of c u l t u r e ) I n fec ted #G418 r CFU-GM C o n t r o l #G418 r CFU-GM T o t a l #CFU-GM {%) T o t a l # CFU-GM PA12/N2 Supernate 10 21 13/5037 (0.25) 9/12900 (0.07) 12/4150 (0 .3 ) 0/4425 0/1847 0/1990 PA12/N2 Supernate 10 21 2/6063 (0.03) 8/15248 . (0.05) 9/6300 (0.14) 0/3780 0/6563 0/3450 PA12/N2 c o - c u l t i v a t i o n 10 21/1124 (1 .9) 18/1340 (1 .3) 2/2568 0/960 139 TABLE XV v - n e o r T i t e r s i n Medium From In fec ted Long-Term C u l t u r e s (LTC)* Marrow Time of A n a l y s i s T i t e r of v - n e o r Sample (day of c u l t u r e ) ( c f u /m l of LTC medium) 10 4 x 1 0 2 21 0 10 32 21 0 10 2.3 x IO3 * I n f e c t i o n s on day 0 were done w i th v i r a l supernatant (marrow samples 1 or 2) or v i r a l producer c e l l s (sample 3) w i th v - n e o r t i t e r of 4 x 10^ c f u / m l . TABLE XVI I n f e c t i o u s Center Assays : G418 r CFU-GM From v - n e o r I n fec ted Long-Term Marrow C u l t u r e s Marrow Time of v - n e o r P o s i t i v e CFU-GM Sample A n a l y s i s (day) T o t a l CFU-GM Tested 1 2 21 21 5/11 7/10 141 To assess the a b i l i t y of the amphotropic v - s r c v i r u s to i n f e c t pr imary human c e l l s and to c rea te human v i r u s - p r o d u c i n g feeders for subsequent exper iments , human s k i n and bone marrow f i b r o b l a s t s were i n f e c t e d w i t h v i r u s -c o n t a i n i n g medium. The c e l l s g r a d u a l l y changed morphology over the two to four weeks f o l l o w i n g i n f e c t i o n , a c q u i r i n g l a r g e n u c l e i w i t h prominent n u c l e o l i , prominent cy top lasmic v a c u o l i z a t i o n , m u l t i p l e cy top lasmic p r o j e c t i o n s , and increased c e l l s i z e . In p lace of an organized u n i c e l l u l a r sheet of sp ind le -shaped c e l l s , as was seen i n un infec ted f i b r o b l a s t c u l t u r e s , d i s o r g a n i z e d s w i r l s and p i l e d heaps of c e l l s were seen i n s r c - i n f e c t e d c u l t u r e s . Approximate ly four weeks a f t e r i n f e c t i o n , clumps of c e l l s began to pee l o f f the t i s s u e c u l t u r e d ishes and f l o a t i n the medium of i n f e c t e d f i b r o b l a s t c u l t u r e s . Assays for t ransforming v i r u s from the medium i n these c u l t u r e s i s shown i n Table X V I I . Both s k i n and marrow f i b r o b l a s t s produced l a r g e amounts of v - s r c a f t e r many weeks i n c u l t u r e . However, i n s p i t e of t h i s and the c e l l s ' morpholog ica l a l t e r a t i o n , there was no major change i n the growth r a t e of the i n f e c t e d f i b r o b l a s t s and a l l c u l t u r e s e v e n t u a l l y senesced a f t e r passage i n c u l t u r e for up to s i x months. The hemopoiet ic c e l l s i n f e c t e d w i t h v - s r c i nc luded f e t a l l i v e r (1 ca se ) , p e r i p h e r a l blood or marrow from pa t i en t s w i t h ch ron ic myelogenous leukemia (CML) (3 ca ses ) , and bone marrow from pa t i en t s w i t h mye lodysp la s i a or pre leukemia (5 ca se s ) . F e t a l l i v e r and CML samples were chosen because they p rov ide a convenient source of l a rge numbers of p rogen i to r s which behave r e l a t i v e l y normal ly i n c u l t u r e , i . e . form morpho log i ca l l y d i s t i n c t hemopoiet ic c o l o n i e s of va r i ous l i neages i n m e t h y l c e l l u l o s e assay and p r o l i f e r a t e i n l o n g -term c u l t u r e ( 1 4 , 1 9 ) . In a d d i t i o n , a l a r g e p ropo r t i on of p rogen i to r s i n f e t a l l i v e r and CML samples are i n the a c t i v e part of the c e l l c y c l e (20 ,21 ) . T h i s i s i n con t ra s t to normal p r i m i t i v e p rogen i to r s that are l a r g e l y quiescent 142 ( 2 2 ) . The e f f i c i e n c y of r e t r o v i r a l i n f e c t i o n i s f e l t to be i n f l uenced by the c y c l i n g s t a tu s of the target c e l l s such that v i r a l i n t e g r a t i o n and r e p r o d u c t i o n i s more e f f i c i e n t i n c y c l i n g c e l l s (23) . The f e t a l l i v e r sample was s p l i t i n t o three p o r t i o n s . One was i n f e c t e d w i t h v - s r c c o n t a i n i n g supernate, one was i n f e c t e d by continuous exposure i n c u l t u r e to a v - s r c producing bone marrow f i b r o b l a s t feeder , and the t h i r d p o r t i o n served as an un in fec ted c o n t r o l . The number of p rogen i to r s i n these c u l t u r e s dropped to undetectable l e v e l s w i t h i n 4 weeks of long- te rm c u l t u r e i n i t i a t i o n . No d i f f e r e n c e was noted between the c o n t r o l and e i t h e r of the i n f e c t e d samples. However, the adherent " f i b r o b l a s t " l a y e r of f e t a l c e l l s i n the i n f e c t e d c u l t u r e s became mor pho log i ca l l y abnormal and q u i c k l y began to p e e l o f f the t i s s u e c u l t u r e d i s h and f l o a t as b a l l s of c e l l s i n the medium. The v i r a l t i t e r assayed i n medium from these i n f e c t e d f e t a l l i v e r c u l t u r e s i s shown on Table XVII and was maintained throughout the l i f e of the c u l t u r e s . The CML samples cons i s t ed of one marrow sample which was i n f e c t e d w i t h v - s r c - c o n t a i n i n g supernatant and 3 samples of CML blood l i g h t d e n s i t y c e l l s (samples were obta ined twice from one p a t i e n t ) . Some of the blood c e l l s were i n f e c t e d w i t h v i r a l supernatant and then placed on p r e - e s t a b l i s h e d bone marrow feeder l a y e r s . The remainder of the c e l l s were e i t h e r i n f e c t e d by co -c u l t i v a t i o n w i t h v - s r c producing bone marrow feeder c e l l s throughout the d u r a t i o n of the long- te rm c u l t u r e or were not i n f e c t e d and placed on bone marrow feeders to serve as c o n t r o l s . Table XVII shows the v i r a l t i t e r recovered from medium i n these c u l t u r e s a f t e r va r ious per iods of t ime. S i g n i f i c a n t t i t e r s were present a f t e r more than 4 months i n c u l t u r e . F igu re 18a shows the non-adherent c e l l counts from long- term c u l t u r e s of two blood samples from one CML p a t i e n t . There i s no s i g n i f i c a n t d i f f e r e n c e between i n f e c t e d and c o n t r o l c u l t u r e s i n e i t h e r sample. F igu re 18b and 18c 143 TABLE XVII Transforming V i r a l Assays From v - s r c In fec ted Human C e l l s Sample Week of C u l t u r e T i t e r ( f f u / m l ) A . Human F i b r o b l a s t s S k i n 8+ 1.5 x 1 0 4 Marrow 8+ 1.7 x 1 0 3 B. Long-Term C u l t u r e s F e t a l L i v e r 2 >10 3 15 >10 3 CML-1 . 17 27 CML-2 2 100 CML-3(a) 2 27 17 56 Preleukemia-1 5 15 144 Chronic Myelogenous Leukemia 10' c a> _c "D 0 c o Z 105-20 10 15 20 800 r 500 5 10 15 w e e k s p o s t i n f e c t i o n I I I 20 FIGURE 18. T o t a l nonadherent c e l l s per long term c u l t u r e (pane l a) and p rogen i to r numbers per 10^ nonadherent c e l l s (pane ls b and c) i n c u l t u r e s i n i t i a t e d w i th p e r i p h e r a l b lood c e l l s from a p a t i e n t w i th CML on normal bone marrow f i b r o b l a s t f eede rs . Some of the c u l t u r e s were i n f e c t e d w i th amphotropic v - s r c - c o n t a i n i n g r e t r o v i r u s . P t . 3a and 3b represent two samples from the same p a t i e n t . ^ . . . . ^ p t 3 b - i n f e c t e d • p t 3 b - c o n t r o l A . . . . A p t 3 a - i n f e c t e d • p t 3 a - c o n t r o l 145 show the number of l a r g e e r y t h r o i d and granulocyte-macrophage c o l o n i e s grown from non-adherent c e l l s i n these c u l t u r e s . Again there i s no s i g n i f i c a n t d i f f e r e n c e between colony numbers i n the i n f e c t e d or c o n t r o l c u l t u r e s . I n f e c t i o n by c o - c u l t i v a t i o n , ra ther than i n f e c t i o u s supernatant , d i d not a l t e r these f i n d i n g s . S i m i l a r r e s u l t s were obta ined w i th the other two CML samples. A f i n a l s e r i e s of experiments were done u s ing bone marrow samples from 5 p a t i e n t s w i t h mye lodysp l a s t i c or preleukemic d i s o r d e r s . These p a t i e n t s a l l had r e f r a c t o r y cytopenias i n two or more c e l l l i n e s i n the absence of bone marrow b l a s t s . Prev ious work i n our own l a b o r a t o r y and others have shown that hemopoiet ic p rogen i to r s from such pa t i en t s f a i l to form e i t h e r q u a n t i t a t i v e l y or q u a l i t a t i v e l y normal c o l o n i e s i n v i t r o . Both e r y t h r o i d and g r a n u l o c y t e -macrophage colony numbers are reduced and the formation of s m a l l c l u s t e r s of u n d i f f e r e n t i a t e d c e l l s i s of ten noted (24-26) . Having f a i l e d to show p e r t u r b a t i o n of hemopoiesis i n human long- term c u l t u r e s e s t a b l i s h e d w i t h r e l a t i v e l y normal c e l l s , i n f e c t i o n of mye lodysp l a s t i c c e l l s was undertaken to see i f the expected abno rma l i t i e s i n i n v i t r o hemopoiesis cou ld be modi f ied i n any way. Table XVII shows an example of one preleukemic c u l t u r e i n which we were ab le to main ta in v i r a l t i t e r s i n c u l t u r e for a reasonable pe r iod of t ime. F igu res 19 and 20a show that the number of non-adherent c e l l s i n pre leukemic long- te rm c u l t u r e s d i d not change whether or not the bone marrow c e l l s were i n f e c t e d w i t h v - s r c . The same r e s u l t s were obta ined w i t h two a d d i t i o n a l samples. S i m i l a r l y i n F igu re 20b, which i l l u s t r a t e s granulocyte-macrophage c o l o n i e s i n one of the preleukemic c u l t u r e s , i n f e c t i o n w i th v - s r c d i d not change the number (or appearance) of the c o l o n i e s . The remaining 4 pre leukemic samples d i d not produce enough scoreable hemopoiet ic c o l o n i e s to a l l o w such an a n a l y s i s to be done. 146 P R E L E U K E M I A : E X P T i w e e k s p o s t i n f e c t i o n FIGURE 19. T o t a l nonadherent c e l l s per long- term bone marrow c u l t u r e from a p a t i e n t w i t h mye lodysp las ia or pre leukemia . ^ ^ v - s r c - c o n t a i n i n g v i r u s i n f e c t e d rj .uninfected c o n t r o l 147 PRELEUKEMIA: EXPT 3 a i o 7 , — : w e e k s p o s t i n f e c t i o n FIGURE 20. T o t a l nonadherent c e l l s per long- term bone marrow c u l t u r e (panel a) or granulocyte/macrophage co lony- fo rming c e l l s (CFU-C) per 10^ nonadherent c e l l s (panel b) from a pa t i en t w i t h mye lodysp las i a or pre leukemia . A .... A v - s r c - c o n t a i n i n g v i r u s i n f e c t e d • . . . . r-j un infec ted c o n t r o l 148 Attempts to d e r i v e immortal c e l l l i n e s independent of the need fo r f i b r o b l a s t feeders from the non-adherent c e l l s i n the v - s r c i n f e c t e d CML or pre leukemic long- te rm c u l t u r e s were unsucces s fu l . S i m i l a r l y , r e p l a t i n g exper iments , i n which hemopoiet ic c o l o n i e s grown from v - s r c i n f e c t e d c e l l s were p lucked , d i spe r sed and r ep l a t ed i n m e t h y l c e l l u l o s e assay, f a i l e d to show any inc rease i n r e p l a t i n g p o t e n t i a l over s i m i l a r c o l o n i e s plucked from c o n t r o l , un in fec ted c u l t u r e s . 4) DISCUSSION The data presented i n t h i s chapter i n d i c a t e that human hemopoiet ic p rogen i to r s that are subsequently maintained i n long- term c u l t u r e can be s u c c e s s f u l l y used as t a rge t s for r e t r o v i r a l - m e d i a t e d gene t r a n s f e r . P rev ious workers have exposed human long- term marrow c u l t u r e s to a number of d i f f e r e n t r e t r o v i r u s e s . Us ing a v i r u s c o n t a i n i n g the human HPRT gene, one group has been ab le to demonstrate both v i r a l r e p l i c a t i o n and s u c c e s s f u l i n f e c t i o n of p rogen i to r s (33) . With v i r u s e s c o n t a i n i n g ' r a s ' oncogenes, r e t r o v i r a l r e p l i c a t i o n was a l s o demonstrated i n long- term c u l t u r e . However, q u a n t i t a t i v e data on the frequency of gene t r ans fe r and express ion was not presented i n the above s t u d i e s . Table XIV shows the p r o p o r t i o n of G418 r p rogen i to r s i s o l a t e d at v a r i o u s times i n long- te rm c u l t u r e i n the cur rent exper iments . Al though the f requencies of G418 r c o l o n i e s i n these experiments us ing n e o r v i r u s i n supernatant were low, they were r ep roduc ib l e and could be improved somewhat by u s i n g c o - c u l t i v a t i o n w i t h v i r a l producer c e l l s as the i n f e c t i o n technique . Data from murine systems have shown frequencies of gene t r ans fe r as h igh as 100% to CFU-S (27 ,28 ) . However, the frequency of gene exp re s s ion , as measured by the a b i l i t y of these c e l l s to form G418 r hemopoiet ic c o l o n i e s , was much lower (10-20%) (29-30) . Th i s d i f f i c u l t y w i t h express ion of the t r a n s f e r r e d 149 gene has a l s o been seen w i t h the human adenosine deaminase gene among o thers i n pr imary hemopoiet ic c e l l s (31) . With the data a v a i l a b l e from the cur ren t s t u d i e s , i t i s not p o s s i b l e to assess whether d i f f i c u l t i e s w i t h gene t r a n s f e r or gene expres s ion was the primary problem l e a d i n g to low numbers of G418 r c o l o n i e s . Most l i k e l y both f ac to r s are c o n t r i b u t i n g . Future experiments to address t h i s problem w i l l i n c lude the use of r e t r o v i r a l vec to r s i n which e x p r e s s i o n of the n e o r gene i s d r i v e n by a promoter other than the v i r a l LTR. For example, the herpes v i r u s thymidine k inase promoter i s f e l t to f u n c t i o n i n p r i m i t i v e t a r g e t s , such as embryonal carcinoma c e l l s , and perhaps hemopoie t ic p rogen i to r s more e f f e c t i v e l y than the r e t r o v i r a l promoter (32) . We had hoped to use the long- term marrow c u l t u r e system to s tudy the s t a b i l i t y of r e t r o v i r a l mediated gene t r ans fe r over s e v e r a l months i n v i t r o . However, the cur ren t experiments were done w i th v i r u s c o n t a i n i n g he lpe r v i r u s as w e l l as the n e o r recombinant v i r u s . Therefore many c e l l s i n f e c t e d at the beg inn ing of the long- term c u l t u r e become i n f e c t i o u s v i r a l producers themselves, making i t imposs ib le to t e l l whether G418 r c o l o n i e s were the r e s u l t of a pr imary or secondary i n f e c t i o n . The use of a h e l p e r - f r e e recombinant v i r u s would overcome t h i s problem. However, our best he lpe r f ree recombinant n e o r v i r u s has a 1 0 - f o l d lower t i t e r than the h e l p e r - c o n t a i n i n g v i r u s (4 x 10^ c fu /ml and 4 x 10^ c fu /ml r e s p e c t i v e l y ) . The use of t h i s he lpe r f ree v i r u s r e s u l t e d i n even lower , unworkable f requencies of G418 r e s i s t a n c e (data not shown). The low frequency of G418 r c o l o n i e s a l s o made i t imposs ib le to cont inue the experiments for per iods longer than 3 weeks. The number of c e l l s i n human long- te rm c u l t u r e s drops p r o g r e s s i v e l y w i t h time i n c u l t u r e . A l a r g e number of c e l l s was necessary for p l a t i n g i n these experiments i n order to recover a measurable number of G418 r c o l o n i e s . These c e l l numbers were not a v a i l a b l e 150 a f t e r 3 weeks. Never the less the c o - c u l t i v a t i o n experiment where the frequency o f GA18 r c o l o n i e s was almost 2% and the a v a i l a b i l i t y of new v i r a l vec to r s g i v e s hope that the problems of low frequency w i l l soon be overcome. The demonstrat ion of s u c c e s s f u l t r ans fe r of the neo r gene to p r o g e n i t o r s i n long- te rm c u l t u r e and our previous success w i t h p rogen i to r s i n sho r t - t e rm m e t h y l c e l l u l o s e assay made i t seem f e a s i b l e to attempt the t r ans f e r of o ther r e t r o v i r a l l y encoded genes to human hemopoiet ic p r o g e n i t o r s . The v - s r c gene was s e l e c t e d because of the genera l i n t e r e s t i n e l u c i d a t i n g the r o l e of known t ransforming oncogenes i n human mal ignancies and the data from murine l o n g -term c u l t u r e s sugges t ing that the v - s r c c o n t a i n i n g v i r u s per turbs hemopoiesis i n that system. Dexter et a l have shown increased colony numbers, i nc reased s e l f - r e n e w a l , and the genera t ion of immorta l , m u l t i p o t e n t , factor-dependent c e l l l i n e s from v - s r c i n f e c t e d murine c u l t u r e s ( 7 , 9 ) . The r o l e of v - s r c i n these changes i s unc lea r as the v i r a l gene i s not present i n the DNA of the c e l l l i n e s (34) . Neve r the l e s s , the e f f e c t s desc r ibed are r e a l and r e p r o d u c i b l e i n o ther l a b o r a t o r i e s (18) . The l i t e r a t u r e d e s c r i b i n g i n f e c t i o n of human long- term c u l t u r e s w i t h r e t r o v i r u s e s c o n t a i n i n g oncogenes i s r e l a t i v e l y sparse ( 8 ) . However, Greenberger et a l were able to demonstrate r e t r o v i r a l i n f e c t i o n of hemopoie t ic c e l l s i n human long- te rm c u l t u r e w i t h K i r s t e n or Harvey r a s - c o n t a i n i n g r e t r o v i r u s e s . The l e v e l of t r ans fe r r ed gene express ion i n these hemopoiet ic c e l l s i s unc lea r from the data presented. Never the le s s , the on ly p e r t u r b a t i o n i n hemopoiesis noted i n the i n f e c t e d c u l t u r e s was the genera t ion of an inc reased number of E b s t e i n - B a r r v i r u s (EBV) transformed B lymphocytes i n the i n f e c t e d c u l t u r e s as compared to c o n t r o l s . In the experiments presented i n t h i s chap te r , we were s i m i l a r l y unable to demonstrate any r e p r o d u c i b l e e f f e c t on human hemopoiesis i n long- term c u l t u r e by v i r u s e s c o n t a i n i n g the v - s r c 151 oncogene. Th i s occurred i n s p i t e of v - s r c v i r a l t i t e r s that p e r s i s t e d i n c u l t u r e fo r many weeks. The absence of e f f ec t may have been due to t e c h n i c a l f a c t o r s r e l a t e d to low l e v e l s of i n f e c t i o n and gene t r ans fe r to hemopoie t ic p r o g e n i t o r s i n the long- term c u l t u r e s or to the r e l a t i v e l y shor t pe r iod of o b s e r v a t i o n i n the human as compared to murine system. A l t e r n a t i v e l y , there are i n t r i n s i c d i f f e r e n c e s between murine and human c e l l s that may a l l o w the behaviour of the former to be more e a s i l y perturbed by the v - s r c oncogene. F i n a l l y , the v - s r c gene alone may be unable to t ransform hemopoiet ic c e l l s and the e f f e c t s seen i n murine c u l t u r e s may be due to the i n d i r e c t e f f e c t s on hemopoiet ic c e l l s of i n f e c t e d s t romal c e l l s . The morpholog ica l a b n o r m a l i t i e s that were noted i n the v - s r c i n f e c t e d human f i b r o b l a s t s i n t h i s repor t are r emin i scen t of changes desc r ibed i n i n f e c t e d murine marrow f i b r o b l a s t s ( 7 ) . Thus the microenvironments i n the i n f e c t e d human, as w e l l as murine long- te rm c u l t u r e s , were abnormal. I t i s not p o s s i b l e to determine from a v a i l a b l e da ta i f these changes were q u a n t i t a t i v e l y or q u a l i t a t i v e l y s i m i l a r i n the two systems. The Moloney leukemia v i r u s which was the he lper v i r u s used i n the murine experiments does not con ta in an oncogene. Neve r the l e s s , i t may have c o n t r i b u t e d i n undetermined ways to the observed e f f e c t s on hemopoiesis i n murine c u l t u r e s . The amphotropic he lper v i r u s used i n the human i n f e c t i o u s i s a non-pathogenic murine v i r u s which may be incapable of p o t e n t i a t i n g the " s r c " e f f e c t . There i s now a cons ide rab le body of exper imenta l data i n d i c a t i n g that more than one oncogene may be necessary to transform primary c e l l s . Weinberg et a l f i r s t demonstrated that ra t embryo f i b r o b l a s t s could be transformed by the ras and myc oncogenes together but not by e i t h e r gene alone (35) . S i m i l a r da ta now e x i s t s for other systems, i n c l u d i n g murine B lymphoblasts i n l o n g -term c u l t u r e (36) . Thus a second gene may be necessary to complete the t r ans fo rma t ion of v - s r c i n f e c t e d hemopoiet ic c e l l s . 152 Al though these experiments have f a i l e d to demonstrate a b i o l o g i c a l e f f e c t f o r a r e t r o v i r a l l y t r ans f e r r ed oncogene on human hemopoiesis i n long- te rm c u l t u r e , they have shown that i t i s p o s s i b l e to main ta in r e t r o v i r a l i n f e c t i o n i n pr imary human c e l l s fo r s i g n i f i c a n t per iods of t ime. The experiments u s i n g the n e o r - c o n t a i n i n g v i r u s have shown that some hemopoiet ic p rogen i to r s i n these c u l t u r e s are s u c c e s s f u l l y i n f e c t e d and express the t r a n s f e r r e d gene. 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M i l l e r AD, Trauber DR, But t imore C. Fac tors i n v o l v e d i n the p roduc t ion of he lpe r v i r u s - f r e e r e t r o v i r u s v e c t o r s . Somatic C e l l Molec Genet 12: 175, 1986. 17. E g l i t i s MA, Kan to f f P, G i l b o a E , Anderson WF. Gene express ion i n mice a f t e r h igh e f f i c i e n c y r e t r o v i r a l - m e d i a t e d gene t r a n s f e r . Sc ience 230: 1395, 1985. 18. Hogge D, Anderson S, Humphries RK. E f f e c t s on av ian v - s r c on murine and human hemopoiesis i n v i t r o . Second Annual Meeting on Oncogenes, p 40, 1986. 19. C a p p e l l i n i MD, P o t t e r CG, Wood WG. Long-term haemopoiesis i n human f e t a l l i v e r c e l l c u l t u r e s . Br J Haematol 57: 61, 1984. 20. Peschle C, M i g l i a c c i o AR, M i g l i a c c i o G, C i c c a r i e l l o R, L e t t i e r i F , Q u a t t r i n S, Russo G, Mastroberardino G. I d e n t i f i c a t i o n and c h a r a c t e r i z a t i o n of three c l a s se s of e r y t h r o i d p rogen i to r s i n human f e t a l l i v e r . Blood 58: 565, 1981. 21. Eaves AC, Cashman JD, Gaboury LA, Kalousek DK, Eaves C J . Unregulated p r o l i f e r a t i o n of p r i m i t i v e ch ron ic myeloid leukemia p rogen i to r s i n the presence of normal adherent c e l l s . Proc N a t l Acad S c i USA 83: 5306, 1986. 22. Cashman J , Eaves AC, Eaves C J . Regulated p r o l i f e r a t i o n of p r i m i t i v e hematopoie t ic p rogen i to r c e l l s i n long- term human marrow c u l t u r e s . B lood 66: 1002, 1985. 23. Varmus H, Swanstrom R. R e p l i c a t i o n of r e t r o v i r u s e s . I n : "RNA Tumor V i r u s e s , 2nd E d i t i o n " , Weiss R, T e i c h N , Varmus H, C o f f i n J ( eds ) , Co ld Spr ing Harbor Labora to ry , Cold Spr ing Harbor, New York , pp 467, 1984. 24. Chui DHK, C l a r k e B J . 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Gruber HE, F i n l e y KD, Hershberg RM, Katzman SS, L a i k i n d PK, S e e g m i l l e r J E , Friedmann T, Yee JK , J o l l y DJ . R e t r o v i r a l vec tor -media ted gene t r ans f e r i n t o human hematopoiet ic p rogen i to r c e l l s . Science 230: 1057, 1985. 34. Wyke J A , Stoker AW, Sear le S, Spooncer E, Simmons P, Dexter TM. Per turbed hemopoiesis and the genera t ion of m u l t i p o t e n t i a l stem c e l l c lones i n s r c - i n f e c t e d bone marrow c u l t u r e s i s an i n d i r e c t or t r a n s i e n t e f f ec t of the oncogene. Molec C e l l B i o l 6: 959, 1986. 35. Land H, Parada L F , Weinberg RA. Tumorigenic convers ion of pr imary embryo f i b r o b l a s t s r equ i r e s at l e a s t two coopera t ing oncogenes. Nature 304: 596, 1983. 36. Schwartz RC, Stanton LW, R i l e y SC, Marcu KB, Wi t t e ON. Synergism of v-myc and v-Ha-ras i n the i n v i t r o n e o p l a s t i c p rogres s ion of murine lymphoid c e l l s . Molec C e l l B i o l 6: 3221, 1986. 156 C H A P T E R VI SUMMARY AND CONCLUSIONS 1) SUMMARY Hemopoiesis i s the r e s u l t of the a c t i v i t i e s of p r i m i t i v e p l u r i p o t e n t c e l l s , hemopoiet ic stem c e l l s . In normal organisms, many of these c e l l s e x i s t and, through the processes of 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 , c o n t r i b u t e to the poo l of mature blood c e l l s . The r e s u l t i s that normal hemopoiesis i s p o l y c l o n a l . Because these c e l l s are able to reproduce themselves, i . e . s e l f -renew, hemopoiesis cont inues throughout the l i f e - s p a n of the an ima l . Hemopoietic mal ignancies a r i s e most commonly i n a s i n g l e c e l l . The c l o n a l i t y of these d i so rde r s has been c o n v i n c i n g l y demonstrated u s i n g cy togene t i c and G6PD isoenzyme a n a l y s i s of malignant c e l l s . The pro to type d i sease for such s t u d i e s was ch ron ic myelogenous leukemia (CML) i n which a p l u r i p o t e n t hemopoiet ic c e l l has been shown to be the target for malignant t r ans fo rmat ion ( 1 , 2 ) . While s i m i l a r data i s a v a i l a b l e for a number of o ther neoplasms, no tab ly other m y e l o p r o l i f e r a t i v e d i s o r d e r s and some cases of acute nonlymphoblas t ic leukemia (ANLL), such a n a l y s i s i s l i m i t e d to d i seases which have cy togene t i c markers and the one t h i r d of b lack women who are heterozygous for two e l e c t r o p h o r e t i c a l l y d i s t i n c t v a r i a n t s of G6PD. Recent developments i n the f i e l d of molecular g e n e t i c s , i n c l u d i n g r e s t r i c t i o n fragment l eng th polymorphism (RFLP) (3) a n a l y s i s , and the use of marker genes such as those encoded by r e t r o v i r u s e s ( 4 , 5 ) , w i l l make d e t a i l e d a n a l y s i s of c l o n a l i t y more w i d e l y a p p l i c a b l e to s c i e n t i f i c r e sea rch . 157 The r e g u l a t i o n of hemopoiesis i s a complex process i n v o l v i n g v a r i o u s p r o t e i n growth f ac to r s and c e l l u l a r i n t e r a c t i o n s w i t h i n the bone marrow microenvironment . The p r e c i s e mechanism of a c t i o n of these f a c t o r s , t h e i r l e v e l of i n t e r a c t i o n and c o n t r o l , and t h e i r r e l a t i v e importance are not p r e s e n t l y understood for normal hemopoiesis . How such r e g u l a t i o n i s per turbed i n hemopoiet ic neoplasms i s even more obscure. However, a l a r g e body of evidence i n d i c a t e s that gene t i c change i s fundamental to the development of mal ignancy. Such change has been demonstrated at the cy togene t i c or chromosomal l e v e l fo r most human cancers ( 6 ) . In the l a s t few yea r s , there has been an exponen t i a l r i s e i n the number of molecular gene t i c a b n o r m a l i t i e s that have been found i n human tumor c e l l s , some of which appear chromosomally normal . Many of these molecular abnorma l i t i e s i n v o l v e c e l l u l a r oncogenes, the d i s c o v e r y of which has a l lowed i n v e s t i g a t o r s to focus the search for c r i t i c a l a l t e r a t i o n s i n cancer c e l l s on a r e l a t i v e l y sma l l number of candidate genes ( 7 ) . Impress ive though, the r e s u l t s to date a re , there are undoubtedly many "oncogenes" w a i t i n g to be d i scovered and the t e c h n i c a l advances of molecu la r b i o l o g y w i l l cont inue to o f f e r new approaches to the study of c a r c i n o g e n e s i s . One such approach i s the technique of gene t r ans fe r us ing recombinant r e t r o v i r a l v e c t o r s . Genes of p o t e n t i a l importance i n the e t i o l o g y of a p a r t i c u l a r tumor could be i n s e r t e d i n t o the appropr ia te vec to r a l l o w i n g the t r a n s f e r of t h i s gene i n t o a s e l e c t e d target c e l l p o p u l a t i o n . Subsequent a n a l y s i s of i n f e c t e d c e l l s may r e v e a l s p e c i f i c changes i n t h e i r behavior or p h y s i c a l or chemica l p r o p e r t i e s that can be a t t r i b u t e d to the presence of the new gene. In the present s t u d i e s , s e v e r a l of the techniques a l l u d e d to above have been used to ana lyze c l o n a l i t y i n human malignant c e l l s . In a d d i t i o n , the technique of r e t r o v i r a l mediated gene t r ans fe r was developed for use on human hemopoiet ic c e l l s . 158 A . Cy togene t i c A n a l y s i s of Hemopoietic Co lon ie s i n P a t i e n t s w i t h J u v e n i l e  Monosomy 7 Syndrome Us ing a technique that a l lows cy togene t i c a n a l y s i s of i n d i v i d u a l hemopoie t ic c o l o n i e s plucked from m e t h y l c e l l u l o s e assay, i t was p o s s i b l e to show that e r y t h r o i d p rogen i to r s and, i n one case, myeloid p rogen i to r s were i n v o l v e d i n the n e o p l a s t i c c lone of two pa t i en t s w i t h j u v e n i l e monosomy 7 syndrome. Thus, t h i s mye lodysp l a s t i c or p r o l i f e r a t i v e d i s o r d e r of ch i ldhood must o r i g i n a t e , at l e a s t i n some cases, i n a p r i m i t i v e , mu l t ipo ten t hemopoiet ic c e l l . Th i s d i s o r d e r bears many c l i n i c a l and cy togene t i c s i m i l a r i t i e s to the more common myelodysplas ias seen i n a d u l t s . In both a d u l t s and c h i l d r e n w i t h monosomy of chromosome 7 i n bone marrow c e l l s , the i n i t i a l cy topen ic or m y e l o p r o l i f e r a t i v e phase of the d i sease t y p i c a l l y evo lves i n t o frank ANLL w i t h i n s e v e r a l years of d i a g n o s i s . De novo ANLL has been shown by G6PD a n a l y s i s to of ten i n v o l v e a p l u r i p o t e n t hemopoiet ic c e l l , p a r t i c u l a r l y i f the d i sease has a smouldering or gradua l onset i n an e l d e r l y p a t i e n t ( 8 ) . Thus, p r i m i t i v e p l u r i p o t e n t hemopoiet ic p rogen i to r s appear to be common t a rge t s for n e o p l a s t i c t rans format ion i n hematologic mal ignancy. Th i s i s t rue i n s p i t e of the fac t that the phenotype of the d isease v a r i e s c o n s i d e r a b l y , i . e . m y e l o p r o l i f e r a t i v e , d y s p l a s t i c or acute leukemia . The p r e c i s e nature of the gene t i c change that occurs i n these mul t ipo ten t malignant c e l l s i s , o f course , of great i n t e r e s t and the subject of in tense i n v e s t i g a t i o n i n many c e n t e r s . 159 B . I d e n t i f i c a t i o n of Hemopoietic P rogen i to r s that Are Not Par t of the Mal ignant Clone i n Long-Term Cu l tu re s from a G6PD Heterozygote w i t h CML In t h i s study G6PD isoenzyme a n a l y s i s was combined w i t h hemopoiet ic colony cy togene t i c s to analyze the c l o n a l i t y of p rogen i to r s i n long- te rm marrow c u l t u r e s from p a t i e n t s w i th CML. Al though 100% of bone marrow metaphase c e l l s i n p a t i e n t s w i t h ch ron ic phase CML t y p i c a l l y show the P h i l a d e l p h i a chromosome abnormal i ty (Ph) ( 1 ) , s t ud i e s i n which p a t i e n t s have r e c e i v e d aggress ive c y t o t o x i c chemotherapy or a i n t e r f e r o n have revea led the presence of Ph nega t ive hemopoiet ic c e l l s i n v i v o ( 9 , 1 0 ) . S i m i l a r l y , Ph-nega t ive hemopoiet ic c o l o n i e s have been recovered from long- te rm c u l t u r e e s t a b l i s h e d w i t h CML marrow c e l l s (11) . The p o l y c l o n a l i t y of the Ph-nega t ive c e l l s i n v i v o has been demonstrated by G6PD a n a l y s i s i n one pa t i en t ( 9 ) . However, i t has been suggested, and some data presented to support the i d e a , that at l e a s t some c y t o g e n e t i c a l l y normal marrow and blood c e l l s i n CML are par t of the n e o p l a s t i c c lone (12) . That i s , the Ph chromosome i s a secondary f i n d i n g that develops r e l a t i v e l y l a t e i n the course of the d i s ea se . The l o n g -term marrow c u l t u r e provides a system i n which one could p o t e n t i a l l y demonstrate t h i s phenomenon of Ph-negat ive c l o n a l i t y . I t i s p o s s i b l e to o b t a i n c y t o g e n e t i c a l l y normal p rogen i to r s from CML long- term c u l t u r e s on a r e l a t i v e l y r o u t i n e bas i s and to study these c o l o n i e s for t h e i r G6PD isoenzyme type i n the appropr i a t e G6PD heterozygous p a t i e n t . Al though c e l l s from one dozen b lack women w i t h CML were screened i n the cur ren t s tudy, on ly two were found to be s u i t a b l e for these exper iments . In c u l t u r e s from one of these p a t i e n t s , i t was p o s s i b l e to demonstrate hemopoiet ic c o l o n i e s de r ived from c e l l s i n long- term c u l t u r e that were not par t of the malignant c l o n e . Al though t h i s does not exclude the p o s s i b i l i t y that a c y t o g e n e t i c a l l y normal c l o n a l popu la t ion of c e l l s a l s o e x i s t s , i t does 160 show that at l e a s t some of the Ph-negat ive c e l l s are t r u l y normal . T h i s f i n d i n g may have p r a c t i c a l importance for c l i n i c i a n s p lann ing to use i n v i t r o techniques such as the long- term c u l t u r e to purge pa t i en t bone marrow of malignant c e l l s p r i o r to autologous marrow t r a n s p l a n t a t i o n (13) . C. R e t r o v i r a l - M e d i a t e d Gene Transfer to Human Hemopoietic C e l l s The technique of r e t r o v i r a l - m e d i a t e d gene t r ans fe r was developed fo r use i n human c e l l s as a t o o l which would a l l o w a n a l y s i s of c l o n a l i t y i n c u l t u r e d c e l l s as w e l l as f u n c t i o n a l s t ud i e s i n t o the gene t i c r e g u l a t i o n of normal and malignant hemopoies is . Short-Term Assays . Using recombinant v i r u s c o n t a i n i n g the n e o r gene, i t was p o s s i b l e to demonstrate e f f i c i e n t gene t r ans fe r to normal and malignant hemopoiet ic c e l l s , both e s t a b l i s h e d c e l l l i n e s and primary p rogen i to r s i n m e t h y l c e l l u l o s e assay. Express ion of the t r ans f e r r ed gene was shown by RNA spot b l o t and G418 r e s i s t a n c e of hemopoiet ic c o l o n i e s formed by the i n f e c t e d c e l l s . The c l o n a l i t y of i s o l a t e d G418 r e s i s t a n t , v i r a l l y - i n f e c t e d K562 c e l l l i n e s was v e r i f i e d by Southern b l o t t i n g w i t h demonstrat ion of a unique s i t e of r e t r o v i r a l i n t e g r a t i o n i n the c e l l u l a r DNA of each c l o n e . A number of v a r i a b l e s were op t imized to increase the frequency of G418 r e s i s t a n t c o l o n i e s and s u c c e s s f u l gene t r ans fe r was obta ined w i t h neo r v i r u s both w i t h and wi thout a s s o c i a t e d he lper v i r u s . Al though these experiments were g e n e r a l l y s u c c e s s f u l , the h ighes t frequency of gene t r ans fe r to primary p rogen i to r s ob t a ined , as assessed by the p r o p o r t i o n of G418 r e s i s t a n t g r a n u l o c y t e -macrophage c o l o n i e s , was 15.7%. While t h i s f i g u r e i s encouraging, i t i s much l e s s than the 60% frequency obta ined w i th K562 c e l l s or the 100% frequency repor ted by others for murine CFU-S (14 ,15) . The reasons for t h i s d i sc repancy cou ld not be p r e c i s e l y determined i n the cur rent study but probably i n c l u d e : 161 ( i ) inadequate express ion of the t r ans fe r r ed gene from the promoter i n the r e t r o v i r a l LTR, ( i i ) i n e f f i c i e n t gene t r ans fe r due to f a c to r s such as the c y c l i n g s t a tus of target c e l l s . Long-Term C u l t u r e s . Having shown that r e t r o v i r a l - m e d i a t e d gene t r a n s f e r to pr imary hemopoiet ic p rogen i to r s was a f e a s i b l e technique, the next phase o f the research focused on the study of v i r a l l y - i n f e c t e d p rogen i to r s mainta ined i n long- te rm c u l t u r e . The purpose of these experiments was to u l t i m a t e l y use the long- te rm c u l t u r e as a system i n which to i n v e s t i g a t e the s t a b i l i t y of gene t r a n s f e r and express ion over t ime. Secondly, p r e l i m i n a r y f u n c t i o n a l s t u d i e s were planned us ing v i r u s c o n t a i n i n g the v - s r c oncogene. ( i ) Experiments u s ing the neo r v i r u s were s u c c e s s f u l i n demonstra t ing G418 r e s i s t a n t hemopoiet ic c o l o n i e s a f t e r per iods of up to three weeks i n c u l t u r e . However, the low frequency of G418 r e s i s t a n t granulocyte-macrophage c o l o n i e s i n these experiments ( l e s s than 2%) made d e f i n i t i v e s t u d i e s on the s t a b i l i t y of the t r ans f e r r ed gene us ing h e l p e r - f r e e v i r u s for longer pe r iods of time i n c u l t u r e p r o h i b i t i v e . ( i i ) I n f e c t i o n s of va r ious hemopoiet ic target c e l l popu la t ions were c a r r i e d out u s ing a recombinant v - s r c - c o n t a i n i n g r e t r o v i r u s pseudo-typed w i t h an amphotropic h e l p e r . P rogen i to r s from f e t a l l i v e r , CML blood or marrow, and m y e l o d y s p l a s t i c marrow were i n f e c t e d and fo l lowed i n long- term c u l t u r e for pe r iods up to 4 1/2 months. Al though no pe r tu rba t ions i n the q u a n t i t a t i v e or q u a l i t a t i v e aspects of hemopoiesis measured i n the c u l t u r e s were observed, i t was p o s s i b l e to show s u c c e s s f u l i n f e c t i o n by the v - s r c v i r u s of pr imary human c e l l s and t h e i r long- te rm maintenance i n these c u l t u r e s . The above experiments represent p r e l i m i n a r y exper ience w i t h the technique of r e t r o v i r a l - m e d i a t e d gene t r a n s f e r . Seve ra l d i f f e r e n t v i r a l vec to r s were 162 used and a v a r i e t y of human c e l l types, i n c l u d i n g e s t a b l i s h e d leukemic c e l l l i n e s , pr imary normal and malignant p r o g e n i t o r s , and bone marrow and s k i n f i b r o b l a s t s were used as t a r g e t s . Two d i f f e r e n t c u l t u r e systems, sho r t - t e rm m e t h y l c e l l u l o s e assay and long- term c u l t u r e s , were used to s tudy the r e s u l t s of v a r i o u s i n f e c t i o n s . Al though not a l l of these experiments were comple te ly s u c c e s s f u l , the o v e r a l l r e s u l t s are s u f f i c i e n t l y encouraging to a l l o w one to proceed i n the expec t a t i on that the m o d i f i c a t i o n of e x i s t i n g techniques and the development of new ones w i l l make future progress p o s s i b l e . A l r e a d y , the a v a i l a b i l i t y of new r e t r o v i r a l vec to r s p r o v i d i n g d i f f e r e n t promoters than that present i n the r e t r o v i r a l LTR, make i t l i k e l y that the l e v e l of gene exp re s s ion f o l l o w i n g s u c c e s s f u l gene t r ans fe r w i l l soon be r o u t i n e l y much h ighe r than was achieved i n the experiments desc r ibed here (16) . Vec to r s c o n t a i n i n g other genes of i n t e r e s t such as hemopoiet ic growth f a c t o r s are be ing cons t ruc t ed . These w i l l be important reagents fo r future experiments designed to exp lo re the importance of va r ious molecules i n normal and malignant hemopoiesis . 2) CONCLUSIONS The s t u d i e s desc r ibed here have used s e v e r a l e s t a b l i s h e d techniques to extend our knowledge of the c l o n a l i t y of hemopoiet ic p rogen i to r s i n human ma l ignanc i e s . In a d d i t i o n to t h e i r t h e o r e t i c a l i n t e r e s t , these f i n d i n g s may have importance fo r c l i n i c i a n s sea rch ing for new therapeu t ic m o d a l i t i e s fo r hemopoie t ic neoplasms. The technique of r e t r o v i r a l - m e d i a t e d gene t r ans fe r has been adapted fo r use i n human hemopoiet ic p r o g e n i t o r s . Al though much remains to be l ea rned about t h i s technique and i t s a p p l i c a t i o n , the exper ience desc r ibed p rov ides a good foundat ion fo r future work. The technique c l e a r l y has e x c i t i n g p o t e n t i a l 163 fo r use as a t o o l i n bas i c and c l i n i c a l r e sea rch . The use of gene t r a n s f e r to d i s s e c t the f ac to r s r e g u l a t i n g va r ious c e l l processes and mark c e l l s fo r s t u d i e s of va r ious c l o n a l popula t ions i s i n i t s in fancy but has a l r eady y i e l d e d important b i o l o g i c a l i n f o r m a t i o n . The e ra of gene therapy fo r i n h e r i t e d d i s o r d e r s w i th molecular gene t i c defec ts i s c l o s e at hand (17) and may u l t i m a t e l y be made p o s s i b l e by techniques such as those desc r ibed i n t h i s work. 164 REFERENCES 1. Whang J , F r e i I I I E , T j i o JH , Carbone PP, Brecher G. The d i s t r i b u t i o n of the P h i l a d e l p h i a chromosome i n pa t i en t s w i t h ch ron ic myelogenous leukemia . Blood 22: 664, 1963. 2. F i a l k o w P J , Jacobson R J , Papayannopoulou T. Chronic m y e l o c y t i c l eukemia . C l o n a l o r i g i n i n a stem c e l l common to the g r anu locy t e , e r y t h r o c y t e , p l a t e l e t and monocyte/macrophage. Am J Med 63: 125, 1977. 3. V o g e l s t e i n B , Fearon ER, Hamil ton SR, Feinberg AP. Use of r e s t r i c t i o n fragment l eng th polymorphisms to determine the c l o n a l o r i g i n of human tumors. Science 227: 642, 1985. 4. 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Lemischka IR, Raule t DH, M u l l i g a n RC. Developmental p o t e n t i a l and dynamic behavior of hematopoiet ic stem c e l l s . C e l l 45: 917, 1986. 16. Franz T, H i l b e r g F, S e l i g e r B, S tock ing C, Os te r tag W. R e t r o v i r a l mutants e f f i c i e n t l y expressed i n embryonal carcinoma c e l l s . Proc N a t l Acad S c i USA 83: 3292, 1986. 17. Anderson WF. Prospects for human gene therapy. Science 226: 401, 1984. 166 APPENDIX I LIST OF ABBREVIATIONS ADA adenosine deaminase AEL acute e ry thro leukemia ALL acute lymphob las t i c leukemia AML acute myelogenous leukemia AMML acute myelomonocytic leukemia AMoL acute monocytic leukemia ANLL acute nonlymphoblas t ic leukemia bcr breakpoint c l u s t e r r eg ion BFU-E burst forming u n i t - e r y t h r o i d bp base p a i r BSA bovine serum albumen C a + + c a l c ium CAT chloramphenicol a c e t y l t r a n s f e r a s e cfu colony forming u n i t CFU-C colony forming u n i t i n c u l t u r e CFU-E colony forming u n i t , e r y t h r o i d CFU-GEMM mixed colony forming u n i t , g r anu locy t e , e r y t h r o i d , macrophage, megakaryocyte CFU-GM granu locy te macrophage colony forming u n i t CFU-M macrophage colony forming u n i t CFU-Meg megakaryocyte colony forming u n i t CFU-S sp leen colony forming u n i t cm cent imeter CML ch ron i c myelogenous leukemia 167 CC>2 carbon d i o x i d e cpm counts per minute CSF-1 colony s t i m u l a t i n g f ac to r 1 DHFR d i h y d r o f o l a t e reductase d l d e c i l i t e r DNA d e o x y r i b o n u c l e i c a c i d EBV E p s t e i n Bar r v i r u s EGF epidermal growth f ac to r env gene encoding r e t r o v i r a l envelope g l y c o p r o t e i n s ep e r y t h r o p o i e t i n FAB F r e n c h - A m e r i c a n - B r i t i s h FCS f e t a l c a l f serum f f u focus forming u n i t s G418 g e n e t i c i n G6PD glucose 6 phosphate dehydrogenase gag gene encoding r e t r o v i r a l coat p ro t e in s G-CSF granu locy te colony s t i m u l a t i n g f a c t o r gm gram GM-CSF granulocyte-macrophage c o l o n y - s t i m u l a t i n g f a c t o r GTP guanid ine t r iphosphate HBSS Hanks balanced s a l t s o l u t i o n HPRT hypoxanthine phosphor ibosy l t rans fe rase HS horse serum IgH immunoglobulin heavy cha in IgK immunoglobulin kappa l i g h t cha in IgX immunoglobulin lambda l i g h t cha in I L - 1 , 2 , 3 i n t e r l e u k i n 1,2,3 Kb k i l o b a s e 168 LTC long term c u l t u r e LTR long te rm ina l repeat M-CSF macrophage co lony s t i m u l a t i n g f a c t o r M:E m y e l o i d : e r y t h r o i d mg m i l l i g r a m ml m i l l i l i t e r mm m i l l i m e t e r MoMuLV Moloney murine leukemia v i r u s MSV murine sarcoma v i r u s MT m e t a l l o t h i o n e i n n e o r neomycin r e s i s t a n c e or neomycin phosphot ransferase PDGF p l a t e l e t de r i ved growth f a c t o r pg picogram Ph P h i l a d e l p h i a chromosome PK p r o t e i n k inase pM p icomolar PNP pur ine nuc leos ide phosphorylase p o l r e t r o v i r a l gene fo r reverse t r a n s c r i p t a s e R rad rbc red b lood c e l l RFLP r e s t r i c t i o n fragment l eng th polymorphism RNA r i b o n u c l e i c a c i d rpm r e v o l u t i o n s per minute RSV Rouse sarcoma v i r u s SDS sodium dodecy l s u l f a t e SV40 s im ian v i r u s 40 TCR.A T c e l l recep to r A cha in TCR.B T c e l l recep to r B cha in 169 ug microgram V - n e o r r e t r o v i r u s encoding the gene f o r neomycin r e s i s t a n c e V - s r c v i r a l s r c oncogene from the Rous sarcoma v i r u s wbc whi te blood c e l l 170 APPENDIX I I  SELECTED CYTOGENETIC NOMENCLATURE The cy togene t i c nomenclature used throughout t h i s work was the I n t e r n a t i o n a l System for Human Cytogene t ic Nomenclature (1978). In the c o n s t r u c t i o n of the karyotype the autosomes are numbered from 1 to 22 as n e a r l y as p o s s i b l e i n descending order of l e n g t h . The sex chromosomes are r e f e r r e d to as X and Y . The symbols p and q are used to des igna te , r e s p e c t i v e l y , the shor t and long arms of each chromosome. A) Normal Karyo types . Normal human karyotypes are designated as f o l l o w s : 46,XX Normal female. 46, XY Normal male. B) Numer ica l Chromosome A b e r r a t i o n s . The + and - s igns are placed before the appropr i a t e symbol to i n d i c a t e a d d i t i o n a l or mi s s ing whole chromosomes, e . g . , 47, XX,+8 47 chromosomes, XX sex chromosomes, an a d d i t i o n a l chromosome #8. 4 5 , X Y , - 7 45 chromosomes, XY sex chromosomes, a m i s s i n g chromosome #7. C) Chromosome Mosa ics . The chromosome c o n s t i t u t i o n of the d i f f e r e n t c e l l l i n e s i n a mosaic are l i s t e d i n order of the predominant c lones . 4 5 , X / 4 6 , X X A chromosome mosaic w i th two c e l l l i n e s , the major one w i th 45 chromosomes and a s i n g l e X , the o ther w i t h a normal female karyo type . 171 (a) R e c i p r o c a l t r a n s l o c a t i o n s are des ignated as f o l l o w s : 4 6 , X Y , t ( 9 ; 2 2 ) Breakage and reun ion have occur red i n chromosomes #9 and #22; 46 ,XY , t (9q ;22q ) Breakage and reunion have occur red i n the long arm of chromosome #9 and the long arm of chromosome #22. 4 6 , X Y , t ( 9 ; 2 2 ) ( q 3 4 ; q l l ) Breakage and reun ion have occur red at bands q34 i n chromosome #9 and q l l i n chromosome #22. 46 ,XY,Ph Abbrev ia ted nomenclature f o r a normal male karyotype w i th a s tandard P h i l a d e l p h i a chromosome r e s u l t i n g from a r e c i p r o c a l t ( 9 ; 2 2 ) ( q 3 4 ; q l l ) . (b) D e l e t i o n s of par t of a chromosome which shor tens the l eng th of one of i t s arms are des ignated as f o l l o w s : 46 ,XX ,de l ( 5 ) (q l 3q31 ) D e l e t i o n of par t of the long arm of chromosome 5 between bands 13 and 31 i n an o therwise normal female ka ryo type . (c ) I nve rs i ons i n which the reg ion of a chromosome around the centromere becomes i nve r t ed r e l a t i v e to the d i s t a l po r t i ons of the long and shor t arms are des ignated as f o l l o w s : 46XX, inv (16) (p l3q22) I nve rs i on of one chromosome 16 between bands p l3 and q22 i n an o therwise normal female karyo type . 172 Reference 1. Report of the s tand ing committee on human cy togene t i c nomenclature. Cytogen C e l l Gen 21: 309, 1978. 173 APPENDIX I I I METHOD FOR ESTABLISHMENT AND MAINTENANCE  OF HUMAN LONG TERM CULTURES ( M o d i f i e d from Reference 1) C e l l s . A l l marrow specimens were c o l l e c t e d i n s t e r i l e tubes c o n t a i n i n g 800 u n i t s of p r e s e r v a t i v e free hepar in i n 1 ml of a-medium. A buffy coat p r e p a r a t i o n of the marrow was made by c e n t r i f u g a t i o n at 200 g x 4^ and h a r v e s t i n g the upper l a y e r of c e l l s . Long-term c u l t u r e s were i n i t i a t e d by p l a c i n g 2 x 10? nuclea ted marrow buffy coat c e l l s i n 8 ml of growth medium i n a 60 mm x 15 mm t i s s u e c u l t u r e d i s h ( F a l c o n ) . Growth Medium, a-medium supplemented w i t h e x t r a glutamine (400 mg/1), i n o s i t o l (40 mg/1), and f o l i c a c i d (10 mg/1) was supplemented w i t h horse serum (HS, 12.5%, Flow L a b o r a t o r i e s ) , f e t a l c a l f serum (FCS, 12.5%, Flow L a b o r a t o r i e s ) , 2-3-mercaptoethanol ( 1 0 _ 4 M ) , and hydrocor t i sone sodium s u c c i n a t e ( 1 0 " 6 M ) . Mercaptoethanol was made up to 1 0 _ 4 M from a f rozen s tock s o l u t i o n of 10 _ 2 M , and hydrocor t i sone from a 10~ 4M s o l u t i o n kept at 4°C and made up f resh every week. S o l u t i o n s of both of these reagents were made up i n a-medium- Both horse and f e t a l c a l f se ra were s e l e c t e d i n i t i a l l y fo r t h e i r a b i l i t y to support hematopoiesis i n c u l t u r e : the horse serum i n mouse CFU-C assays , and the f e t a l c a l f serum i n mouse and human CFU-E and BFU-E assays . I n i t i a t i o n of Long Term C u l t u r e s . Dishes were incubated d u r i n g the f i r s t 3-4 days at e i t h e r 37°C and then t r ans f e r r ed to 33°C, i n a l l cases i n a h u m i d i f i e d atmosphere of 5% CO2 i n a i r . At day 3 or 4 a t o t a l change of the growth medium was performed before the dishes were t r ans f e r r ed to 33°C where they remained through the du ra t i on of the c u l t u r e . Maintenance. At weekly i n t e r v a l s , the h a l f growth medium was rep laced w i t h f resh medium and h a l f of the non-adherent c e l l s were removed w i t h the 174 spent medium. These c e l l s were used for nuc lea ted c e l l counts , and fo r assessment of the p rogen i to r content by c l o n i n g a l i q u o t s i n s tandard m e t h y l c e l l u l o s e c u l t u r e s . Enzymatic Detachment of the Adherent Layer . B a c t e r i a l co l l agenase type I (200 uni t s /mg) p r o t e i n , Sigma Chemicals) was d i s s o l v e d i n ca l c ium and magnesium-free Hanks balanced s a l t s o l u t i o n (HBSS-Ca-Mg) to a f i n a l c o n c e n t r a t i o n of 0.13% and the s o l u t i o n s t e r i l i z e d by passage through a 0. 22u m i l l i p o r e f i l t e r . Th i s s o l u t i o n was kept at 4°C and used w i t h i n 48 hours . Jus t before use, FCS was added to g ive a f i n a l concen t r a t i on of 20% FCS and 0.10% co l l agenase . To detach adherent c e l l s , non-adherent c e l l s and a l l the growth medium were f i r s t removed from the c u l t u r e d i s h . C u l t u r e s were then v i g o r o u s l y washed twice w i th f resh HBSS-Ca-Mg, and the a d d i t i o n a l detached c e l l s then added to the non-adherent suspension. Ten ml of 20% FCS i n 0.10% co l l agenase s o l u t i o n were then p ipe t t ed onto the adherent l a y e r and the c u l t u r e s incubated undis turbed for 3 hours at 37°C i n an atmosphere of 5% CO2 i n a i r . At the end of the i ncuba t i on p e r i o d , many c e l l s were comple te ly detached and most of the remaining adherent c e l l s could be r e a d i l y resuspended by g e n t l e but sus t a ined p i p e t t i n g . Adherent c e l l s were cen t r i fuged at 180g for 10 min at room temperature, and then washed twice i n 2% FCS i n HBSS-Ca-Mg. A f t e r each c e n t r i f u g a t i o n , the c e l l p e l l e t was c a r e f u l l y resuspended to minimize the format ion of clumps. Adherent c e l l s were then assayed for p rogen i to r content i n s tandard m e t h y l c e l l u l o s e assay. Reference 1. Coulombel L , Eaves AC, Eaves C J . Enzymatic treatment of long- te rm human marrow c u l t u r e s r evea l s the p r e f e r e n t i a l l o c a t i o n of p r i m i t i v e hemopoiet ic p rogen i to r s i n the adherent l a y e r . Blood 62: 291, 1983. 175 APPENDIX IV METHOD FOR HEMOPOIETIC COLONY ASSAYS IN METHYLCELLULOSE CULTURE ( M o d i f i e d from Reference 1) E r y t h r o p o i e t i c (CFU-E and BFU-E) and g r a n u l o p o i e t i c (CFU-GM) p r o g e n i t o r s were assayed i n 0.8% m e t h y l c e l l u l o s e i n a-medium or I s c o v e ' s medium, supplemented w i t h 30% FCS, 1% de ion ized bovine serum albumen (BSA) buffered w i t h b icarbonate (1 ml of 7% bicarbonate fo r 40 ml of 10% BSA), 1 0 - 4 M 2-3-mercaptoethanol and 200 mM L-Glu tamine . 3 u n i t s per ml of human e r y t h r o p o i e t i n (Ep, p u r i f i e d i n the Ter ry Fox Laboratory to a s p e c i f i c a c t i v i t y of at l e a s t 100 u n i t s per mg of p r o t e i n ) , was added to assays to assess both e r y t h r o i d and g r a n u l o p o i e t i c p r o g e n i t o r s . 10% of phytohemagglut in in (PHA)-s t imula ted human leukocyte cond i t i oned medium was added to a l l assay d i s h e s . PHA-condi t ioned medium was prepared by i n c u b a t i n g blood buffy coat l eukocytes (separated i n 0.1% m e t h y l c e l l u l o s e ) at a c o n c e n t r a t i o n of 1 x 10^ c e l l s per ml w i t h 0.5% PHA (Gibco , M- type) , i n I s c o v e ' s or a-medium w i t h 10% FCS. Incuba t ion was performed at 37°C d u r i n g 7 days, and at the end of the i ncuba t i on p e r i o d , the medium was c o l l e c t e d , spun at 2000 rpm to remove c e l l s and c e l l d e b r i s , and then s to red i n f rozen a l i q u o t s . E r y t h r o p o i e t i n and cond i t i oned medium prepara t ions were both c a l i b r a t e d aga ins t s tandard prepara t ions to ensure equiva lence of a c t i v i t y i n d i f f e r e n t batches . C e l l s were p l a t ed i n m e t h y l c e l l u l o s e assays at a f i n a l c o n c e n t r a t i o n of 0.5 to 2 x 10^ c e l l s per 1.1 ml of c u l t u r e . Buffy coat marrow c e l l s were p l a t e d at 2 x 10^ c e l l s per 1.1 ml ; F i c o l l hypaque separated blood c e l l s at 4 x 10^ per 1.1 m l . Non-adherent c e l l s from long- term c u l t u r e s were u s u a l l y 176 p l a t e d at 0.5 x 10^ c e l l s per 1.1 ml dur ing the f i r s t 2-3 weeks, because of the inc reased concen t r a t i on of CFU-GM i n the non-adherent f r a c t i o n d u r i n g that p e r i o d , and then subsequently at 1 x 10^ c e l l s per 1.1 m l . A f t e r 6-8 weeks, the number of c e l l s recovered i n the non-adherent f r a c t i o n was of ten reduced so that as few as 1 to 2 x 10* c e l l s was a l l that could be p l a t e d . Adherent c e l l s were p l a t e d at 1 x 10^ or 0.5 x 10^ c e l l s per 1.1 m l . As the adherent c e l l suspension was a mixture of some hemopoiet ic c e l l s and many non-hemopoiet ic c e l l s ( i n c l u d i n g f i b r o b l a s t s ) , i t was found that i t was important to s e l e c t assay p e t r i d ishes that s t r o n g l y prevented any spreading of the adherent c e l l s du r ing the 3 week pe r iod requ i red for hemopoiet ic co lony growth. I f t h i s was not done, f i b r o b l a s t p r o l i f e r a t i o n was ex tens ive and i n h i b i t i o n of e r y t h r o i d , and to a l e s s e r extent of g r a n u l o c y t i c , co lony growth r e s u l t e d . Each assay was set up i n d u p l i c a t e or quad rup l i ca t e and scored 2 times to o b t a i n r e l i a b l e counts of both s m a l l and l a r g e e r y t h r o i d c o l o n i e s u s i n g the f o l l o w i n g c r i t e r i a : i s o l a t e d s i n g l e or pa i red c l u s t e r s of CFU-E de r i ved e r y t h r o i d c e l l s were counted at day 7 or 8 a f t e r p l a t i n g . Burs t s c o n t a i n i n g 3-8 c l u s t e r s were scored at day 12-14, and burs ts c o n t a i n i n g more than 8 c l u s t e r s a f t e r 16-18 days. However, i n most i n s t ances , BFU-E numbers have been added together and presented as a s i n g l e v a l u e . C o l o n i e s of g ranu locy tes and macrophages (from CFU-GM) c o n t a i n i n g more than 20 c e l l s were scored at day 12-14, sometimes i n the same assay d ishes as used fo r the CFU-E and BFU-E es t imate but more of ten i n assays wi thout added e r y t h r o p o i e t i n . Reference 1. Gregory C J , Eaves AC. Human marrow c e l l s capable of e r y t h r o p o i e t i c d i f f e r e n t i a t i o n i n v i t r o : D e f i n i t i o n of three e r y t h r o i d colony responses . Blood 49: 855, 1977. 177 APPENDIX V METHOD FOR CYTOGENETIC ANALYSIS OF METAPHASES FROM  CELLS IN INDIVIDUAL HEMOPOIETIC COLONIES ( M o d i f i e d from Reference 1) Hemopoietic c o l o n i e s were plucked from m e t h y l c e l l u l o s e assays w i t h a f i n e l y drawn out g l a s s p i p e t . Each set of assay c u l t u r e s was monitored at frequent i n t e r v a l s to s e l e c t the op t ima l time for p l u c k i n g i . e . , when l a r g e c o l o n i e s had matured to the point where they could be i d e n t i f i e d w i t h conf idence as e r y t h r o i d , g r a n u l o c y t i c or mixed, but colony growth had not yet s topped. Th i s u s u a l l y occurred a f t e r 9-12 days for BFU-E and up to 15 days fo r CFU-C. As a r e s u l t , d i f f e r e n t types of c o l o n i e s were u s u a l l y harves ted at d i f f e r e n t times i n d i f f e r e n t assay d i s h e s . One hour p r i o r to h a r v e s t i n g , 0 . 1 ml of Colcemid (1 u g / m l ) , was evenly d i s t r i b u t e d over the m e t h y l c e l l u l o s e sur face w i t h a 26 gauge needle (7-8 d rops ) . Se lec ted c o l o n i e s were then p lucked , and d i spe r sed i n a s m a l l volume of 0.075 M KCL i n a round-bottomed w e l l o f m i c r o t i t e r p l a t e (20 u l / m i c r o w e l l ) . A f t e r 15-20 minutes i n hypo ton ic KC1, c o l o n i e s were t r ans f e r r ed one by one onto p o l y l y s i n e - c o a t e d s l i d e s and a l lowed to s e t t l e fo r 10 minutes . The c e l l s were then f i x e d by dropping a mix ture of 3:1 me thano l : ace t i c a c i d onto the s l i d e and b l o t t i n g excess mois ture from the edges of the s l i d e w i t h a co t ton swab. The s l i d e s were d r i e d and s t a i n e d for Giemsa banding. Only c o l o n i e s i n which at l e a s t 2 metaphases cou ld be analyzed were accepted. Reference 1. Dube ID, Eaves C J , Kalousek DK, Eaves AC. A method fo r o b t a i n i n g h igh q u a l i t y chromosome prepara t ions from s i n g l e hemopoiet ic c o l o n i e s on a r o u t i n e b a s i s . Cancer Genet Cytogenet 4: 157, 1981. 

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