Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A Scid mouse model of human acute myelogenous leukemia (AML) Moore, Carolyn J. L. 1996

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-ubc_1996-0393.pdf [ 5.15MB ]
JSON: 831-1.0087207.json
JSON-LD: 831-1.0087207-ld.json
RDF/XML (Pretty): 831-1.0087207-rdf.xml
RDF/JSON: 831-1.0087207-rdf.json
Turtle: 831-1.0087207-turtle.txt
N-Triples: 831-1.0087207-rdf-ntriples.txt
Original Record: 831-1.0087207-source.json
Full Text

Full Text

A Scid Mouse Model of Human Acute Myelogenous Leukemia (AML) by Carolyn J. L. Moore B. Sc. Hon. University of Western Ontario, 1992  A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science  in  The Faculty of Graduate Studies, Genetics Graduate Program and the Department of Medicine '  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 25, 1996 © Carolyn J. L. Moore, 1996  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  this thesis for  department  or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying of  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be granted her  for  It  is  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ABSTRACT A mouse model of human acute myelogenous leukemia (AML) was achieved using 400 cgy of total body irradiation of low dose -Cesium radiation (1.48 Rads/min.) to 137  Scid mice which were injected intravenously with human AML cells. Demonstrated engraftment in mice of an AML cell line, M07eJ-IL-3, which harbours a retroviral construct producing human IL-3 and neomycin (G418) resistance, was determined by Southern analysis of mouse tissues for human DNA and G418 resistance of cells from mousetissues.Nine AML peripheral blood patient samples were intravenously injected into mice following 400 cgy radiation and subsequent injections of 6 ug human interleukin-3 (IL-3) and 10 ug stem cell factor (SCF) intraperitoneally every other day. Five out of 9 patients samples engrafted in the Scid mice as assessed by DNA analysis using a human specific HERV-H probe, May-Grunwald Giemsa staining for leukemic cell morphology in mousetissuesand fluorescence in situ hybridization (FISH) for cytogenetic abnormalities in the patients. Scid-hu patient #8 demonstrated high levels of engraftment . in the bone marrow 4 weeks post injection while later time points demonstrated dissemination of the human cells in to the bone marrow, spleen, peripheral blood and tumours. These results suggest that there is heterogeneity in the degree to which patient samples will engraft if at all. This model may aid the studies of the AML stem cell biology and be useful for preclinical studies of novel therapies for this disease.  TABLE OF CONTENTS  Page  ABSTRACT  ii  List of Tables  vi-vu  List of Figures  y'dirxx  Acknowledgments  X  1. INTRODUCTION  1  1.1. : The Hematopoietic System  1  1.1.1: Normal Development 1.1.2: Growth Factors (cytokines) 1.1.3: Abnormal Development 1.2. : Acute Myelogenous Leukemia  1 .  4 7 8  1.2.1: Growth Factors and A M L  11  1.2.2: Mouse Models of Human AML  11  1.3. : Experimental design and objectives 1.3.1: Optimizing the Protocol for Engraftment of A M L  16 16  cells in Scid Mice 1.3.2: Determination of Engraftment of Human A M L Cell lines in the Scid Mice  17  1.3.3: Studies with Primary Patient Samples  2. M A T E R I A L S A N D M E T H O D S  18  19  2.1. : Scid Mice  19  2.2. : Cells  20  2.2.1: Cell lines  20  2.2.2: Primary Human AML samples  20  2.3. : May-Grunwald Staining  21  2.4. : Southern Analysis  21  2.5. : MethylCellulose Culture  22  2.6. : Fluorescence in situ Hybridization (FISH)  23  2.6.1: Digoxigenin-labeled Probe  23  2.6.2: Preparation of Slides  23  2.6.3: Hybridization  24  2.6.4: Probe Detection  25  3. R E S U L T S  26  3.1: Optimization of Conditioning of Scid Mice to Allow  26  Engraftment of Human AML Cells 3.1.1: Suspension Culture  28  3.1.2: DNA Analysis of Cultured Tissues of Mice  35  3.2: Demonstration of Engraftment in Scid Mice using AML Cell Lines  IV  38  3.2.1: Suspension Culture 3.2.2: DNA Analysis 3.3.: Human Patient Samples in Scid Mice treated with 400 cgy  41 ,  4  4 47  of Total Body Irradiation 3.3.1: DNA Analysis  47  3.3.2: Morphology of Cells from Mouse Tissues  50  3.3:3: FISH  56  4. DISCUSSION  65  5. REFERENCES  68  LIST OF TABLES  - . Table 1 .•'  Table 2  .  Page  M07e and TF1 cells isolated in suspension culture from  32  sacrificed mouse tissue after various weeks post injection  G418 resistance of cells isolated in suspension culture from sacrificed Scid mouse tissues previously injected with10  33 •  7  , cells M07eJ-EL-3 after various; weeks postihjection  Table.3 ;  G418 resistance of cells isolated in suspension culture from sacrificed Scid mouse tissues previously injected with 10 cells. 7  34 -" •"•  TF1J-GM-CSF cells after various weeks post injection  Table 4  G418 resistance of cells isolated in suspension culture from .  .'•  ' 42  sacrificed Scid mouse tissues previously injected with 10 cells 7  M07eJ-IL-3 after various weeks post injection  Table 5  Southern analysis of summary of Scid mouse tissue's, treated with or without .4 ug of cyclophosphamide and 400 cgy total . body irradiation  ,vc .  ;  . 43  Table 6  AML patient background- their AML French-American-British  48  classification, cytogenetics, factor responsiveness to IL-3 and SCF above control values and their engraftment in Scid-hu mice with the patient samples  Table 7  Summary of the AML patient samples engrafting in Scid mouse tissues from DNA, FISH and morphological analysis  . 4 9  LIST OF FIGURES  Page  Figure 1  The hemopoietic system  Figure 2 .  The percentage survival of Scid mice treated with 4-6 ug of .  .  2-3  29-30  cyclophosphamide and 450 cgy prior to intravenous injection of 10  7  cells of M07e, M07eJ-IL-3, TF1 and TF1 J-GM-CSF  Figure 3  Southern analysis of a Scid mouse treated with 6 ug of cyclophosphamide'and 450 cgy priortomtravenous'tail vein injectionof  /  '\  10 cells of TF1 J-GM-CSF after 4 weeks post injection: •  '  ,  7  Figure 4  36-37  Percentage survival of Scid.mice, treated with or without 4 ug of  39-40  cyclophosphamide prior to intravenous injection of 5xl0 M07e 6  or M07eJ-IL-3 cells  Figure 5  ,  1  Southern analysis of a Scid mouse receiving 400 cgy prior to intravenous injection of 5xl0 M07eJ-EL-3 cells after 5 weeks post 6  injection  VUL  45-46  Figure 6  Southern hybridization of EcbRl digested DNA from  51-52  patient #5 and #8 in sublethally irradiated Scid mice using a human specific HERV-H probe  Figure?  May-Grunwald Giemsa staining of a Scid mouse injected  54-55  with 10 primary human patient #8 cells after 5 and 6 weeks 8  post injection of cells  Figure 8 "  Figure 9  .  Fluorescence in situ hybridization of normal human and mouse  57-58  cell control samples using a human centromeric chromosome 8 probe  Fluorescence in situ hybridization using a centromeric human  59-60  chromosome 8 probe on a Scid-hu patient #8 mouse at 6 weeks post injection  Figure 10  Photographs of FISH analysis on methylcellulbse cultures of .-., Scid-hu patient #8 at 6 weeks post injection  63-64 '  ACKNOWLEDGMENTS  I would like to thank the following people for their technical assistance and kind gifts to make this Master's project successful: members of my lab, Laurie Allies, Gitte Gerhard, and Donna Hogge for her guidance and support; Dr. Gerry Krystal for the MEL cells and Dr. Dixie Mager for the HERV-H probe. To the members of my committee Drs. Connie Eaves, Rob McMaster and John Carlson, many thanks for your suggestions, revisions, criticisms and assistance in the completion of this thesis. '  '•>.••'.•  Finally, a special thanks to my family, K. Grindstaff, P. Durante, K. Reesor, R. Leushner and M. Rivardfor their undying support and motivation.  In memory of E. C. L. Moore.  •  x..  L  INTRODUCTION  1.1  The Hematopoietic System  1.1.1  Normal Development Hematopoiesis or the blood forming process which is continual throughout life, follows a  complex hierarchical system. The totipotent stem cells which hold the potential to differentiate into all mature blood cells, have limited numbers and are located in the bone marrow or peripheral blood.  M o s t of the stem cells are normally quiescent but can be activated with appropriate  cytokines or differentiation factors via the surrounding microenvironment (Quesenberry, 1990; Abbas et al, 1991; Lichtman, 1990) to allow their proliferation and differentiation into progenitor cells (Figure 1,(Abbas et al, 1991)). The lymphoid lineage begins when a pluripotent stem cell produces a lymphoid progenitor to differentiate into B , T and Natural Killer cells ( N K ) upon stimulation with various interleukins (JL-; such as IL-7). The pluripotent stem cell gives rise to both lymphoid and myeloid progenitors.  Through this passage it decreases its proliferative  capacity and becomes more committed to one lineage. Lineage-committed myeloid progenitors, when induced to differentiate by growth factors such as erythropoietin, Granulocyte-macrophage colony  stimulating  factor  (GM-CSF),  I L - 3 , IL-1  or  I L - 6 can  produce  erythrocytes,  megakaryocytes and mast cells, basophils, eosinophils, neutrophils and monocytes (Figure 1). There is thus a continuous spectrum of cells in the hematopoietic hierarchy which can be arbitrarily subdivided into the stem cell, progenitor and differentiated cell compartments based on functional assays, cell surface phenotype and morphology. However, it is likely that there is a more continuous gradation of various properties of the cells as they divide and differentiate. The  1  F i g u r e 1- T h e hematopoietic system (Abbas et a l , 1991)  The maturation of different lineage's of blood cells from their precursors regulated by various cytokines. CFU = Colony forming unit; IL = interleukin; GM-CSF = Granulocyte macrophage colony stimulating factor.  2  mechanisms by which the proliferation and differentiation of normal stem cells are regulated remains poorly defined although interactions with growth factors and inhibitors in the microerivironment of the marrow are known to be important.  1.1.2 G r o w t h factors (cytokines)  Regulators of the hematopoietic system termed growth factors or cytokines can have either positive or negative influences on stem cells. Such factors act in concert with one another and can determine the passage of a newly derived pluripotent stem cell through the hematopoietic tree.  The presence and concentration of Stem Cell Factor (SCF), Granulocyte-macrophage  colony stimulating factor (GM-CSF), G-CSF, Interleukin 3 (IL-3) and others appear to play a role in the proliferation and/or differentiation of the progenitors into the myeloid compartment of mature cells. (Broxmeyer et al, 1991; Koike et al, 1986; Metcalf & Burgess, 1982). There are numerous cytokines which effect the proliferation and differentiation of the hematopoietic cascade. Of particular interest are SCF, GM-CSF and IL-3. Mouse strains carrying the White spotting (W) and Steel (SI) mutation have a characteristic phenotype of anemia, with defective pigmentation and gametogenesis (Broxmeyer et al, 1991; Witte, 1990). These mice are defective in their production of functionally competent stem cells and bone marrow stromal cells, respectively. When either mutation is homozygous, embryonic lethality ensues. The c-kit gene has been mapped to the W locus on chromosome 5 and is a transmembrane tyrosine kinase receptor (Chabot et al, 1988). Several groups identified the c-kit ligand as a soluble growth factor called Stem Cell Factor, SCF (Zsebo et al, 1990), Mast  4  cell.Growth Factor , MGF (Williams et al, 1990; Anderson et al, 1990), the kit ligand, KL (Huang et al 1990) and Steel Factor , SLF (Witte, 1990; Flanagan & Leder, 1990) each by different methods. SCF has a range of biological activities such as its affects on primitive progenitors in particular when synergizing with GM-CSF and IL-7, or GM-CSF, IL-3 or G-CSF (Broxmeyer et al, 1991) to give rise to an increased number and size of hematopoietic colonies in vitro. With erythropoietin, a cytokine which differentiates erythfoid progenitors into erythrocytes (Figure 1), SCF synergizes in vitro to enhance the formation of multi-lineage colony forming units (CFUGEMM) and burst forming erythroid units (BFU-E) (Broxmeyer et al, 1991). In combination with other cytokines SCF acts directly on stem cells (Brandt et al, 1994). Growth factors capable of stimulating stem cell differentiation and maintenance have been identified through long-term culture analysis. Primitive hematopoietic progenitors that initiate long term hematopoiesis in vitro, long term culture initiating cells (LTC-IC), can be maintained by Steel factor alone, G-CSF and IL-3 alone, or by culturing target cells with Sl/Sl murine fibroblasts, genetically incapable of Steel factor production (Sutherland et al, 1993). Otsuka et al, 1991, demonstrated that progenitor maintenance was enhanced in LTC providing a continuous source of EL-3 and IL-6 to primitive hematopoietic cells. The combination of G-CSF, IL-3 and SCF increased the output of clonogenic cells from LTC-IC s which display many of the characteristics expected of pluripotent stem cells (Sutherland et al, 1993). In vitro experiments with the human AML cell line, M07-e, (as well as normal progenitors) has determined that SCF induces proliferation in combination with IL-3 and/or GMCSF (Broxmeyer et al, 1991; Horie & Broxmeyer, 1993; Hallek et al, 1994). Molecular mechanisms underlying this synergism, have suggested that SCF and GM-CSF or IL-3 act on  5  immediate early genes in the cell cycle regulating c-fos, junB, egr-1 and c-myc mRNA's (Hallek et al, 1994; Horie & Broxmeyer 1993) through the mitogen- activated protein (MAP) kinase pathway (Raf-1 etc.). SCF stimulation of its receptor induces autophosphorylation of tyrosine residues (and subsequent binding of phosphotidylinositol 3'-kinase (PI3K) and phospholipase Cgamma-1 (PLC-l-g).) These growth factors thus appear to regulate the expression of these genes in M07e cells in vitro by stabilizing junB mRNA and inducing the transcription of egr-1 and c-fos (Horie & Broxmeyer, 1993). Investigation of the levels of c-kit mRNA expression in various human leukemic cell lines showed that the following exposure to IL-3, GM-CSF, and insulin like growth factor, the highest c-kit mRNA levels were seen in M07e cells and other megakaryocytic cell lines (Hu et al, 1994). Human IL-3, cloned by Yang et al, 1986, has been demonstrated to stimulate the growth of erythroid and myeloid precursors (Morstyn & Burgess, 1988). IL-3 is the product of CD4+ T cells and acts on the most primitive cells in the hematopoietic hierarchy.  The human  megakaryocytic AML cell line, M07e described by Avanzi et al, 1988, became IL-3 responsive after transferring to serum-free media. IL-3 also increased the cloning efficiency of these cells in methylcellulose more than any other cytokine (Avanzi et al, 1988). IL-3 and GM-CSF are factors which can cross-compete for AML cell binding. High-affinity GM-CSF and IL-3 receptors are heteromeric structures of unique alpha chains and a common beta chain, (reviewed in Lowenberg & Touw, 1993). GM-CSF acts locally through its production by activated T cells, mononuclear phagocytes, vascular endothelial cells and fibroblasts (Abbas et al, 1991). Potential target cells include both myeloid progenitors and mature leukocytes. Both IL-3 and GM-CSF overlap in their  6  effects on myeloid target cells demonstrating the redundancy of these hematopoietic growth factors.  1.1.3  A b n o r m a l development  Abnormal hematopoiesis can occur when stem cells are deregulated in any of a variety of ways. Deregulation of stem cells can cause two types of stem cell disorders; aplastic anemia, caused by hematopoietic stem cell failure, and clonal hemopathies, the result of an injury stem cell resulting in abnormal stem cell proliferative activity (Lichtman, 1990). Abnormal development of the stem cell arises through genetic changes, radiation or chemotherapy causing the stem cell to uncontrollably divide and block the differentiation pathway (Lichtman, 1990; Lichtman & Henderson, 1990; Sawyers et al, 1991) as seen in Acute myelogenous leukemia (AML)(Smith et al, 1992; Taylor et al, 1992). Chronic myelogenous leukemia (CML) also has a clonal nature. In this case the initial stem cell always has the same chromosomal abnormality, the Philadelphia chromosome, t(9;22)(q34;ql 1), which then marks all subsequent cells arising from it. The cells, however, do eventually differentiate into essentially normal mature cells (Till & McCullogh, 1980). Thus CML and AML both arise from stem cells and the different cellular characteristics of these diseases can be attributed to the retention and loss respectively of the ability of the mutated stem cells to differentiate (Till & McCullogh, 1980). The exact stage of hematopoiesis from which AML originates has been controversial. It has been suggested that AML could be derived from a transformed totipotent stem cell as in CML, but may also arise at later stages in hematopoiesis (Griffin & Lowenberg, 1986). Glucose6-phosphate dehydrogenase (G6PD) isoenzyme analysis allows the detection of clonal populations  7  in females who are heterozygotes at this X-linked gene (Fialkow et al, 1981). When G6PD analysis was performed on G6PD heterozygous women that had AML, the leukemic cells showed expression of only one isoenzyme (monoclonal population) while when in remission analysis of their normal blood cells demonstrated both isoenzymes (polyclonal population)(Till & McCullogh, 1980; Fialkow et al, 1981). Further analysis of the clonal nature of AML was reported by Fearon et al, 1986, who used recombinant DNA markers and chromosomal abnormalities as markers in patients. He analyzed X chromosome- linked DNA polymorphisms in the hypoxanthine phosphoribosyl transferase (HPRT) gene in females heterozygous for this feature. Such studies showed that the mature granulocytes in some patients in remission could also be derived from the same stem cell as the leukemic blasts. These results were also confirmed by studies of other genetic markers.  1.2 Acute Myelogenous Leukemia AML is a disease which can affect persons of all ages, however, the incidence increases markedly with age. Symptoms of the disease may include weakness, fatigue, weight loss, easy bruising, prolonged bleeding from skin injuries and fever. Further clinical inspection often shows splenomegaly, hepatomegaly, anemia and reduced platelet numbers. These features are directly related to the malignant stem cell's inability to produce functional mature blood cells and its uncontrolled proliferation (Lichtman, 1990; Lichtman & Henderson, 1990). AML is a group of diseases where the single abnormal clone is severely defective and leads to an accumulation of leukemic blast cells in the marrow which are unable to differentiate into mature cells (Lichtman & Henderson, 1990). There are, however, many - morphological variants as a result of differences in the ability of the leukemic clone to undergo normal  8  ' •  '  hematopoietic differentiation and maturation. These variants are classified by morphology of the cells, cytogenetic abnormalities and cell surface antigen expression. The 7 phenotypes or variants of AML have been classified in the French American British classification system. The FAB types are listed according to morphology of the dominant cell type, histochemical characteristics of cells on blood and marrow stains and somewhat by the reactivity pattern of the blast cells to monoclonal antibodies for specific surface epitopes on myeloid or lymphoid cells.  The acute leukemia variants listed are in order of FAB classification: M l  myeloblastic without maturation, M2 myeloblastic with granulocytic maturation, M3 promyelocytic,  M4 myelomonocytic,  M5 monocytic,  M6 erythroleukemia and M7  megakaryocytic. These subtypes of leukemias differ in frequency among patients with AML in the population with M l or M2 myeloblastic leukemia with or without maturation being the most prevalent (roughly 50%). Some of these particular FAB subtypes have their own characteristic cytogenetic features.  M2 is often associated with the t(8;21)(q22;q22) karyotype. Acute  promyelocytic leukemia cells resemble promyelocytes, which contain large granules. Peroxidase stains these cells intensely. This type of AML is associated with a t(15;17)(q22;q21) karyotype. Myeloblastic and monocytic cells are dominant in the M4 subtype while a subvariant M4 (eo) may present with eosinophilia in the bone marrow. The subvariant is usually associated with inversions in chromosome 16. M5 monocytic variant associations with chromosome 11 abnormalities have been made (e.g. t(9;ll),  t(ll;17)).  The monocytes appear large and nucleus convoluted.  Erythroleukemia is hallmarked by nucleated red cells in the blood. Megakaryoblastic leukemia usually has high blast cell counts and severe myelofibrosis with the budding cytoplasm from the cells (Lichtman & Henderson, 1990).  9  Recently, the pathogenic relationship between one of these cytogenetic abnormalities, the inv(16), has been elucidated (Liu et al, 1993). The breakpoint genes on 16p and 16q have been shown to be MYH11, a smooth muscle myosin heavy chain gene, and CBFB, a human counterpart of mouse core binding factor (CBFbeta), a DNA binding factor. The fusion protein of the 5' portion of CBFB and 3' portion of MYH11 may dimerize to form a more stable complex with the alpha subunit than that formed by wild-type CBFB, and prevent binding of the normal complex to target sequences, thereby acquiring some new or inappropriate activity in transcriptional regulation (Liu etal, 1993). The t(8;21) common abnormality in AML patients represents 40% of cytogenetic abnormalities in M2 patients (Hogge, 1994).  The M2 subtype has tumour mass forming  tendencies outside the bone marrow and has a marked in vitro response to IL-5. The genes involved in this translocation are ETO (eight twenty-one) on chromosome 8q22 and AML1 on chromosome 21q22.3 (Erickson et al, 1994; Miyoshi et al, 1993). The ETO gene has been determined to be a brain specific transcription factor which is only expressed in t(8;21) AML's while the AML1 gene, important in hematopoiesis as a transcription factor, is present in all AML's lacking the t(8;21). The aberrant fusion protein of AML 1-ETO has been demonstrated to involve the transactivating domain of ETO with the DNA binding domain of AML 1 (Erickson et al, 1994). The indirect or direct consequences of the aberrant protein in explaining the IL-5 response or tumour masses outside of the bone marrow or on target genes has yet to be determined.  Clinical monitoring of AML in complete remission patients suggests that the  AML1/ETO fusion RNA persists in the patients that have undergone Autologous Bone marrow transplantation or chemotherapy (Kusec et al, 1994).  10  1.2.1 G r o w t h factors a n d A M L  From culture techniques developed for the analysis of clonal proliferation of normal murine and human marrow progenitor cells (Lowenberg & Touw, 1993), it became apparent through culturing that cytokines were required for the survival and proliferation of AML progenitors. IL-3, GM-CSF, G-CSF and SCF were found to induce leukemic colony formation or activate DNA synthesis in leukemic blasts from more than 80% of AML patients (Lowenberg & Touw, 1993). Studying the effects of IL-3, GM-CSF and G-CSF on leukemic clonogenic cells in vitro, Vellenga et al, 1987, determined that, in general, these factors promoted AML-CFC selfrenewal and that the combination of these 3 growth factors supported their prolonged survival of AML progenitors (Vellenga et al, 1987). However, there is considerable heterogeneity in the factor responsiveness of AML cells from different patients (Till & McCullogh, 1980). In vitro proliferation of AML cells is limited as the number of progenitors are low and most AML are not cycling or mature (Lowenberg & Touw, 1993).  1.2.2. M o u s e models of h u m a n A M L  In vitro assays for hematopoietic progenitors have been used to elucidate, some of the growth factor requirements of primary human cells and tq characterize the progenitor content of normal human bone marrow and blood. As discussed above, in semisolid colony assays, when supplemented with cytokines, both leukemic and normal clonogenic cells grow as both are largely growth factor dependent. Normal and leukemic clusters can be similar in size and shape. In addition, such assays are unable to detect either normal or malignant stem cells (Metcalf, 1991).  11  Rarely will AML samples adapt to continuous growth in vitro. When this occurs it may represent the expression of a genetic alteration which has occurred during culture and allows the outgrowth of a subclone in the leukemic population or immortalization (Gluck et al, 1989). AML cells often die out over several weeks in standard long term culture which allow the maintenance of normal hematopoietic cells.  An in vivo murine model of human A M L could . potentially allow  reproducible, reliable and sensitive investigation of the biology of normal and leukemic human stem cells, characterization of cellular interactions with the surrounding environment and the basis' of disease homing patterns which cannot be studied ex vivo.  Immunosuppressed athymic nude mice have previously been investigated as a human model of AML (Thacker & Hogge, 1994). In this model 8-10 week old mice were treated with 6 mg of cyclophosphamide and 650 cgy. of total body irradiation prior to intravenous injection of factor-dependent human leukemic cell lines, M07e and TF-1, engineered with recombinant retroviruses to produce EL-3 and GM-CSF respectively with a neomycin resistance gene marker (neo). The mice injected with factor producing cell lines demonstrated hind limb paralysis by 7 weeks and the pattern of tumour cell dissemination in the animals resembled that of human AML. Cell lines which did not produce growth factors did not proliferate in mice unless exogenous growth factors were provided. Neither uninfected cell lines or neo marked cell lines exhibited symptoms such as hind limb paralysis up to 30 weeks post injection.  The use of nude mice has limitations since they are difficult to breed. However more importantly these mice have normal, functional B cells, macrophages and NK cells and thus are capable of mounting an immune response which will inhibit the engraftment of human cells. Therefore Scid (severe combined immune deficient) mice were chosen to expand upon the  12  research of Mr.Thacker at the Terry Fox Lab. Scid mice lack B as well as T cells (Bosma & Carroll, 1991; Bosma et al, 1983), and therefore are more severely immunodeficient than nude mice. In addition Scid mice have a decreased capacity to fight infections and are sensitive to irradiation. The autosomal recessive scid mutation impairs the joining of the T cell antigen receptor genes during their recombination in developing T cells and similarly affects irrrmunoglobulin gene rearrangement in developing B cells. As a result the development of both cell types are arrested. However, other hematopoietic cell types such as macrophages and NK cells are not affected (Bosma & Carroll, 1993). Scid mice should allow better engraftment levels of human AML cell lines and primary human AML cells in vivo due to their more severe immune deficiency. To further immunocompromise these mice, we anticipated that cyclophosphamide could be given since this drug had been demonstrated to decrease in vivo NK cell activity (Mayumi & Good, 1989) although the effect is short-lived.  Scid mice have been manipulated to understand the biology of human stem cells and the leukemic growth and progression of various diseases as well as to provide a vehicle to assess therapeutic strategies. An Acute lymphoblastic leukemia (ALL) cell line (A-l) was intravenously injected and demonstrated the engraftment of the A-l cells in the bone marrow and spleen at 8 weeks post injection. Two out of 3 primary non T-ALL patient bone marrow samples(KamelReid et al, 1989) engrafted in the bone marrow (by 4 weeks post i.v. injection) and spleen (7 to 10 weeks post injection). The pattern of dissemination in the tissues of the Scid mice resembled that of the final stages of the human disease.  Normal human bone marrow was successfully  transplanted into Scid mice with or without growth factors by Lapidot et al, 1992. Immature human cells engrafted in the murine microenvironment and repopulated the animal with human  13  progenitors and mature cells of the myeloid, lymphoid and erythroid lineages after administration of mast cell growth factor (MGF), PIXY (a fusion protein of GM-CSF and IL-3) and erythropoietin (Lapidot et al, 1992). DNA analysis, short term culture and FACS analysis were used to determine the levels of engraftment attained and to provide evidence of in vivo differentiation of injected human progenitors into various lineages.  Using a similar protocol  human cord blood cells were also shown to be able to engraft irradiated Scid mice for 14 weeks, in this case even without the use of human growth factor injections (Vormoor et al, 1994). Examination of childhood AML in Scid mice by morphological studies and PCR detection of the human beta-globin gene has demonstrated limited engraftment of the leukemic cells for 17-19 weeks after i.v. injection whereas their transplantation under the kidney capsule did not lead to overt leukemia (Chelstrom et al, 1994). Scid mice treated with 200 cgy total body irradiation and given no growth factors have developed disseminated human leukemia of childhood AML. Use of the maximal dose of irradiation that Scid mice will tolerate (400 cgy) (Fulop & Phillips, 1989) plus cytokine treatment might have allowed better growth of the AML cells. ALL, AML and CML cell lines and a few primary ALL, AML and CML patient samples using i.v. or i.p. injections or kidney capsule implants were investigated by Sawyers et al, 1992, using 300 cgy irradiated Scid mice as recipients. Dissemination of tissues with cells from a CML blast crisis patient was seen but mice given primary AML cells were not highly engrafted.  Other mutant mice with relevant immune defects have also been investigated as recipients of human AML cells. Beige mice have impaired NK cell function (Roder & Duwe, 1979) and these mice have been crossed with Scid mice to produce homozygous scid/beige mice in the F2  14  progeny. These mice have a <2% incidence of leakiness in B and T cell production as compared to Scid mice in which production of some B and T cells does occur (Mosier et al, 1993).  Bg/nu/xid mice bred to reduce NK cells (beige mutation), lymphokine-activated killer (LAK) cells (xid mutation) and T cells (athymic- nude mutation) have been given 400 cgy irradiation and i.v. injection of normal human bone marrow following subcutaneous implantation of an osmotic minipump which continuously released human GM-CSF and IL-3 (Kamel-Reid & Dick, 1988). They reported that engraftment was rapid and that a large proportion of the seeded cells must have been progenitors to give rise to the human macrophage colony-forming units (MCFU) seen in the mice. They also claimed that the injection of human growth factors was not necessary in these mice. In subsequent experiments by the same group bg/nu/xid mice were irradiated with 400 cgys followed by i.v. injection of retrovirally infected human bone marrow. They found that retrovirally- marked human cells could be detected in the mice 4 and 16 weeks after being transplanted (Dick et al, 1991).  To produce another type of additionally immunocompromised mouse, C.B 17 scid/scid mice were backcrossed for ten generations onto the NOD/Lt (Non obese diabetic-T cell-mediated autoimmune, insulin- dependent diabetes mellitis) strain background (Shultz et al, 1995). This new NOD-Scid mouse strain exhibits the B and T cell deficiency of the scid mutation and also had the reduced NK cells, lack of circulating complement and nonfunctional antigen presenting cells (APC's) characteristic of the NOD mouse. The leakiness of the C.B.-17-scid mice is virtually abolished in the NOD-Scid mice as no mature B cells are found to develop. However, Shultz et al also reported that these mice show a high incidence of spontaneous thymic lymphoma development which limits their use for long term experiments (Schultz et al, 1995).  15  1.3 E x p e r i m e n t a l Design a n d Objectives 1.3.1 O p t i m i z i n g the protocol for engraftment of h u m a n A M L cells i n S c i d mice  The protocol which allowed engraftment of AML cell lines in nude mice consisted of 650 cgy irradiation with 6 mg of cyclophosphamide i.p. injected 24 hours prior to administering 10  7  M07e or TF-1 cells which had been previously infected with a growth factor-producing retrovirus (Thacker & Hogge, 1994). However, AML cells directly isolated from patients did not engraft in nude mice treated in this way. Thus, Scid mice were chosen as an alternative host for these primary human cells. My initial objective was to determine the maximum dose of low dose cesium radiation (1.48 Rads/min.) with or without cyclophosphamide that the Scid mice would tolerate to permit suppression of the NK cells and engraftment of the factor-producing Mo7eJ-IL3 or TF1J-GM-CSF cell lines with their factor dependent counterparts used as Controls. Four to six mg of cyclophosphamide was injected intraperitoneally and 450 cgy of total body irradiation given 24 hours prior to intravenous injection of the leukemic cells. Some of the mice did not survive the regimen and thus further modification of the protocol was warranted. The irradiation was reduced to 400 cgy and the cyclophosphamide reduced to 4 or 0 mg. Work by others had suggested that the injection of some cyclophosphamide might optimize the approach (Mayumi & Good, 1989) while other investigators had found that 400 cgy total body irradiation was the maximal tolerable dose for Scids (Fulop & Phillips, 1989; Kamel-Reid et al, 1989; Lapidot et al, 1992). The use of AML cell lines for these studies was logical as they are easily cultured, and enabled the fine tuning of a protocol before using it for primary patient cells which are not as readily available.  16  1.3.2 Determination of engraftment o f h u m a n A M L cell lines i n the S c i d mice The M 0 7 e A M L cell line was originally derived from a child with Acute megakaryoblastic leukemia ( F A B M 7 ) (Avanzi et al, 1988). The cell line is factor dependent on I L - 3 , G M - C S F and/or Stem Cell Factor. These cells were transfected with a recombinant retrovirus allowing transfer and expression of a c D N A for human I L - 3 and neomycin phosphotransferase (gene for neomycin resistance) making the resulting M 0 7 e J - I L - 3 cells factor independent and G418 resistant (an analog of neomycin) (Thacker & Hogge, 1994). A n erythroleukemic (M6) cell line called TF-1 was derived from a Japanese A M L patient (Kitamura et al, 1988). A s with M 0 7 e cells, these cells are factor dependent, but responsive to human G M - C S F and I L - 3 only. These cells were transfected with a G M - C S F Tk-neo construct rendering T F 1 J - G M - C S F  factor  independent by the production of human G M - C S F and G418 resistant (Thacker & Hogge, 1994). M y plan was to sacrifice the Scid mouse recipients at 4 week intervals or when any mice were sick.  Suspension cultures would then be set up for the cells removed using media  appropriate for M 0 7 e , M 0 7 e J - J L - 3 , TF-1 and T F 1 J - G M - C S F cell lines. M 0 7 e J - J L - 3 and T F 1 J G M - C S F cultures also contained G418 to select for neo resistant cells. Twenty per cent of the cells in the bone marrow, hind and front, spleen and peripheral blood tissues from the mice were to be cultured while the remaining 80% would be pelleted for analysis of human D N A content with a human specific H E R V - H probe.  The probe in an E c o R l fragment from a human  endogenous retroviral like element which in its full length, spans about 1 kb and is found in roughly 1000 copies per haploid human genome (Mager & Henthorn, 1984; Mager & Freeman, 1987). Thus this probe was used as a sensitive method for detecting human D N A .  17  1.3.3  Studies w i t h P r i m a r y Patient Samples  Utilizing the protocol established with the AML cell lines in which 400 cgy of total body irradiation was given prior to i.v. injection of the cells, 10 primary AML cells were injected i.v. 8  into Scid mice 24 hours after their irradiation. Frozen patient samples were thawed slowly in alpha-MEM and 5% FCS, assessed for viability with a Trypan blue on a hemocytometer and then injected into irradiated mice. Growth factors were utilized to enhance the engraftment of the AML cells as the previous work in our lab had suggested that growth factors might be required for the successful engraftment in athymic nude mice (Thacker & Hogge, 1994).  Human IL-3 and SCF were  injected intraperitoneally every other day. These cytokines were used because studies in our laboratory (Hogge, unpublished data) had indicated that the majority of AML patient samples respond (in proliferation assays) to a greater extent to this combination in comparison to IL-3, SCF or GM-CSF as single factors or in various other combinations. In these experiments we were able to demonstrate the engraftment of primary AML cells in Scid mice with a pattern of dissemination in marrow, blood and spleen that resembled the human disease. Thus, the feasibility of using this model to study the biology and treatment of human AML was established.  18  2. MATERIALS AND METHODS 2A Scid Mice Scid mice were given to the Terry Fox Laboratory by Dr. John Dick (University of Toronto) and maintained in the animal facility of the British Columbia Cancer Research Centre under pathogen free conditions approved by the University of British Columbia's Animal Care Committee. To induce further immune suppression particularly of natural killer cell activity, Scid mice between the ages of 4 weeks and 6 months were treated with or without 4-6 mg cyclophosphamide injected intraperitoneally, along with 400-450 cgy of total body irradiation at 1.48 Rads/minute prior to injection of human cells. Twenty-four hours after irradiation human leukemic cells were injected via tail vein at a dose of 0.5-1.0 x 10 cells for cell lines ( M07e, 7  Mo7eJ-IL-3, TF-1 and or TF1J-GM-CSF) and at 0.5-1.0 x 10 cells for primary AML cells from 8  patients. Mice which had received injections of patient cells also received intraperitoneal injection of 6 ug hIL-3 (Sandoz, Basel, Switzerland) and 10 ug hSCF (PEG-rhSCF Lot #4657-45 Amgen, Thousand Oaks, CA., USA.) every 48 hours. Mice were sacrificed at 4 week intervals post injection or when they appeared ill. Mouse tissues were collected from the bone marrow, spleen, peripheral blood and any tumours that appeared. Some of the cells from these tissues were spread onto slides for morphological analysis, some were cultured in methylcellulose with growth factors to generate cells for analysis by fluorescence in situ hybridization (FISH) and most (~ 80% of the cells) were pelleted for DNA analysis.  Slides were stained with May-Grunwald-Giemsa.  Colonies arising in the  methylcellulose cultures 10-14 days after plating were plucked and hybridized to 6 or 10 well slides and FISH analysis performed. Pelleted cells were frozen at -20°C until phenol-chloroform  19  extractions were performed. Extracted DNA was then digested with EcoRl, run on a 0. agarose gel and Southern blotted using the human specific HERV-H probe.  2,2 Cells 2.2.1  Cell Lines:  The human growth factor-dependent cell lines M07e (Avanzi et al, 1988) and TF-1 (Kitamura et al, 1989) were provided by Drs. L. Pegoraro and T. Kitamura respectively. TF1 cells were maintained in RPMI with 10% Fetal Calf Serum (FCS) and 5 ng/ml of rhGM-CSF (Sandoz, Basel, Switzerland).  M07e cells were cultured in DMEM and 10% FCS and  supplemented with 5 ng/ml of rhJL-3 (Sandoz, Basel, Switzerland). Factor independent lines of TF-1 and M07e cells had been previously generated by transducing the parental cell lines with human GM-CSF or IL-3 cDNA-containing retroviruses respectively (Thacker & Hogge, 1994). These cells did not require addition of growth factors to their growth media.  2.2.2  P r i m a r y H u m a n A M L Cells:  At the Cell Separator Unit, Vancouver Hospital, human AML cells were obtained from consenting adults undergoing therapeutic leukapheresis, after approval of the Clinical Screening Committee for Research Involving Human subjects of the University of British Columbia. The human primary AML cells were washed twice in alpha-MEM with 5% FCS and either injected immediately into mice or cryopreserved at -135°C in 10% DMSO in alphaMEM and 50% FCS until required. Frozen stocks were thawed slowly in alpha-MEM + 5% FCS, counted-on a hemacytometer and assessed for viability with 0.1% Trypan Blue dye  20  exclusion. The cells were then pelleted by centrifugation at 1.5xl0 rpm for 15 minutes and 3  resuspended in a-MEM and 5% FCS at the required concentration for injection into animals.  2.3 M a v - G r u n w a l d Staining  Fresh tissue slide preparations were fixed in 100% methanol prior to May-Grunwald staining, using standard protocols. The stained slides were viewed by a light microscope and scored for the presence and percentage of leukemic cells by morphology.  2.4 Southern Analysis  Detection of proviral DNA within retrovirally infected cells was by Southern analysis using the neo gene. A human specific endogenous retroviral DNA sequence (HERV-H, r  RT10EA) was used to detect human cells in injected mice (RT10EA probe generously given by Dr. Dixie Mager; Mager and Freeman, 1987) also by Southern analysis. Isolation of genomic DNA from sacrificed mouse tissues was done by cell lysis, proteinase K digestion and phenol/chloroform extraction (Sambrook et al, 1989). 10-15 ug of genomic DNA was digested with either Xbal or EcoRl overnight at 37°C, electrophoresed on a 0.8-1.0% agarose gel, and subsequently transferred to Zetaprobe membrane (Biorad, Mississauga, Ontario, Canada). Xbal was used in restriction enzyme digests of tissue where the presence of neo-virus-infected cells was to be detected since there are cleavage sites for this enzyme within each proviral LTR which allow it to release a 4 kbp proviral band. EcoRl digested DNA was hybridized with the HERV-H probe since this enzyme releases an internal conserved EcoRl fragment from some HERV-H elements of about 1 kbp in size (Mager & Henthorn, 1984; Mager & Freeman, 1987). A P - labeled neo specific or HERV-H specific 32  21  r  probe generated with random primer labeling (Feinberg & Vogelstein, 1984), was hybridized to Southern blots using standard techniques (Sambrook et al, 1989).  2.5 Methylcellulose C u l t u r e  Two methylcellulose mixtures were used to plate the cells from mice injected with human AML patients samples depending on the response of the primary human AML patient samples originally. Mixture A and B were prepared by adding the following components to alpha medium of 40% methocult H4100 (Stemcell Technologies Inc., Vancouver, B.C., Canada), 10" M beta-mercaptoethanol, 30% fetal calf serum (FCS), 0.2 ml of L-Glutamine, 4  1% of 10% BSA and 7% Sodium Bicarbonate. Mixture A also contained 10% Agar-LCM (Stemcell Technologies Inc.), 3 U/ml Epo (Lot# H C4F20MD45-Stemcell Technologies Inc.). Mixture B contained 10 ng/ml SCF ( Amgen), 1 ng/ml of hIL-3 ( Sandoz), 1 ng/ml hGM-CSF (Sandoz). Mixtures A and B were aliquotted into polypropylene 14 ml Falcon tubes (Becton Dickinson) at 3.0 ml per tube and either used fresh or stored in a -20°C freezer. Nucleated cellsfromsacrificed mouse tissues were counted on a hemacytometer after lysing red blood cells with 3% Acetic Acid. These nucleated cells from mouse tissues were added to either methylcellulose mixture to yield a final concentration in alpha medium of 0.55.0xl0 cells/ml and 1 ml of the mixture plated in 35 mm petri dishes (Greiner Laboratories, 5  GMBH, Nurtingen). Duplicate cultures were plated for each tissue sample. They were incubated at 37°C for 2-3 weeks and scored for the presence of colonies using inverted light microscopy.  22  2.6 Fluorescence i n situ H y b r i d i z a t i o n ( F I S H ) 2.6.1  Digoxigenin -labeled probe  The human centromeric 8 (D8Z2-ATCC, Rockville, Maryland) FISH probe was nick translated using the following protocol:  10% of 10X buffer (0.5M Tris pH 8.0, 0.05M  MgCl , 7xl0" beta-mercaptoethanol, 0.1 mg/ml BSA, 3xl0" M each of dATP, dGTP, dCTP, 3  3  2  10" M dTTP and 431 ul ddH20) with 3% Digoxigenin-11-UTP (Dig-ll-UTP, Boehringer 3  Mannheim), 2 ug probe DNA, 10% enzyme mix (0.5 U/ul DNA pol I, 0.0075 U/ul DNAse I, 50 mM Tris (pH 7.5), 5 mM Magnesium acetate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM beta-mercaptoethanol, 50% glycerol (v/v) and 100 ug/ml Bovine Serum Albumin), 2% (1:10) DNA polymerase 1( Gibco-BRL) and ddH20 to 100 ul was combined and incubated at 16°C for 20 minutes. To the probe mixture, 10 ul of stop buffer (300 mM EDTA) was added and the volume brought up to 300 ul with ddH20 and then added to a Sephadex G-50 in TE (Tris-EDTA) column.  Unincorporated nucleotides were removed by passing the nick  translated probe over a Sephadex G-50 column and a sample of the probe electrophoresed on a 1.5% agarose gel and the DNA stained with ethidium bromide and visualized under UV light to estimate average fragment size and determine the concentration of probe DNA.  2.6.2  P r e p a r a t i o n of Slides  Colonies were pooled and/ or individually plucked from 2-3 week old methylcellulose cultures of cells from mousetissuesusing sterile, finely drawn out glass pipettes into wells of 96-well microtitre dishes (Falcon) for incubation in 0.075 M KC1 solution for 20 minutes to allow hypotonic lysis of cytoplasmic membranes. The contents of the wells were then pipetted onto poly-L-lysine coated slide wells and left for 10 minutes to allow the cells to settle. After  23  '  removing hypotonic solution from slide wells with absorbent material, a drop of 20% fixative (3:1 methanol: acetic acid) was added to each well and incubated for 3 minutes at room temperature before the slides were submersed in 100% fix in Copeland jars for 10 minutes. Slides were dried at room temperature and stored in a -20°C freezer until required.  2.6.3  Hybridization  Prepared slides were washed in Copeland jars for 30 minutes in 2XSSC (0.355 M sodium chloride and 0.0355 M sodium citrate) at 37°C.  Cell DNA was denatured by  immersing slides in 70% deionized formamide in 4XSSC at 70°C for 2 minutes.  Slide  preparations were then dehydrated by sequential ethanol washes were performed at 70%, 80%, 95% and 100% ethanol for 2 minutes each at room temperature and the slides allowed to air dry. The hybridization mix consisted of the probe at a final concentration of 2 ng/ml with 10% salmon sperm DNA, 70% hybridization solution (5.5 ml Formamide, lg dextran sulfate, 0.5 ml 20XSSC and ddH20 to 7 ml) and ddH20 making up the difference to the total volume. The probe was denatured at 70°C for 5 minutes and then put on ice. Slides were warmed to 37°C before applying 10 ul of probe per slide, coverslipping the slides and sealing the edges of the coverslip with rubber cement. Slides were incubated overnight at 37-39°C in humidified containers. The following day, the coverslips were removed and the slides washed in 50% formamide in 4XSSC at 45°C for 15 minutes. 55% formamide in 4XSSC wash was done twice at 45°C for 15 minutes each followed by a 2XSSC and 2-0.1XSSC washes at the same temperature and duration.  24  2.6.4  P r o b e Detection  To block nonspecific antibody binding 100 ul of 0.1% BSA in 4XSSC was added to each slide under a coverslip and incubated at room temperature for 5 minutes. The rest of the procedure was performed in the dark. The primary antibody was made at a 1: 25 dilution of Anti- Digoxigenin fluorescein Fab fragments (Boehringer Mannheim) in filtered 0.1 %BSA in 4XSSC. Under a coverslip 100 ul of primary antibody was placed on each slide and incubated at 37°C for approximately 1 hour and 15 minutes.  Coverslips were removed and slides  washed once in 4XSSC, once in 0.1% Triton X-100 with 4XSSC and once in PN buffer (0.1M  NaH2P04/Na2HP04, pH 8.0 and 1% NP-40) for 10 minutes each at room  temperature on a shaker bath. Before adding 100 ul of secondary fluorescein labeled rabbit anti-sheep-FITC antibody (Dimension.Lab Inc.) at 1:50 dilution in filtered PNM buffer (PN buffer plus 5% non-fat dry milk powder and 0.05% sodium azide), the slides were blocked with PNM buffer at room temperature for 5 minutes. The second antibody was added to each slide coverslipped and incubated for 1 hour at 37°C. Three 5 minute PN buffer washes of the slides were done at room temperature in shaking water bath. Two 2 minute 0.1 % Triton X100 in 4XSSC washes warmed to 40°C were done at room temperature followed by another two at 40°C. Nuclei were counterstained by placing 2 ul of 0.1% propidium iodide in antifade (0.23g DABCO (1,4 - Diazabicyclo-[2.2.2] octane (Sigma)) onto each slide. Cell nuclei were scored for the number of hybridization signals using an epifluorescence microscope (Axioplan Zeiss, Germany). A minimum of 25 nuclei per well per sample were scored and representative photographs taken using Fugichrome 400 RH135 film at various magnifications and exposure times.  25  3.  RESULTS  3.1 O p t i m i z a t i o n of conditioning of S c i d mice to allow engraftment of h u m a n A M L cells :  Previous work by Thacker & Hogge, 1994, had shown that growth factor-dependent human AML cell lines will engraft in irradiated cyclophosphamide -treated nude mice when provided with a source of human factor on which their growth is dependent either by repeated i.p. injections of the growth factors or by rendering the cells autostimulatory through infecting them with a retrovirus allowing the transfer and expression of cDNAs for those same factor(s). However the use of nude mice was unsatisfactory because pf the difficulty of breeding them " in house" and, more importantly, because of their residual immune function (B cells, macrophages, NK cells) which made engraftment of even cell lines slow and unreliable. Scid mice, which lack both T and B cell function should theoretically provide less resistance to xenografting of human cells. Indeed, other investigators have had success in using these animals as recipients for a variety of human hematopoietic cells (Lapidot et al, 1992: Dick et al, 1991; Kamel-Reid et al, 1989, Lapidot; 1994). However, Scid mice are known to tolerate radiation poorly due to their genetic abnormality which inhibits DNA repair. Thus, it was likely that the radiation and cyclophosphamide regimen that was tolerated as immunosuppression, by nude mice would not be tolerated by Scids. N  Nevertheless,  it seemed reasonable to expect that more fully  immunosuppressed mice with depressed NK cell and macrophage function (as well as B and T cells) might be better recipients.  A series of initial experiments were therefore designed to  determine the maximal dose of total body irradiation with or without cyclophosphamide that would be tolerated by Scid mice injected with human AML cells.  26  To determine if this regimen was sufficiently immunosuppressive to permit engraftment of human leukemic cells, the immunosuppressed mice were injected with human AML cell lines. Cell lines were chosen for these initial studies rather than primary patient AML cells because they are available in unlimited quantities and do not have the inherent variability of different patient samples. As a result of previous studies (Thacker & Hogge, 1994) both growth factor dependent and factor independent variants of the human AML cell lines M07e and TF-1 were available for these experiments. The factor-independent cells had been created by transducing the parental factor- dependent cells with recombinant retrovirus encoding the human IL-3 cDNA (for M07e cells) or GM-CSF cDNA (for TF-1 cells). Comparison of the growth of factor-independent, autocrine growth factor stimulated cell lines with that of the factor-dependent, parental cell lines in mice would potentially allow us to determine if the presence of human growth factor was as necessary for growth of these human AML cells in Scid mice as it had been in nude mice. Most primary human AML cells exhibit factor-dependent growth in vitro. This finding suggests the need for human growth factors to allow engraftment of such cells in Scid animals. To determine the appropriate protocol to grow AML cell lines in the Scid mice, the same regimen was performed on the four to eighteen week old Scid mice as the nude mice previously studied in our lab (Thacker & Hogge, 1994). Thirty-two Scid mice were treated intraperitoneally with 4-6 mg of cyclophosphamide and then 450 cgy of total body irradiation 24 hours prior to intravenous injection of 10 cells of the factor-dependent parental AML cell lines, M07e and TF7  1. Thirty-eight Scid mice treated with cyclophosphamide and radiation as mentioned above, were also intravenously injected with lxlO cells of Mo7eJ-IL-3 (22 mice) or TF1 J-GM-CSF (16 mice) 7  which had been rendered growth factor-independent by retroviral infection with recombinant  27  retroviruses containing cDNAs for human IL-3 or GM-CSF respectively. Mice were sacrificed at 4 week intervals post injection of human cells. Mice were also sacrificed if they appeared sick. Figure 2 demonstrates the survival of the percentage of Scid mice after treatment with 450 cgy and 4-6 mg cyclophosphamide. Within the first three weeks, greater than 75% of the mice had expired. Thirteen were sacrificed in the various experiments prior to 4 weeks post injection due to severe illness ( yellow and disheveled coats and loss of normal activity). Experiments in nude mice had indicated that proliferation of IL-3 producing M07e cells required at least 6 to 8 weeks to produce lethal toxicity. Symptoms were usually first manifest by hind limb paralysis or the appearance of tumours. It therefore appeared likely that the symptoms observed in the present experiments were caused by toxicity from the cyclophosphamide or radiation or both. The protocol was subsequently modified to determine which of these factors was causing the high death rate of the Scid mice. However, tissues from the Scid mice sacrificed after treatment with this first regimen were analyzed first to see if engraftment of human AML cells had been achieved.  3.1.1  Suspension C u l t u r e :  To determine whether there were AML ceils in the hematopoietic tissues from the sacrificed mice prior to 4 weeks post injection, the cells were cultured in media that supported the growth of the corresponding human cells. Cells from the tissues of mice injected with M07e cells were cultured in M07e medium and 6 ng/ml rhIL-3 while cells from mice injected with M07eJIL-3 cells were cultured in DMEM, 10% FCS and 0.6 mg/ml G418 (w/v). Tissues from TF-1 injected mice were cultured in RPMI with 10% FCS and 2 ng/ml rhGM-CSF, whereas those injected with TF1J-GM-CSF cells were cultured in the same liquid medium with 0.8 mg/ml G418  28  F i g u r e 2i The percentage s u r v i v a l of S c i d mice after various weeks post injection treated w i t h 450 cgy a n d 4-6 m g of cyclophosphamide p r i o r to intravenous injection of 10 cells of h u m a n A M L cell lines. M Q 7 e . MQ7e.T-IL-3. T F 1 a n d T F 1 . T - G M - C S F . 32 Scid mice, four to eighteen weeks old, were treated with 450 cgy total body irradiation and 4-6 rhg of i.p injected cyclophosphamide 24 hours prior to intravenous tail vein injection of M 0 7 e and T F 1 cells. 38 Scid mice treated with 4-6 mg of cyclophosphamide i.p. and 450 cgy 24 hours prior to intravenous tail vein injection of 10 M 0 7 e J - I L - 3 (22 mice) or T F 1 J - G M - C S F (16 mice) cells. 13 mice were sacrificed in these experiments prior to 4 weeks post injection due to severe illness. M 0 7 e (16 mice) -circle; M 0 7 e J - I L - 3 (22 mice)- diamond; T F 1 (16 mice)- triangle; T F 1 J - G M C S F (16 mice)- square. 7  7  29  without added growth factor. Cells from the bone marrow, spleen and peripheral blood of each mouse were cultured. Cultures were discarded if no increase in ceil numbers was seen after 6 weeks incubation. Those cultures where cell numbers increased over 4-6 weeks were harvested and some cells saved for DNA analysis. Table 1 illustrates the response of the murine hematopoietic tissues in suspension culture at various weeks post injection with M07e and TF1. Four mice out of 16 injected with M07e were sacrificed at 1 and 2 weeks post injection and demonstrated no increase in cell numbers in suspension except one mouse at 2 weeks post injection. All of the remaining 12 mice died before tissues could be cultured. One mouse injected with TF1 cells and sacrificed at 2 weeks post injection demonstrated no increase in cell number in any of the hematopoietic tissues. The remaining mice injected with TF1 cells (13 mice) all died of toxicity before any tissues could be cultured. Therefore cells from suspension culture were recovered from the hind bone marrow of only 1 out of 7 mice sacrificed which were injected with factor-dependent cells. Since leukemic cells could not be demonstrated in the remaining 6 animals, their deaths were most likely due to toxicity from their radiation and/ or cyclophosphamide treatment. The presence of factor-producing AML cell lines in hematopoietic tissues was detected in cultures from mice sacrificed 2 weeks post injection as indicated by growth of G418 resistant cells in suspension culture.  These cultures were grown in suspension with G418, an analog of  • neomycin (Table 2, 3). G418 resistant cells were recovered from suspension cultures of tissues from mice previously injected with the growth factor-producing retrovirally-infected cell lines containing the neo gene, Mo7eJ-IL-3 (Table 2) and TF1 J-GM-CSF (Table 3). G418 resistant cells were first observed in the bone marrow of both mice and spleen of 1 of 2 mice sacrificed 2  31  Table 1  Weeks Post Iniection of 10 Cells 7  M O T e Cells  T F 1 Cells  M o u s e Tissue  Week 1  Week 2  Week 1  Week 2  BMHind  0/2  1/2  0/1  0/2  B M Front  0/2  0/2  0/1  0/2  Spleen  0/2  0/2  0/1  0/2  | Blood  0/2  0/2  0/1  0/2  T A B L E 1: M Q 7 e a n d T F 1 cells isolated | n suspension culture f r o m sacrificed mouse tissue after various weeks post i n i e c t i o n . S c i d mice treated w i t h 450 cgy a n d 4-6 m g cyclophosphamide were intravenously injected w i t h 10 cells M 0 7 e o r T F 1 without h u m a n growth factors. T h e mice were sacrificed a n d the mouse tissues c u l t u r e d after various weeks post injection o f the A M L cell lines. Suspension culture conditions as follows: M 0 7 e , M 0 7 e m e d i a +5 n g / m l r h I L - 3 ; T F 1 , R P M I +10% F C S a n d 2 n g / m l r h G M - C S F . C u l t u r e s incubated at 37°C for u p to 6 weeks after sacrifice. C u l t u r e s were pelleted a n d resuspended every week w i t h new media. Positive cultures demonstrated by continuous increase i n cell numbers a n d h i g h cell viability. Fractions indicate the n u m b e r o f mice where cultures showed cell g r o w t h by 6 weeks o f culture over the n u m b e r o f mice analyzed. 7  32  Table 2  Weeks Post Iniection of 10 Mo7e.T-IL-3 Cells 7  M o u s e Tissue  Week 2  Week 4  B M Hind  2/2  1/1  B M Front  2/2  1/1  Spleen  1/2  1/1  Blood  0/2  1/1  T A B L E 2: G 4 1 8 Resistance of cells isolated i n suspension culture f r o m sacrificed S c i d mouse tissues previously injected w i t h 10 cells MQ7e-.TIL-3 after various weeks post iniection. S c i d mice treated w i t h 450 cgy a n d 4-6 m g cyclophosphamide were intravenously injected w i t h 10 cells M 0 7 e - J I L - 3 were analyzed after several weeks post injection o f cells. C u l t u r e conditions for M 0 7 e - J I L - 3 cells f r o m mouse tissues was D M E M +10% F C S 401 w i t h 0.6 m g / m l G 4 1 8 (w/v) i n 10 m l flasks incubated at 37°C. C u l t u r e s were maintained for u p to 6 weeks after sacrifice before d i s c a r d i n g as negative cell g r o w t h . Cultures were pelleted a n d resuspended every week w i t h new m e d i a . Positive cultures demonstrated by continuous increase i n cell numbers a n d h i g h cell viability. Fractions indicate the n u m b e r o f mice where cultures showed cell g r o w t h b y 6 weeks o f culture over the n u m b e r o f mice analyzed. 7  7  33  Table 3  W e e k s Post Iniection o f 10 T F 1 - . T G M - C S F Cells 7  M o u s e Tissue  Weekl  Week 2  Week 4  B M Hind  0/2  2/2  1/1  B M Front  0/2  0/2  1/1  Spleen  0/2  0/2  1/1  Blood  0/2  0/2  0/1  T A B L E 3: G 4 1 8 Resistance of cells isolated i n suspension culture f r o m sacrificed S c i d mouse tissues previously injected w i t h 10 T F 1 - . T G M - C S F cells after various weeks post iniection. S c i d mice treated w i t h 450 cgy a n d 4-6 m g cyclophosphamide were intravenously injected w i t h 10 cells T F 1 - J G M - C S F were analyzed after several weeks post injection o f cells. C u l t u r e conditions for T F 1 - J G M - C S F cells f r o m mouse tissues was R P M I +10% F C S 401 w i t h 0.8 m g / m l G 4 1 8 (w/v) i n 10 m l flasks incubated at 37°C. Cultures were maintained for u p to 6 weeks after sacrifice before d i s c a r d i n g as negative cell g r o w t h . Cultures were pelleted a n d resuspended every week w i t h new m e d i a . Positive cultures demonstrated by continuous increase i n cell numbers a n d h i g h cell viability. Fractions indicate the n u m b e r o f mice where cultures showed cell g r o w t h b y 6 weeks of culture over the n u m b e r of mice analyzed. 7  7  34  weeks post injection of Mo7eJ-IL-3 cells. The peripheral blood yielded no increase in cell number and hence no selection of these cells (Table 2). Cultures from tissues of mice injected with TF1J-GM-CSF cells demonstrated G418 resistance in cells from the hind bone marrow of both mice analyzed at 2 weeks post injection (Table 3).  By 4 weeks post injection, all tissues except blood from the analyzed mouse  demonstrated G418 resistant cells (Table 3). The absence of TF1 J-GM-CSF cells in the tissues of the 2 mice sacrificed at week one post injection may signify that more time is required for these human cells to grow within the mouse tissues for detection via suspension culture. '  3.1.2  -  • >  D N A Analysis of cultured tissues of mice:  Cultured tissue samples from sacrificed Scid mice which increased in cell number were expanded and genomic DNA extracted for Southern blot analysis. DNA from G418 resistant cells were digested with Xbal and analyzed by Southern blot analysis using a neo probe (Figure 3). Digestion with Xbal releases the 4 kb retroviral insert. In Figure 3, dilutionsfrom0% to 100% of DNA from TF1J-GM-CSF cells in DNA from the parental TF-1 cells were compared to DNA from mouse tissues 4 weeks post injection of TF1 J-GM-CSF cells; (hind and front bone marrow and spleen), after an Xbal digest of the DNA and Southern blotting. All samples were hybridized to the neo probe. The sensitivity of the probe varied but it was usually able to detect approximately 4% neo positive DNA. The 2 week and 4 week Mo7eJ-IL-3 mouse cultures displayed similar results to Figure 3 i.e. 100% hybridization of DNA from G418 resistant, cultured cells with the neo probe.  :35  .  F i g u r e 3 ; Southern analysis o f a S c i d mouse treated w i t h 450 cgy a n d 6 m g of cyclophosphamide p r i o r to intravenous t a i l vein iniection o f 10 T F 1 J - G M - C S F cells after 4 weeks post iniection. 7  A) Scid mice were treated with 450 cgy of total body irradiation and 6 mg cyclophosphamide intraperitoneally injected 24 hours before 10 TF1J-GM-CSF cells were intravenously injected into the tail vein. After 4 weeks post injection a Scid mouse was sacrificed by cervical dislocation and the front and hind bone marrow, spleen and peripheral blood tissues cultured in suspension in RPMI and 10% FCS without growth factor. Cultures which divided within 6 weeks of incubation were expanded and the others discarded. Suspension cultures were pelleted after expanding for 2 weeks and DNA purified. DNA was digested with Xbal and run on a 1% agarose gel at 14 Volts overnight. Dilution samples of TF1J-GM-CSF: TF1 were made at 100%, 33%, 11%, 4% and 0% of TF1J-GM-CSF DNA and digested with Xbal and run beside the suspension culture samples to determine the percentage of TF1J-GM-CSF DNA in the samples. A positive control, the neo probe (1 kb in length) was run in the final lane of the gel. Zetaprobe membrane after southern blotting was hybridized with the neo probe. X-ray film was exposed for 14 days in a minus 20°C freezer. Lanes 1-5: Dilutions of TF1J-GM-CSF: TF1 DNA as labeled above. Lanes 6-8: DNA of cultured tissues from the front bone marrow (front BM), hind bone marrow (hind BM) and spleen, 4 weeks post injection of a Scid mouse injected with 10 TF1J-GM-CSF cells. B) Structure of the Tk-neo construct. The retroviral construct which was infected into TF-1 parental cell line to render them GM-CSF factor producing and neomycin resistant. The 5' and 3' Long terminal repeats (LTRs) contain Xbal sites within them to release a fragment 4 kb in length. 7  7  36  37  These experiments demonstrated that the human AML cell lines M07e and TF-1 show cytokine-dependent engraftment in Scid mice similar to that previously observed in nude animals. However, the toxicity of 450 cgy with 4-6 mg of cyclophosphamide resulted in excessive early mortality in the treated animals.  3.2 Demonstration of engraftment i n S c i d mice using A M L cell lines:  As the first regimen was toxic, with 75% of the Scid mice dying before the third week post injection, the protocol was altered to determine whether or not cyclophosphamide should be utilized in conjunction with a lower dose of irradiation. Four sets of 5 mice aged 4 -8 weeks bid, were treated with or without 4 mg of cyclophosphamide and 400 cgy of total body irradiation prior to intravenous injection of 5xl0 M07e or Mo7eJ-IL-3 cells. The results are depicted in 6  Figure 4 and show that by the third week post injection, all 15 mice treated except mice injected with Mo7eJ-JL-3 cells and receiving 400 cgy only had died. The mice receiving M07e cells and 400 cgy with or without cyclophosphamide died less than a week after treatment. It is possible that these, mice died due to infection from microorganisms introduced at the time of M07e cell injection. All 5 of the mice treated with 400 cgy plus cyclophosphamide and M07e-JJL-3 cells had died before their third week post injection. The mice receiving 400 cgy only followed by injection of Mo7eJ-IL-3 cells survived for at least 5 weeks post treatment unless sacrificed earlier for analysis of engraftment of leukemic cells (beginning 4 weeks post injection). One mouse died at 5 weeks post injection. A mouse was then sacrificed for thistimepoint.It is presumed that the previous mouse died due to leukemia as the mouse sacrificed at 5 weeks post injection showed hind limb paralysis and a internal tumour upon sacrifice. At 8 weeks post injection, only 1 mouse  ;  .  38  -  .  : F i g u r e 4 ; Percentage s u r v i v a l of mice receiving 400 cgy w i t h o r without cyclophosphamide p r i o r to intravenous iniection of 5 x l 0 M Q 7 e o r MQ7e.T-IL-3 cells. 6  Four sets of 4-5 mice aged 4-8 weeks old were treated with or without 4 mg of cyclophosphamide i.p. followed by 400 cgy 24 hours prior to i.v. injection of 5xl0 M07e or M07eJ-IL-3 cells. Mice were sacrificed at various weeks post injection of cells unless they expired first. M07e with cyclo + cgy (4 mice)- circle; M07e with cgy only (4 mice)-square; M07eJ-IL-3 with cyclo + cgy (5 mice)- triangle; M07eJ-IL-3 with cgy only (4 mice)- diamond 6  39  % Survival of Mice after Treatment  40  remained.  This remaining mouse was sacrificed and demonstrated a severe arch and slow  movement. It was concluded that the 4 to 8 week old Scid mice could tolerate 400 cgy of total body irradiation. Administration of higher doses of irradiation or cyclophosphamide with the irradiation caused early (before 3 weeks post injection) death of a large number of these mice.  3.2.1  Suspension C u l t u r e :  Twenty percent of the viable cells in the hematopoietic tissues (bone marrow, spleen and peripheral blood) derived from sacrificed mice were suspended in culture and the growth monitored for 6 weeks. G418 resistant cells from the Mo7eJ-IL-3 injected mice were analyzed as before in the previous protocol (Table 4). Table 4 shows the cultured samples of mice injected with 5xl0 cells of Mo7eJ-IL-3. The cultures indicated that engraftment of these cells in the bone 6  marrow of the mouse treated with 400 cgy and 4 mg of cyclophosphamide can be detected by 2 weeks post injection. In mice treated with 400 cgy, dissemination to all culturedtissueshad occurred by 4,weeks post injection. However, the cultures oftissuesfrom the mouse treated with 400 cgy and sacrificed at 8 weeks demonstrated growth in the bone marrowtissuesonly. The soft tumour from the mouse sacrificed at 5 weeks, which was situated medially to both kidneys, was 0.8 cm in width and height with only 0.2 cm in depth.  3.2.2  ,  D N A Analysis:  Table 5 shows the results of Southern analysis of DNA directly isolated from the tissues of 4 mice injected with Mo7eJ-IL-3 cells and sacrificed at varioustimespost injection. Using the EcoRl HERV-H fragment as a probe to determine the percentage of human AML cell DNA  41  Table 4  Weeks Post Iniection o f 10 M Q 7 e J I L - 3 Cells 7  Mouse Tissue  Week 2 400 cgy cyclo  Week 4 400 cgy only  Week 5 400 cgy only  Week 8 400 cgy only  B M Hind  1/1  1/1  B M Front  1/1  1/1  1/1 1/1  1/1 1/1  Spleen  0/1  1/1  1/1  0/1  Blood  0/1  1/1  1/1  0/1  Tumour  -  -  1/1  -  +  T A B L E 4: G 4 1 8 Resistance of cells isolated i n suspension culture f r o m sacrificed S c i d mouse tissues previously injected w i t h 10 cells M o 7 e J - I L - 3 after various weeks post injection. S c i d mice treated w i t h 400 cgy w i t h o r without 4 m g o f cyclophosphamide were intravenously injected w i t h 10 cells M o 7 e J - I L - 3 were analyzed after several weeks post injection o f cells. C u l t u r e conditions for M o 7 e J - I L - 3 cells f r o m mouse tissues was D M E M + 1 0 % F C S 401 w i t h 0.6 m g / m l G418 (w/v) i n 10 m l flasks incubated at 37°C. Cultures were maintained for u p to 6 weeks after sacrifice before discarding as negative cell growth. C u l t u r e s were pelleted a n d resuspended every week w i t h new m e d i a . Positive cultures demonstrated b y continuous increase i n cell numbers a n d h i g h cell viability. Fractions indicate the n u m b e r o f mice where cultures showed cell g r o w t h by 6 weeks Of culture over the n u m b e r o f mice analyzed. 7  7  42  TABLE 5  M o u s e Tissues w i t h H u m a n D N A iri % Treatment  Weeks Post Injection  BM Hind  BM Front  Spleen  Peripheral Blood  Tumour  H e a l t h of Mouse  400 cgy + 4 u g cyclophosphamide  2  <50  10  -  -  NA  Sick  400 cgy only  4  100  100  100  -100  NA  Sick  400 cgy only  5  100  100  100  -100  100  Sick  400 cgy only  8  100  10  <1  <1  NA  Slow Moving  T A B L E 5: Southern analysis s u m m a r y o f S c i d mouse tissues treated w i t h o r without 4 u g o f cyclophosphamide a n d 400 cgy total body i r r a d i a t i o n . S c i d mice were treated w i t h 400 cgy w i t h o r without 4 u g cyclophosphamide p r i o r to intravenous injection of 5x10* cells o f M 0 7 e o r M 0 7 e - J I L - 3 . A f t e r several weeks post injection o f the cell lines, mice were sacrificed a n d 8 0 % o f the cells f r o m the tissues were analyzed for h u m a n specific D N A using a H u m a n Endogenous R e t r o v i r u s l i k e element ( H E R V - H ) , an E c o R l fragment o f approximately 1 k b w h i c h is present i n the h u m a n genome i n - 1000 copies ( M a g e r & H e n t h o r n , 1984; M a g e r & F r e e m a n , 1987). Sensitivity is based o n the percentage o f h u m a n D N A w h i c h can be detected i n serial dilutions o f h u m a n i n mouse D N A r u n o n the same gel as the mouse tissue D N A samples. H e a l t h o f mouse: S i c k - mouse shows h i n d l i m b paralysis, weight loss, shortness o f breath a n d lethargy; S l o w M o v i n g - mouse displays lethargy otherwise n o r m a l . M o 7 e J - I L - 3 mice only demonstrated engraftment as M 0 7 e mice i n this regimen died i n the first week post injection.  43  within the tissues allowed a more sensitive measure of human-derived DNA in comparison with the neo probe. The HERV-H elements have been shown to be present in the human genome in approximately 1000 copies while only one copy of the neo marker was present in the genome of the retrovirally marked AML cells. HERV-H probe could detect as little as 0.1% human DNA in human: mouse mixtures while the neo probe sensitivity was only at 5% and only useful in detecting cells marked with this gene. Table 5 demonstrates that human DNA was found in all hematopoietic tissues at 4 weeks and 5 weeks post injection of AML cells while engraftment was seen only in the marrow of the mouse sacrificed at 8 weeks. These data confirm the results of the suspension cultures of the same tissues where G418 resistant cells were detected (Table 5). Figure 5 depicts Southern blotting of EcoRl digests of the tissues of the Scid mouse sacrificed 5 weeks post injection with M07eJ-IL-3 cells along with human/mouse DNA dilutions from 100% to 0%. This blot was hybridized using the HERV-H element as a probe (RT10EA). All tissues probably had 100% hybridization to the probe although the signal is weaker in lanes with DNA from front bone marrow and blood that from other tissues due to underloading of DNA as detected by the ethidium bromide stained gel (not shown). The human: mouse DNA dilution series indicates that the sensitivity of the probe for detection of human DNA was approximately 1% which was more sensitive than the neo probe. In summary, these experiments studying the engraftment of human AML cell lines in Scid mice determined the most aggressive immunosuppressive regimen that the animals would tolerate and demonstrated the feasibility of engrafting human leukemic cells in Scid mice. The next series of experiments examined the ability of AML cells directly isolatedfrompatients to grow in these mice treated with 400 cgy.  44  Figure iv Southern hybridization of a Scid mouse receiving 400 cgy prior to intravenous iniection of 5 x l 0 MQ7e.T-IL-3 cells after 5 weeks post iniection of cells. 6  A) Scid mice received 400 cgy prior to intravenous injection of 5xl0 M07eJ-IL-3 cells via the tail vein. After 5 weeks post injection of cells, one Scid mouse was sacrificed by cervical dislocation artd the tissues samples collected from the front and hind bone marrow, spleen, peripheral blood and any tumours. DNA was extracted from the pelleted tissues, cut with EcoRl and electrophoresed on a 0.8% agarose gel with human (M07e) /mouse (MEL) dilutions at 100, 25, 10, 5, 1 and 0% human DNA along with 25 pg of positive control probe (RT10EA). The southern blot was hybridized with a human specific HERV-H probe called RT10EA. X-Ray film was exposed to the hybridized Zetaprobe membrane for 10 days in a -20°C freezer.,. B) Diagram of the human endogenous retroviral (HERV-H ) element. The full length form of the HERV-H element which is present in roughly 1000 copies in every human haploid genome. The RT10EA EcoRl fragment is 1 kb in length and is contained within the full length copy of the HERV-H element between the flanking long terminal repeats represented by the dark arrows at each end of the element. A limited restriction map of the element is also shown. St - Stul; E - EcoRl; B-Bgl 11. 6  45  DILUTIONS  SCID5WK M07EJIL3 400 Rads only  ECORI DIGEST RT10EA PROBE  46  3.3 Human Patient Samples in Scid mice treated with 400 cgy of total body irradiation 3.3.1  DNA Analysis A total of nine different patient samples were tested for their engraftment in the  immunocompromised Scid mice. 0.5 to 1.0 xlO cells were injected i.v. into irradiated mice 7  which then received i.p. injections of hIL-3 and hSCF every two days. Table 6 shows the FAB type and karyotype of the leukemic blasts from the 9 patients and the ability of these cells to grow in mice. Cells of a variety of FAB types and different cytogenetic abnormalities engrafted with variable success.  Of the nine patient samples, 5 of them demonstrated more than 10%  engraftment levels as determined by the human specific HERV-H probe (Table 6 and 7). Table 7 summarizes all the Southern analysis performed on the mice where patient samples successfully engrafted. Engraftment was shown by 4 weeks post injection in 3 of the positive patient samples and by 8 weeks in the other 2 positive patient samples. Those that did show engraftment in the bone marrow, spleen and blood were mice that displayed symptoms of human AML such as hind limb paralysis, weakness, weight loss and laboured breathing. One of the positive patients (patient #8) demonstrated 100% engraftment in bone marrow tissues by 4 weeks post injection while the spleen and bloodtissuehad 10% engraftment only. By 6 weeks post injection these lattertissuesfrom the same patient had risen to 100% engraftment levels by Southern analysis using the human specific probe. At 8 weeks post injection of patient cells, the Scid-hu mouse demonstrated many internal tumours, in the right and left kidneys towards the midline, and a subdermal rear tumour not more than 0.5 cm found at the site of 3  injection. Thus, these Scid-hu mice manifested a similar pattern of dissemination in thetissuesas human AML.  47  Patient #  FAB Type. ___  1  M5a  2 • 3  AML-NOS M4  4  CML-myeloid Blast Crisis  5 6  M4 M4 or M5a  7 8  M5b M5a  9  Ml  Cytogenetics  46, X Y ; 48, X Y , +8, +8 Unknown 47, X X , +13; 46, X X 46-7, X Y , der(5), t(5,?)(pl3,?), t(9;22)(q34;qll) . del(ll)(q23),+der(22), t(9;22)(q34;qll) 47, X X , +8 46, X Y 46, X Y ? dell6q22 46, X Y 47, X X , -7, +8, +8, t(9;ll)(p22;q23) 49, X X , +4, +8, +8, t(9;ll)(p22;q23) 46, X Y  Factor Responsiveness  Growth in Mice  19X  negative  6X 10X  negative positive  6X  negative.  24X 5X  positive positive  not done 7X  negative positive  14X  positive  >  Table 6; A M L patient background, their A M L French-American-British classification, cytogenetics, factor responsiveness to IL-3 and SCF above control values and their engraftment in Scid-hu mice injected with the patient samples. Patients diagnosed in the Vancouver Health Sciences Hospital at the Cell Separator Unit undergoing leukapheresis, samples were obtained from consenting adults after approval of the Clinical Screening Committee for Research Involving Humans of the University of British Columbia. F A B and cytogenetic analysis was done at the Vancouver Health Sciences Hospital. Factor responsiveness of patients cells to IL-3 and SCF is expressed as fold increase in H - thymidine incorporation as compared to control samples without growth factor stimulation. 3  48  % Human DNA in Murine Tissues ffind • Bone Marrow  Front Bone Marrow  Patient #  Weeks Post Injection  3  4  0  0  0  8  100  100  0  14  100  100  5  6  S  9  Spleen  Blood  Marrow Morphology FISH Data Analysis (AML cells)  Tumour  Accuracy Level  Health of Mouse  0  n/a  0.1%  normal  0  n/a  1%  enlarged spleen  not  1  0  n/a  0.1%  enlarged spleen  done  18  100  100  0  100  n/a  0.1%  4  -10  -10  0  0  n/a  10%  normal  n/a  1%  sick  -50%  positive Hind B M  ' sick  >50% • not done  6  -40  -10  <1  <1  12  0  0  0  0  100  0.1%  normal  <10%  not done  14  0  0  0  0  n/a  0.1%  normal  <10%  not done  3  100  -10  -10  <10  n/a  10%  enlarged spleen  10  0  0  0  0  n/a  0.1%  normal  14  5  1  0  0  n/a  0.1%  normal  4  -100  -100  5  -10  6A  100  6B  not <10%  done  -10  -10  n/a  1%  sick  >50%  not done  <1  10  100  1%  sick  -10%  positive  100  100  100  n/a  1%  sick  -100 %  positive  100  100  100  100  n/a  1%  sick  8  100  -100  30  100  100,10 0  1%  sick.  4 with gf  50  1  0  0  n/a  1%  weak  4 no gf  0  0  0  0  n/a  1%  normal  8 with gf  100  100  0  5  n/a  10%  sick  8 no gf  90  50  0  0  n/a  10%  normal  >50%  12 with gf  100  100  100  100  100  10%  sick  >50%  positive >90%  not done  0%  not done  Table 7- Summary of the A M L patient samples engrafting in the Scid mouse tissues from DNA. FISH and morphological analysis Five of 9 patient samples that had been intravenously injected into Scid mice after 400 cgy engrafted in the mice as demonstrated through DNA analysis of bone marrow, spleen, blood and any tumours as well as morphology of cells from slides and FISH analysis. The percentage of human DNA from Southern hybridization analysis with a human specific HERV-H probe was recorded from the various murine tissues by comparison with human: mouse cell dilutions. The sensitivity of the individual blots, determined by the lowest percentage of human DNA detectable from the human: mouse dilutions, was recorded under "level of accuracy." Health of the mouse was reported at time of sacrifice. Visual inspection of internal organs was also noted in this column. Morphology data from May-Grunwald Giemsa staining procedures was summarized here according to the % of AML blasts observed from the bone marrow. Any FISH analysis of the human patients was recorded either as positive, whereby the chromosome 8 centromeric probe hybridized to methylcellulose colonies, or as not done, n/a - not applicable; ~ -approximately; < - less than; > - greater than. gf= q.2 d. injections i.p. of hIL-3 and hSCF  49  Figure 6 depicts an autoradiograph of the southern hybridization of the patients #5 and #8 after 6 and 5 weeks post injection of primary human AML cells into Scid mice respectively. The autoradiograph clearly detected 1% of human DNA through the human/ mouse dilution series. The tissue samples of patient #5 depicted roughly 40% human DNA in the hind bone marrow while the front bone marrow was less than 10% human DNA. Spleen and peripheral blood samples indicated no hybridization as no signal as great as the 1% human DNA dilution could be observed. - Mouse tissue samples from a Scid-hu mouse injected, with patient #8 cells, demonstrated engraftment in the hind bone marrow (-40%), peripheral blood (5%) and tumour (100%) whereas the spleen did not show hybridization at all. The front bone marrow sample which had not properly been solubilized before electrophoresis, could not be analyzed. The peripheral blood sample shows about 5% engraftment in comparison with the human DNA dilutions, this sample was severely underloaded and its actual engraftment level is probably closer to 100%.  3.3.2 Morphology of cells from mouse tissues Human AML cells were detected on May-Grunwald-Giemsa stained slides from mouse tissues harvested from Scid mice that were very sick and/ or demonstrated also a high percentage of human DNA in the various tissues (see Table 6). At 18 weeks post injection of patient #3 cells into a Scid mouse, morphological analysis revealed 80-90% of AML blasts in the bone marrow and peripheral blood tissues. Scid-hu mouse of patient #5 demonstrated after 6 weeks post injection roughly 10% AML blasts in peripheral blood, 50% in bone marrow and 20% in the spleen. At .12 weeks post injection, morphology analysis indicated approximately 30% in marrow  50  Figure 6_; Southern hybridization of E c o R l digested D N A from patient #5 and #8 in sublethallv irradiated Scid mice using a human specific H E R V - H probe.  A) Scid mice irradiated with 400 cgy of total body irradiation were injected intravenously via tail vein with 10 primary human AML cells from the peripheral blood of patients #5 and #8. After 6 weeks post injection (patient #5) and 5 weeks post injection (patient #8), one Scid mouse from these patients was sacrificed and 80% of the tissue samples from the hind and front bone marrow, spleen and peripheral blood as well as any tumours were pelleted for DNA analysis. DNA was purified by standard protocols and 15 ug of DNA from each tissue was EcoRl digested. A 0.8% agarose gel was used to electrophorese these samples along with EcoRl digested human/mouse dilutions of 100-0% human DNA and a positive probe control at 14 Volts overnight. The Zetaprobe membrane with the RT10EA probe was hybridized at 60°C in a shaker bath for 6-8 hours and washed in 2XSSC twice. X-ray film was exposed to the Zetaprobe membrane for 14 days. Lanes 1-4 labeled 100, 10, 1 and 0: Dilutions of human (M07e cell-a human AML cell line) and mouse DNA (MEL cell-murine erythroleukemia cell line) were made at 100%, 10%, 1% and 0% human DNA. Lanes 5-9 labeled H, F, S and B: Patient #5 (FAB-M4)-Tissue sample DNA of a Scid mouse after 6 weeks post injection of 10 primary human AML cells from the hind bone marrow (H), front bone marrow (F), Spleen (S) and peripheral blood (B). Lanes 9-13 labeled as H, F, S, B and T: Patient #8 (FAB-M5a)- Tissue sample DNA of a Scid mouse after 5 weeks post injection of 10 primary human AML cells from the hind bone marrow(H), front bone marrow (F), spleen (S), peripheral blood (B) and tumour (T). Lane 14 +ve: 50 pg of RT 10EA probe DNA (1 kb in length). B) Diagram of the HERV-H element represented in full length with approximately 1000 copies in the human haploid genome . The RT10EA EcoRl fragment lies in the last third of the HERVH element shown with the Long Terminal Repeats (LTR's) as black arrows at either end and a limited restriction map of the element. E-EcoRl, St-Stul and B-Bgl 11. 8  8  8  51  52  and spleen. The mouse at 14 weeks post injection displayed less than 10% in all tissues. In the 10 and 14 week post injection Scid-hu micefrompatient #6, morphology of the slides were good however only less than 10% of AML blasts were observed in all tissues. Morphology analysis of Scid-hu mice with patient #7 cells showed no AML blasts at either time point in any tissues. Scid-hu mice injected with patient #8 cells demonstrated good morphology and staining with a high proportion of AML blasts in all slides. At 5 weeks post injection, spleen and tumour morphology indicated 100% AML blasts, while bone marrow. and peripheral blood samples demonstrated 10% and 30% respectively. At 6 weeks post injection, bone marrow and peripheral blood demonstrated 100% AML blasts while the spleen indicated roughly 30% (Table 7, Figure 7). Figure 7 reveals the bone marrow cells from Scid mice injected with AML patient #8 at 5 and 6 weeks post injection by May-Grunwald Giemsa stain. Differences in the morphology of the AML cells in comparison to the murine neutrophil was apparent in nuclear and cellular size, and high nuclear to cytoplasmic ratio of the AML blasts; Tissues were taken directlyfromthe hind bone marrow of the sacrificed mouse and spread onto the slide without dilution to obtain a representative picture of the marrow morphology. At 8 weeks post injection of patient #8 cells, almost 100% of human AML cells were revealed in every tissue. Tumour slides of the meninges and subdermal tumour as well as the left and right kidney tumours had 100 and 80% human AML. blasts respectively. Hind bone marrow and front bone marrow had roughly 90% of AML blasts present while the spleen only half of the cells were human AML cells.  53  Figure 7: Mav-Grunwald Giemsa staining of a Scid mouse injected with 10 primary human patient #8 cells after 5 and 6 weeks post iniection of cells. A) Photograph of the Scid-hu patient #8 bone marrow after 5 weeks post injection of 10 primary human A M L cells after staining by May-Grunwald Giemsa protocol. The arrows indicate the A M L blasts amidst the murine cells in the bone marrow. The mouse neutrophil is clearly recognized by its donut-shaped nucleus and small size. Morphological scoring on this slide was approximately 10% human A M L blasts. B) Photograph of the Scid-hu patient #8 bone marrow after 6 weeks post injection of cells. The morphological scoring of this slide was determined as 100% human A M L blasts within the bone marrow of this Scid-hu mouse as no mouse neutrophils or mouse cells were observed. 8  54  55  Patient #9 Scid-hu mice who were treated with or without growth factor, demonstrated with growth factor injected at 4 weeks post injection, roughly 10% of AML blasts in spleen. Bone marrow and peripheral blood were unable to be scored due to poor slide morphology. Without growth factor, the mouse tissues showed only normal mouse cells. At 8 weeks post injection,  peripheral blood slides showed 30% AML blasts with growth factor and 0% without growth factor injected. Bone marrow morphology was limited by number of intact cells in the both mice with and without growth factor. Spleen morphology was scored as approximately 20% in the growth factor injected mouse. At 12 weeks post injection there was only one mouse injected with patient cells and it was given growth factors. Morphology of the cranial"tumour demonstrated 100% of AML blasts while bone marrow, spleen and peripheral blood had 50%, 50% and 60% respectively.  3.3.3  FISH  Colonies and clusters of cells growing in methylcellulose assay of cells from tissues from 2 mice injected with cells from patient #8 and one mouse injected with cells from patient #5 were plucked and pooled on glass microscope slides for FISH analysis. Cells from these mice were chosen for analysis because they had shown 100% engraftment with human cells and the patient AML. samples had detectable cytogenetic abnormalities (Table 6).  The two patient samples  selected for analysis had tetrasomy (patient #8) and' trisomy (patient #5) for chromosome 8 respectively. The results of the hybridization of slides from mice injected with these AML cells  56  Figure 8^ Fluorescence in situ hybridization of normal human and mouse cell control samples using a human centromeric chromosome 8 probe. Slides of normal human (46 XX) and mouse (MEL) cells were cytospun onto slides and hybridized following FISH protocols (see materials and methods). Slides were observed on a photomicroscope at 40X and the signals counted for 50 cells. The total number of signals observed for each number of possible signals was divided by the total number of cells scored and multiplied by 100.  57  % of Signals in Total Cells Scored  Figure % Fluorescence in situ hybridization using a centromeric human chromosome 8 probe on a Scid-hu patient #8 mouse at 6 weeks post iniection. Hind and front bone marrow, spleen and blood tissues were cultured in methylcellulose directly after sacrificing a Scid-hu patient #8 mouse after 6 weeks post injection. Methylcellulose colonies were plucked from 2-3 week old plates, the cells lysed in hypotonic KC1 solution and put onto 6 or 10 welled slides. Hybridization of the slides were as described in materials and methods. Nuclei were observed at 40X on a photomicroscope, 25 random cells per well counted for number of signals and the number of cells with 0-5 signals were scored as a percentage of the total number of cells counted.  59  % of Signals in Total Cells Scored co O a •  IE C  3  a oo  o  c m  H  co c/> c m CO  TI  —*  o  3 <-+•  rjj  T|  Jj  co  )  H  TJ >  D  m  H  =8=  00  3 o o  CL  77 C/)  60  from patient #8 with a highly repetitive centromere probe for human chromosome 8 and the control slide results are presented in Figures 8 and 9. Control slides were analyzed by counting 25 random cells per well for the number of visible signals which were hybridized to each nucleus for normal mouse slides (negative control) and normal human slides (positive control). The percentage of signals for the normal human slide was approximately 90% for 2 signals indicative of a normal human when hybridizing with a human centromeric 8 probe. Normal mouse samples demonstrated 100% of 0 positive signals. FISH analysis of the 6 weeks post injection of patient #8 Scid-hu mouse demonstrated that 4 positive signals were observed in majority of cells in every mouse tissue. The bone marrow shows approximately 95% of tetrasomy in the tissues. Spleen tissue demonstrates only 80% of 4 positive signals while the peripheral blood samples demonstrate roughly 90% of the time indicating 4 positive signals. Three signals were also picked up in a thesetissuesbut that may be an inherent fault in the protocol were a signal is present but can not be completely discerned by the eye because it could be on the opposite side of the nucleus. The three dimensional nature of the protocol may limit the exact number of signals which should be seen. The other possibility is that the slides may have more background (dust and dirt) that may attract the hybridization of the probe. These FISH slides, however, were very clean and had very little background. Figure 10 depicts FISH photographs of methylcellulose colonies from the 6 week Scid-hu mouse treated with patient #8 primary AML cells probed with Digoxigenin-labeled centromere probe for human chromosome 8 detected with FTTC-labeled anti-digoxigenin and propidium iodide counterstain. The control female photo A, demonstrates in interphase and metaphase only 2 positive signals hybridizing to the two chromosome 8's. The hind bone marrow clearly defined  61  4 positive probes in all cells from the slides and in this photo. The bright red halos around these interphase nuclei are due to excess propidium iodide. Front bone marrow in metaphase and interphase also demonstrates 4 positive probes and in the blood sample too 4 positive probes were demonstrated indicating that the extra two chromosome 8's were detected in every tissue. In Table 7, the results of both of the FISH experiments revealed a correlation with the Southern analysis in both patients. However, FISH analysis of samples from mice injected with cells from patient #5 showed that the majority of the colonies in all of the wells, did not hybridize with the FISH probe. Thus, the frequency of leukemic cells detected in this case where the malignant cells were trisomic for chromosome 8 was lower than that found with the Southern analysis. Nevertheless, cells with 3 chromosomes 8 were detected in hind marrow preparations from mice injected with this patient sample. Although each FISH experiment used the same probe, the slides from mice injected with cells from patient #5 were older and showed higher background nonspecific labeling with probe perhaps explaining the less successful results in this case.  62  Figure 10- Photographs of FISH analysis on methylcellulose cultures of Scid-hu patient #8 at 6 weeks post iniection. Methylcellulose pooled colonies from 2-3 week old cultures were fixed to 6 or 10 welled slides. Hybridization of the slides were as described in materials and methods. Slides were hybridized with human chromosome 8 an alpha satellite centromeric DNA probe labeled with AntiDigoxigenin fluorescein Fab fragments (Boehringer Mannheim) and rabbit anti-sheep-FITC antibody (Dimension Lab Inc.). Patient #8 cytogenetics showed tetrasomy for chromosome 8 (47, XX, -7,+8,+8, t(9;ll)(p22;q23) and 49, XX, +4, +8, +8, t(9;ll)(p22;q23). Nuclei were observed at 40X on a photomicroscope. Photographs were taken on Axioplan Zeiss, Germany, using Fugichrome 400 RH135 film at various magnifications and exposures. A) Normal human control - interphase and metaphase, B) Hind bone marrow, C) Peripheral blood, D) Front bone marrow -metaphase, and E) Front bone marrow- interphase.  63  A  64  4.  DISCUSSION  Scid mice tolerated only 400 cgy of total body irradiation without subsequent injection of cyclophosphamide. A higher dosage of irradiation with cyclophosphamide, resulted in premature death of the mice from toxicity. Engraftment in this murine model has been demonstrated using the human AML cell lines M07eJ-LL-3 and TF1J-GM-CSF. The M07e and TF1 cell lines were factor-dependent and, in general, did not engraft in these mice as no additional human factors were injected into the mice.  DNA analysis of  mouse tissue cultures suggested that the  engraftment of the M07eJ-LL-3 cells progressed faster than the TF1J-GM-CSF cells as tissue dissemination appeared more wide spread in the M07eJ-LL-3 mice. The optimized protocol of 400 cgy was sufficient enough to allow 5 out of 9 AML patient samples to engraft into bone marrow, 3/5 by 4 weeks post injection and the remaining 2/5 Scid-hu mice by 8 weeks post injection. The dissemination in thetissueswas demonstrated by DNA and morphology analysis where the varioustimepoints indicated that engraftment was first detected in the bone marrow, then the spleen and finally to peripheral blood and or tumours. Some variability in extent of engraftment of a single patient sample was seen between different mice. In some cases the concentration of cells on intravenous injection into the tail vein of the mice may not have been uniform which could cause one mouse out of a patient group to have more cells than another. However, it is likely that other factors also influenced this variability. The Scid mice, while immunodeficient in B and T cells, still have a functional myeloid lineage and NK cell activity. These immune cells are still capable of fighting the foreign human cells which have been injected, perhaps explaining why some of the AML cells failed to engraft or engrafted inconsistently.  Scid-hu mouse injected with patient #6 cells demonstrated this  65  phenomenon with DNA and morphology analysis (Table 6). At 3 weeks post injection, hind bone marrow is demonstrated to be roughly 100% while the 10 and 14 week time period displayed very little engraftment in any tissues. As well, the M07eJ-IL-3 mouse at 8 weeks post injection treated with 400 cgy only demonstrated engraftment only in the bone marrow and not in the spleen or peripheral blood as had been observed in previous time points (Table 4). The ScidTNOD mice promises to be a more reliable and reproducible model for AML patient cell engraftment (in house comparison with Scid, Scid/NOD, Rag2, and Beige/nu/xid mice; Blair et al, 1996; Ailles et al, 1996). However, the different success of engraftment with the different patient samples suggests that inherent heterogeneityfromone patient to the next may also play a role in the success of these experiments. The growth factor supplements were provided every other day, while other labs have achieved in vivo support of human growth factors by using a minipump regulating the concentration of growth factors to the AML cells (Kamel-Reid & Dick, 1988) This would be beneficial so as to reduce the amount of injury and stress to the animals. However, recent studies suggested that the use of growth factors is not required for the in vivo growth of primary human cells in Scid mice ( Vormoor et al, 1994; Kamel-Reid & Dick, 1991 ; Lapidot et al, 1994). This was suggested by Scid-hu patient #9 as there was no engraftment at 4 weeks post injection without cytokines however by 8 weeks, engraftment was visible through DNA analysis in the bone marrow. While engraftment will occur for some patients without the addition of human growth factors this may not be true for all patient samples, again due to patient heterogeneity. Patient sample #9 datafromthe Scid-hu mice suggest that the cells engrafted more quickly with exogenous cytokine treatment (Table 6).  66  The DNA analysis using the HERV-H probe was quite sensitive and could demonstrate 0.1% engraftment levels of human cells in a mouse tissue sample. The fluorescence in situ hybridization analysis also proved to be very sensitive for detection of malignant cells. Probing with alpha satellite DNA for human chromosome 8 has been demonstrated by other labs (Jenkins et al, 1992; De Vita et al, 1993; Pagliaro & Stanley, 1993) to be an excellent way of detecting trisomy 8 (Kibbelaar et al, 1993; Chen et al, 1992). This Scid-hu mouse model of AML demonstrates a viable means of isolating, characterizing and understanding the AML stem cell. In conjunction with techniques such as retroviral marking biological characterization of these cells and their normal counterparts may be possible in mice. The in vivo model will be useful for detection of minimal residual disease of these patients as well as new testing of drug therapies for AML patients. Current research involves the Scid-Nod mice which have shown an increased efficiency of human AML cell engraftment (Ailles et al, 1996; Blair et al, 1996). The possibility of characterizing the AML stem cell for each patient through limiting dilution assays, the self renewal capacity through secondary transplantation and novel therapeutic strategies are possible with this in vivo Scid model of human AML.  67  5. REFERENCES  Abbas, A., K., Lichtman, A. H. and Pober, J. S. eds. In cellular and molecular immunology. W. B. Saunders Company, USA 1991. Ailles, L.E., Gerhard, B. and Hogge, D.E. 1996. Retroviral marking and engraftment of human AML cells in NOD/Scid mice. Abstract submitted. Anderson, D.M., Lyman, S.D., Baird, A., Wignall, J.M., Eisenman, J., Rauch, C , March, C.J., Boswell, H.S., Gimpel, S.D., Cosman, D. and Williams, D.E. 1990. Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63: 235-243. Avanzi, G . C , Lista, P., Giovinazzo, B., Miniero, R., Saglio, G., Benetton, G., Coda, R., Cattoretti, G. and Pegoraro, L. 1987. Selective growth response to IL-3 of a human leukaemic cell line with megakaryoblastic features. Br. J. Haematol. 69: 359-366. Blair, A., Ailles, L.E., Hogge, D.E. and Sutherland, H.J. 1996. Phenotype of primitive AML cells which engraft NOD/Scid mice. Abstract submitted. Bosma, G.C, Custer, R.P., and Bosma, M.J. 1983. A severe combined immunodeficiency mutation in the mouse. Nature 301: 527-530. Bosma, M.J. and Carroll, A.M. 1991. The scid mouse mutant: definition, characterization, and potential uses. Annu. Rev. Immunol. 9: 323-350. Brandt, J.E., Bhalla, K. and Hoffman, R. 1994. Effects of Interleukin-3 and c-kit ligand on the survival of various classes of human Hematopoietic progenitor cells. Blood 83: 15071514. Broxmeyer, H.E., Maze, R., Miyazawa, K., Carow, C , Hendrie, P.C, Cooper, S., Hangoc, G., Vadhan-Raj, S. and Lu, L. 1991. The kit receptor and its ligand, steel factor, as regulators of hematopoiesis. Cancer Cells 3: 480-487. Cesano, A., Hoxie, J.A., Lange, B., Nowell, P.C, Bishop, J. and Santoli, D. 1992. The severe combined immunodeficient (SCID) mouse as a model for human myeloid leukemias. Oncogene 7: 827-836. Chabot, B., Stephenson, D.A., Chapman, V.M., Besmer, P. and Bernstein, A. 1988. The protooncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335: 88-89.  68  Chelstrom, L.M., Gunther, R., Simon, J., Raimondi, S.C., Krance, R., Crist, W.M. and Uckun, F.M. 1994. Childhood acute myeloid leukemia in mice with severe combined immunodeficiency. Blood 84: 20-26. Chen, Z., Morgan, R., Berger, CS. and Sandberg, A.A. 1992. Application of fluorescence in situ hybridization in hematological disorders. Cancer Genet. Cytogenet. 63: 62-69. DeVita, V.T, Hellmamn, S and Rosenberg, S.A eds. In Important Advances in Oncology. Fluorescence in situ hybridization in cancer diagnosis. M. MiLeBeau pp. 29-45. Philadelphia. 1993. Dick, J.E. 1991. Immune-deficient mice as models of normal and leukemic human hematopoiesis. Cancer Cells 3: 39-48. Dick, J.E., Kamel-Reid, S., Murdoch, B. and Doedens, M. 1991. Gene transfer into normal human Hematopoietic cells using in vitro and in vivo assays. Blood 78: 624-634. Erickson, P.F., Robinson, M., Owens, G. and Drabkin, H.A. 1994. The ETO portion of acute myeloid leukemia t(8;21) fusion transcript encodes a highly evolutionarily conserved, putative transcription factor. Cancer Res. 54: 1782-1786. Fearon, E.R., Burke, P.J., Schiffer, C.A., Zehnbauer, B.A. and Vogelstein, B. 1986. Differentiation of leukemia cells to polymorphonuclear leukocytes in patients with acute nonlymphocytic leukemia. N. Eng. J. Med. 315: 15-24. Feinberg, A. P. and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specificity activity. Anal. Biochem. 132: 6-13. Fialkow, P.J., Singer, J.W., Adamson, J.W., Vaidya, K., Dow, L. W. and Moehr, J.W. 1981. Acute nonlymphocytic leukemia: heterogeneity of stem cell origin. Blood 57: 10681073. Flanagan, J.G. and Leder, P. 1990. The kit ligand: a cell surface molecule altered in steel mutant fibroblasts. Cell 63: 185-194. Fulop, G.M. and Phillips, R.A. 1989. Use of scid mice to identify and quantitate lymphoidrestricted stem cells in long-term bone marrow cultures. Blood 74: 1537-1544. Gluck, U., Zipori, D., Wetzler, M., Berrebi, A., Shaklai, M., Drezen, O., Zaizov, R., Luria, D., Marcelle, C , Stark, B. and Umiel, T. 1989. Longterm proliferation of human leukemia cells induced by mouse stroma. Exp Hematol. 17: 398-402. Griffin, J.D. and Lowenberg, B. 1986. Clonogenic cells in acute myeloblastic leukemia. Blood 68: 1185-1195.  69  Hallek, M., Ando, K., Eder, M., Slattery, K., Ajchenbaum-cybalista, F. and Griffin, J.D. 1994. Signal transduction of steel factor and granulocyte macrophage colony-stimulating factor: differential regulation of transcription factor and GI cyclin gene expression, and of proliferation in the human factor-dependent cell line M07. Leukemia 8: 740-748. Hogge, D.E. 1994. Cytogenetics and oncogenes in leukemia. Curr. Opinions Oncol. 6: 3-13. Horie, M. and Broxmerger, H.E. 1993. Involvement of immediate-early gene expression in the synergistic effects of steel factor in combination with granulocyte-macrophage colonystimulating factor or interleukin -3 on proliferation of a human factor- dependent cell line. J.Biol Chem. 268: 968-973. Hu, Z., M, W., Uphoff, C.C., Quentmeier, H. and Drexler, H.G. 1994. c-kit expression in human megakaryoblastic leukemia cell lines. Blood 83: 2133-2144. Huang, E., Nocka, K., Beier, D.R., Chu, T., Buck, J., Lahm, H., Wellner, D., Leder, P. and Besmer, P. 1990. The hemopoietic growth factor KL is encoded by the SI locus and is the ligand of the c-kit receptor, the gene product of the W locus. Cell 63: 225-233. Jenkins, R.B., Le Beau, M.M., Kraker, W.J., Borell, T.J., Stalboerger, P.G., Davis, E.M., Penland, L., Fernald, A., Espinosa 111, R., Schaid, D.J., Noel, P. and Dewald, G.W. 1992. Fluorescence in situ hybridization: a sensitive method for trisomy 8 detection in bone marrow specimens. Blood 79: 3307-3315. Kamel-Reid, S. and Dick, J.E. 1988. Engraftment of immune-deficient mice with human hematopoietic stem cells. Science 242: 1706-1709. Kamel-Reid, S., Letarte, M., Sirard, C , Doedens, M., Grunberger, T., Fulop, G., Freedman, M.H., Phillips, R.A. and Dick, J.E. 1989. A model of human acute lymphoblastic leukemia in immune-deficient SCID mice. Science 246: 1597-1600. Koike, K., Stanley, E.R., Ihle, J.N. and Ogawa, M. 1986. Macrophage colony formation supported by purified CSF-1 and/or interleukin-3 in serum free culture: evidence for hierarchical difference in macrophage colony forming cells. Blood 67: 859-864. Kusec, R., Laczika, K., Knobl, P., Friedl, J., Greiniz, H., Kahls,P., Linkesch, W., Schwarzinger, I., Mitterbauer, G., Purtscher, B., Haas, O.A., Lechner, K., and Jaeger, U. 1994. AML1/ETO fusion mRNA can be detected in remission blood samples of all patients with t(8;21) acute myeloid leukemia after chemotherapy or autologous bone marrow transplantation. Leukemia 8: 735-739. Lapidot, T., Pflumio, F., Doedens, M., Murdoch, B., Williams, D.E. and Dick, J.E. 1992. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 255: 1137-1141.  70  Lapidot, T., Sirard, C , Vormoor, J., Murdoch, B., Hoang, T., Caceres-Cortes, J., Minden, M., Patterson, B., Caligiuri, M.A. and Dick, J.E. 1994. Nature 367: 645-648. Lichtman, M.A. In Hematology 4th ed. Classification and clinical manifestations of the hemopoietic stem cell disorders, eds. W. J Williams, E. Beutler, A.J. Erslev and M.A. Lichtman; pp.148-157. McGraw-Hill, 1990. Lichtman, M.A. and Henderson, E.S. In Hematology 4th ed. Acute myelogenous leukemia, eds. W.J. Williams, E. Beutler, A.J. Erslev and M.A. Lichtman;pp.251-271. McGraw-Hill, 1990. Liu, P., Tarle, S.A., Hajra, A., Claxton, D.F., Marlton, P., Freedman, M., Siciliano, M.J. and Collins, F.S. 1993. Fusion between transcription factor CBFB/PEBP2 B and a myosin heavy chain in acute myeloid leukemia. Science 261: 1041-1044. Lowenberg, B. and Touw, LP. 1993. Hematopoietic growth factor and their receptors in acute leukemia. Blood 81: 281-292. Mager, D.L. and Henthorn, P.S. 1984. Identification of a retrovirus-like repetitive element in human DNA. Proc. Natl. Acad. Sci; USA 81: 7510-7514. Mager, D.L and Freeman, J.D. 1987. Human endogenous retroviruslike genome with type C pol sequences and gag sequences related to human T-cell lymphotropic viruses. J. Virol. 61; 4060-4066. Mayumi, H. and Good, R.A. 1989. Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide. J. Exp. Med. 169: 213-238. Metcalf, D. and Burgess, A. W. 1982. Clonal analysis of progenitor cell commitment to granulocyte or macrophage production. J. Cell Physiol. I l l : 275-283. Metcalf, D. The clonal culture in vitro of human myeloid leukemic cells, in The leukemic cell 2nd ed. ed. Catovsky, D. Churchill Livingstone. 1991. Miyoshi, H., Kozu, T., Shimizu, K.^ Enomoto, K., Maseki, N., Kaneko, Y., Kamada, N. and Ohki, M. 1993. The t(8;21) translocation in acute myeloid leukemia results in production of an AML1- MTG8 fusion transcript. EMBO J. 12: 2715-2721. Morstyn, G. and Burgess, A.W. 1988. Hemopoietic growth factors: a review. Cancer Res. 48:5624-5637. Mosier, D.E., Stell, L.L., Gulizia, R.J., Torbett, B.E. and Gilmore, G.L. 1993. Homozygous scid/scid, beige/beige mice have low levels of spontaneous or neonatal T cell-induced B cell generation. J. Exp. Med. 177: 191-194.  71  Otsuka, T., Thacker, J.D. and Hogge, D.E. 1991. The effects of interleukin 6 and interleukin 3 on early hematopoietic events in long-term cultures of human marrow. Exp. Hematol. 19: 1042-1048. Pagliaro, L.C. and Stanley, W.S. 1993. Interphase FISH and morphologic analysis of AML. Cancer Genet. Cytogenet. 67: 95-100. Quesenberry, P.J. In Hematology 4th ed. Hemopoietic stem cells, progenitor cells, and growth factors, eds. W.J. Williams, E. Beutler, A.J. Erslev and M.A. Lichtman; pp. 129-147. McGraw-Hill, 1990. Roder, J. and Duwe, A. 1979. The beige mutation in the mouse selectively impairs natural killer cell function. Nature 278: 451-456. Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular cloning. A laboratory mamual. Cold Spring Harbour: Cold Spring Harbour Laboratory Press. 1989. Sawyers, C L , Denny, CT. and Witte, D.N. 1991. Leukemia and the disruption of normal hematopoiesis. Cell 64: 337-350. Sawyers, C.L., Gishizky, M.L., Quan, S., Golde, D.W. and Witte, O.N. 1992. Propagation of human blastic myeloid leukemias in the SCID mouse. Blood 79: 2089-2098. Shultz, L.D., Schweitzer, P.A., Christianson, S.W., Gott, B., Schweitzer, I.B., Tennent, B., McKenna, S., Mobraaten, L., Rajan, T.V., Greiner, D.L. and Leiter, E.H. 1995. J. Immunol. 154: 180-191. Smith, M.T. Wiemels, J., Rothman, N and Linet, M.S. 1992. Chemical exposure, ras oncogene activation, and acute myeloide leukemia. J. Natl.Cancer Inst. 84: 1614-1615. Sutherland, H.J., Hogge, D.E., Cook, D. and Eaves, CJ. 1993. Alterative mechanisms with and without steel factor support primitive human hematopoiesis. Blood 81: 1465-1470. Taylor, J.A.; Sandler, D.P., Bloomfield, CD., Shore, D.L., Ball, E.D., Neubauer, A., Mclntyre, O.R., and Liu, E. 1992. ras oncogene activation and occupational exposures in acute myeloid leukemia. J.Natl.Cancer. Inst. 84: 1626-1632. Thacker, J.D. and Hogge, D.E. 1994. Cytokine-dependent engraftment of human myeloid leukemic cell lines in immunosuppressed nude mice. Leukemia 8: 871-877. Till, J.E. and McCullogh, E.A. 1980. Hemopoietic Stem cell differentiation. Biochim. Biophys. Acta. 605: 431-459.  72  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items