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Expression and function of Hox homeobox transcription factors in early hematopoiesis Sauvageau, Guy 1995

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EXPRESSION AND FUNCTION OF HOXHOMEOBOX TRANSCRIPTION FACTORS INEARLY HEMATOPOIESISbyGuy SauvageauM.D., Université de Montréal, 1987M.Sc., Université de Montréal, 1988A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESExperimental MedicineWe accept this thesis as conforminglardTHI OF BRITISH COLUMBIAJune 1995@ Guy Sauvageau, 1995In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)Department of_________________The University of British ColumbiaVancouver, CanadaDate 4 /,9’SDE-6 (2188)ABSTRACTThe Hox genes, first identified in Drosophila, are now recognized as keydeterminants of mammalian development. Recent evidence of Hox geneexpression in leukemic cell lines have suggested that these transcription factorsalso play important roles in regulation of hematopoiesis. Using an improvedRT/PCR technique which enables the generation of extended-length andrepresentative cDNA from fewer than 1,000 cells, the expression pattern of Hoxgenes in highly purified primitive hematopoietic subpopulations was examined.Two different approaches were used. The first exploited the presence of ahighly conserved region in the DNA-binding domain of Hox genes that could betargeted with degenerate primers for amplification from cDNA obtained fromeach purified population and the amplified produced were subsequentlysubcloned and sequenced. Over 150 Hox sequence containing clones werecharacterized. HOXA9, A6, A5, A4, A2, B3, B7 B9 and C9 were detected in asubpopulation which was highly enriched for very primitive cells detected aslong-term culture-initiating cells (LTC-IC) and depleted for clonogenicprogenitors. HOXA1O, A9, A7 A5, A4 and B7were detected in a subpopulationhighly enriched for clonogenic myeloid progenitors, and HOXA1O, A9, A7, A6,A5, B7and C8 were found in an erythroid progenitor enriched subpopulation.These data suggested that HOX A cluster genes are widely expressed amongstearly hematopoietic subpopulations; in contrast, some genes of the B clusterappear to be restricted to the more primitive LTC-lC containing subpopulation.To better assess the potential differential expression of Hox genes, the initialamplified cDNA from five purified CD34 subpopulations of bone marrow cellswas analyzed by Southern blot using probes for specific Hox genes. With thisapproach, it was possible to show that expression of HOXB3 and B4 wasIIImarkedly higher (up to 40 fold) in the most primitive subpopulation than in themore mature subpopulations whereas that of other Hox genes such asHOXA1O, A9 and B9 was constant in all populations. Taken together, these datasuggest that many Hox genes are active in early hematopoiesis and that someof them (i.e. HOXB3 and B4) are possible candidates for regulating stem cellfunction. To gain further insight into the role Hox genes may play in earlyhematopoiesis, the effect of overexpression of HOXB4 was studied in a murinemodel using a myeloproliferative sarcoma-based retroviral vector carrying thehuman HOXB4 cDNA under the control of the 5’ viral long terminal repeat.Overexpression of HOXB4 had proliferative effects on clonogenic progenitors,day 12 CFU-S and cells with marrow repopulating ability (MRA) as assessed bycolony replating or recovery from seven day liquid cultures (up to 200 fold overneo-transduced control). To study the possible effects of HOXB4overexpression on hematopoietic cells maintained for prolonged periods invivo, HOXB4 or nec-transduced marrow cells were transplanted into lethallyirradiated syngeneic recipients and reconstitution of various hematopoieticpopulations analyzed. At 20 weeks post-transplantation, recipients of HOXB4-transduced marrow had -5 fold more clonogenic progenitors per femur thanneo controls. To determine if HOXB4 overexpression affected the expansion ofthe earliest hematopoietic stem cells (HSC), their numbers were determined byusing the competitive repopulation unit (CRU) assay. CRU numbers inrecipients of HOXB4-transduced bone marrow cells had recovered to 140% ofnormal levels found in untransplanted mice or some 50 fold higher than inrecipients of nec-transduced marrow; all recipients of HOXB4-transducedmarrow however had normal peripheral blood counts. Southern blot analysis ofunique proviral integration patterns in DNA isolated from bone marrow andthymus of secondary recipients confirmed that HQXB4-transducedivhematopoietic repopulating cells were totipotent and that they extensively selfrenewed. This dramatic effect of HOXB4 on HSC self-renewal was maintainedin serial transplantation experiments. Together, these results indicate HOXB4 tobe an important regulator of very early but not late hematopoietic cellproliferation. The ability of HOXB4 to reverse the severe decline in HSCnumbers suggest an exciting new approach to the controlled amplification ofgenetically modified hematopoietic stem cell populations.VTABLE OF CONTENTSTITLEABSTRACT iiTABLE OF CONTENTS vLIST OF TABLES AND FIGURES viiiABBREVIATIONS xiACKNOWLEDGMENTS xivCHAPTER 1. Introduction1.1 Overview 11.2 Hematopoiesis, its cellular constituents and regulation 21.2.1 Ontogeny of the hematopoietic system 21.2.2 Cellular constituents of the mammalian hematopoieticsystem The hematopoietic stem cell (HSC) Definition Quantitation of HSC Properties of HSC Cycling status of HSC HSC self-renewal potential Repopulating potential of HSC Stem cell “aging” Heterogeneity of the HSC 1 Purification of murine HSC 1 Towards defining human HSC and their properties 1 41 .2.3 Regulation of hematopoiesis 1 Regulation of hematopoiesis by externalfactors Regulation of hematopoiesis by intracellularfactors 181.3 Homeodomain-containing transcription factors 241.3.1 Definition, classification and chromosomal organization ofthe homeobox genes 241.3.2 Regulation by homeobox proteins of embryonic cell growthand differentiation 281.3.3 Upstream regulation of homeobox (HOM-C/Hox) geneexpression 311.3.4 Downstream targets of HOM-C I Hox genes 321.3.5 Hox gene expression in post-developmental normal andneoplastic tissues 331.3.6 Homeobox gene expression in normal and leukemichematopoietic cells 341.3.6.1 Hox gene expression in leukemic cells 341 .3.6.2 Hox gene expression and function in normalhematopoietic cells 371.4 Thesis Objectives 39viCHAPTER 2. A RT/PCR method to generate representativecDNA from small number of hematopoietic cells 412.1 Summary 412.2 Introduction 422.3 Materials and Methods 442.3.1 RNA extraction and first strand eDNA synthesis 442.3.2 Homopolymeric dA Tailing 452.3.3 Amplification of the cDNA 452.3.4 Southern Analysis of Amplified cDNA 462.4 Results and Discussion 482.4.lCondition Adjustments 482.4.2 Presence in Amplified cDNA of Sequences Distantfrom the 3’ end of mRNA 512.4.2 Sensitivity and Representativeness of the cDNA 54CHAPTER 3. Differential expression of Hox homeobox genesin functionally distinct CD34+ subpopulations of human bonemarrrow cells 583.1 Introduction 593.2 Materials and Methods 603.2.1 Cell Purification 603.2.2 Clonogenic Progenitor Assays 603.2.3 Long-term Culture 613.2.4 cDNA Generation and Amplification 613.2.5 PCR Amplification of Hox Homeodomains 613.2.6 Southern Blot Analysis of Total Amplified cDNA 623.3 Results 633.3.1 Characterization of Purified CD34+ Bone Marrow CellSubpopulations 633.3.2 Hox Gene Expression in CD34+ Subpopulations 653.3.3 cDNA Southern Blot Analysis of Hox Gene Expressionin CD34+ Subpopulations 683.3.4 cDNA Southern Analysis of Hox Gene Expression inCD34- Cells 733.4 Discussion 75CHAPTER 4. Overexpression of HOXB4 in HematopoieticCells Causes the Selective Expansion of More PrimitivePopulations in Vitro and in Vivo 774.1 Introduction 784.2 Materials And Methods 804.2.1 Animals 804.2.2 Retroviral Generation 804.2.3 Cell Lines 814.2.4 Retroviral Infection of Primary Bone Marrow Cells andHematopoietic Cell Lines 824.2.5 Transplantation of Retrovirally Transduced BoneMarrow 834.2.6 In Vitro Clonogenic Progenitor Assays 834.2.7 CFU-S 84VII4.2.8 Marrow Repopulating Ability (MRA.854.2.9 Competitive Repopulating Unit (CRU 854.2.10 DNA and RNA Analyses 864.2.11 Western Analysis 864.3 Results 874.3.1 Retroviral-Mediated Transduction of HOXB4 to MurineBone Marrow Cells 874.3.2 Increased Proliferative Activity in Vitro of ClonogenicProgenitors Overexpressing HOXB4 904.3.3 HOXB4 Effects on the Maintenance in Vitro of Day 12CFU-S and Cells with Marrow Repopulating Ability (MRA) 934.3.4 HOXB4-lnduced Expansion in Vivo of ClonogenicProgenitors and Day 12 CFU-S 954.3.5 HOXB4-lnduced Expansion in Vivo of Cells WithLong-Term Lympho-Myeloid Repopulating Ability 1004.4 Discussion 108CHAPTER 5. General Discussion and Conclusions 1135.1 Hox genes are expressed in normal early hematopoietic cells 1135.2 HOXB4 overexpression causes expansion of earlyhematopoietic cells 1145.3 Hox genes: Master regulators of hematopoiesis9 1155.4 Future studies 116CHAPTER 6. References 120vi”LIST OF TABLES AND FIGURESChapter 1Figure 1.1 Schematic representation of the hematopoietic hierarchy 5Figure 1.2. Schematic representation of initiation of transcription by theRNA polymerase II 19Figure 1.3. Possible hierarchical organization of some transcriptionfactors involved in hematopoietic development 23Figure 1.4. Relationship between the HOM-C Drosophila Homeoboxgenes of the Antennapedia and Bithorax cluster complex andthe various mammalian Hox genes 27Figure 1.5. Three sets of genes must be sequentially activated for properdifferentiation of a specific body part 29Figure 1.6. Regulation of HOM-C I Hox gene expression 32Chapter 2Figure 2.1. Evaluation of PCR amplified total cDNA obtained from limitednumbers of HL-60 or low density human bone marrow cells 50Figure 2.2. Comparative expression of c-myc in normal bone marrow,K562 and HL-60 cells by Northern blot analysis 52Figure 2.3. Southern blot analysis using a neor specific probe to test thesensitivity of PCR cDNA amplification 55Figure 2.4. Southern blot analysis of PCR amplified cDNA to detectexpression of HOXB4 in HL-60 versus K562 cells 56Chapter 3Figure 3.1. FACS profiles of the CD34-’- subpopulations isolated frombone marrows no.1 (CAD3) and no.2 (CAD6) 64Table 3.1. Clonogenic progenitor and LTC-IC content of the differentpurified CD34+ bone marrow subpopulations 65Table 3.2. Expression of Hox genes in purified bone marrowsubpopulations and in hemopoietic cell lines determined bysequence analyses of amplified homeobox containing cDNA 68Figure 3.2A. Southern blot analysis of the total amplified cDNA derivedfrom each CD34+ subpopulation 70Figure 3.2B. Southern blot analysis of the total amplified cDNA derivedfrom each CD34 subpopulation 71ixFigure 3.3. Southern blot analysis of total amplified cDNA derived fromthe low density cells fraction of three different marrows 74Chapter 4Figure 4.1. Structure and expression of HOXB4 and control neoretroviruses used in this study 88Figure 4.2. Overview of the experiments and assays used in thesestudies 89Table 4.1. Clonogenic progenitor frequencies in bone marrow cellsimmediately post infection with HOXB4-neo or neoretroviruses 91Figure 4.3. The effect of HOXB4 overexpression on the ability ofindividual methylcellulose colonies to form secondarycolonies upon replating 92Figure 4.4. HOXB4 effects on day 12 CFU-S in vitro 94Figure 4.5. HOXB4 effects on cells with marrow repopulating ability(MRA) 95Figure 4.6. Northern and Southern blot analyses to demonstratehematopoietic reconstitution by HOXB4- or neo-transducedbone marrow cells 97Table 4.2. Mice transplanted with HOXB4 transduced cells haveincreased myeloid and pre-B lymphoid clonogenicprogenitors in bone marrow and spleen 99Table 4.3. Evaluation by limiting dilution analysis of competitive long-term repopulating cells (CRU) in mice transplanted withHOXB4 versus neo control transduced bone marrow cells 102Figure 4.7. Southern blot analysis of proviral integration patterns in bonemarrow and thymic DNA isolated from secondary recipientstransplanted with varying numbers of HOXB4 or neotransduced bone marrow cells 104Table 4.4. Expansion of donor-derived CRU in primary and secondaryrecipients of HOXB4- or neo-transduced bone marrow cells 106Table 4.5. Enumeration of bone marrow, spleen and peripheral bloodcell counts of primary mice transplanted 16-34 weeks earlierwith HOXB4- or neo-transduced cells 107Table 4.8. Schematic depiction of the sizes of various hematopoieticpopulations reconstituted in primary recipients of HOXB4- orxneo-transduced bone marrow cells compared to normal(unmanipulated) mice 108xiABBREVIATIONSAMV avian mye lopro liferative virusATCC American Tissue Culture Collectionbp base pairBD Becton DickinsonBFU-E burst forming unit-erythroidBMT bone marrow transplantationBSA bovine serum albumincDNA complementary deoxyribonucleic acidCFU colony forming unitCFU-E colony forming unit-erythroidCFU-GEMM CFU- granulocyte, erythrocyte, macrophage, megakaryocyteCFU-GM CFU-granulocyte, macrophageCFU-S colony forming unit-spleencGy centiGrayCLL chronic lymphocytic leukemiaCRU competitive repopulation unitdATP deoxyadenosine triphosphatedCTP deoxycytidine triphosphatedGTP deoxyguanosine triphosphateDNA deoxyribonucleic aciddNTPs deoxynucleotide triphosphatesDTT 1 ,4-dithioerythritoldTTP deoxythymidine triphosphateEpo erythropoietinES embryonic stem (cells)xiiFAB French American BritishFCS fetal calf serum5-FU 5-fluorouracilG-CSF granulocyte-colony-stimulating factorGM-CSF granulocyte-macrophage-colony-stimulating factorHGF hematopoietic growth factorHSC hematopoietic stem cellHXM hypoxanthine-xanthine-mycophenolic acidlAP intracisternal A particleIL interleukinKCl potassium chlorideLTC long-term cultureLTC-IC long-term culture-initiating cellLTR long terminal repeatMDR-i multi-drug resistance-i or P-glycoprotein13-ME f3-mercaptoethanolMgCl2 magnesium chlorideMIP-la macrophage inhibitory protein-i alphaMRA marrow repopulating abilitymRNA messenger ribonucleic acidMSCV murine stem cell virusNCS newborn calf serumOSM oncostatin-Mpgk phosphoglycerate kinasePHA phytohemagglutininR-PE R-phycoerythrinRNA ribonucleic acidRU repopulation unitS.D. standard deviationSCCM spleen cell conditioned mediumSDS sodium dodecyl sulfateTaq Thermus AquaticusTBP Tata-binding proteinTdT terminaldeoxyribonucleotidyl transferaseTGF-1 tumor growth factor-beta 1U unitsxl”xivACKNOWLEDGMENTSI am very grateful to my supervisor Dr. Keith Humphries for the constantsupport, availability and freedom that he gave me throughout this work. Hissense of judgment combined with his incredible knowledge of hematopoiesishave profoundly imprinted the making of this project.I would also like to thank my co-supervisor Dr. Peter Lansdorp not only forhis support and ideas but also for the help he has been providing in one of themost important resource for this project: state of the art purification ofsubpopulations of human bone marrow cells.I also want to thank all the members of Keith’s laboratory (Patty, Jana,Margaret, Rob, Sue, Cheryl and Sean) for their constant help and pleasant time.A very special thank to Unnur Thorsteinsdottir whom I have worked in a closeand pleasant collaboration and who, through her careful and intelligent way,has made possible a lot of the work presented in this thesis.Additionally, I would like to thank Vishia Dragowska, Diane Reid, GayleThornbury, Maya St-Clair, Stacey Grant, Fred Janson and Rob Schamborzki fortheir valuable assistance.My sincere thanks to our collaborators Drs. Corey Largman and JefferyLawrence with whom I have had a lot a pleasant discussions and who haveintroduced me to the complex world of homeobox genes. Their contribution to alot of the experiments described in this thesis has proven to be invaluable.I would also like to thank Dr. Connie Eaves for her help and support in thiswork and also for the time she has spent in helping to write some of the morexvdifficult manuscripts. I also thank Dr. Allan Eaves, who through his dedication tothe Terry Fox laboratory, has made all of these necessary collaborationspossible. Finally I would like to thank my wife Chantal Lacroix and also all themembers of our families for their constant support during the more difficult times.The financial support during this period of time was provided by a ClinicianScientist Award from the Medical Research Council of Canada.1CHAPTER 1Introduction11 OverviewEvery day billions of mature specialized cells found in the peripheral blood of anormal human adult need to be replaced. The burden of the life-long productionof these mature elements is carried out by a small number of primitive bonemarrow cells constituting less than 0.01% of the total marrow. These primitivecells have the key properties of being able to undergo self-renewal, that isdivide and give rise to progeny cells with essentially the same biologicalproperties or they can differentiate and give rise to “committed progenitors” thatprogressively acquire the specialized characteristics of one of the 10 lineageswhich constitute human hematopoiesis. This impressive machinery appears tobe highly controlled at multiple levels by both positive and negative regulatorsand, under normal circumstances, assures the life long maintenance of variousperipheral blood cell numbers within a relatively narrow range.While as reviewed below, much progress has now been made inidentifying a variety of cytokines that can regulate the cycling status of primitivehematopoietic cells, little is known yet about the genetic mechanismsresponsible for the self-renewal and differentiation outcomes of these earlycells. Accumulating evidence however points to transcription factors such asIkaros, PU-i and GATA-2 as key regulators of early hematopoietic cellsfunction. A group of transcription regulators initially described in the fruit fly asmaster regulators of embryonic cell proliferation and differentiation was recentlyfound to be expressed in hematopoietic cell lines. These genes contain a helixturn-helix DNA-binding domain termed the “homeobox” and by analogy to their2function in Drosophila, it has been hypothesized that homeobox genes mayalso play central roles in the regulation of early hematopoiesis.As a test of this hypothesis, the research described in this thesis wasaimed at identifying whether and which homeobox genes are differentiallyexpressed in normal primitive hematopoietic cells and, subsequently testing thepossibles roles such genes may play in early hematopoiesis using retroviralgene transfer to engineer overexpression in murine bone marrow cells.Following a brief review of the current understanding of the organization andregulation of the hematopoietic system and its cellular constituents, the nextsections of the introduction focus on two topics that are central to this work: Hoxhomeobox genes and hematopoietic stem cells.1.2 Hematopoiesis, its cellular constituents and regulation1.2.1 Ontogeny of the hematopoietic systemIn mammals, hematopolesis appears to derive from the mesodermal layer of theforming embryo. Blood-forming cells originate in the yolk sac (extra-embryonic)and, at specific times, migrate to the fetal liver and ultimately to the bone cavities(bone marrow) (Moore and Metcalf, 1970; Toles et al., 1989). The extra-embryonic origin of the definitive hematopoietic system has recently beenchallenged however by studies showing a potential early intraembryonic site ofhematopoiesis (Godin et al., 1993; Medvinsky et al., 1993).1.2.2 Cellular constituents of the mammalian hematopoletic systemA multiplicity of both in vivo and in vitro assays are now available for thedetection of hematopoietic cells found at various levels within the hematopoietichierarchy (reviewed in Eaves, 1995). One of the first hematopoietic assays to be3developed was the spleen-colony-forming-unit (CFU-S) assay (Till andMcCulloch, 1961). This assay detects cells present at a low frequency in murinebone marrow that, when intravenously injected into myelo-ablated recipients,can generate macroscopic hematopoietic nodules on the spleen 9-14 dayslater. These colonies were shown to be clonal (Wu et al., 1968), to containmultiple myeloid lineages (Till and McCulloch, 1961) and, importantly, in manycases were able to generate similar colonies upon transplantation intosecondary recipients (Siminovitch et al., 1963).CFU-S were thus initially considered as HSC because they sharedimportant characteristics which were attributed to these cells including highproliferative potential, multipotentiality and self-renewal ability (Si minovitch etal., 1963). It is now clear that most CFU-S cells are committed myeloidprogenitors (Jones et al., 1989) that can be physically separated from cells withgreater long-term repopulating potential (Mulder and Visser, 1987; Spangrudeet al., 1991; van der Loo et al., 1994; Visser and de Vries, 1988). Neverthelessstudies focusing on CFU-S have played key roles in developing concepts of theorganization and regulation of mammalian hematopoiesis (Till et al., 1964).A wide range of myeloid cells with various potential for proliferation anddifferentiation can be quantitated based on their ability to form various types ofcolonies in semi-solid culture supplemented with different cocktails of crude orpurified hematopoietic growth factors (reviewed in Eaves, 1995). Such cellshave been operationally defined as “clonogenic progenitors” and have beeninstrumental in the characterization of cytokines acting on hematopoletic cells.Using unmanipulated bone marrow cells, most progenitors that can berecognized in these cultures are oligo- or uni-potential and have limitedproliferative potential with no or minimal self-renewal potential. More recently4additional in vitro assays have opened up the identification of bone marrowcells with B but not T lymphoid potential (Suda et al., 1989).Another group of in vitro assays aimed at detecting earlier myeloid cellsrely on in vitro simulation of the bone marrow microenvironment by “feedercells”. This group of assays pioneered by Dexter (Dexter et al., 1977) is basedon the concept that cells with limited proliferative potential (i.e. the clonogenicprogenitors described above) will be “extinguished” after a prolonged period ofculture (weeks) whereas clonogenic progenitors present at the end of these“long-term cultures” (LTC) must be derived from a more primitive populationwhich can be accurately quantitated by limiting dilution analysis procedures(Ploemacher et al., 1989; Sutherland et al., 1990). More recently the possibilityof detecting early lympho-myeloid hematopoietic cells was demonstrated by theidentification of cells with myeloid B lymphoid potential which can bemaintained in a two-step long-term “switch” culture assay (Lemieux et al., 1995).Other in vitro and in vivo assays that also appear to recognize primitivehematopoietic cells (Breems et al., 1994; Gordon et al., 1987; Harrison et al.,1993; Hodgson, 1979; Leary, 1987; McNiece et al., 1989; Ploemacher et al.,1991; Szilvassy et al., 1990) are listed in Figure 1.1 but are not explored in thisthesis and therefore are not further described.Although it is clearly possible to assay hematopoietic cells with differentpotential for proliferation and differentiation, it is important to stress that aspecific cell can probably be detected in more than one of the available assays(Figure 1 .1). As an example of this, a subset of clonogenic progenitors identifiedin a direct colony assay can also give rise to spleen colonies (Humphries et al.,1979).5DAY 12 CFU-SMRALTC-ICBLAST COLONYCAFC‘Multi-potentialprogenitorsFigure 1.1 Schematic representation of the hematopoietlc hierarchy.Some of the assays used to evaluate various progenitor poois are depicted in bold. HSC,hematopoletic stem cells; MRA, cells with short-term marrow repopulating ability; BCA, blastcolony assay; LTC-lC, in vitro assay for cells with ability to initiate in vitro long-term cultures; CAFC,in vitro assay for cobblestone forming area; CRU, competitive repopulation unit assay; RU,repopulation unit assay; day 12 CFU-S, colony-forming-unit spleen obtained 12 days afterinoculation. The hematopoietic stem cell (HSC) DefinitionThe HSC can be operationally defined as a long-term repopulating cell which,in most cases, has lymphoid (T and B) and myeloid potential (Orlic and Bodine,1994). Such a definition has relevance in the context of bone marrowtransplantation (BMT) and gene therapy and will be retained through the thesis.Early demonstration of the existence of such cells includes detection ofcommon unique chromosomal markers in lymphoid and myeloid transplantedbone marrow cells (Abramson et al., 1977; Wu et al., 1968). Exploiting the abilityDirect colony assaysCRURUMorphology©0Uni-potentialprogenitorsILymphoidprogenitorsFicuuiii’._ ji.iiAL6of retroviruses to randomly integrate in the genome, many investigators havenow used replication-defective retroviral vectors to infect HSC and document bySouthern blot analysis their lymphoid (T and B) and myeloid cell repopulatingpotential (Jordan and Lemischka, 1990; Keller and Snodgrass, 1990). Quantitation of HSCAt the present time, it is not possible to directly quantitate HSC by the presenceof unique or combinations of surface markers and physical properties althoughpurification strategies based on such properties enable significant enrichment(discussed below) (Uchida et al., 1993). Detection and quantification of suchcells therefore must still rely on functional assays (Orlic and Bodine, 1994).Strict adherence to the rigorous operational definition of a long-termrepopulating cell however requires that recipients are observed for prolongedperiods of time (many months). One approach to accelerate evaluation of HSChas been the measurement of the graft radioprotective ability (Jones et al.,1989; Spangrude and Johnson, 1990). Such assays however primarily reflectthe short-term radioprotective ability of the graft and further, it has now beendocumented that such cells can be physically separated from those with long-term repopulating ability (Jones et al., 1990). Life-sparing assays furthermoredo not necessarily take into account host contributions to hematopoieticrecovery which in some situations can be substantial.One powerful approach to quantitate HSC based on principles ofcompetitive repopulation was developed by Harrison ((Harrison et al., 1988);see Harrison et al., 1993 for review).This assay consists of comparing the longterm repopulating abilities of two populations of transplanted hematopoieticcells that can be distinguished by phenotypic, genetic or biochemical markers.7The first of these populations is called the “competitor” and normally consists ofa predetermined number of normal bone marrow cells that, by themselves willreadily repopulate the recipient. Various numbers of bone marrow cells from asecond population termed the “donor” are simultaneously injected with highnumbers (generally x 106) of competitors. The frequency of HSC or“repopulation units (RU)” in the “donor” is unknown and thus evaluated by therelative contribution to hematopoiesis of both cell populations by using the ratioof donor to competitor-derived hematopoiesis1 (Harrison et al., 1993). Incontrast to the CRU assay which uses limit dilution principles (see below), thisprocedure uses high cell numbers and thus reduces the demand for short-termrepopulation imposed on the “tested” population. Therefore, this assay isparticularly suited for the comparison of the relative numbers of RU in twodifferent purified subpopulations of hematopoietic cells (Harrison et al., 1993).In an effort to obtain a quick and accurate quantitative evaluation of HSC,Szilvassy et al., (Szilvassy et al., 1990) developed an assay that combineslimiting dilution analysis procedures and competitive repopulation to quantitateHSC frequency in unknown “test” population. In its original description, variousnumbers of test cells were co-injected with “helper compromised” syngeneiccells into multiple irradiated recipients. The helper cells assure short termhematopoietic reconstitution and are said to be “compromised” because theyhave lost most of their long-term repopulation ability as a result of serialtransplantation (see later in text for further explanation of this phenomenon).Because lympho-myeloid elements that originate from the “test” cells can beidentified either by genetic markers (Y probe for male graft into femalerecipients (Szilvassy et al., 1990)) or by cell surface antigens (Ly5.1 or Ly5.2:1The following formula is used to calculate RU frequency: RU= (% of hematopoiesis which isdonor-derived / % competitor) x (# of competitor cells injected /1 O)8(Rebel et al., 1994; Spangrude and Scollay, 1990; Szilvassy and Cory, 1993), itis possible to identify recipients that are significantly (i.e. >5%) repopulated by alympho-myeloid “test” cell which contributes to long-term repopulation. TheHSC operationally defined by this assay is termed a CRU (competitiverepopulation unit) and its frequency is established from the proportion of micethat meet the repopulation criteria described above when limiting dilution isreached based on Poison statistics. More precisely, the exact frequency of CRUin a test population is: CRU frequency = 1 / (number of bone marrow “test” cellsthat repopulated exactly 63% of the irradiated recipients). Interestingly, CRUfrequencies determined five weeks after transplantation were shown to bevirtually identical to those obtained at later time points (Szilvassy and Cory,1993) suggesting that this assay can be used to evaluate true long-termrepopulating HSC after a little more than a month. For simplicity, the assay wasrecently modified to allow use of normal bone marrow cells (i.e. fromunmanipulated normal donors) as helper cells and, after adjustment of therepopulation criteria, this has proved to be just as reliable as the method initiallydescribed ((Rebel et al., 1994), see also chapter 4).Together, the availability of assays to identify and quantitate HSC hasopened the way to characterize their properties, phenotype and regulationwhich will be discussed in the following sections. Properties of HSC1 . Cycling status of HSCOne characteristic that distinguishes HSC from other more mature populationssuch as day 12 CFU-S is their cycling state. The majority of HSC underhomeostatic condition are quiescent as elegantly demonstrated by studies9using an S phase-specific agent (5-FU) in which HSC detected by competitiverepopulation assay but not CFU-S, are spared by a single injection of this agent(Hodgson and Bradley, 1979; Lerner and Harrison, 1990). Interestingly, 5-FUappears to recruit HSC into cycle since they become very sensitive to a seconddose of 5-FU given 3 to 5 days later (Harrison and Lerner, 1991). HSC self-renewal potential.Several studies utilizing retroviral marking have now unequivocallydemonstrated by the presence of identical proviral integration patterns in bonemarrow and thymus tissue of primary and secondary recipients that HSC canself-renew (Jordan and Lemischka, 1990; Keller and Snodgrass, 1990;Lemischka et al., 1986).Some HSC that have been “tracked” in these studies were proven to haveextensive proliferative potential. Keller and Snodgrass, (1990) for exampledemonstrated that a single HSC could completely reconstitute all lineages of arecipient for more than 10 months and further that transplantation of this marrowinto a secondary recipient resulted in almost complete reconstitution (>80%) bythis same clone for another 17 months. Although studies such as these haveclearly documented self-renewal division in the HSC compartment, they failedto quantitate the magnitude of self-renewal events because the absolutenumber of stem cell regenerated in recipients was not evaluated. Repopulating potential of HSC.Although HSC can self-renew, there is now considerable evidence to suggestthat this property is not unlimited.Transplantation of small (105) or moderate (107) numbers of murine bonemarrow cells into lethally irradiated recipients rapidly results in normal bonemarrow cellularity (-2-2.5 x iO cells/femur) which persists for the life (>9months) of the animal (Mauch and Hellman, 1989). Day-8 CFU-S also recover10to near normal levels after transplantation of 106107 cells (Harrison et al.,1978; Mauch and Hellman, 1989) but are reduced to -‘65% of normal values inrecipients of 10 bone marrow cells (Mauch and Hellman, 1989). Althoughbone marrow cellularity and day-8 CFU-S are regenerated to normal numbersfollowing BMT with 106 cells or more, studies using competitive repopulationas described before (see section have shown a dramatic (i.e. 3-20fold) reduction in proliferative ability of HSC recovered even after a singletransplantation of 10-10 cells (Harrison, 1982; Harrison et al., 1978).Interestingly, a lesser but still significant reduction was observed when a highernumber of cells was transplanted (108 cells which represent about 1/2 of thehematopoietic system of an adult mouse). It has been argued that this loss oflong-term repopulating ability may result from damage to the recipient’smicroenvironment inflicted by the conditioning regimen (i.e. irradiation, etc.). Inorder to exclude this possibility, experiments have also been conducted using arecipient mouse strain known to have a normal microenvironment but poorlycompetitive early hematopoietic cells (McCulloch et al., 1964) that are nowknown to have a defective c-Kit ligand receptor (Chabot et al., 1988).Transplantation into this anemic strain (W/VV”) can therefore be done withoutconditioning regimens and this was used by various investigators to prove thatthe loss of long-term repopulating ability of transplanted marrow is due not to amicroenvironmental defect induced by the conditioning regimen (Gardner et al.,1 988; Harrison, 1982) but rather to the transplanted cells themselves.The mechanisms for this apparent reduction in repopulating abilityfollowing bone marrow transplantation is not clear. Whether this reduction isdue to a depletion in the number of HSC or to a general decrease in theirproliferative ability for example, remains unresolved.11One possibility to explain this loss in repopulating ability of transplantedHSC is that the proliferative stress imposed upon newly generated HSC shiftsthe balance from self-renewal division to differentiation and that the HSC pool isnot fully recovered. One recent intriguing hypothesis to explain the decreasedproliferative ability of transplanted HSC arises from accumulating evidenceindicating that telomere length reduction occurs with every hematopoietic celldivision and hence could contribute to the decreased proliferative potential oftransplanted HSC (Vaziri et al., 1994). However, telomere shortening has notbeen demonstrated in the inbred mice typically used in experimentalhematology (Greider, 1995; Kipling and Cooke, 1990).Together these results have relevance to clinical bone marrowtransplantation studies and in fact predict the possibility of a persistent declinein HSC in recipients. Since we are still in the early days of clinical BMT (‘20-25years), it will be important to continue to monitor recipients of a bone marrowgraft in order to rule out that HSC depletion will not eventually lead to bonemarrow failure. Stem cell “aciinçi”.The demonstrated decline in HSC repopulating ability after bone marrowtransplantation raised the question whether such a phenomenon would also beobserved in unmanipulated HSC derived from “old” untransplanted mice.By using a technique of competitive repopulation, it was found that bonemarrow from old mice (2 to 2.5 years) compete equally well against “competitorcells” as that of young (3-6 months) donors (Harrison, 1982). This equalcompetitiveness was demonstrated at various time points (including 18 monthsafter transplantation). Furthermore, it was also shown that bone marrow cellsderived from “old” mice performed equally in serial transplantation experiments12as those derived from younger animals. For example, repopulation levels below5% are frequent after two serial transplantations with young or old cells.These results however are at the population level and using cell numbersthat are in large excess of the minimum requirements leave open the possibilitythat individual HSC may have finite self-renewal capacity but that in the normallifespan their limit is not approached. Heteroçeneity of the HSC.By several criteria, HSC appear to be heterogeneous. Fetal HSC, for example,manifest increased ability compared to adults to competitively repopulatemyeloablated recipients (Rebel, 1995). HSC are also heterogeneous in theirexpression of surface molecules. Studies of Spangrude et al. (1988) andUchida and Weissman (1992) for example showed that long-term repopulatingcells are found within a minute (<0.05%) population of bone marrow cells thatexpress Sca-1 Thy1 .1 I0Lin (see next section). Further purification of these cellsinto functionally distinct populations was possible based on their expression ofMac-i, CD4 and c-kit (Morrison and Weissman, 1994). Although the Sca1Thy1.1°Lirrc-kitMac-1CD4 population was shown to be highly enriched forlong-term repopulating cells and fairly depleted of cells with short-termrepopulating potential it was still possible to detect, albeit at lesser frequency,cells with long-term repopulating potential in the two other populations (Mac-i Icand Mac1I0CD4l0) (Morrison and Weissman, 1994) suggesting that HSC areheterogeneous in their expression of certain surface molecules (also reviewedin Uchida et al. (1993)).Heterogeneity in the contribution of some HSC to specific lineages hasalso been documented. Jordan and Lemischka carefully followed, by retroviraltagging, the behavior over time of 142 HSC in 63 different recipients and foundby blood sampling (from 4 to 16 months post-transplantation) that a significant13proportion of HSC were not contributing equally to lymphoid and myeloidrepopulation and further that some HSC showed unstable behavior (i.e. anapparently myeloid-restricted clone can also show lymphoid differentiation at alater time point, etc.). Importantly however, the most frequent HSC behavior(35% of the clones) observed in their studies was that of a cell that persistentlyand equally repopulated the lymphoid and myeloid “arm” of the hematopoieticsystem.HSC may be also heterogeneous in their potential for long-termreconstitution (proliferation?). Experiments utilizing retroviral tagging haverevealed that some HSC only contribute to repopulation for the first 3-9 monthsthat follow transplantation whereas others contribute for the duration of therecipient’s life (Jordan and Lemischka, 1990; Keller and Snodgrass, 1994).Whether the heterogeneity observed, however, represents true or intrinsicdifferences in potential versus differences in the expression of this potential(e.g. premature differentiation) due to stochastic processes remains unclear.Further clarification of the nature of the observed heterogeneity will undoubtedlybenefit from studies utilizing improved purified preparations. Purification of murine HSCThe isolation of pure HSC has been long sought by several groups as a way ofultimately defining I quantitating HSC by phenotype and facilitating moredetailed characterization of their properties. Importantly, the availability of suchpurified cells would open the way to studies aimed at identifying the geneticdeterminants that characterize a stem cell. Numerous strategies to isolate thesecells have been successfully explored. These strategies include: 1- purificationby physical differences between HSC and more mature cells (Jones et al.,1989), 2- the presence of high levels of P-glycoprotein products on the surface14of these cells which is involved in exclusion of the vital dye rhodamine-123(Ploemacher and Brons, 1988b; Spangrude and Johnson, 1990) and, 3- theuse of differentially expressed surface molecules that can interact with variouslectins (Ploemacher and Brons, 1988a) or that can be recognized bymonoclonal antibodies (Morrison and Weissman, 1994; Spangrude et al., 1988;Szilvassy and Cory, 1993). The combined use of some of these approachesnow allows the enrichment of HSC by hundreds of fold to levels of purity thatcan in some instances reach 10% (Morrison and Weissman, 1994)Although variability in the expression of some of the cell surface antigenscan occur between different mouse strains (Spangrude and Brooks, 1993), thefollowing surface markers are usually found on murine cells with long-termrepopulating potential: Ly6A/E (Sca-1), Thy-1’°, lineag& (Mac-f, Gr-f, Ly-f,B220): (Spangrude et al., 1988) , wheat germ agglutinin, (Ploemacher andBrons, 1988a; Visser et al., 1984), c-kit (Morrison and Weissman, 1994; Okadaet al., 1992) and very likely CD34 (Krause et al., 1994). Towards defining human HSC and their propertiesThe in vivo assays for murine HSC described above are not currently availablefor evaluation of human HSC. The recent development of immunocompromisedmice which support the growth of normal human (Kamel-Reid and Dick, 1988;Kyoizumi et al., 1992; Lapidot etal., 1994) and leukemic (Lapidot et al., 1994)cells however, may now open the way for eventual in vivo quantitation of humanHSC using a “humanized” CRU assay.Methods for evaluation of primitive human hematopoietic cells have beendeveloped based on the detection of cells capable of initiating and sustainingclonogenic progenitor output in appropriate long-term culture conditions15analogous to that described for mice in section 1.2.2. Evidence that cells whichinitiate these cultures (called LTC-lC for LTC-initiating cells) overlap with HSCinclude their relative insensitivity to cell-cycle agents and the possibility toselectively enrich these by purification strategies (see below). Further supportcomes from murine studies which have shown LTC-lC frequency to be similar tothat of CRU (Lemieux et al., 1995) and also that CRU can self-renew under thesame LTC conditions that enables detection of LTC-lC (Fraser et al., 1992).Considerable progress has been made in the methods and reagentsavailable for purification of primitive human bone marrow cells (reviewed inLansdorp and Thomas, 1991). The discovery of a sialomucin surface antigen,CD34, (Civin et al., 1984) expressed on 1-4% of bone marrow cells was amajor step toward purification of primitive human bone marrow cells. Cellslacking CD34 fail to give rise to colonies in direct assay, cannot initiate long-term cultures and have no long-term repopulating ability as assessed in nonhuman primates (Berenson et al., 1988).Following the discovery of the CD34 surface antigen, much effort has beeninvested in the further refinement of the separation of this population intofunctionally distinct subpopulations (Lansdorp et al., 1990). For example, it wasshown that CD34+ cells that co-expressed CD45RA and low levels of CD71were highly and selectively enriched in granulocyte-macrophage clonogenicprogenitors and that cells which did not express any of these two cell surfacemolecules were highly enriched for LTC-IC. Further discrimination of thisCD34CD45RkCD71 - population was recently made possible by the use of anantibody directed against the glycoprotein, CD38, (Reinherz et al., 1980). Lowlevels of CD38 expression correlated with cells giving rise to undifferentiatedcolonies in liquid cultures (Terstappen et al., 1991) and also with cells which16preferentially responded to a combination of hematopoietic growth factors(Rusten et al., 1994). As shown in the results section of chapter 3, CD34iinCD3&0 bone marrow cells are also highly enriched in LTC-lC and relativelydepleted in clonogenic progenitors. Other surface antigens such as ckitb0 (Gunjiet al., 1993), HLADRb0 (Brandt et al., 1990; Sutherland et al., 1989), Thy-1(Baum et al., 1992; Craig et al., 1993) and CD4I0 (Louache et al., 1994) havebeen shown to be present on primitive human bone marrow cells and useful fortheir purification.1.2.3 Regulation of hematopoiesisAs discussed above, steady state hematopoiesis requires the daily productionof an enormous number of blood elements (-P5 x 108 and 5 x 1011 respectivelyfor adult mouse and human, (Abkowitz et al., 1995)) which originate from a verysmall population of bone marrow cells. Regulation of this complex process canbe simplistically pictured as the combined effects of external influences (cell-cell, cell receptor-growth factor ligand and cell-matrix interactions), andintracellular signaling events and consequent transcription factors regulationwhich can initiate or repress transcription of multiple genes whose products areultimately the effectors of proliferation and differentiation (Orkin, 1995). Therelative contribution of each of these various levels of control is not clear. Forexample, to what extent do external factors determine versus permitdifferentiation? Conversely, are there key events only determined by intrinsicprocesses? Is the role of external factors only to promote survival and triggerproliferation? Likely central to these regulatory mechanisms however are themolecules that control gene expression, the transcription factors. The followingsections provide an overview of some of these regulators and examines in17detail a group of transcription factors belonging to the homeobox gene familythat are strong candidates for being hematopoietic regulators. Regulation of hematopoiesis by external factorsHematopoietic growth factors (HGF) currently represent the most extensivelydescribed external regulators of the hematopoietic system (see Metcalf, 1993for a detailed review). HGF are necessary for proliferation, survival and possiblydifferentiation of hematopoietic cells (Metcalf, 1993). The number ofcharacterized HGF now exceed 20 and the list is still growing (Eaves, 1995;Metcalf, 1993). Besides erythropoietin (Krantz, 1991; Spivak, 1986) and alsopossibly thrombopoietin (Bartley et al., 1994; Foster et al., 1994; Gurney et al.,1995; Lok et al., 1994), the majority of these factors appear to have overlappingfunctions with the potential to act on progenitors of different committed lineages(Metcalf, 1993). For some of these factors, the nature of this “redundancy” maybe due to the sharing of a common receptor subunit (e.g. IL-3, IL-5, GM-CSFand IL-6, IL-il, LIF, OSM respectively share a unique common p-subunit:(Miyajima et al., 1993; Taga and Kishimoto, 1992)) which appears to beinvolved in intracellular signaling.An interesting notion that has come out of the numerous studies done ongrowth factors is that primitive hematopoietic cells only proliferate in thepresence of two (or more) growth factors (recruitment) (Migliaccio et al., 1991;Miura et al., 1993) whereas more mature progenitors can respond to a singlegrowth factor (Metcalf, 1993). Although primitive hematopoietic cells can berecruited into proliferation by the combination of various cytokines, netexpansion of HSC has not yet been documented (Bodine et al., 1992; Li andJohnson, 1994; Muench et al., 1993; Sutherland et aL, 1993). This may implythe existence of as yet unidentified HGF, but also raises the possibility that HSC18self-renewal may be predominantly regulated by other mechanisms such assignaling through adhesion molecules and / or by poorly understood “internalregulators”.Molecules with the potential to inhibit primitive hematopoietic cellproliferation have also recently emerged as key regulators (Graham et al.,1990). TGF-j31 and MIP-la have been best characterized and shown to havepotent inhibitory effects on CFU-S cycling in vivo (Lord et al., 1992), in vitro(Bodine et al., 1992), and on primitive hematopoietic progenitors (Cashman etaL, 1990; Eaves, 1991; Hatzfeld et al., 1991; Jacobsen et al., 1994).Therefore through both positive and negative regulators, proliferation ofhematopoietic cells can be externally controlled by various cytokines. Thesecytokines exert their proliferative / inhibitory effects through cellular receptorswhich upon activation transmit the appropriate signals to activate nuclearfactors via a complex signaling system (signal transduction). The nuclearfactors that activate “effector” genes involved in proliferation and differentiationof hematopoietic cells can therefore be “influenced” by external regulators but,in some instances, may also independently regulate these processes. After abrief overview of the role these nuclear factors play in the initiation oftranscription, the next section will concentrate on some of the transcriptionfactors that have been implicated in the regulation of proliferation anddifferentiation of mammalian hematopoietià cells. Regulation of hematopolesis by intracellular factorsUnlike procaryotic organisms, eukaryotic RNA polymerase II - the enzymeresponsible for transcription of messenger RNA - cannot initiate transcription byitself. It requires several general transcription factors (TFIIA to TFIIJ, see Figure191 .2) for DNA binding and transcription initiation. For most genes, the corepromotor includes a TATA box (TATAAA) which is located approximately 25 bpupstream of the transcriptional start site (Breathnach and Chambon, 1981).Thus, the TATA box represents the minimal promotor to allow transcription.Additional levels of regulation are the result of other nuclear factors such astranscriptional activators that bind to a multiplicity of regulatory regions some ofwhich are found in the proximity of the TATA box while others can be verydistant, and together recruit the RNA polymerase II complex to initiatetranscription as illustrated in Figure 1.2 (see Ernst and Smale, 1995 for adetailed review). Other nuclear factors termed transcriptional repressors cannegatively regulate gene expression via several mechanisms. For example,transcriptional repressor of the Id gene family can repress transcription bydimerization with known activators such as E2A (Kreider et al., 1992).ActivatorSeciuence ç7TFHD is the TATA binding TEITFllA stabilizeITATA binding,holoenzymeFigure 1.2. Schematic representation of initiation of transcription by the RNApolymerase II.The activator sequence (black box) binds transcription activators (rectangle) which, in its mostsimple form contains a DNA-binding domain and an “activator” domain that recruits thetranscriptional complex through TFIIB (black circle) to the TATA box. TFIIE is involved in initiationof transcription. Abbreviations: pci., polymerase; TF1IA to llH, transcription factor hA to IIH; TFIID isthe TATA-binding protein (TBP). Adapted from Molecular Biology of the Cell 3rd ed., Chapter 9,Garland publishing N.Y.(and probably the rest of thepolymerase complex)20The transcriptional activators (and also some repressors)characteristically contain distinct DNA-binding domains which have been usedto group them into various families. Some of the well characterized familiesinclude the zinc finger proteins which includes members such as Krox-20,Ikaros, etc., (see later in text); basic leucine zipper proteins (e.g. Fos, Jun, etc);the helix-loop-helix proteins such as SCL (see later); the helix-turn-helixproteins (homeobox genes, see later); and many more (reviewed in Ernst andSmale, 1995; Harrison, 1991; Nichols and Nimer, 1992)The recognition of the involvement of some of these nuclear factors invarious leukemias has provided strong arguments implicating them inhematopoietic cell growth and differentiation (reviewed in Nichols and Nimer,1992). In several hematopoietic malignancies for example, the products fromthese genes were either found at increased and / or persistent levels due totheir transcriptional activation by various mechanisms (chromosomaltranslocations e.g. c-myc under the control of an immunoglobulin promotor inBurkitt lymphoma: t(8;14) (Cesarman et al., 1987); retroviral insertion e.g. Spi-1(Moreau-Gachelin et al., 1988)) or found in the form of a fusion proteins (e.g. t(1;19): E2A-PBX, (Kamps et al., 1990)) resulting from chromosomaltranslocations that unite two normally distant genes (reviewed in Nichols andNimer, 1992). Recently, more direct strategies aimed at detecting proteins thatdisplay DNA-binding activity to specific regulatory sequences of erythroidspecific genes previously identified as being mutated in a hereditaryhemoglobin regulation disease (Martin et al., 1989) have been successfullyused to identify hematopoietic “active” transcription factors (e.g. GATA-1, (Tsaiet al., 1989)).21Further insights into the roles such transcription factors may play inhematopoiesis have been obtained from the modulation of their expressionusing gene disruption or overexpression strategies. For example GATA-1deficient cells could not contribute to erythropoiesis in chimeric mice (Pevny etal., 1991) and studies of in vitro differentiation of GATA-1 deficient (X-linked)embryonic stem (ES) cells have demonstrated the absolute requirement for thisfactor in early hematopoiesis. Interestingly these same studies showed thatdefinitive hematopoiesis could be partially rescued by other GATA-likecontaining proteins (Weiss et al., 1994). Similar in vivo experiments havedemonstrated the absolute requirement for the transcription factor NF-E2 inmegakaryocytic development (Shivdasani, 1995). Genetic disruptionexperiments have also demonstrated the necessity for rbtn2 in primitiveerythropoiesis (Warren, 1994) and of SCL / Tal-1 in primitive and definitiveerythropoiesis (Shivdasani et al., 1995). Genetic disruption of another GATAprotein, GATA-2 proved to be essential for definitive multilineage developmentsuggesting a role for GATA-2 in early hematopoietic cells (Figure 1 .3) (Tsai etal., 1994). The possible function(s) of many other transcription factors such asIkaros (see later), PU.1 (see later), Pax-5 and E2A, (Bain et al., 1994); c-myb,(Mucenski et al., 1991); Idl, (Sun, 1994) have now been analyzed using similarsystems (reviewed in Orkin, 1995).Although studies of transcription factors active in hematopoietic tissueshave only begun recently, no single factor that shows strict specificity for thehematopoietic system has yet emerged (Georgopoulos et al., 1992; Yomogidaet al., 1994). Thus, as for most other mammalian differentiation systems, theconcept has emerged that cellular identity is defined by the combinatorial actionof regulatory molecules expressed in a time and site specific manner and alsoby the accessibility of the DNA targets they regulate.22Results obtained from these studies however point to the existence of ahierarchy of transcription factors in the hematopoietic system. Two factorspotentially highly positioned in this hypothetical hierarchy are Ikaros and PU.1.Ikaros is a member of the zinc-finger family of transcription factors and hasprotein sequence similarity (57 amino-acids) to Hunchback— the product of oneof Drosophila’s segmentation genes (see below; Georgopoulos et al., 1992).The Ikaros gene gives rise to five or six alternate transcripts differentiallyexpressed in lymphoid cells (Georgopoulos et al., 1994; Hahm et al., 1994;Molnar and Georgopoulos, 1994) and can activate transcription of lymphoidspecific genes such as CD36 chain and terminal deoxynucleotidyltransferase(TdT) (Georgopoulos et aL, 1992; Hahm et al., 1994). Genetic disruption ofIkaros by gene targeting in embryonic stem cells has resulted in completedisappearance of T, B and NK cells in the homozygous Ikaros deficient micesuggesting that this gene is obligatory for the generation of early lymphoidrepopulating cells (Georgopoulos et al., 1994).PU.1 is the product of the proto-oncogene Spi-1 (mutagenesis site forFriend virus) and is a member of the Ets family of transcription factors (Paul etal., 1991). It is expressed in monocytic, B lymphoid and erythroid cells (Galsonet al., 1993; Klemsz et al., 1990). PU.1 has been implicated in the control ofexpression of lineage-specific genes such CD11b and the CSF-1 receptor (cfms) (PahI et al., 1993; Zhang et al., 1994) and in immunoglobin gene activationin B cells (Pongubala et al., 1992). It may also be involved in the regulation of -globin gene expression since binding sites for this factor are present in intron 2of this gene (Galson et al., 1993) and, PU.1 overexpression can transformerythroblasts (Schuetze et al., 1993). This gene thus appears to regulate targetsfound in more than three lineages. In accordance with these findings, embryoswith genetic disruption of PU.1 have severely impaired hematopoiesis in all23lineages (except megakarypoiesis) and die at ‘-‘day 17 post conception (Scott etal., 1994). The exact nature of death is uncertain but is likely caused by hydrops(severe anemia) in a significant proportion of the homozygous mice.Therefore loss of function of Ikaros or PU.1 causes major perturbations inmultiple lineages. This is strikingly different from the case of GATA-1 or NFE-2whose effects appear to be more limited (see Figure 1.3).Totipotent HSClymphoidIkaros progenitorsG ATA- 2CFU-GM CFU-Meg. CFU-Ery. B cell prog. Tcell prog.Figure 1.3. Possible hierarchical organization of some transcription factorsinvolved in hematopoletic development.Abbreviations: prog., progenitors; CFU, colony-forming-unit; GM, granulocytes-macrophages;Meg., megakaryocytes; Ery., erythrocytes.This picture however is likely far from complete, particularly from the pointof view of understanding those factors involved in the earliest stages ofhematopoiesis. Several investigators have now speculated that strongcandidates would be those transcription factors conserved through evolutionand implicated in embryonic development (Kreider et al., 1992; Krumlauf, 1994;Tanigawa et al., 1993). Such genes include members of the zinc finger familysuch as Krox genes (Swiatek and Gridley, 1993), Trithorax, some segmentationgenes and, of the helix-turn-helix family such as the homeobox genes. Themyeloidprogenitors24homeobox gene family has recently come under scrutiny and initial analysis ofhematopoietic cell lines has revealed the expression of more than 40 distinctmembers of this family (reviewed in Lawrence and Largman, 1992).Furthermore, some of these genes have now been clearly implicated in humanleukemias (Lawrence and Largman, 1992). The following section reviews someof the current knowledge about the classification, function, regulation andtargets of these homeobox genes and evidence implicating them inhe matopoi esi s.1.3 Homeodomain-containing transcription factors1.3.1 Definition, classification and chromosomal organization of thehomeobox genesFor years geneticists working with fruit flies suspected that a special phenotypedescribed as “something that has changed into the likeness of something else”or homeotic mutation (Bateson, 1894) was the result of a single gene mutation(Bridges and Morgan, 1923). One of the most famous of these, initially calledNasobemia, (later called Antennapedia: leg transformation of an antenna(Duboule, 1994)) lead Gehring and others on the challenging path of identifyingthe mutations responsible for these phenotypes. Many years later, Lewis(Lewis, 1978) identified a gene complex (called bithorax) on Drosophilachromosome 3 which encoded for “substances controlling levels of thoracic andabdominal development”. Chromosome walk studies performed on thischromosome (Garber et al., 1983) yielded the first isolation of a homeotic genelater called Antennapedia. cDNA clones that hybridized to this new gene alsocross-hybridized to genomic D.NA outside the Antennapedia gene regionsuggesting the presence of other related genes perhaps constituting a new25gene family (Garber et al., 1983; McGinnis et al., 1984). Subsequently, it wasfound that this cross-hybridization was caused by a shared highly-conserved180 bp sequence in these genes that encoded a 60 amino acid domain referredto as the homeodomain (McGinnis et al., 1984). Thus the discovery of the firstDrosophila homeobox gene is barely 10 years old (McGinnis et al., 1984; Scottand Weiner, 1984). The 60 amino acid homeodomain forms a helix-turn-helixstructure that is believed to bind DNA (Hoey and Levine, 1988; Laughon andScott, 1984; Otting et al., 1990). In Drosophila, these genes are clustered in 2groups (Antennapedia and Bithorax) which together form a large complextermed HOM-C (Figure 1.4).Homeobox genes were also found in vertebrates including mice and man(McGinnis et al., 1984). The homeodomain of the first cloned mouse homeoboxgene was found to be very similar to that of the Drosophila (Carrasco et al.,1984). This discovery was among the first to suggest that the genetic controlsimplicated in development are evolutionary conserved. Interestingly, multiplemammalian homeobox genes were found to be clustered (Rabin et al., 1985)similar to that of the fruit fly and further their relative chromosomal positions arepreserved ((Boncinelli et al., 1989); see also Figure 1.4). A distinguishingfeature of the mammalian homeobox genes is their organization as four clusterseach containing 9 to 11 different genes located on separate chromosomes (7,17, 12 and 2 in man). It is believe that each cluster has originated by duplicationof a common yet unidentified ancestral cluster (Martin, 1987). The relativeposition of individual genes in each cluster has been conserved and memberslocated at the same relative position show the highest similarity constituting aso-called paralog group ((Boncinelli et al., 1989); Figure 1.4).26The name “Hox” was given to the 38 mammalian homeobox genes foundin the four clusters. The nomenclature of the Hox genes was initially confusingsince each gene was labeled according to its discovery (Martin, 1987). A newnomenclature based on the relative position of each gene was coined at thethird Homeobox workshop (Scott, 1992). The old and new nomenclature ofeach Hox gene has been reported elsewhere (Scott, 1992) and only the newnomenclature is shown in Figure 1 .4 and used throughout this thesis.Within the homeobox gene complex of the fruit fly, there are a series ofgenes which also include a homeodomain but fail to cause homeotictransformation (DeRobertis, 1994). These genes have divergenthomeodomains. Divergent Homeobox genes are also present in themammalian genome but they are not found within any of the four Hox clusters(Boncinelli et al., 1989). These divergent genes are numerous and have beenclassified into more than 12 different “classes” and “families” some of which aredescribed here: the Pax/Prd genes are distinguished by a 182 bp DNA-bindingdomain which flanks the homeodomain, (Gruss, 1992); the POU genes containa distinctive domain of 15O amino acids just before the divergenthomeodomain and include the Pit-i gene responsible for murine dwarfism,(Rosenfeld, 1991); the Jjyj proteins contain two tandemly arranged cysteineand histidine-rich regions, (Karlsson et al., 1990); the Msx family, for muscle-specific homeobox, are recognized for their role in limb bud formation, (Robertet al., 1991); the HNF-1 -like factors, for hepatocyte-derived nuclear factors, aredistinguished by an extra-long homeodomain and an ability to dimerize andcontrol the transcription of albumin and other liver-specific genes (Frain et al.,1989); the endobox genes are known for their unique endodermal expression(Wright et aL, 1988); the J homeobox genes are highly conserved andclustered but with still unknown functions (Kim and Niremberg, 1989); the PBX27family was identified by the involvement of one of its members in a translocationassociated with a human lymphoblastic leukemia (Kamps et al., 1990); and, thejj class was identified by their expression in hematopoietic cells (Deguchi andKehrl, 1991; Deguchi et al., 1991). Further there are a series of “orphan”homeobox genes some of which will likely form new classes (DeRobertis,1994).Drosophila(HOM-C lab pb Did Scr Antp Ubx abd-A Abd-B-E1-Ei--Ei-Li-Ei-i’fi-Ei-Ei-1! 11 IVertebrates(H ox) Hox AHox Bj—If-I Hox CHox DParalog# 1 2 3 4 5 6 7 8 9 10 11 1213Hindbrain Trunk3 Direction of transcription of each gene 5,Direction of activation with retinoic acids treatmentRelative binding affinity to TAAT core sequenceFigure 1.4. Relationship between the HOM-C Drosophila Homeobox genes ofthe Antennapedia and Bithorax cluster complex and the various mammalian Hoxgenes.From left to nght the Drosophila members of the HOM-C complex are labial (lab) which is similar tothe group 1 paralog in vertebrate; proboscipedia (pb) with group 2; Deformed (Did) with group 4;Sex combs related (Scr) with group 5; Antennapedia (Ant), Ultrabithorax (Ubx), Abdominal-A (abdA) which have no mammalian homologs; and abdominal-B (abd-B) with group 9 to 13. Only therecent nomenclature (Ascona Workshop, Scott, 1992) is shown. As discussed later in the text,genes located at the 3’ end of the Hox clusters are expressed earlier and in more anteriorstructures of the embryo than those found at the 5’ end of the clusters. lndividualHox genes havethe same direction of transcription as indicated by one of the two arrows. Genes located at the 3’end of the cluster respond early to retinoic acid and at a lower concentration than that required toactivate 5’ genes. The ability of Hox genes to bind to the core DNA sequence C/GTAATTG alsodecreases 3’ to 5’ (Pellerin et al., 1994).281.3.2 Regulation by homeobox proteins of embryonic cell growthand differentiation.In Drosophila embryogenesis the HOM-C genes are major determinants ofsegment identity (Lewis, 1978). However genes of the HOM-C complex onlybecome active after two important sets of genes have pre-established someessential embryonic structures called parasegments2.Before and afterfertilization, products derived from more than 12 different genes are active inestablishing dorso-ventral and antero-posterior axis. These genes are calledmaternal-effect genes because the phenotype of the embryo is only dependenton the maternal alleles (Anderson et al., 1993; St. Johnston and NussleinVolhard, 1992). These genes are true morphogens since they regulate geneexpression in a gradient-dependent manner that is established by variousmechanisms such as mRNA localization (e.g. bicoid for anterior boundary;oskar for posterior boundary (Ephrussi and Lehmann, 1992)) or transmembranereceptors (“ Toll—dorsal—cactus—decapentaplegic” sequential activation forventral boundary which is involved in determination of the embryonic layers(Roth et al., 1989; Rushlow et al., 1989)).Once body polarity is established, a second set of at least 25 differentgenes regulated by the maternal-derived genes described above becomesactivated. These genes of “second order” are termed segmentation genes andfall into three groups (Kornberg, 1993; Nusslein-Volhard and Wieschaus, 1980):i) the gap genes which include the well studied zinc-finger-containingtranscription factors hunchback (which as said before shares similarity to Ikaros)and KrQppel that, in a cross-regulatory manner, delineates anterior segmentsfrom more posterior ones (Hulskamp and Tautz, 1991); ii) the pair-rule genes2 In this text, segment and parasegment have been used loosely. A parasegment is the basic unitof organization of the Drosophila body and does not correspond exactly to the segmentsobserved on the adult’s body.29such as even-skipped, paired, hairy and fushi tarazu, deletion of which inducesthe loss of every other segment (Akam, 1987); and iii) the segment-polaritygenes involved as indicated by their name in the polarity of individualsegments. Together these genes establish the various parasegments whereinthe HOM-C homeobox genes become active and lead to differentiation of eachparasegment (Figure 1.5)._ pior Maternal genesanteriorA1MM7::1tatboIparasegmentCell fate of individual parasegment:One pair of halters on T3 for the HOM-Cparasegment shown above Homeobox genesFigure 1.5. Three sets of genes must be sequentially activated for properdifferentiation of a specific body part.First maternal genes are establishing the antero-posterior and dorso-ventral axis. This occursthrough external influences on the oocyte by the maternal follicular cells (see arrows). Aconcentration gradient of a gene (bicaid) involved in the setting of the anterior pole is shown.Then segmentation genes become activated. The cell fate of individual parasegments isdetermined by the HOM-C homeobox gene.Thus HOM-C genes appear to be master regulators of differentiation.However, they need to be activated in a time and site specific manner. Failure todo so generally results in a homeotic transformation. Contrary to the DrosophilaHOM-C complex, very little is known about embryonic activation of mammalianHox genes besides these three characteristics. First there is a strict correlation30between the position each Hox gene occupies on the chromosome and its siteof expression. Genes found at the 3’ region of the Hox cluster (e.g. first paraloggroup: Al, Bi, Dl, see Figure 1 .4) are expressed in the anterior region of theembryo whereas genes found at the 5’ end are expressed in more posteriorregions. The expression “spatial colinearity” has been coined for thisphenomenon (Duboule and Dolle, 1989; Graham et al., 1989). Second, Hoxgenes from the different paralog groups are sequentially transcribed from the 3’to the 5’ region of the chromosome, a pattern reminiscent of globin activation.This phenomenon has been referred to as “temporal colinearity” (lzpisuaBelmonte et al., 1991). The third interesting feature about Hox gene expressionis termed posterior dominance. Hox genes expressed in the anterior region ofthe embryo will also be expressed, at lesser levels, in the more posteriorregions so that a complex array of Hox genes are expressed in more posteriorregions. The gene that has the greatest influence on cell fate of any posteriorregion is normally the one which shows no anterior overlap in expression (thelatest to be expressed). Based on this pattern of expression, loss of expressionof a Hox gene will generally result in “anteriorisation” of the structures normallyfound at this position and gain of function causes “posteriorisation” due to thesame phenomenon (see Krumlauf (1994) and McGinnis and Krumlauf, (1992))for a summary of loss- and gain- of function phenotypes of Drosophila andmouse HOM-C / Hox genes).During development Hox gene expression is not ubiquitous but ratherthese genes are expressed in paraxial mesoderm, neural tube, neural crest,hindbrain segments, surface ectoderm, branchial arches, gut, gonadal tissuesand limbs (reviewed in Krumlauf, 1994). In limb budding the expression of Hoxgene forms a two-dimensional network which again follows spatial andtemporal co-linearity patterns (Duboule, 1992).311.3.3 Upstream regulation of homeobox (HOM-C/Hox) geneexpression.Upstream cis-regulators of Hox gene expression are still poorly defined.Retinoic acids clearly upregulate Hox gene expression (Langston and Gudas,1992; Popperl and Featherstone, 1993). Hox genes themselves appear tocross- and auto-regulate their expression (Zappavigna et al., 1991).Segmentation genes also appear to regulate Hox gene expression (Kennison,1993). Zinc-finger-containing genes some of which are believed to beanalogous to gap segmentation genes (Krox-20) can directly regulate theexpression of specific Hox genes (Krox-20: (Schneider-Maunoury et al., 1993;Swiatek and Gridley, 1993); kreisler: (Frohman et al., 1993; McKay et al.,1994)). Recently GA TA-i was shown to possibly regulate HoxB2 (VieilleGrosjean and Huber, 1995) a gene apparently involved in globin regulation(Sengupta et al., 1994).A number of “trans-regulators” that function in chromatin structurepatterning have also been identified. The polycomb group are negativeregulators of HOM-C expression (Paro, 1990; Paro, 1993). They appear tofunction by inducing heterochromatin formation thus stabilizing suppression ofHOM-C genes expression (Kraumlauf, 1994). Interestingly, genetic disruption ofa recently characterized vertebrate polycomb gene, bmi-1, causes posteriorskeletal transformation and hematopoietic defects (van der Lugt et al., 1994).Another group of trans-regulators of HOM-C I Hox genes is the Trithoraxgroup (Kennison, 1993). These genes are involved in chromatin structureregulation3 but unlike the polycomb group, they appear to positively regulateHOM-C gene expression (Krumlauf, 1994). In Drosophila, loss-of-functionThe Trithorax genes are capable of transvection i.e. they can allow interaction of elements foundon two homologous chromosome32mutations of Trithorax result in phenotypes that mimic the loss of function ofmultiple ANT-C and BX-C genes (Kennison, 1993). Interestingly, one vertebratemember of the Trithorax group—HRX—is rearranged in a type of human acutelymphocytic leukemia involving chromosome 11 (Gu et al., 1992; Tkachuk et al.,1992). A summary of some of the known positive and negative regulators of theHOM-C / Hox gene complex is shown in Figure 1.6.Trithorax Retinoic Others? T rfamily acids a ge+geneChromatin— HOM-C / Hox,%- ,%Polycomb Othergroup homeoboxgenesFigure 1.6. Regulation of HOM-C / Hox gene expression.Both the Trithorax and polycomb group of genes affect chromatin structure. Cis-regulators of thecomplex (retinoic acids, Krox-20 and other homeobox genes) are shown. As presented later inthe text, specificity of Hox gene to their targets appears to be mediated by co-binding of anotherfactor (dark circle) which can be another Hox gene (Zappavigna et al., 1991), or anotherhomeodomain-containing molecules such as extradenticle (van Dijk and Murre, 1994) or itsvertebrate homolog PBX-1 (Chang et al., 1995).1.3.4 Downstream targets of HOM-C I Hox genes.Very little is yet known about the target genes regulated by genes of the HOMC / Hox clusters. Both in Drosophila and vertebrates, three HOM-C / Hox targetshave been identified by immunoprecipitation strategies involving Hox proteinbinding to genomic DNA targets (Gould et al., 1990; Graba et al., 1992;Tomotsune et al., 1993). Interestingly two of these three identified targets fallinto the category of adhesion molecules (Gould et al., 1990; Tomotsune et al.,1993). Other studies have demonstrated the interaction of various Hox proteinswith regulatory regions of some adhesion molecules (Edelman and Jones,1993; Hirsch et al., 1990; Hirsch et al., 1991; Jones, 1993; Jones et al., 1992;33Jones et al., 1992). Recently a growth factor member of the TGF-13 family(decapentaplegic: dpp) was shown to be directly regulated by Ultrabithorax, amember of the HOM-C complex (Sun et al., 1995). However these authors alsoshowed that at least one other factor (possibly extradenticle) was required forspecific and full activation of dpp by Ultrabithorax (Sun et al., 1995).Through their homeodomain, Hox genes bind to a tetranucleotide core(ATTA or ATAA; (Desplan et al., 1988)). Since there is such high similarity in thedifferent Hox gene homeodomains, questions have been raised about theability of specific Hox genes to activate unique and different targets. Clues tothese important questions have been found again in the Drosophila modelwhere it was recently shown that extradenticle (a non HOM-C homeodomaincontaining gene closely related to PBX-7) dramatically raises the DNA-bindingspecificity of Ultrabithorax (a HOM-C gene) to an oligonucleotide derived fromthe enhancer region of dpp (Chan et al., 1994; van Dijk and Murre, 1994).Similar findings were recently obtained with PBX-1 and multiple Hox proteins(Chang et al., 1995). Other studies have shown that Hox gene binding to targetDNA fragments can be modulated by Hox-Hox interaction (Zappavigna et al.,1994) and / or by different affinities exhibited by the various Hox proteinstowards the same target (see Figure 1.4, Pellerin et al., 1994). It remains to beproven whether these results will be confirmed in studies with native chromatin.1.3.5 Hox gene expression in post-developmental normal andneoplastic tissuesThere is accumulating evidence to suggest that Hox genes are also postdevelopmentally expressed in germinal and somatic adult mammalian tissues(Barba et al., 1993; Friedmann et al., 1994; Ko et al., 1988; Kongsuwan et al.,1988; Watrin and Wolgemuth, 1993).34Most (n=30) of the 38 vertebrate Hox genes are expressed in normalhuman adult kidney in a tissue-specific pattern (Barba et al., 1993; Cub et al.,1993). A difference in the relative levels of Hox expression and transcript size isobserved between medullary and cortical kidney tissues. Renal cell carcinomasalso express Hox genes but with a more variable pattern (Barba et aL, 1993).Cells from normal and neoplastic human colonic mucosa express mostHox genes (n=29, (De Vita et al., 1993)). Expression of various Hox genes canalso be found in other tissues such as liver, lungs (Barba et al., 1993) breast(Friedmann et al., 1994) and testis (Watrin and Wolgemuth, 1993). Althougheach tissue appears to have its own “Hox fingerprint” (Barba et al., 1993) thenature of this difference is less clear. It may, for example, simply reflect adifference in mature to primitive cell content of each specific tissue.1.3.6 Homeobox gene expression in normal and leukemichematopoietic cells1.3.6.1 Hox gene expression in leukemic cellsThe idea that Hox genes may be expressed and therefore regulatehematopoiesis originated from studies which analyzed the expression of alimited number of Hox genes in various leukemic cell lines (Kongsuwan et al.,1988; Lonai et al., 1987). Subsequent extensive studies of the expression of all38 Hox genes in four hematopoietic cell lines led to the identification of apeculiar pattern of Hox gene expression-activation which was not reminiscent ofthat found in embryonic development (Magli et al., 1991). In this study, theauthors found that cell lines with erythroid features (K562 and OCIM2)coordinately expressed most of the Hox B and C cluster genes and that celllines with myeboid features (HL-60 and U937) expressed predominantly A andC cluster genes. Therefore the “paralogous” Hox gene expression patterns as35found in embryonic development was not seen in these hematopoletic cell linesbut rather whole clusters (or large regions of a cluster) were turned on. It wasalso found that cluster D Hox genes were almost completely silent in every cellline analyzed (Magli et al., 1991). Subsequent studies confirmed this cluster-associated pattern of expression where cells with erythroid (K562, HEL, MBO2)but not myeloid (KG1a, KG1, HL-60, U937, THP1, PLB985, ML3, TMM) featuresexpressed all of the B cluster genes but one (HOXB1: Mathews et al., 1991;Vieille-Grosjean et al., 1992) and also that D cluster genes were poorlyexpressed (reviewed in Lawrence and Largman, 1992).Hox genes are also expressed in lymphoid cell lines (Lawrence et al.,1993; Petrini et al., 1992) but apparently not in resting B or T lymphocytes (Careet al., 1994; Petrini et al., 1992). Observed exceptions to this rule includeHOXB7 expression in CD8 human T cells (Inamori et al., 1993) and HOXC4 intotal T lymphocytes (Lawrence et al., 1993; Meazza et al., 1995). HOXC4expression is unusual because it appears to be restricted to lymphoid but notmyeloid cells lines (Lawrence et aL, 1993) and further it was recently shown thatthe HOXC4 protein migrates from the cytoplasm to the nucleus in newlyactivated lymphocytes (Meazza et al., 1995).Analysis of Hox gene expression in primary human leukemias (Celetti etal., 1993; Lawrence et al., 1995) has revealed expression patterns that appearto be specific for leukemia subtypes as described by the French-AmericanBritish (FAB) classification. For example, B cell chronic lymphocytic leukemias(CLL) express HOXB4 and 87 whereas T cell CLL also express HOXB2 (Celettiet al., 1993). One striking example is that of HOXA1O which was foundexpressed in all acute myeloid leukemias examined but not in the promyelocyticsubtype (FAB M3) (Lawrence et al., 1995). Although expressed in leukemiccells, Hox genes have not yet been directly implicated in human leukemia. The36only report implicating a Hox gene in leukemic transformation is that of HoxB8which together with IL-3 is overexpressed in the murine leukemic cell lineWEHI-3B due to activation by insertion of an endogenous intracisternal Aparticle (lAP) sequence upstream of both genes (Blatt and Sachs, 1988). HoxB8and IL-3 when co-transfected into murine bone marrow cells was also found tobe highly leukemogenic (Perkins et al., 1990) whereas alone neither causedleukemia (Perkins and Cory, 1993; Wong et al., 1989).While Hox genes have not yet been shown to be involved in humanleukemias, non-clustered homeobox genes with a divergent homeodomain aremajor participants. PBX1, like extradenticle, appears to cooperate with Hox IHOM-C genes for DNA binding (see above). Fusion of the PBX DNA-bindingdomain, as a result of a chromosomal translocation t(1;19), with thetransactivation domain of another transcription factor (E2A) is associated withan aggressive human pre-B leukemia (Kamps et al., 1990). Another example isTCL3 (former HOX-li) a gene involved in T cell acute lymphoblastic leukemiaby virtue of translocation t(10;14) which juxtaposes the T-cell receptor S genepromotor to a region upstream of TCL-3 resulting in ectopic expression of thisgene (Hatano et al., 1991). TCL-3 function is still unknown but interestingly,mice with genetic disruption of this gene were found to be asplenic (Roberts etal., 1994).Numerous other divergent homeobox genes are expressed inhematopoietic cells. These include genes such as HLX (previously HB24), firstidentified as expressed in mitogen-stimulated human B lymphocytes and later inCD34-’- bone marrow cells (Deguchi and Kehrl, 1991a; Deguchi and Kehrl,1991b; Deguchi et al., 1991); the HOP gene found in HL-60 cells (Hromas,1992); the Prh gene isolated from AMy-transformed chicken monoblasts37(Crompton et al., 1992); and the HEX gene whose expression seems to berestricted to the hemopoietic system (Bedford et al., 1993). Hox gene expression and function in normalhematopoietic cells.At the time the research presented in this thesis was initiated, Hox geneexpression in normal hematopoletic cells had not been systematically studied.The reasons for this included the recent recognition of Hox genes as possibleregulators of hematopoietic cells, the technical difficulties in obtaining highnumbers of “pure” functionally and phenotypically-defined bone marrowsubpopulations, the low level of expression of these genes and the absence ofavailable full sequences of most Hox cDNA. Direct evidence for Hox proteinfunction in hematopoiesis has also only recently become available. Indeed onlyone report was published when this project was initiated (see later, (Shen et al.,1992)).There are now however several lines of evidence that implicate Hox genesin the regulation of hematopoiesis. Three studies have described the use ofantisense oligonucleotides to down-regulate mRNA levels of specific Hoxgenes (Care et al., 1994; Takeshita et al., 1993; Wu et al., 1992). In the first ofthese studies (Wu et al., 1992) murine bone marrow cells were first exposed forfour hours to HoxB7 antisense oligonucleotides and then plated in semi-solidcultures supplemented with serum and growth factors. Granulocytemacrophage colony-forming-cells were found to be reduced by approximatelyfour-fold in the cultures initiated with the cells treated with the antisenseoligonucleotide. Erythroid and megakaryocytic progenitors were unaffected bythis oligo. Effects of antisense oligonucleotides directed against HOXC6 wereexamined and shown to suppress the formation of colony-forming unit-erythroid(CFU-E) without affecting earlier erythroid progenitors (BFU-E) or myeloid38derived colonies (Takeshita et al., 1993). Evidence for Hox gene involvement inhuman T cell proliferation has also been provided recently (Care et al., 1994).Upon phytohemagglutinin (PHA) stimulation, mature human T lymphocytessequentially express Hox B cluster genes (3’ to 5’) (Care et al., 1994). Antisensetreatment of PHA-stimulated T cells with HOXB2 and HOXB4 (two 3’ genes)markedly inhibited proliferation.Several studies have now been reported in which the effect ofoverexpression of a Hox gene was examined in cell lines or in primary cells.Cells of the HL-60 line can be induced to differentiate into morphologicallyidentifiable granulocytes or monocytes by treatment with differentiating agentssuch as retinoic acids or dimethyl sulfoxide for granulocytic differentiation orwith phorbol ester or vitamin D3 for monocytic differentiation (Lill et al., 1995).HOXB7is not expressed in undifferentiated HL-60 cells (Magli et al., 1992) butis readily detectable following monocytic differentiation with vitamin D3 andremains undetectable in cells undergoing granulocytic differentiation (Lill et al.,1995). Overexpression of HOXB7in this cell line inhibited granulocytic but notmonocytic differentiation. Together with the antisense study of Wu et al.described above, these findings implicate HOXB7 as an important regulator ofmyeloid differentiation of human (Lill et al., 1995) and murine (Wu et al., 1992)hematopoietic cells. In another study, HOXB6 was overexpressed in K562 cellsand resulted in the reduction of erythroid features of this cell line characterizedby decrease globin synthesis, heme content and surface glycophorinexpression (Shen et al., 1992).HoxB8, the immediate neighbor of HoxB7 was overexpressed in murinebone marrow cells (Perkins and Cory, 1993). In this study the authors used areplication-defective retroviral vector to infect murine bone marrow cells39(Perkins and Cory, 1993). Generation of non-tumorigenic IL-3-dependentmyelomonocytic cell lines was readily obtained by simply growing cells in highconcentration of lL-3. The cell lines would however differentiate upon reductionof lL-3 concentration. Most mice reconstituted with these transduced cells werefree of leukemia for at least seven months but approximately 20% eventuallydeveloped a myeloid leukemia which in some cases was associated withrearrangement of the lL-3 gene.Studies involving the divergent homeobox gene HLX (murine hix: (Allenand Adams, 1993; Allen et al., 1995) human HLX: (Deguchi et al., 1992))showed that it may have important roles in both myeloid (Deguchi et al., 1992)and lymphoid (Allen et al., 1995) cell proliferation and differentiation. RecentlyTCL-3 was shown to also generate IL-3-dependent immortal cell lines whenoverexpressed in murine bone marrow cells (Hawley et al., 1994). These celllines were not leukemogenic when reinjected into syngenic recipientssuggesting that a secondary “hit” may be necessary in order to allow fulltransformation as found in the t(1 0;1 4) human T cell leukemia.1.4 Thesis ObjectivesAs reviewed in the previous sections, several lines of evidence point to Hoxhomeobox genes as important regulators of hematopoiesis. These include theobserved expression of multiple Hox genes in primary leukemias andleukemic cell lines analyzed so far; the perturbation in hematopoietic cellgrowth and / or differentiation upon induced alteration in specific Hox geneexpression; and, finally by their clear involvement in cell growth anddifferentiation in embryonic development and hence the likelihood the playsimilar roles in the continuous process of hematopoietic development.Nevertheless at the time this project was initiated, there was extremely limited40direct evidence of Hox gene expression and function in normal primitivehematopoietic cells. Three major questions were thus address during thecourse of this research:1- Are Hox homeobox genes expressed in CD34 human bone marrowcells?2- If so, are they differentially expressed in functionally and phenotypically“distinct” subpopulations of CD34+ cells?3- What role do Hox genes play in the regulation of proliferation anddifferentiation of early hematopoietic cells?To address these questions, work was carried out towards three specificaims as presented in chapters 2 to 4.1- Generation of representative and extended-length cDNA from purifiedsubpopulations of human hematopoietic cells to enable characterization of Hoxgene expression patterns (Chapter 2).2- Characterization of Hox gene expression in purified human bone marrow CD34subpopulations and identification of novel and / or differentially expressed Hoxgenes in these purified subpopulations (Chapter 3).3- Examination of the functional role of candidate Hox genes in the regulation ofproliferation and differentiation of primitive hematopoietic cells by modulatingtheir expression in primary bone marrow cells (Chapter 4).41CHAPTER 2.A RT/PCR method to generate representative cDNA from smallnumber of hematopoletic cells42.1 SummaryThe study of Hox gene expression in rare hematopoietic populations (<0.01% ofbone marrow cells: less than 5,000 cells available) is a formidable challengethat requires the use of PCR-based techniques. Available methods foramplification of total cDNA from small cell numbers remain however suboptimalwith respect to yields due to frequent manipulations, representation of raretranscripts and generation of products spanning longer mRNA. This chapterdescribes the optimization of an RT-PCR method that overcomes some of theselimitations. The resulting methodology is demonstrated to maintain relativelevels of total cDNA and yield products that extend at least 1 .75 kb 5’ of thepolyadenylation site of even rare transcripts from cell numbers ranging fromhundreds to thousands.4 The method validated in this chapter has been used and described in these two publishedmanuscripts:1-Sauvageau G. et al., (1994). Differential expression of homeobox genes in functionallydistinct CD34 subpopulations of human bone marrow cells. Proc. NatI. Acad. Sd. USA 91:12223-12227 (see next chapter).2-Lawrence H.J., Sauvageau G., Ahmadi N., Lau T., Lopez A.R., LeBeau M.M., Link M.,Humphries R.K. and Largman C. (1995). Stage and lineage-specific expression of the HOXA1Ohomeobox gene in normal and leukemic hematopoietic cells. In press, Experimental hematology.It was also extensively used in the two following manuscripts recently submitted:1- Helgason C., Sauvageau G., Lawrence H.J., Largman C. and Humphries, R.K. (1995).Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cellsdifferentiated in vitro. Manuscript Submitted2-Hough M.R., Chappel M.S., Sauvageau, G., Takei F., Kay R. and Humphries, R.K.(1995). Dramatic reduction in lL-7 responsive clonogenic progenitors in mice over-expressing theHeat-Stable antigen. Manuscript Submitted.42A RT/PCR method to generate representative cDNA from smallnumber of hematopoietic cells2.2 IntroductionThe continuing evolution of cell purification strategies and PCR have greatlyenhanced the accessibility of molecular analysis of populations of cells found ata low frequency. As described in the introduction, primitive hemopoietic cellsfound at frequencies of less than one in ten thousand bone marrow cells cannow be enriched by cell sorting techniques to near homogeneity. However thecurrent low yields of these highly purified cells (hundreds to thousands)continue to pose significant hurdles to the quantitative analysis of geneexpression.Several strategies have now been described for using PCR methods toamplify cDNA from a low number of cells (Belyavsky et al., 1989; Brady et al.,1990; Doherty et al., 1989; Domec et al., 1990; Don et al., 1993; Dumas et al.,1991; Froussard, 1992; Lambert and Williamson, 1993; Rappolee et al., 1989).Losses of nucleic acids during multiple manipulations and difficulties inobtaining amplified cDNA that adequately represents the initial mRNA remainmajor concerns (Lambert and Williamson, 1993). One difficulty encountered hasbeen that of striking a balance between obtaining cDNA that preserves relativelevels of even rare transcripts versus generating the longest possible cDNA.Using a procedure which minimizes precipitation steps and employs suboptimalreverse transcriptase conditions, Brady et al. have reported generation ofrepresentative cDNA from as few as one hematopoietic cell (Brady et al., 1990).However, this method, by design, limits the transcript size to approximately 700bp or less, which can hamper identification of coding regions or conserveddomains (such as homeodomains) that may be distant from the polyadenylation43site. Previous efforts to obtain cDNAs encompassing 5’ mRNA sequences havegenerally increased the difficulty of the technique mainly because ofincompatible buffer components. This has resulted in complicated protocols thatcan affect reproducibility and yield (Belyavsky et al., 1989). Single tubeconditions (Don et al., 1993) have recently been described but these can becomplicated by the necessity of polylinker addition to the double strandedcDNA. Procedures have been described for obtaining more extended amplifiedcDNA (Belyavsky et al., 1989) but their applicability to limited cell numbers iscompromised by frequent manipulations and precipitation steps which can leadto significant loss of the nucleic acids.Most recently, random primers have been used during first strandsynthesis of cDNA and in subsequent amplification by PCR (Froussard, 1992).By eliminating tailing or linker addition steps, this approach decreases thecomplexity of the technique and should generate products spanning 5’ and 3’region cDNA sequences. However, the random primers have the potential toamplify genomic DNA and ribosomal RNA which may prove problematic in thestudy of gene expression.This chapter describes a reproducible and relatively straight-forwardmethod for the generation of amplified cDNA from a low number of cells thatprovides requisite sensitivity, representativeness and the capacity forgenerating cDNA that encompasses the more 5’ region of mRNA. The resultingprotocol has been tested using limiting cell numbers and mixtures ofhemopoietic cell lines known to differ in expression of specific genes. Theseresults demonstrate that this method enables the generation of cDNA thatretains adequate relative message abundance of even rare transcripts asexemplified by Hox genes, and includes sequences at least 1 .75 kb 5’ to the44end of mRNA. By its simplicity and reliability, this optimized method shouldenhance the opportunities for comparative gene expression studies betweendifferent subpopulations of cells in instances where cell number are limiting andrepresentation of long-length mRNAs is required.2.3 Materials and Methods2.3.1 RNA extraction and first strand cDNA synthesis.The choice of RNA extraction was made on comparison of published methods(Chomczynski and Sacchi, 1987; Cumano et al., 1992; Domec et al., 1990) anda commercially available kit (Pharmacia QuickPrep Micro mRNA PurificationKit). Two of these methods, one based on affinity purification (PharmaciaQuickPrep mRNA micro purification), the other as described by Cumano et al.(1992) gave consistent recovery of the RNA starting with 1,000 to 10,000 cells(data not shown). Because of its simplicity the latter method was chosen. Inbrief, 1,000 to 10,000 hematopoletic cells were pelleted and the supernatantremoved carefully. Cells were lysed in 50 .iI of a guanidium isothiocyanatesolution (5 M GIT, 20 mM 1,4-dithioerythritol (DTT), 25 mM sodium citrate pH7.0, 0.5% Sarcosyl) and the nucleic acids precipitated by adding 25 .tl of 7.5 Mammonium acetate containing 40 mg of glycogen as carrier and 2 volumes of95% ethanol. After two 70% ethanol washes, the pellet was dried at roomtemperature and resuspended in 3 of DEPC-treated water, heated at 65°Cfor 5 minutes and left at 22°C for 3 minutes. Reverse transcription conditionswere scaled down and modified from those previously described (Ausubel etal., 1991). To the 3 l.tI of RNA, the reverse transcription mixture consisting of 3 .tlof DEPC treated water, 2 .il of 5X RT buffer (GIBCO, BRL), 1 i.l of 0.1 M DTT, 0.2tl of 25 mM dNTPs (500 mM), 0.2 p.1 of the oligo dT primer at 1 mg/mI (6omers:5’CATGTCGTCCAGGCCGCTCTGGAC-AAAATATGAATTCT(4)-3’as described45by Brady eta!. (Brady et al., 1990), Oligos Etc., Portland, Oregon) and 0.1 jil ofplacental RNAase inhibitor (10 U/, GIBCO, BRL) was added. This mixture wasincubated at 37°C for 5 minutes and 0.5 jil of MMLV Superscript reversetranscriptase (200 U/I, GIBCO, BRL) with or without 0.1 ml of AMV (20 u/I,Pharmacia) was added. The reaction was left at 40 to 42°C for 1 hour and heatinactivated at 70°C for 10 minutes.2.3.2 Homopolymeric dA Tailing.After heat inactivation of the reverse transcriptase(s), the cDNA was precipitatedwith 5 of 7.5 M ammonium acetate and 30 .il of ethanol as described abovewith the help of a linear polyacrylamide carrier (Gaillard and Strauss, 1990).This carrier was chosen because when compared to tRNA, or to the absence ofcarrier, more consistent results were obtained. This is in accordance with resultsalready published (Gaillard and Strauss, 1990). When compared to mockcontrols, it was also found that this carrier did not interfere with the enzymaticactivity of all enzymes (including Taq polymerase, terminal deoxynucletidyltransferase (TdT) and the reverse transcriptases) used in this protocol (data notshown). The pellet was washed once with 70% ethanol, dried at roomtemperature and resuspended in 5 l of a tailing solution consisting of 1 p1 of5X tailing buffer (GIBCO, BRL), 0.5 .il of 100mM dATP (10 mM final, Pharmacia)and 3.5 j.iI of distilled water. After the pellet was resuspended, 0.5 p1 of TdT (15U/p1, GIBCO, BRL) was added to the solution and incubated at 37°C for 15minutes followed by a 10 minutes heat inactivation of the enzyme at 70°C.2.3,3 Amplification of the cDNA.PCR conditions for amplification of cDNA were optimized as described inresults. Final conditions were as follows: To the 5 p1 of tailed cDNA, 36.75 p1 ofa PCR solution were added. This solution consisted of 25 p1 of a 2X buffer (2046mM Tris pH 8.8, 100 mM KCI, 10 mM MgCI2), 4 ti of the oligo dT primerdescribed earlier (1 mg/mI in Tris pH 7.5), 0.5 iI of nuclease free BSA(10mg/mi, Sigma), 0.25 j.ii of triton X-100, 5 p.1 of water and 2 p.1 of a nucleotidemix consisting of dCTP, dGTP and dTTP each at 25mM to provide 1.0 mM finalof each nucieotide including the dATP previously added in the tailing reaction.The tubes were incubated at 92°C for 2 minutes before the addition of 1.0 p.1 ofgene 32 protein (Pharmacia), cooled on ice for 2 minutes and 1 p.1 of Taqpolymerase (5U/1.ti, G1BCO, BRL) was added to the reaction. The cDNA wasamplified using an Ericomp thermal cycier (Ericomp, San Diego, CA) with thefollowing parameters: 94°C for 1 mm., 55°C for 2 mm. and 72°C for 10 mm. for40 cycles except for the first cycle which was performed at an annealingtemperature of 370C. The ramp temperatures were found to be critical in orderto get good amplification and for this cycler were: 72°C-94°C, 0.32°C/sec;94°C-55°C, 0.44°C/sec; 55°C-72C, 0.28°C/sec.2.3.4 Southern Analysis of Amplified cDNA.A 10 p.1 aliquot of the total amplified cDNA was electrophoresed in a 1%agarose gel and passively transferred for 6 hours onto an ionic nylonmembrane (Zeta-probe, BioRad). Probes were labeled with 32P-dCTP (3000Ci/mmol; Amersham) by random priming and purified on a Sephadex-G50column before hybridization. Blots were hybridized for 20 hours at 60°C in 4.4 XSSC, 7.5% formamide, 0.75% SDS, 1.5mM EDTA, 0.75% skim milk, 370 mg/mIof salmon sperm DNA and 7.5% Dextran sulfate. Membranes were thenwashed twice at 60°C for 30 mm. each in 0.3 X SSC, 0.1% SDS and 1 mg/mi ofsodium pyrophosphate. When necessary, greater stringency washes wereperformed (0.1 X SSC, 0.1% SDS and 1 mg/mi of sodium pyrophosphate). Forreprobing, membranes were stripped in a 1 % SDS solution at 95°C for 20 to 3047minutes and tested for the absence of a signal by overnight exposure to KodakX-OMAT XAR5 film. Probes were as follow: HOX B4 an EcoRI/ Xbal 0.75 kbfragment of the genomic sequence obtained from the American Type CultureCollection (ATCC, Rockville, MD); HB24, a full-length cDNA including thedivergent homeodomain kindly provided by Dr. J. Kehrl; neomycinphosphoribosyl-transferase (neor), a 1 kb fragment derived from pMC1 Neo(Thomas and Capecchi, 1987); and f3-actin, a 2.0 kb chicken cDNA probe. Forthe detection of c-myc expression 2 probes subcloned from a c-myc genomicsequence (ar-kFkushdi et al., 1983) were used: a 5’ probe spanningnucleotides 67 to 455 of c-myc cDNA is a Sac-l/ Xhol 773bp fragment and thesecond is a 1 .9kb PvuIl 3’ probe spanning the last 563 nucleotides (exon 3) ofc-myc cDNA.482.4 Results and DiscussionIn an effort to develop a reliable method for obtaining representative andextended-length amplified cDNA from small cell numbers, I have focused bothon optimizing PCR conditions in order to reduce the number of PCR cycles andon reducing the manipulation of the nucleic acids to avoid potential loss. Keyconsiderations for the choice of a final protocol were simplicity andreproducibility together with the generation of products that included raremessages that spanned as much 5’ mRNA sequence as possible.2.4.1 Condition Adjustments.Several known critical variables affecting PCR amplification including pH,magnesium, deoxynucleotide and primer concentrations were first optimized.First strand cDNA was prepared from O.lj.ig of total RNA previously extractedfrom HL-60 cells by cesium chloride gradient and amplified under conditionsinitially based on these as described by Brady et al. (1990) and subsequentlymodified as described below. Amplified cDNA was evaluated fromquadruplicate preparations for each condition by Southern blot analysis using a13-actin probe. Optimal cDNA amplification was found with pH8.8 and with dNTPand magnesium concentrations of 1 and 5mM respectively. Primerconcentration as tested was not found to be an important determinant ofamplification (data not shown). Even under these optimized conditions it wasstill necessary to use at least 100 PCR cycles (Taq DNA polymerase was readded at cycle no. 50) to obtain sufficient material for Southern analysis of raretranscripts (data not shown).To make this method readily usable for the analysis of rare transcripts, itwas necessary to seek to further improve the efficiency of the amplification by49studying the effect of other factors such as the addition of gene 32 protein(Schwarz et al., 1990) and the use of a low KCI buffer (Ponce and Micol, 1992).Although gene 32 protein of bacteriophage T4 was shown to improve theefficiency of Taq polymerase in its ability to generate longer PCR fragmentsusing specific primers, its value in amplification of total cDNA using oligo dTprimers has not been reported. As shown in Figure 2.1B to 2.1D, the addition of1.0 p1 of gene 32 protein to PCR reactions from 25,000 total human bonemarrow and RNA from 3,000 HL-60 cells resulted in an appreciable increase inthe amount (approx. 1 log) and average size range (approx. 2 fold) of the cDNAas assessed by ethidium bromide stain (Figure 2.1B) and, as discussed ingreater detail below by Southern blot analysis using probes for the 5’ region(Figure 2.1C) or 3’region of c-myc (Figure 2.1D). Variations of KCI and Trisconcentrations in the PCR buffer were also assessed. The low KCI bufferdescribed by Ponce and Micol (Ponce and Micol, 1992) was less efficient in oursetting. However, using a buffer with a modest increase in Tris and KCIconcentrations (respectively 12 and 62 mM instead of 10 and 50 mM)noticeably and reproducibly improved the yield of the amplified cDNA (data notshown).50Figure 2.1. Evaluation of PCR amplified total cDNA obtained from limitednumbers of HL-60 or low density human bone marrow cells.(A) c-myc 2.2 kb cDNA schematic showing the respective location of the 5’ (black box) and 3’probes (hatched box). The facing arrows indicate relative location of the c-myc specific primers. (B)Agarose gel electrophoresis and ethidium bromide staining of aliquot (10 ii.I) of the cDNAprepared from 3,000 HL-60 or 25,000 unseparated low-density human bone marrow cells usingthe amplification strategy described in the Methods section. (C) Southern blot analysis of theamplified cDNA with a probe to the 5’ region of c-myc (autoradiogram exposed for 1 day at -70°C).(D) Membrane of panel C rehybridized to a probe to the 3’ region of c-myc (exposed for 35minutes). (E) Membrane of panel D rehybridized to a full-length homeobox gene HB24 probe(exposure for 6 hours at room temperature). (F) Product of amplified HL-60 cDNA reamplifiedusing c-myc specific primers flanking an intron. Panels B,C,D,E; lane 1, lambda marker cut withHindlll + EcoRl; lane 2, human bone marrow cells amplified without gene 32 protein; lane 3,human bone marrow cells amplified with gene 32 protein; lane 4, HL-60 cells; lane 5, HL-60 cellreaction without reverse transcriptase; lane 6, HL-60 cell reaction without tailing; lane 7, HL-60amplified with gene 32 protein. Panel F: lane 1, HL-60 cells; lane 2, HL-60 cell reaction withoutreverse transcriptase; lane 3, HL-60 cells without tailing; lane 4, 123 bp ladder.2.2 kb c-myc cDNAA. 511234567 1234B.C.D.E.--F.4L 950560‘Al 95056051Further modifications, after RNA purification, included reduction in thenumber of precipitation steps to one using a very reliable linear polyacrylamidecarrier. A precipitation step between the tailing reaction and the PCRamplification has been omitted by: i) performing a precipitation with ammoniumacetate after the reverse transcription to resolve buffer incompatibility and, ii)minimizing the volume of the tailing solution thus allowing adjustment of thetailing deoxynucleotide to high concentration that assures an homopolymericdeoxyadenosine tail of the first cDNA strand. The final concentration of thedNTP in the PCR reaction was adjusted by taking into account the highconcentration of the dATP in the tailing reaction (which is theoretically onlymarginally consumed in the tailing process) explaining the absence of thisnucleotide in the PCR mix.Together, the improvements made to minimize nucleic acid loss and tooptimize the amplification efficiency resulted in high yield of amplified cDNAthereby allowing a decrease in the number of PCR cycles to 40 from 100 ormore.2.4.2 Presence in Amplified cDNA of Sequences Distant fromthe 3f end of mRNA.Gel electrophoresis and ethidium bromide staining of amplified cDNA preparedas above revealed an average size of approximately 800-900 bp (Figure 2.1 B).To better assess if sequence distant from the 3’ end of mRNA was represented,Southern blot analysis was performed on amplified eDNA prepared from HL-60cells and normal human bone marrow cells using probes for 5’ versus 3’ c-mycsequences (Figure 2.1C and D respectively), and, second by attempts toreamplify cDNAs with primers directed at a 191 bp region located 712 bp fromthe c-myc polyadenylation site (Figure 2.1F). As shown in Figure 2.1C and522.1 D, both the 5’ c-myc region probe (which spans nucleotides 67 to 455 of the2.2 kb c-myc cDNA), and a 3’ c-myc region probe (which spans the last 563nucleotides (exon 3) of the 2.2 kb c-myc cDNA) hybridized to amplified cDNAfrom HL-60 or bone marrow. Significantly, higher signals were observed for HL60 cells consistent with the known overexpression of c-myc in these cells asdetectable by standard Northern analyses of total cellular RNA (Figure 2.2) andsuggestive of preservation of relative mRNA levels by this procedure.1 2 3Figure 2.2. Comparative expression of c-myc in normal bone marrow, K562 andHL-60 cells by Northern blot analysis.5 pg of total cellular RNA was loaded in each lane. HL-60 (lane 1), K562 (lane 2), or total humanbone marrow (lane 3) cells. Probes were to the 5’ region of c-myc (upper panel) as described inFigure 2.1, orto f3-actin (lower panel).In both bone marrow and HL-60, signal intensities for 5’ region sequencewere very strong indicating significant levels of amplified cDNA extending aminimum of 1.75 kb 5’ of the polyadenylation site. Further amplification of cDNAusing c-myc specific primers, resulted in the expected 191 bp product (Figure2.1 F), again confirming the presence of amplified cDNA sequence at least 700bp from the polyadenylation site. It is noteworthy that amplified cDNA obtainedfrom reverse transcription reactions in which homopolymeric tailing withdeoxyadenosine was omitted did hybridize to the 3’ but not 5’ c-myc region53probe (lane 6 of Figure 2.1C versus 2.1D); this material also did not give rise toa PCR product with specific c-myc primers (Figure 2.1 F, lane 3), suggesting thattailing is important to obtain maximal length products but also that some degreeof amplification can occur in the absence of tailing.Based on melting temperature, I hypothesized that hybridization of theoligo dT-based primer to poly-adenosine (polyA) stretches of greater than 4-5 inlength occuring in the second strand synthesis (37°C) was responsible for thisresult. This hypothesis was based on the fact that c-myc has a long polythymidine stretch in the 3’ region of its mRNA and that this region is spanned bythe 3’ probe used here. After reverse transcription of the mRNA, the thymidinestretch is converted to an adenosine one which could become a potential targetfor internal priming by the oligo dT primer. This is most likely during the secondstrand synthesis which is done at low annealing temperature (37°C). To test thishypothesis, this same blot shown in Figure 1 was rehybridized to a full-lengthcDNA probe for HB24, a homeobox gene which is devoid of such thymidinetracks (Deguchi et al., 1991a). As predicted, no signal was obtained with thisprobe in amplified cDNA prepared without tailing (Figure 2.1E, lane 6) but astrong signal was found in every instance in which samples were subjected toreverse transcripton and deoxyadenosine tailing. This result was furtherconfirmed by attempts to reamplify eDNA using c-myc specific primers located 5’to the polythymidine stretch; an amplified product was readily apparent forcDNA prepared with tailing (lane 1, Figure 2.1F) but was not observed in theabsence of tailing (lane 3, Figure 2.1 F).As illustrated by the Southern blot analyses in Figures 2.1 smears but notbands were obtained following hybridization. Similar results were observedwith all probes tested so far. This phenomenon is common to other PCR54methods amplifying total cDNA published so far (Belyavsky et al., 1989; Bradyet al., 1990; Domec et aL, 1990; Don et al., 1993; Dumas et al., 1991; Froussard,1992; Lambert and Williamson, 1993) and likely represents prematuretermination of the first strand synthesis and of later PCR amplification products.Internal priming during first and second strand synthesis may also contribute tothe range of sizes.2.4.2 Sensitivity and Representativeness of the cDNA.To test the sensitivity of this method for detecting moderate to rare abundancetranscripts, 10,000 HL-60 cells were mixed with murine hemopoietic BaJF3 cellsthat were previously transfected with a neomycin resistance gene. The level ofneor gene expression in these latter cells has been evaluated by Northernanalysis to be about 1% of the total messages (data not shown). As shown inFigure 2.3, amplified neor specific cDNA sequence was detected when as fewas 100 neo transfected cells were present in 10,000 HL-60 cells (1%) thussuggesting this method enabled detection of messages with a frequency ofabout 0.01%. Moreover, the neo’ signal intensities correlated well with theproportion of neo-resistant positive BaJF3 cells mixed with HL-60.5512345678Figure 2.3. Southern blot analysis using a near specific probe to test thesensitivity of PCR cDNA amplification.Ten thousand HL-60 cells were mixed with different numbers of neor -expressing BaIF3 cells andamplified cDNA prepared: lane 1, 10,000 HL-60 cells alone with no reverse transcriptase; lane 2,HL-60 cells alone with no tailing; lanes 3 to 8, 10,000 HL-60 cells mixed with 1 0, 1 0, 100, 10, 1,or no neor positive Ba/F3 cells. Autoradiographic exposure was 16 hours at -70°C.To further evaluate this method for the detection of rare messages, aprobe for the homeobox HOXB4 gene was hybridized to a nylon membranecontaining different amplified cDNA derived from a mixture of HL-60 (negativefor HOXB4 expression (Magli et al., 1991)) and K-562 cells (positive for lowlevel expression of HOXB4 (Magli et al., 1991)). As shown by Southern blotanalysis (Figure 2.4A), the intensity of the HOXB4 signal was directly correlatedwith the number of K-562 cells added and was absent in HL-60 cells alone.HOXB4 signal was detected with as few as 100 K-562 cells suggesting thismethod has the potential to detect rare messages present in a small proportionof the cells.56Figure 2.4. Southern blot analysis of PCR amplified cDNA to detect expressionof HOXB4 in HL-60 versus K562 cells.Panel A, hybridization to HOXB4 specific probe (exposure for 96 hours at -70°C); panel B,hybridization to 5’ c-myc specific probe (exposure for 72 hours at -70°C). Lane 1, 10,000 HL-60cells no tailing; lane2, 10,000 HL-60 cells alone; lanes 3-6, 10,000 HL-60 cells mixed with100 or 10 K562 cells respectively.Together these results suggest that this method provides a simplifiedapproach to obtain amplified cDNA from small cell number that enables thedetection of even low abundance message and the quantitative comparison ofexpression level of a given gene in different cell populations by Southernanalysis.It should be noted that internal priming on poly-adenosine tracts during thefirst strand cDNA synthesis would likely affect representation of a messagesince such cDNA would subsequently be “tailed” and PCR-amplified. Thus, for• 1 2 3 4 5 6B./57those transcripts with poly-adenosine stretches, there is a likelihood that theywill be over-represented as various partial cDNAs. This problem is likely to becommon to any oligo dT primer-based cDNA amplification procedure.Moreover, the chances of occurrence of internal priming are high during thegeneration of the second cDNA strand since it is performed at a lowtemperature (37°C). This is likely to result in loss of full-length product for mRNAthat have very long poly-thymidine tracts since the amplification of the smallerfragments would be more effective. For these two reasons, extreme care shouldbe used in comparing amplified cDNA signal intensities between two differentgenes or for the same gene when using two different probes. Nevertheless,these concerns should not apply when using the same probe to study therelative level of expression of a given gene in different cell populations.In conclusion, this simple method generates amplified cDNA thatencompasses sequence at least 1.75 kb distant from the polyadenylation site ofmRNA and preserves relative amplification of individual messages byoptimization of PCR amplification which includes the use of gene 32 protein andby minimization of internal priming by using only one primer and high annealingtemperature. Such a procedure was successfully used in the studies describedin the next chapter to characterize Hox gene expression in purified CD34-’-human bone cell subpopulations.58CHAPTER 3Differential expression of Hox homeobox genes infunctionally distinct CD34 subpopulations of human bonemarrrow cells55 The material presented in this chapter is essentially as described in:Sauvageau G., Lansdorp, P.M., Eaves C.J., Hogge D., Dragowska W.H., Reid D.S., Largman C.,Lawrence H.J. and Humphries, R.K. (1994). Differential expression of Hox homeobox genes infunctionally distinct CD34-’- subpopulations of human bone marrrow cells. Proc. Natl. Acad. Sci.USA 91: 12223-12227.Ms Vishia Dragowska and Dianne Reid are gratefully acknowledged for their help in cell purificationand culture, respectively.59Differential expression of Hox homeobox genes infunctionally distinct CD34 subpopulations of human bonemarrrow cells3.1 IntroductionStudies of Hox gene expression during the differentiation of primitive normalhematopoietic cells have been hampered by technical difficulties not only inobtaining the relevant subpopulations of appropriate phenotypic andfunctionally defined purity but also in the low numbers of such cells that wouldbe available (<104) given their known frequencies, making standard mRNAanalyses impossible. The recent characterization of the pattern of CD45RA andCD71 expression on CD34+ cells in normal human bone marrow now allowsthe reproducible isolation of relatively homogeneous progenitor populations oflineage-restricted erythroid, granulopoietic and more primitive cells (Lansdorpet al., 1990; Lansdorp and Thomas, 1991). Such populations might therefore beexpected to provide suitable starting material for investigating possible changesin Hox gene expression during early stages of hematopoiesis. Using a modifiedRT-PCR procedure that allows representative amplification of extended-lengthcDNAs from a few thousand cells (chapter 2), evidence for the expression ofmultiple Hox genes in primitive normal hematopoietic cells in now presentedtogether with the description of striking differences in the patterns of expressionof certain Hox genes within functionally distinct subpopulations of these cells.603.2 Materials and Methods3.2.1 Cell Purification.Low density cells (<1 .077g/cm3)of 5 different heparinized cadaveric humanbone marrows (CAD3, CAD6, CAD7, CAD9, CAD1O) were isolated bycentrifugation on Ficoll-Paque (Pharmacia LKB, Uppsala, Sweden) and keptfrozen in Iscove’s medium containing 2.5% human serum albumin and 7.5%DMSO. Cells from CAD3, CAD6 and CAD9 were thawed and stained withdirectly conjugated fluorescent antibodies to CD34 (8G1 2-Cy5), CD45RA (8d2-R-phycoerythrin, R-PE) and CD71 (OKT9-fluorescein isothiocyanate) washedtwice and resuspended in 2 mg/mI of Propidium Iodide (P-5264; SigmaChemical Co.) prior to sorting on a FACStarPlus® (Becton-DickinsonImmunocytometry, San Jose, CA) as previously described (Lansdorp andDragowska, 1992). After initial separation of CD34 cells on the basis ofCD45RA and CD71 expression (Figure 3.1A and 3.1C), CD34 CD45RAI0CD7110cells were stained with the CD38 (Leul 7-PE, BD)-conjugated antibodyand further separated into subpopulations expressing different levels of CD38(Figure 3.1 B and 3.1 D). Each subpopulation was subjected to two rounds ofsorting and cell purity was assessed after the first sort. Cells from each of the 5marrows were also stained with 8G12-Cy5 alone and separated into totalCD34 and CD34- subpopulations.3.2.2 Clonogenic Progenitor Assays.Aliquots from purified CD34+ subpopulations were assayed for coloniesderived from CFU-E, mature BFU-E, primitive BFU-E, CFU-GM or CFU-GEMM inmethylcellulose cultures containing fetal calf serum, 3 U/mI of highly purifiedhuman erythropoietin (100,000 units/mg, Stem Cell Technologies mc,Vancouver, B.C.), 50 ng/ml of human Steel factor (Amgen), 20 ng/ml each of61human IL-6 (Immunex), human GM-CSF (Sandoz), human G-CSF (Amgen) andhuman IL-3 (Sandoz) (Eaves, 1985).3.2.3 Long-term Culture.The long term culture-initiating cells (LTC-IC) content of each CD34subpopulation was evaluated essentially as described previously by placing2,400 cells on irradiated (8000 cGy) murine fibroblast feeder layers consistingof 1 .5x1 0 cells per 35 mm dish of both M2-1 0B4 cells (Lemoine et al., 1988)and SI/SI cells (Sutherland et al., 1990) engineered by retroviral gene transferto produce 10 ng/ml of human lL-3, 130 ng/ml of human G-CSF and 10 ng/ml ofhuman Steel factor. After 6 weeks, the clonogenic progenitor content of the LTCwas determined and the number of LTC-IC calculated by dividing this numberby 4 based on previous studies (Sutherland et al., 1990) and also confirmedhere by limiting dilution analysis (data not shown).3.2.4 cDNA Generation and Amplification.The generation and amplification of total cDNA obtained from each bonemarrow subpopulations was performed exactly as described in chapter PCR Amplification of Hox Homeodomains.The amplified total cDNA derived from each different bone marrowsubpopulation or cell line was subjected to a second round of PCR amplificationusing degenerate primer sets designed to match all of the different sequencesrepresented at the conserved ce-1 helix (5’(TC)(TCGA)GA(AG)(TC)T(CG)GA-(AG)AA(AG)GA(AG)TT 3’) and the x-3 helix (5’ C(GT)(ACGT)C(GT)(AG)TTCTG(AG)AACCA(ACGT)A 3’) regions of the human Hox gene homeodomains(Vieille-Grosjean et al., 1992). One percent of the total eDNA (0.5 iii) of eachsubpopulation was amplified and the resulting 118 bp products were subclonedand sequenced. Control preparations from original RNA not reverse transcribed62failed to yield a band indicating that contamination by genomic DNA was notencountered.3.2.6 Southern Blot Analysis of Total Amplified cDNA.One fifth of the total amplified cDNA prepared from each purified subpopulationor from cell lines was analyzed by Southern blot (see Chapter 2).Homeodomain free probes for all Hox genes but HOXB3 and B4 were preparedfrom subcloned sequences of cDNA or genomic clones initially kindly providedby Dr. E. Boncinelli. Probes for HOXB3, HOXB4, CD71 and MDR1 were derivedfrom clones obtained from the American Type Culture Collection (ATCC,Rockville, MD). Full length HB24 cDNA and CD34 cDNA sequences were kindlyprovided by Drs J. Kehrl and B. Seed respectively. Densitometric analysis wasperformed by using two different autoradiogram exposure times for each blot toensure linearity of the results.633.3 Results3.3.1 Characterization of Purified CD34 Bone Marrow CellSubpopulations.Antibodies directed against CD34, CD45RA and CD71 were used to isolate byFACS 3 phenotypically and functionally distinct subpopulations (I, IIM and IllE)from the low density fraction of 3 different normal human bone marrow samples(CAD3, CAD6 and CAD9). Figure 3.1A and 3.1C shows the FACS profile of 2 ofthese 3 marrows; CAD3 and CAD6 (CAD9 profiles were virtuallysuperimposable to CAD6, not shown). In each case, subpopulation I was furthersubdivided into two (CAD3, Figure 3.1B) orthree (CAD6, Figure 3.1D; CAD9,data not shown) subpopulations by sorting the cells based on their surfaceexpression of the CD38 antigen. After the first found of sorting, each of thesesubpopulations was analyzed and found to be over> 98% pure.0 I,,0 ,l ,2 ,oI 0,01.Z:;. •j IA•00 ,. 1. 1.2 1. • ,i 1Q IIC071 - FITCFigure 3.1. FACS profiles of the CD34 subpopulations isolated from bonemarrows no.1 (CAD3) and no.2 (CAD6).For both marrows, the cells in subpopulation I (CD34 CD45RA CD71 ) shown in Panels A and Cwere further fractionated according to their expression of CD38 into 2 fractions for marrow no.1(1.1 and 1.2) (Panel B) and into 3 fractions for marrow no. 2 (IA, lB, IC) (Panel D). Each of thesesubpopulations represent between 0.4 and 3% of the low density bone marrow cells and are>99% pure.For CAD3 and CAD6, functional assays were performed on aliquots fromeach of the purified subpopulations (Table 3.1). For both marrows, long termculture-initiating cells (LTC-IC) were detected exclusively in subpopulation I andsubdivision of these cells according to their expression of CD38 revealedfurther segregation of the LTC-IC to the CD38I0w-med cells. In contrast, cells ofsubpopulation I capable of proliferating in semi-solid assays in response to apotent cocktail of soluble growth factors (Steel factor + G-CSF + GM-CSF + IL-3+ IL-6) were more concentrated in the D3med-high fraction. As a result, forboth marrows it was possible to demonstrate that in the CD3810W fractions,LTC-IC constituted the major class of detectable hematopoietic cells and atleast 50% (CAD3; population 1.1) and up to 98% (CAD6; population IA) of theseBone marrow 1 Bone marrow 264w000CIIMIIIE100 t, 1.2II’____________BUi01.2.C’)00 ‘65could not be detected as clonogenic cells in a 4 week direct assay. Those fewclonogenic cells that were identified as co-purifying with the LTC-IC in theCD3810W fraction, although classified as CFU-GM, all showed delayed initiationand generated very large colonies of cells of a morphology in situ (i.e. withoutstaining) of granulocytes and macrophages. Subpopulations IIM and IllE werehighly and differentially enriched in granulopoietic and erythroid clonogeniccells, respectively, in each case to a purity of 20% with 5% of clonogenic cellsof the opposite lineage still present.Table 3.1. Clonogenic progenitora and LTC.ICb content of the different purifiedCD34+ bone marrow subpopulations.Fractions CFU-E BFU-E CFU-GM CFU-GEMM LTC-ICSorted(%purity) Enrich- (%purity) Enrich- (%purity) Enrich- (%purity) Enrich- (%purity) Enrichment ment ment ment mentCAD3:1.1 0 0 0 0 2.5 2 0 0 4.3 >21001.2 0 0 8.4 9 26 24 5.1 113 5.3 >2600IIM 0 0 0 0 26 24 0 0 0 <20IIIE 4.3 13 13 14 6.5 6 5.6 124 0 <20TotalMarrowC 0.3 1 0.95 1 1.1 1 0.05 1 <0002d 1CADS:IA 0 0 0 0 0.5 1 0 0 29 1450lB 0 0 1.5 3 5.8 8 0.5 167 29 1450IC 0.8 1 4 7 17 24 0.5 167 0 0IIM 0 0 0 0 24 34 0 0 0 0IIIE 14 18 22 37 2.8 4 0 0 0 0TotalMarrow 0.8 1 0.6 1 0.7 1 0.003 1 0.02 1aBased on analysis of 400 cells.bBased on analysis of 2400 and 1000 cells for marrow 1 and 2 respectively.°This part of the experiment was performed on a different aliquot of the same marrow thawed and assayed ata later time.dBased on the analysis of 6.6 x 1 4 cells.3.3.2 Hox Gene Expression in CD34 Subpopulations.To assess Hox gene expression in purified bone marrow cell subpopulations,total amplified cDNA were generated from 1,000 to 5,000 cell aliquots using theRT-PCR procedure described in chapter 2. Amplified total cDNA from thepurified CD34+ subpopulations were analyzed for Hox gene expression usingtwo different approaches. The first of these was aimed at obtaining an initial66indication of the range and potential pattern of Hox gene expression in theCD34 purified subpopulations. It was performed on one marrow (CAD3) andconsisted of reamplifying the cDNA derived from each subpopulation with a setof Hox gene specific degenerate primers that spanned a 118 bp region of thehomeodomain. Resulting products were subcloned and a total of 92 cloneswere sequenced. As summarized in Table 3.2, this analysis revealed theexpression of 12 different Hox genes in the various CD34+ subpopulations:seven from cluster A, three from cluster B and two from cluster C. No expressionof any cluster D genes was detected in these cells. In all, 82 of the 92 clonessequenced were found to belong to cluster A. This difference probably reflects ahigher expression of cluster A genes in these cells since, as reinforced by theanalysis of the cell line data described below, it is unlikely that the degenerateprimers used preferentially amplified this particular group of Hox genes.Interestingly, most of the cluster A sequences detected (34/46; 76%) in the mostprimitive subpopulations (1.1 and 1.2) were found to be within the 3’ region (Al toA6) of this cluster, whereas 27 of the 38 (71%) cluster A sequences found in themore differentiated subpopulations (IIM and IllE) were concentrated at the 5’region of this cluster (A7 to Al3). Also, of the 8 sequences belonging to clusterB, 6 were limited to subpopulation 1.1 and 1.2 suggesting a more restrictedexpression of this Hox cluster to the most primitive hematopoletic cells.For comparison, and to assess both the sensitivity and specificity of thisapproach, a similar analysis was carried out using equivalent numbers of cellsfrom the K562 (erythroleukemic) and HL-60 (promyelocytic) cell lines.Homeodomain sequences were obtained from 27 clones derived from K562cells and 22 clones derived from HL-60 cells. As also shown in Table 3.2, five ofthe seven expressed Hox genes detected in HL-60 were Hox A genes, while sixof the eight genes detected in K562 were Hox B genes. These results are in67agreement with previous reports of Hox gene expression in these 2 cell lines(Magli et al., 1991). Moreover, they are consistent with previous observations ofpreferential expression of Hox A genes in myeloid cell leukemias and Hox Bgenes in leukemic cell lines with erythroid phenotypes (Lawrence andLargman, 1992).68Table 3.2. Expression of Hox genes in purified bone marrow subpopulationsand in hemopoletic cell lines determined by sequence analyses of amplifiedhomeobox containing cDNAHox Purified subpopulationsb Cell linesgenesa (CAD3)1.1 1.2 IIM IIIE HL-60 K562Al +(2)A2 +(1)A3A4 +(3) +(6) +(1)A5 +(5) +(11) +(7) +(2) +(5)A6 ÷(4) +(3) +(1) ÷(1)A7 +(6) +(5) +(8)A9 +(5) +(6) +(8) +(4)AlO +(1) +(3) +(2)AllA13BiB2 +(2)B3 ÷(1) +(3)B4B5 +(2)8687 +(4) +(1) +(1) +(2)B8 +(2)B9 +(1) +(3)C4-6C8 +(1) +(1)C9 ÷(1) +(4) +(12)ClO-13Dl-13Total: 19 32 24 17 22 27aFor each cluster, the genes are listed as found 3’ to 5’ on their respective chromosome.bFunctional characteristics of each of these subpopulations are presented in Table 3.1.c.+.. indicates detection of one or more cDNA clones containing Hox gene specific homeobox sequence.The total number of independent clones identified for a specific gene is shown between parenthesis.3.3.3 eDNA Southern Blot Analysis of Hox Gene Expression inCD34 Subpopulations.To characterize further the extent and possible differential expression of Hoxgenes in early hematopoietic cells, the initial amplified total cDNA preparedfrom the various CD34 subpopulations was also analyzed by Southern blotusing homeodomain-free Hox gene specific probes. For marrow CAD6 the five69CD34 subpopulations were analysed using 11 different Hox gene probes(Figure 3.2A): 7 of the 9 known cluster B genes (B2, B3, B4, B5, B6, B8, B9), 4 ofthe 11 cluster A genes (A4, A5, A9, AlO) and one non-cluster homeodomaincontaining gene (HB24: Figure 3.2B) whose expression was recently describedas restricted to CD34 cells (Deguchi et al., 1992). Expression of 10 of these 11Hox genes was detected in all subpopulations with the exception of B6 whichwas not detected in any of the primitive subpopulations nor in HL-60 or K562cells (Figure 3.2A). On the other hand, HB24 appeared to be equally expressedin all subpopulations paralleling that of actin (Figure 3.2B). Densitometricanalysis of these blots showed that HOXB9, which is located towards the 5’ endof the B cluster, was expressed at similar levels in all CD34+ subpopulations,whereas B3 and B4 which are more 3’ cluster B genes were expressed at muchhigher levels (17 and 6-fold respectively) in subpopulation IA, which is highlyenriched in LTC-IC, than in IIM, which is enriched in myeloid progenitors anddevoid of LTC-IC. This differential expression of 3’ cluster B genes was similarlyobserved when subpopulation IA was compared with the erythroid cell enrichedsubpopulation IllE. These results suggest that the 3’ genes are preferentiallyexpressed in the most primitive hemopoietic cells in contrast to more 5’ geneswhich appear to be more uniformly expressed at least in the early stages ofdifferentiation. This finding was reinforced by the findings for Hox genes locatedin the middle of the B cluster (B5 and B7) whose expression was only slightlymore restricted to the more primitive subpopulations IA and lB.70B2B3A41•.B41A100.1. I0.060.140.I7 10.32— 11—0.6Figure 3.2A. Southern blot analysis of the total amplified cDNA derived fromeach CD34 subpopulation.Bone marrow no.2 (CAD6) and from HL-60 and K562 cells. Exposure times were adjusted tofacilitate comparison of the expression of individual Hox genes between the 5 subpopulationsstudied. For HOXA4, hybridization to HL-60 or K562 was not done. Vertical alignment of the blotsfollows the relative positions 3’ to 5’ of each Hox gene in its cluster. Each blot is accompanied by adisplay (right panel) that shows the normalized expression (to actin and to subpopulation IA) of thedifferent Hox genes found in subpopulations IA and IIM as determined by densitometric analysis.(B) The same membrane was also hybridized to probes for actin, CD34, -globin, P-glycoprotein(MDR1), CD71 and HB24 (see next page).A “Cta1.5-0.500.3b *A5 ..:‘B8 I .1A9B971B——i.Aj IaACTIN-GLOBINI‘ ;121 ICD34MDRCD71HB24Figure 3.2B. Southern blot analysis of the total amplified cDNA derived fromeach CD34 subpopulation.See legend of Figure 3.2A for abbreviations.This pattern also extended to the cluster A genes. As shown in the rightpanel of Figure 3.2A, expression of the 3’ gene HOXA4 was 7-fold higher insubpopulation IA than in IIM and, a relatively uniform expression for the 5’genes A9 and AlO was found. Again, for a gene from the middle of the cluster,HOXA5, expression was moderately elevated in subpopulation IA (3-fold higherthan in subpopulation IIM). Moreover, the relative expression of the six paralogs72analyzed (i.e., A4 and B4, A5 and B5 and, A9 and B9) was strikingly similar ineach of the five subpopulations suggesting that, as in embryonic development,a simultaneous expression of the Hox paralogs may occur during hematopoleticcell maturation.In order to examine the reproducibility of this apparent trend, Southern blotanalyses were similarly carried out on the four subpopulations of CAD3 and the5 subpopulations of a third marrow (CAD9) fractionated exactly as for CAD6(Figure 3.1). Two selected Hox probes (one 3’- HOXB3, and one 5’- HOXA1O)were used for this analysis (data not shown). Densitometric analysis of Hoxgene expression normalized to actin revealed again that HOXB3 expressionwas mainly limited to the most primitive subpopulations (15 and 28 fold higherin the CD34 CD38- cells of subpopulation IA than in subpopulation IIMrespectively for CAD9 and CAD3) and that HOXA1O expression was invariantamong all of the subpopulations.The significance of these patterns of Hox gene expression in the variousCD34+ populations analyzed was reinforced by several controls. First, the levelof CD34 expression in these subpopulations as detected by eDNA Southernanalysis was found to correlate with the surface expression of this antigen asstudied by FACS analysis with a progressive decline: lA>IB>IC>IIM=lllE (seeFigure 3.2B for cDNA Southern results). MDR1 expression was also higher inthe more primitive cell subpopulations, was not found in HL-60 and was presentat high level in K562 consistent with the expected expression pattern for thisgene (Chaudhary and Roninson, 1991). Similarly, CD71 expressiondetermined by cDNA analysis correlated with its surface expression (i.e., moreexpressed in the lIM and IllE subfractions, Figure 3.2B) and only the73subpopulation enriched for late erythroid progenitors (CFU-E, subpopulationIIIE) was found to express significant levels of 13-globin mRNA.3.3.4 cDNA Southern Analysis of Hox Gene Expression inCD34 Cells.To explore the possibility of additional changes in Hox gene expression withfurther hematopoietic differentiation, expression of two selected Hox genes,HOXB3 and HOXA1O, was analyzed in the CD34- fraction of mononuclear cellsfrom five different marrows compared to the total or CD34 fractions. Results ofSouthern blot analysis of total amplified cDNA generated from the variousfractions of 3 representative marows are shown in Figure 3.3 (CAD7, CAD9 andCAD1O). As expected from the analysis of CD34 subpopulations, expression ofboth HOXB3 and HOXA 1 0 was readily detected in the total CD34mononuclear cell fraction of the three bone marrows. In sharp contrast however,expression of both genes was virtually extinguished in the CD34- fraction.Consistent with these observations, the signal intensity from total mononuclearcells was lower in proportion to the percentage of CD34 cells.74B.M. 1 B.N4. 2 l1.N’l. 3 CELl, LINESI+,II+,II+,II_•••_— r ‘— r — r rInccc - z z— —Figure 3.3. Southern blot analysis of total amplified cDNA derived from the lowdensity cells fraction of three different marrows.Cells were sorted in unfractionated, CD34-enriched and CD34-depleted subpopulations. CD34-’-content of the unfractionated, CD34 and CD34- fractions were 5.5% / 95.8% / 0.4% for marrow 1(CAD7); 16.1% / 95.2% / 1.8% for marrow 2 (CAD9) and 7.3%; /95.5% / 0.7% for marrow 3(CAD1 0). Viability was over 95% in each fraction. No RT, no reverse transcriptase.I Ilifthll IS .iACTINHOX B3HOX AlO753.4 DiscussionThese studies document expression of at least 16 different Hox genes in highlypurified subpopulations of primitive CD34 human marrow cells. Moreover,these data suggest that the lineage- subfractions of these primitive cells arecharacterized by markedly higher levels of expression of certain Hox genes incomparison to their levels in Iineage subfractions. Detailed functionalcharacterization of these cell subpopulations indicates that the preferentialexpression is associated with a very primitive CD34 subpopulation containingall LTC-lC and relatively few lineage-restricted progenitors detectable in directcolony assays. This enhanced expression appears to involve genes located inthe 3’ regions of the A and B clusters. On the other hand, 5’ located genes wereexpressed at relatively equal levels in each of the primitive CD34subpopulations analyzed. Our results suggest that early differentiation eventsinvolve or are accompanied by a down-regulation (potentially 3’ to 5’) of class IHox genes. For some, such as HOXB3, this down regulation appears tocoincide with the earliest stages of hematopoietic differentiation whereas forothers, such as HOXA 1 0, expression may persist to later stages ofdevelopment. Interestingly, however, for at least two Hox genes examined here,expression was virtually extinguished with further progression to the CD34stage.Using various cell lines as prototypes of cells at different stages ofhemopoietic maturation, it has been suggested that expression of some Hoxgenes may be lineage-restricted (Lawrence and Largman, 1992; Magli et al.,1991; Mathews et al., 1991). For example, HOXB3 has been described aserythroid-specific because it is expressed in both OCIM2 and K562 cells, andboth of these cell lines exhibit some erythroid features (Magli et al., 1991;76Mathews et al., 1991). Our results failed to demonstrate preferential expressionof HOXB3 in normal human erythroid progenitors (subpopulation tIlE). In fact,this gene was expressed at about 20-fold higher levels in more primitivesubpopulations in which no CFU-E or BFU-E were detectable by comparison tothe only subpopulation in which these erythroid progenitors were found.Another Hox gene, HOXA1O has been similarly described as myeloid-restricted(Magli et al., 1991; Shen et al., 1989). However, in the present study, we foundthis gene to be equally expressed in all of the CD34 subpopulations.Using a growth factor-stimulated peripheral blood-derived CD34 purifiedsubpopulation, Giampaolo et al. (1994) concomitantly reported that HOXB3 isthe only Hox gene expressed in this “quiescent hemopoietic progenitor cellpopulation” and that there is a sequential activation (3’ to 5’) of the B clustergenes which follows growth factor stimulation of this purified population(Giampaolo et al., 1994). These results differs from those described in our studyin that we have observed down-regulation of Hox gene expression withmaturation of bone marrow cells. It thus appear that Hox gene expression differsin peripheral blood progenitors and that growth factor stimulation maysequentially recruit the clustered homeobox genes.Taken together our results combined with that of Giampaolo point to the‘xistence of a highly regulated program of Hox gene expression at the earlieststages of hemopoietic cell differentiation. The exact relationship between thesepatterns of mRNA expression and actual proteins levels will need to be clarified.Nevertheless, such findings set the stage for future identification of mechanismsregulating hematopoietic cell proliferation and lineage determination.77CHAPTER 4Overexpression of HOXB4 in Hematopoletic Cells Causes theSelective Expansion of More Primitive Populations in Vitro and inVivo6.6 The material presented in this Chapter is essentially as descirbed:Sauvageau G., Thorsteinsdottir U., Eaves C.J., Lawrence H.J., Largman C., Lansdorp P.M. and.Humphries R.K. (1995). Overexpression of HOXB4in Hematopoietic Cells Causes the SelectiveExpansion of More Primitive Populations in Vitro and in Vivo. Genes and Dev. 9; 1753-1 765.Unnur Thorsteinsdottir significant contribution to the work involving the CRU evaluation of themice transplanted with HOXB4-transduced cells is gratefully acknowledged.78Overexpression of HOXB4 in Hematopoietic Cells Causes theSelective Expansion of More Primitive Populations in Vitro and inVivo4.1 IntroductionIn the previous chapter, it was shown that most Hox A and B cluster genes areexpressed in CD34+ normal human bone marrow cells (Sauvageau et al.,1994). Purification of this CD34 bone marrow fraction into functionally distinctsubpopulations and RT-PCR-based analysis of Hox gene expression in thesecells has revealed two patterns of expression: one in which the level ofexpression of a given Hox gene (e.g. HOXAlO, B9, C8) was essentiallyinvariant in the different subpopulations and the other in which the expressionlevel was much higher (up to 40-fold) in subpopulations containing the mostprimitive hematopoletic cells (e.g. HOXB3, B4). No gene was found to be up-regulated in the more mature CD34+ cell subpopulations. Comparison of thelevels of expression of selected Hox genes in CD34+ and CD34 cells showedthat Hox gene expression was higher in the CD34+ cells and lower orundetectable in the CD34- cells. Together, these data suggest that Hox genesundergo down-regulation of expression with hematopoietic differentiation andfurther that some, such as HOXB3 and HOXB4, are almost exclusivelyexpressed in the most primitive bone marrow cells. Interestingly, this apparentlyhighly regulated program of Hox gene expression in hematopoiesis has strikingparallels with changes in expression associated with embryonic development(3’ to 5’) and is different from that reported in cell lines (i.e. paralogs, seegeneral introduction). Based on these results, the hypothesis that the patternsand levels of Hox gene expression play critical roles in determining primitivehematopoietic cell properties was generated.79To test this hypothesis, HOXB4 overexpression was engineered in murinebone marrow cells by retroviral-mediated gene transfer. The effects producedby this manipulation on the subsequent behaviour of the transduced cells andtheir progeny in vitro and in vivo was then analyzed. The results of theseexperiments show that the enhanced expression of HOXB4 can profoundly andselectively increase the proliferative potential of primitive hematopoietic cellswithout detectable effects on the relative or absolute numbers of mature endcells they generate in vivo.804.2 Materials And Methods4.2.1 AnimalsMice used as recipients were 7 to 12 week old (C57B1/6J x C3H/HeJ) Fl((B6C3)F1) and those used as bone marrow donors (C57Bl/6Ly-Pep3b xC3H/HeJ) Fl ((PepC3)F1) were male and female mice bred and maintained inthe animal facility of British Columbia Cancer Research Center from parentalstrain breeders originally obtained from The Jackson Laboratories (Bar Harbor,MA). (B6C3)F1 and (PepC3)F1 mice are phenotypically distinguishable on thebasis of allelic differences at the Ly5 locus: (B6C3)F1 mice are Ly5.2homozygotes and (PepC3)F1 mice are Ly5.1/Ly5.2 heterozygotes. All animalswere housed in microisolator cages and provided with sterilized food andacidified water.4.2.2 Retro viral GenerationThe MSCV 2.1 retroviral vector (kindly provided by Dr. R. Hawley; SunnybrookResearch Institute, Toronto, Ontario) contains a polylinker cloning siteimmediately 5’ to a murine pgk promoter-neo cassette (Hawley et al., 1992).The HOXB4 cDNA region encompassing the complete coding sequence, wasisolated as a BamHl fragment from a plasmid (kindly provided by Dr. E.Boncinelli, Ospedale S. Faffaele, Milan, Italy) and cloned upstream of the pgkneo cassette at the Xbal site of MSCV 2.1 by blunt-end ligation using standardprocedures (Davis et al., 1994b). To produce helper-free recombinantretroviruses, 10 tg of MSCV 2.1 and MSCV 2.1-HOXB4 plasmid vector DNAwere first transfected using calcium phosphate precipitation into both theecotropic GP+E-86 (Markowitz et al., 1988b) and the amphotropicGP+envAMl2 (Markowitz et al., 1988a) packaging cell lines. Virus containingsupernatant were harvested 24-48 hours after the transfection and used tocross-infect the amphotropic and ecotropic packaging cells in the presence of 681p.g/ml polybrene (Sigma Chemical Co., St. Louis MO). Infected cells were thenselected in 1 mg/mI G418 (Gibco/BRL Canada; Burlington, Ontario) to obtain apolyclonal population of amphotropic and ecotropic viral producing cells. Toincrease the viral titer of these cells, filtered supernatant from ecotropic andamphotropic virus producing cells harboring the same retroviral construct wereused to cross-infect these same cells 4 times. The viral titers of the GP+E-86-MSCV2. 1 -pgk-neo and GP+E-86-MSCV2. 1 -HOXB4-pgk-neo virus (hereaftercalled neo and HOXB4-neo) producer cells were 3-5 X 1 o6 CFU/ml and 3-5 Xio CFU/ml respectively as assessed by transfer of G418 resistance to NIH 3T3cells (Cone and Mulligan, 1984). Absence of helper virus generation in theHOXB4-neo viral producer cells was verified by failure to serially transfer virusconferring G418 resistance to NIH 3T3 cells (Cone and Mulligan, 1984). Theabsence of helper virus in serum of 5 mice transplanted with HOXB4-neo-transduced bone marrow (5 mice analyzed) was also confirmed using a rescueassay.4.2.3 Cell LinesThe ecotropic packaging cell lines, GP+E-86 and the amphotropic cell line,GP+envAMl2, used to generate the recombinant retroviruses were maintainedin HXM medium which consists of Dulbecco’s Modified Eagles Medium(DMEM), 10% heat-inactivated (55°C for 30 minutes) newborn calf serum(NCS) (Gibco/BRL), 15 jig/mI hypoxanthine (Sigma), 250 pg/ml xanthine(Sigma) and 25 .tg/ml mycophenolic acid (Sigma). Virus producing cells weremaintained in HXM medium supplemented with 1 mg/mI of neomycin analogG418 and for the amphotropic cell line in addition 200 ig/ml hygromycin(Sigma). Twenty-four hours prior to harvest of viral supernatant or co-cultivationwith bone marrow cells, virus producer cells were cultured in RPMI with 10%fetal calf serum (FCS) or DMEM with 10% NCS respectively. The murine82hematopoietic cell line FDC-P1 (Dexter et al., 1980) was maintained in RPMIwith 10% fetal calf serum and supplemented with 5% mouse pokeweedstimulated spleen cell conditioned medium (SCCM). The human hematopoieticcell line K562 (Lozzio and Lozzio, 1975) was maintained in RPMI with 10%FCS. Unless specified otherwise all cultures were maintained at 37°C in ahumidified atmosphere of 5% C02 in air. All media, serum and growth factorsunless otherwise specified were obtained from StemCeIl Technologies Inc.,Vancouver, B.C., Canada.4.2.4 Retro viral Infection of Primary Bone Marrow Cells andHematopoietic Cell LinesBone marrow cells were obtained from (PepC3)F1 (Ly5.1/Ly5.2) mice injectedintravenously 4 days previously with 150 mg/kg body weight of 5-fluorouracil (5-FU) in phosphate-buffered saline, by flushing femurs and tibias with DMEM 2%FCS using a 21 gauge needle. Single cell suspensions of 1-5 x io cells/miwere then cultured on a petri dish for 48 hours in DMEM containing 15% FCS,10 ng/ml human interleukin-6 (IL-6), 6 ng/ml murine IL-3 and 100 ng/mI murineSteel factor. All cells were then harvested and plated at 1-2 x io cells/mi in theabove medium supplemented with 6 ig/ml polybrene on viral producer cellmonolayers irradiated (1500 cGy X-ray ) the same day at 80-100% confluence.Cells were co-cultured for 48 hours with a medium change after 24 hours.Loosely adherent and non-adherent cells were recovered from the co-culturesby agitation and repeated washing of dishes with Hank’s balanced salt solutioncontaining 2% FCS. Recovered bone marrow cells were washed once and thencounted using a hemocytometer. All growth factors were used as dilutedsupernatant from transfected COS cells prepared in the Terry Fox Laboratory.Murine and human hematopoietic cell lines FDC-P1 and K562 were infectedby exposure to filtered (0.22 jim, low protein binding filter, Millipore, Bedford,83MA) viral supernatant from the ecotropic or amphotropic virus producer cellsrespectively. The viral supernatants were supplemented with 6 pg/ml polybreneand FCS to give final concentrations of 20%. For FDC-P1 cell infection, virussupernatant was additionally supplemented with 5% SCCM. Transduced cellswere selected and maintained in 1 mg/mI of G418.4.2.5 Transplantation of Retro virally Transduced Bone MarrowLethally irradiated 7-10 week old (B6C3)F1 (Ly5.2) mice (950 cGy, 11 OcGy/min,137Cs gamma- rays) were injected intravenously with 2 x io bone marrowcells derived from (PepC3)F1 (Ly5.1/Ly5.2) immediately after co-cultivation ofthese cells with HOXB4-neo or neo viral producer cells. The levels of Ly5.1donor-derived repopulation in recipients were assessed 12, 20 and 34 weekspost transplantation by flow cytometric analysis of peripheral blood samplesobtained by tail vein puncture (Rebel et al., 1994). In all animals > 86% of theperipheral blood leukocytes were of donor Ly5.1 origin. At these same timepoints peripheral blood cell counts and hematocrit were determined for someof these animals.4.2.6 In Vitro Clonogenic Progenitor AssaysFor myeloid clonogenic progenitor assays, cells were plated on 35mm petridishes (Greiner, Germany ) in a 1.1 ml culture mixture containing 0.8%methylcellulose in alpha medium supplemented with 30% FCS, 1% bovineserum albumin (BSA),104M (3-mercaptoethanol (13-ME), 3 U/mI human urinaryerythropoietin (Epo) and 2% SCCM in the presence or absence of 1 .4 mg/mI ofG418. To ensure random colony selection in single colony replatingexperiments, cultures were also supplemented with 500 ng/ml murine IL-3,which abrogates the size difference between colonies. Bone marrow cellsharvested from the co-cultivation with virus producer cells or from reconstitutedtransplant animals were plated at a concentration of 2 x io cells/dish or 2-4 x841 o cells/dish respectively. Spleen cells from animals transplanted withHOXB4-neo- or neo-transduced cells were plated 3 x io cells/dish or 3 x io6cells/dish respectively. Colonies were scored on day 12-14 of incubation asderived from CFU-GM, BFU-E or CFU-GEMM according to standard criteria(Humphries et al., 1981). In two experiments identification of colony types wasconfirmed by Wright staining of cytospin preparations of colonies. For pre-Bclonogenic progenitor assays, cells were plated in 0.8% methylcellulose inalpha medium supplemented with 30% FSC, io M f3-ME and 0.2 ng/ml of IL-7. Pre-B colonies were scored on day 7 of incubation.4.2.7 CFU-S AssayDay 4 5-FU bone marrow cells were harvested after infection by co-cultivationwith viral producers and injected immediately into lethally irradiated (B6C3)F1mice or after 1 week culture at an initial density of 1-5 x io cells/mI in 30%FCS, 1% BSA, io M 13-ME, 3 U/mI of Epo, 2% SCCM with or without 1.4mg/mI of G418. The number of cells that each mouse received was adjusted togive 10-15 macroscopic spleen colonies (2-4 x io bone marrow cellsharvested from co-cultivation with virus producer cells or a proportion of the 1week old liquid cultures, described above, corresponding to 2 x io or 1 xHOXB4- or neo-transduced input cells, respectively). CFU-S content of bonemarrow cells obtained from mice transplanted 20 weeks earlier with neo- orHOXB4-infected cells was also evaluated by intravenous injection of 2 x ioor 2 x io bone marrow cells/mouse respectively. Untransplanted lethallyirradiated mice were tested in each experiment for endogenous CFU-Ssurviving irradiation and consistently gave no spleen colonies. Twelve daysafter injection, animals were sacrificed by neck dislocation and the number ofmacroscopic colonies on the spleen were evaluated after fixation inTelleyesniczky’s solution854.2.8 Marrow Repopulating Ability (MRA) AssayLethally irradiated (B6C3)F1 mice were injected intravenously with 2 x io day4 5-FU bone marrow cells directly after they were harvested from the cocultivation with viral producer cells or with a proportion of these cells kept for 7days in the liquid culture described above, corresponding to 1.5 x io neo- orHOXB4-infected input cells. Thirteen days later 3 mice per group weresacrificed and femoral cells harvested, counted and pooled. Dilutionscorresponding to various proportions of a femur were then injectedintravenously into lethally irradiated recipients for macroscopic spleen colonyevaluation as described above. As a control to determine endogenous MRAsurviving irradiation in primary recipients, half a femur, pooled from twountransplanted lethally irradiated mice was assayed in three secondaryrecipients.4.2.9 Competitive Repopulating Unit (CRU) AssayBone marrow cells pooled from 3-4 mice transplanted 12, 16 or 20 weeksearlier with neo- or HOXB4-transduced cells derived from (PepC3)F1(Ly5.1/Ly5.2) mice were injected at different dilutions into lethally irradiated(B6C3)F1 (Ly5.2) mice together with a lifesparing dose of 1 x io competitorbone marrow cells from (B6C3)F1 (Ly5.2) mice. The level of lympho-myeloidrepopulation with Ly5.1 donor derived cells in these secondary recipients wasevaluated >13 weeks later by flow cytometric analysis of peripheral blood asdescribed (Rebel et al., 1994) in all experiments but for the secondary recipientswhere it was evaluated at 5 weeks post reconstitution. Recipients with 1%donor (Ly5.1) derived peripheral blood lymphoid and myeloid leukocytes asdetermined by the side scatter distribution of Ly5.1 + cells, were considered tobe repopulated by at least one CRU. CRU frequency in the test cell population86was calculated by applying Poisson statistics to the proportion of negativerecipients at different dilutions as described before (Szilvassy et al., 1990).4.2.10 DNA and RNA AnalysesSouthern blot analyses to assess proviral integration were performed aspreviously reported (Pawliuk et al., 1994) using standard techniques. High-molecular weight DNA was digested with Sstl which cuts in the LTRs toreleases the proviral genome or with EcoRl which cuts the provirus once torelease DNA fragments specific to the proviral integration site(s). Total cellularRNA was isolated using TRIzol (Gibco/BRL) and separated usingformaldehyde/agarose gel electrophoresis. The RNA was transferred to nylonmembrane (Zeta-Probe; Bio-Rad) prehybridized, hybridized and washed asdescribed (Davis et al., 1994a). Probes used were a Xhol/Sall fragment ofpMClneo (Thomas and Capecchi, 1987), Kpnl/Msel fragment of pXM(ER)-190which releases the full-length erythropoietin receptor cDNA, (kindly provided byA. D’Andrea) and full-length HOXB4 cDNA labeled as described (Lawrence etal., 1995).4.2.11 Western AnalysisTo detect HOXB4 protein, FDC-P1 and K562 transfected cells were harvestedand lysed in cracking buffer (1% SDS, 6 M urea, 1% 6-ME, 0.01 M sodiumphosphate, pH 7.2). Proteins (5 .tg) were subjected to SDS-PAGE in a 12.5%gel and transferred to nitrocellulose membrane. Membranes were incubatedwith a mixture of two polyclonal antisera (1/5,000 dilution for each) raisedagainst peptides deduced from the N-terminal and C-terminal regions flankingthe homeodomain of the HOXB4 protein, respectively (BAbC0, Richmond, CA),and incubation with secondary antibody coupled to alkaline phosphatase. Eachpeptide antisera was previously shown to specifically detect HOXB4 expressedas a bacterial fusion protein.874.3 Results4.3.1 Retro viral-Mediated Transduction of HOXB4 to MurineBone Marrow CellsIn an effort to achieve increased and persistent expression of HOXB4 inprimitive hematopoietic cells, the human HOXB4 cDNA was introduced intomurine bone marrow cells by retroviral-mediated gene transfer. This HOXB4cDNA was chosen based on its availability and its derivation from ahematopoletic tissue (Piverali et al., 1990). Of the 361 amino acids found in theHOXB4 protein, only 9 are divergent between human and mouse and none ofthese occur within the homeodomain. This high degree of similarity (97%) madeit very likely that murine and human HOXB4 would be interchangeable(Bachiller et al., 1994).The HOXB4 cDNA (Piverali et al., 1990) was inserted into the MSCVretroviral vector 5-prime to a phosphoglycerate kinase promoter (pgk)-drivenneo gene such that HOXB4 expression was driven from the promotor-enhancersequences contained within the viral long terminal repeat (LTR) (Figure 4.1A).The LTR sequences of MSCV were derived from a myeloproliferative sarcomavirus modified to show enhanced activity in embryonic stem cell lines andtherefore likely to have similar activities in primitive hematopoleitic cells (Grez etal., 1990; Hawley et al., 1992). High titer polyclonal viral producer cells weregenerated from the GP÷E-86 ecotropic packaging cell line using standardmethods. Integrity of the HOXB4-neo retrovirus was verified by Northern blotanalysis to detect the expected mRNA in viral producer cells (Figure 4.1 B) andby Western blot analysis to detect HOXB4 protein in transduced murine (FDCP1) and human (K562) hematopoietic cell lines (Figure 4.1C).FDC-P1 K5620 0 XG) 0 G) 0CFigure 4.1. Structure and expression of HOXB4 and control neo retrovirusesused in this study.(A) Diagrammatic representation of the integrated HOXB4-neo and the neo proviruses.Constructions were based on the MSCV2.1 vector in which the neo gene is linked to an internalmurine pgk promotor. Expected size of full-length viral transcripts and also those initiated from thepgk promoter are shown. SD and SA denote splice donor and splice acceptor sites. Alternatetranscripts derived from these sites are not shown. Restriction sites indicated are EcoRl (E), andSstl (Ss). (B) Northern blot analysis of the neo and HOXB4-neo viral producer cell lines. Themembrane was sequentially hybridized to a probe specific for neo or HOXB4. (C) Western blotanalysis of K562 and FDC-P1 cell lines transduced with the HOXB4-neo or the neo virus andprobed with a polyclonal antibody directed against a HOXB4 synthetic oligopeptide.88DHOXB4-neo3.9 kb1.3kbSs E SscD SA_______Ipgk-neoI neo2.7 kb1.3kb0 XG) 0C X— 3.9 kb— 2.7kb neo*— 1.3kb__— 3.9kbI-IOXB4BC—46kDaI[_j. — 3OkDa89The experimental strategy used to study the effects of HOXB4overexpression on the behaviour of primitive hematopoietic cells and theirprogeny is depicted schematically in Figure 4.2.5-FU injection Bone marrow Viral infectionharvestIn vitro cultures4d>II95Q cGyTransplantationA. In vitro culturesDay 0 G418 selection Day 7h ‘ ‘— repeat MR A and C F U - SMRA 13 d > 12 d>assayCFU-S % 12Iassay ColonyI clonogenic_____G-41 replatingI I I• •ILprogenitorassayB. In vivo transplantationl2to2OwksTransplantationPrimary recipient clonogenicprogenitorassayCRU assay CFU-S_______l6wksassaySecondaryCRU assayrecipientsTertiaryrecipientsFigure 4.2. Overview of the experiments and assays used in these studies.904.3.2 Increased Proliferative Activity in Vitro of ClonogenicProgenitors 0verexpressing H0XB4Initial studies examined possible effects of HOXB4 overexpression in committedclonogenic progenitor cells detected by their ability to give rise to myeloid,erythroid or myeloid/erythroid colonies in semi-solid culture. Bone marrow cellsfrom mice treated 4 days previously with 5-fluorouracil (5-FU) were co-cultivatedwith HOXB4 or control neo viral producer cells and 48 hours post infection wereplated in methylcellulose cultures. In three independent experiments, the genetransfer efficiency to clonogenic progenitors was similar for HOXB4 and controlneo viruses, with 58-70% of colonies showing G418 resistance (Table 4.1).Neo- or HOXB4-transduced bone marrow cells did not give rise to colonies inthe absence of exogenous growth factors showing that HOXB4 overexpressiondid not render clonogenic cells growth factor-independent. Neither were thereany differences detected in the proportions of different colony types generatedby HOXB4- or neo-infected cells (assessed by both in situ scoring and byWright staining of cytospin preparations of individually plucked colonies, Table4.1). This suggests that HOXB4 overexpression also does not alter the ability ofcommitted clonogenic progenitors to complete their differentiation into differenttypes of mature blood cells. However, HOXB4-infected cells did give rise tosignificantly more large colonies (containing >1000 granulocytes andmacrophages) than what was observed in control cultures containing neotransduced cells. The majority of these large colonies had a diffuse morphology.Only rarely could the type of dense colonies with a diffuse halo described byPerkins and Cory (1993) who studied bone marrow cells overexpressingHoxB8, be identified in our experiments.91Table 4.1. Clonogenic progenitor frequencies in bone marrow cellsimmediately post infection with HOXB4-neo or neo retrovirusesaVirus Total CFCb G-41 8r Distribution of Progenitor Classes (%± SD)per 1 0 cells (%± SD)CFU-GM CFU-GM BFU-E CFU-GEMMsmall largeneo 55±47 70±6.7 72± 11 16± 14 2.5± 2.5 9.0± 1.5HOXB4- 50 ± 31 58 ± 7.6 51 ± 8.5 41 ± l4 1.4 ± 2.4 6.9 ± 4.5neoa Results are expressed as mean ± S.D. from 3 experiments. In situ colony scoring wasconfirmed by microscopic examination of Wright stained cytopsin preparations of arepresentative sampling of individually plucked colonies.b Abbreviations: CFC, colony-forming ceHs; CFU-GM, colony-forming unit granulocytesmacrophages; CFU-GM large= more than 1000 cells per colony; BFU-E, burst forming uniterythroid; CFU-GEMM, mixed myeloid,erythroid colony forming unitc Significantly different from the neo control (Student t test, p<0.05)To further characterize the proliferative capacity of the HOXB4-infected cells,whole methylcellulose cultures were harvested seven days after plating andvarious proportions assayed in secondary methylcellulose cultures. The resultsobtained from two separate experiments revealed that the cells harvested fromthe primary assays of HOXB4-infected cells were able to generate two to threefold more secondary colonies than neo-transduced control cells obtained fromprimary assays initiated with the same number of original input cells.Furthermore, one third (32 ± 8%) of the colonies obtained in the secondarycultures of HOXB4-transduced cells were large in size (>1O cells/colony)whereas the proportion of such colonies in the assays of the replated neotransduced cells was much lower (3.5 ± 0.5%). To assess whether this increasein proliferation reflected a generalized effect of HOXB4 on the majority ofclonogenic progenitors, well isolated HOXB4- and neo-transduced G418-92resistant colonies were picked at random either 7 (Figure 4.3A) or 11 days(Figure 4.3B) after initiation of the primary cultures and one third of each colonythen individually replated into secondary cultures. Under these conditions,-60% of neo-transduced colonies did not replate whereas 80% of theindividually analyzed HOXB4-transduced colonies replated generating amedian of 160 and 450 secondary colonies per clone, respectively, in these 2experiments.A B> 3000 - 3000 02000- 20001000 - 1000_____0Cu 0• 400- 8 4000____1 0 00 0300- 30082 o o200 200_____>‘1100100 10 0 —===———--o—neo HOXB4 neo HOXB4Figure 4.3. The effect of HOXB4 overexpression on the ability of individualmethylcellulose colonies to form secondary colonies upon replating.In 2 independent experiments, well isolated HOXB4- or neo-transduced colonies from primarycultures were randomly picked after seven (A) or eleven days (B) of G418 selection inmethylcellulose cultures. Each dot represents the number of secondary colonies generated fromeach primary colony that was picked and replated in the same culture conditions described above.The calculated mean number of secondary colonies obtained per primary colony is indicated bythe broad dash. The difference in means was significant to p<O.005 for A and B (Two tailedStudent t test).934.3,3 HOXB4 Effects on the Maintenance in Vitro of Day 12CFU-S and Cells with Marrow Repopulating Ability (MRA)Based on the observed increase in the proliferative ability of progenitors with invitro clonogenic potential following their transduction with HOXB4, additionalexperiments were performed to determine whether similar effects on thebehaviour of earlier cells detectable as day 12 CFU-S or as cells, with marrowrepopulating ability (MRA) could be seen. For these studies, infected bonemarrow cells were assayed for CFU-S and MRA content immediately after theperiod of co-cultivation with viral producer cells and again after an additionalseven days in liquid culture in the presence of 1 .4 mg/mI of G41 8.Day 12 CFU-S frequencies of cells harvested immediately after cocultivation with HOXB4 viral producers were similar to those obtained for theneo control (Figure 4.4, day 0). However, after maintaining the HOXB4- or theneo-transduced cells in liquid culture for 1 week, the CFU-S content of thecultures initiated with the neo-transduced cells decreased to less than 1% ofinput (Figure 4.4, day 7). In contrast, the day 12 CFU-S content of the culturesinitiated with HOXB4-transduced cells increased to 200 - 500% of input (day 0)values (Figure 4.4). As a result, there were ‘-200 fold more day 12 CFU-S incultures initiated with the HOXB4-transduced bone marrow cells as comparedto neo-transduced controls at the end of a seven day period of incubation.Southern blot analysis of DNA extracted from 23 well isolated spleen coloniesproduced in recipients of HOXB4-transduced cells showed each of the 23colonies to be uniquely retrovirally marked indicating that the 2 to 5-fold netexpansion of day 12 CFU-S observed in these cultures was due to a polyclonalexpansion of HOXB4-transduced day 12 CFU-S or even an earlier cell type(data not shown). Wright staining of cell preparations obtained from thesespleen colonies showed a similar spectrum of late erythroid and myeloid94elements compared to neo control colonies (data not shown), indicating thatHOXB4 overexpression does not affect the pattern of differentiation that day 12CFU-S undergo during spleen colony formation in vivo.1o101LlCI)oleci0I...G)Figure 4.4. HOXB4 effects on day 12 CFU-S in vitro.Day 4 5-EU bone marrow cells were prestimulated and co-cultivated for a total of 4 days with theHOXB4 or control neo viral producer cells. The CEU-S content of recovered cells was assessedimmediately after co-cultivation (day 0) and also after seven days in liquid culture and is expressedas day 12 CFU-S colony numbers per 1 starting day 0 cells. Results shown represent the mean± S.D. from 4 independent experiments.The MRA is an assay that measures the ability of a test cell population toregenerate day 12 CFU-S in the bone marrow of a lethally irradiated recipientstransplanted 13 days earlier (Figure 4.2). The cells thus detected share withHSC a resistance to cycle-active chemotherapeutic drugs (Hodgson andBradley, 1984) and are thought to be precursors of most CFU-S (Mauch andHellman, 1989). MRA measured immediately after infection of bone marrowcells with the HOXB4-virus (day 0 after co-cultivation) was 10-fold higher thanthat measured for the nec-infected cells (Figure 4.5). Following seven days ofliquid culture, the MRA content of the neo-transduced cells was undetectable(i.e. less than two day 12 CFU-S per femur = background) suggesting that, asfor the CFU-S, the maintenance of MRA was compromised under the cultureconditions used. In contrast, the MRA of the cells present in the day 7 cultures ofStarting cells Post in Vitro Cultures(Day 0) (Day 7)95Figure 4.5. HOXB4 effects on cells with marrow repopulating ability (MRA).MRA was assayed by the content of day 12 CFU-S per femur present in recipients transplanted13 days previously with HOXB4- (black) or neo-transduced (hatched) bone marrow cells. The MRAof 2 x 1 cells was determined immediately after viral infections (day 0) or after their culture for 7days. Results shown are the mean ± S.D. of day 12 CFU-S determined in a minimum of 10recipients.Thus, it appears that the marked (i.e. > 2 log) reduction in day 12 CFU-Snumbers that occurred when nec-transduced cells were maintained in vitro forseven days was accompanied by a similar reduction in MRA. In contrast,HOXB4 overexpression reversed this decline leading to a net increase in CFUS content and a near maintenance of MRA.4.3.4 HOXB4-Induced Expansion in Vivo of ClonogenicProgenitors and Dày 12 CFU-STo assess possible effects of HOXB4 overexpression on hematopoietic cellsmaintained for prolonged periods in vivo, HOXB4- or nec-transduced bonemarrow cells were transplanted immediately after infection into lethallyHOXB4-transduced cells was maintained at readily detectable levels andalthough reduced to 25 % of input (day 0), this level was still >200 fold higherthan that present at day 7 in control cultures of neo-transduced cells (Figure4.5).1EcEOC41>‘ca•01000.100-10-1-fr4T LStarting Cells Post in Vitro Cultures(Day 0) (Day 7)96irradiated syngeneic recipients and reconstitution of various hematopoieticpopulations evaluated (Figure 4.2). Each mouse received an innoculum of 2 x1 0 marrow cells estimated to contain 30-40 competitive repopulating units(CRU), as subsequently determined (see Table 4.4). Gene transfer efficienciesin the transplant marrow as assessed by the proportion of G41 8-resistant in vitroclonogenic progenitors was 58±8% and 70±7% for HOXB4- and neotransduced cells, respectively (Table 4.1). This suggests that less than half ofthe CRU in the transplant were transduced since frequencies of retroviralinfection into these cells are typically lower than into in vitro clonogenicprogenitors (Fraser et al., 1993). The extent of donor-derived reconstitution ofperipheral blood leukocytes measured 12 or 20 weeks post-transplantation wassimilar (>86%) for animals transplanted with either HOXB4- or neo-transducedmarrow cells. Reconstitution of the hematopoietic system of recipients with bothtypes of transduced cells was confirmed by Southern blot analysis of DNAextracted from bone marrow and spleen cells of mice sacrificed 20 weeks aftertransplantation (Figure 4.6). Northern blot analyses of RNA obtained from thesetissues showed high levels of expression of HOXB4 and neo (Figure 4.6).Hematopoietic reconstitution by transduced cells was also confirmed by thehigh frequency of splenic and bone marrow G41 8-resistant myeloid clonogenicprogenitors (75±17% and 42±18%, respectively, for recipients of HOXB4- andneo-infected cells).97week 20 post-BMTneo HOXB4Co Cl) Cl) CO Cl)neo4i aW$ —5.0HOXB4• —week 12 post-BMT week 20 post-BMTneo HOXB4 neo HOXB4.-CJC?’,C* (* C* C* C* C*neo4___• — 1.3— IHOXB4L.Figure 4.6. Northern and Southern blot analyses to demonstrate hematopoieticreconstitution by HOXB4- or neo-transduced bone marrow cells.Upper panel: Southern blot analysis of DNA from bone marrow and spleen cells to demonstratethe presence of integrated virus. DNA was digested with Sstl and membranes hybridized toprobes specific for neo or HOXB4. Sstl releases the integrated HOXB4 (3.9 kb) and neo (2.7 kb)proviruses. A 5kb band derived from the endogenous murine HOXB4 gene cross hybridizing tothe human HOXB4 probe provides a single gene copy control of loading. Lower panel: Northernblot analysis to detect expression of transduced HOXB4 and neo genes. Total RNA (5 jig)isolated from bone marrow and spleen cells obtained from mice sacrificed at 12 or 20 weeks post-transplantation was sequentially hybridized to a neo and a HOXB4 probe. In addition to full lengthtranscripts, an expected 1 .3 kb transcript corresponding to the pgk-driven neo gene is observedat lower levels in both neo- and HOXB4-transduced cells. An additional transcript of -. 2.7 kb isalso observed using a neo but not full length HOXB4 probe in the cells transduced with theHOXB4 virus (this transcript is also observed in HOXB4-neo viral producer cells; see Figure 4.1 B);this likely represents a spliced transcipt arising from use of a splice donor site in the MSCV2.1vector (Hawley et al., 1994). Each number assigned to the various lanes identifies a specificmouse which also corresponds to the same mice depicted in Table 4.2. Abbreviations: BMT,bone marrow transplantation; BM, bone marrow; Sp, spleen.98The pre-B and myeloid clonogenic progenitor content of bone marrow 12weeks post-transplantation with HOXB4-transduced cells was on average twofold higher than that of recipients of neo-infected cells (Table 4.2). Myeloidclonogenic progenitor numbers in the spleen were also elevated —ten fold in theone mouse analyzed (Table 4.2). Twenty weeks post-transplantation withHOXB4-transduced marrow, an even greater increase in bone marrow andsplenic clonogenic progenitor numbers was evident (5 and 32-fold higher,respectively, than in nec-transduced marrow recipients).99Table 4.2. Mice transplanted with HOXB4 transduced cells have increasedmyeloid and pre-B lymphoid clonogenic progenitors in bone marrow andspleen.Mouse # Myeloid clonogenic Pre-B clonogenic Myeloid clonogenic PreB clonogenicprogenitors / femur progenitors / femur progenitors / spleen progenitors I spleen(1012 weeks post transplantation.neo-1 22 1,500neo-2 45 2,300neo-3 60 2,000neo-4 38 5,100 595 210mean±SD 41± 16 2,700±1600HOXB4-1 89 5,000HOXB4-2 55 5,400HOXB4-3 150 7,000HOXB4-4 58 5,900 6,650 420mean±SD 88± 44a ,800±a20 weeks post transplantation.neo-5 45 173neo-6 48 250neo-7 29 163mean±SD 41± 10 195± 47HOXB4-5 250 5,453HOXB4-6 190 6,583HOXB4-7 230 >100,000mean±SD 223± 30b 6,018±asignificantly different from neo control (p<0.O5)bsignificantly different from neo control (p.cO.01)Cmouse HQXB4-7 not included in this calculationIn order to evaluate whether the increase in clonogenic progenitor cellnumbers was accompanied by an expansion of an earlier cell type, day 12CFU-S frequencies were also assessed in two independent experiments. Ineach of these, marrow cells from three mice transplanted 16 or 20 weeks earlierwith either HOXB4- or neo-transduced cells were pooled and then assayed. By16 to 20 weeks the frequency of CFU-S in the recipients of control cells wasback to normal (pretranspiantation) levels (i.e., 1 .0 ± 0.3/1 O cells; (Chang andJohnson, 1991)) whereas in mice reconstituted with HOXB4-transduced bone100marrow cells, the frequency of CFU-S was 5.0 and 7.6-fold higher, respectively,for the two time points.4.3.5 HOXB4-Induced Expansion in Vivo of Cells With Long-Term Lympho -Myeloid Repopula ting AbilityTo determine if HOXB4 overexpression affects the expansion of the earliesthematopoietic cells, their numbers were quantified by limiting dilution analysisusing the competitive repopulating unit (CRU) assay (Figure 4.2) (Szilvassy etal., 1990). For this purpose, various numbers of bone marrow cells pooled fromthree mice transplanted either with HOXB4- or neo-transduced marrow cells 12or 20 weeks earlier were transplanted into lethally irradiated recipients. Thepresence or absence of lympho-myeloid repopulation (>1%) with donor-derivedLy5.14-cells in these mice was then evaluated 13 or more weeks later and CRUfrequencies calculated using Poisson statistics, from the proportion of recipientsnegative for donor-derived lympho-myeloid repopulation at different cellinnocula (Szilvassy et al., 1990).By 12 weeks post-transplantation, recipients of neo-transduced marrow hadreconstituted CRU to a frequency of 0.6 in bone marrow cells (Table 4.3) oronly 6% that of normal (unmanipulated) mouse marrow (Szilvassy et al., 1990).This low CRU frequency is consistent with the previously well documentedfinding that even non-retrovirally infected marrow cells will not regenerate CRUnumbers to >10% of pre-transplant values ((Harrison, 1982; Harrison et al.,1990); R. Pawliuk and R.K.H., unpublished observations). For recipients ofHOXB4-transduced marrow a limiting dilution was not reached suggesting aCRU frequency of > 2.9 in bone marrow cells or at least 29% of normallevels and > 4 fold that seen in the recipients of neo-infected cells (Table 4.3).CRU frequencies in the marrow were also measured in primary recipientssacrificed 20 weeks post-transplantation. The CRU frequency of the mice101transplanted with neo-transduced marrow was similar to that obtained at 12weeks or -3% of normal values (Table 4.3). In contrast, the frequency of CRU inrecipients of HOXB4-infected marrow was 14 per 1 O cells. This represents a1.4 fold increase above normal marrow values and a 47-fold increase abovethe CRU frequency measured in the marrow of recipients of control cells (Table4.3).102Table 4.3. Evaluation by limiting dilution analysis of competitive long-termrepopulating cells (CRU) in mice transplanted with HOXB4 versus neo controltransduced bone marrow cellsCRU evaluation of primary recipientsanumber of cells 12 week post-transplantation 20 week post-transplantationinjected intosecondaryrecipients neo HOXB4 neo HOXB46,700,000 7/7 (30±12) 5/5 (76±21) n.d. n.d.670,000 8/8 (11±8) 5/5 (63±17) 4/4(2O±l6)d n.cJ.67, 000 2/7 (3±0) 3/3 (25±3) 0/6 (<1) n.d.33,500 n.d. n.d. n.d. 7/7 (37±27Y’11,000 n.d. n.d. n.d. 5/7(15±12)6,600 n.d. n.d. n.d. 4/6 (14±20)CRU frequency er10 cells (range) 0.6 (0.25-1.6) >2.9 0.3 (0.2-0.5) 14 (10-20)Relative to normal (%)C 6 >29 3 1 40CRU evaluation of secondary recipientsanumber of cells 16 week post-transplantationinjected intotertiaryrecipients neo HOXB415,000,000 2/3 (3±2) 5/5 (54±5)1,500,000 3/5 (5±3) 5/5 (31±11)150,000 1/5 (5±6) 6/6 (8±4)15,000 n.d. 1/6 (1±1)1,500 n.d. 0/5(<1)CRU frequency er 0.02 (0.01 -0.04) 2.1 (0.8-5.3)io cells (range)Relative to normal (%)C 0.2 21aResults are expressed as number of mice repopulated with donor-derived cells (Ly5.1 j overtotal. Numbers in parentheses represent the mean % ± S.D. of peripheral blood Ly5.1 cellsfound in the transplant recipients.bCRU frequency was calculated using limiting dilution analysis (see Experimental Procedures).cCompared to control values (Szilvassy et al., 1990).dmice used for evaluaton of CRU amplification in secondary animals selected from these groupsand are identifiable in Figure 4.7 as mice number 25.1, 25.2, 25.3, 29.1, 29.2, 29.3, 30.1 and30.3.103Recipient mice used to quantitate CRU (20 week posttransplant, Table 4.3)were further assessed by Southern blot analysis to identify those repopulatedby transduced CRU. Of 14 mice analyzed that received 5 or fewer CRU frommice initially transplanted with HOXB4-infected marrow, 12 were positive forproviral integration in thymic and / or bone marrow tissue, all of which hadpreviously been scored as positive for donor-derived Ly5.1 lympho-myeloidrepopulation in the CRU assay (Figure 4.7). Moreover, common proviralintegration patterns for thymic and bone marrow tissue were clearly apparent infive of these secondary recipients confirming the Iympho-myeloid repopulatingpotential of the regenerated CRU. The intensities of the proviral integrationsignals also roughly correlated with the percentages of donor-derived Ly5.1cells in the peripheral blood (e.g., compare the intense signal for mouse 29.2(81% Ly5.1+) vs. a much reduced signal for mouse 29.1 (30% Ly-5.1+)) and fortwo mice (31.3 and 32.1) who were scored as negative for donor-derivedlymph-myeloid repopulation, proviral integrants were not detected. Thiscorrelation strongly indicates that HOXB4-transduced HSC can terminallydifferentiate in vivo as shown above for day 12 CFU-S and in vitro clonogenicprogenitors. Together these results strongly suggest that the measured CRUexpansion in vivo was due to the selective expansion of HOXB4-transducedCRU. In contrast, four recipients of marrow from primary mice initiallytransplanted with neo-infected bone marrow were all negative for proviralintegrants (3 of 4 mice shown, Figure 4.7) but positive for donor Ly5.1 cellsindicating that CRU regeneration in the primary mice included nontransducedCRU.10425.1 25.2 25.3 29.1 29.2 29.3 30.1 30.3 31.1 31.2 31.3 31.4 31.5 32.1 32.2 33.1 33.2TBMTBMTBMTBMTBMTBMTBMTBM TBM TBMTBMTBMTBMT BM TBMTBMTBMI•1. 0iøeaAaUeUIFigure 4.7. Southern blot analysis of proviral integration patterns in bonemarrow and thymic DNA isolated from secondary recipients transplanted withvarying numbers of HOXB4 or neo-transduced bone marrow. cells.DNA was digested with EcoRl which cuts the integrated MSCV provirus once generating DNAfragments specific to each integration site. The membranes were first hybridized to a probe forneo (top) to identify proviral fragments. The membranes were rehybridized to a probe for theerythropoietin receptor (bottom) to provide a control for DNA loading. Exposure times wereequivalent for both probes (‘-1 day). Primary mice used as donors were those sacrificed 20 weekspost transplantation and are described in Table 4.2 and 4.5. The secondary recipients analyzedare as presented in Table 4.3. The number of bone marrow cells injected into each secondaryrecipient is indicated at the top of the Figure. Each mouse is identified with a specific identificationnumber. Percentage Ly5.1 cells in peripheral blood of the mice transplanted with HQXB4-transduced marrow are; 29.1(30%), 29.2(81%), 29.3(39%), 30.1(62%), 30.3(30%), 31 .1(1 6%),31.2(3.2%), 31.3(2%), 31.4(29%), 31.5(9%), 32.1(1.1%), 32.2(9%), 33.1(44%) and 33.2(6%).Abbreviations: T, thymus; BM, bone marrow. Fragment sizes range from over 12 kb (upper tick onthe left of the Figure) to a little less than 4 kb (second tick from the top).Self-renewal of HOXB4-transduced CRU was also demonstrated bydetection of the same pattern of proviral DNA integration in thymus and bonemarrow cells of four different secondary recipients, mice 29.1, 30.3, 33.1 and30.1 (Figure 4.7). Another uniquely identified totipotent CRU was detected inneo667,000 cellsHOXB433,000 cellsHOXB411,000 cellsHOXB46,700 cells——recipient 29.2. Self-renewal of a CRU with apparent subsequent restriction to105the marrow was detected in secondary recipients 31.2, 31.5, 32.2 and 33.2.These latter mice, however, showed lympho-myeloid repopulation by FAGSanalysis of Ly-5.1 positive peripheral blood leukocytes suggesting that thisclone had B lymphoid and myeloid potential. The degree of self-renewaldetected is consistent with the marked expansion of CRU observed in primaryanimals (nearly 900-fold, Table 4.4) and the fact that mice were initiallytransplanted with small numbers of CRU (approximately 32 of which at most halfwould be estimated to have been transduced).To further assess the regenerative capacity of HOXB4-transduced CRU,their expansion in secondary recipients was also evaluated. Bone marrow cellswere harvested from secondary recipients (n=3 for neo and 5 for HOXB4)transplanted 16 weeks earlier with -2 to 5 Ly5.1 CRU (Table 4.3) and CRUfrequencies measured by limit dilution analysis in tertiary recipients. CRUfrequency in the secondary recipients of neo-transduced marrow was 1 in 4.8 X106 cells (Table 4.3) or less than 0.2% of that found in normal unmanipulatedmarrow and indicative of a 17 fold expansion over input (summarized in Table4.4). In sharp contrast, secondary recipients of HOXB4-transduced marrow hada CRU frequency 130 times higher or -‘20% of normal levels and indicative of afurther 900 fold expansion over input (Table 4.4).106Table 4.4. Expansion of donor-derived CRU in primary and secondary recipientsof HOXB4- or neo-transduced bone marrow cellsDonor-derived CRU content per mouseaPiimary recipents Secondary recipientsVirus#0fCRU #0fCRU #ofCRU #ofCRUtransplantedb 20 weeks post TxC transplanted 16 weeks post TxCneo 32 600 2 35HOXB4-neo 32 28,000 5 4,700a Results are expressed as number of Ly5.1+ CRU content per mouse considering that onefemur represents approximately 10% of the total marrow of a mouse.bCRU frequency of 5-FU-treated bone marrow cells cells after co-cultivation was measured in asubsequent experiment and was 1 in 6,000 cells (95% confidence interval: 1 in 2,000 to 1 in12,000).CEstimates based on data presented in Table 4.3.Despite this dramatic expansion of HOXB4-transduced HSC, it is noteworthythat the relative numbers of the various types of in vitro myeloid clonogenicprogenitors (i.e. CFU-GM, BFU-E and CFU-GEMM) in primary and secondaryrecipients of HOXB4-infected cells were the same as in recipients of controlcells. In addition, the total cellularity of the bone marrow was also not differentand the red blood cell, white blood cell and differential counts were also withinthe normal range (Table 4.5, data for secondary recipients not shown). Thusdespite a marked and sustained effect of HOXB4 overexpression on thenumbers of myeloid and lymphoid clonogenic progenitors as well as day 12CFU-S, there was no gross effect on lineage determination nor evidence of aconsequent expansion of later cell types. Moreover, despite a significantexpansion in the numbers of the most primitive hematopoietic cells, none of theprimary recipients (n>.25) of HOXB4-transduced marrow have developed107leukemia or any other obvious blood dyscrasia for up to 12 months posttransplantation.Table 4.5. Enumeration of bone marrow, spleen and peripheral blood cell countsof primary mice transplanted 16-34 weeks earlier with HOXB4- or neo-transducedcelisaGene Cells! Cells! RBCb Hb wbc lym.c gran. mo baso!femur splen (106411) (gIdl) (1O411) % % eosi.(10) (10) %neo 1.9±0.2 3.3±0.5 7.9±0.4 14.8±1.2 7.9±2.5 70±6 22±7 7±1 1±2HOXB4-neo 2.0±0.2 3.6±0.6 7.6±1.0 13.8±1.7 7.8±2.2 75±11 21±6 6±4 0Results are expressed as mean ± S.D. for 9 primary recipients except for bone marrow and spleencellularity measured on 3 mice.bAbbreviations RBC, red blood cells; Hb, hemoglobin; wbc, white blood cells; lym., lymphocytes;ran., granulocytes, mo, monocyctes; baso/eosi., basophils/ eosinophils.All differential counts were done manually on 200 cells (Wright stain).RBC, WBC and Hb were done with a CBC5 Coulter counter using 40 p.1 of hepannized blood.1084.4 DiscussionOur previous work established that HOXB4 and several other Hox A and Bgenes are preferentially expressed in the most primitive purified subpopulationsof CD34+ bone marrow cells. We now demonstrate, using an in vivo murinemodel, that the engineered overexpression of HOXB4 can have profoundeffects on the proliferation of long-term in vivo repopulating hematopoietic stemcells and to a lesser extent on the proliferation of intermediate types ofhematopoietic progenitor cells including both lymphoid (pre-B) and myeloidrestricted populations (Figure 4.8). Nevertheless, this deregulation of primitiveprogenitor cell amplification does not appear to lead to leukemia and is nottranslated into an altered output of any type of mature blood cell or an alteredcommitment to any specific blood cell lineage.I CRU I CFU-S I Clonogenic Mature bloodprogenitors cellsNormal-_>untransplanted________neo-transplant —— —>HOXB4- transplant._>._ ._>HOXB4- transplant__________Figure 4.8. Schematic depiction of the sizes of various hematopoieticpopulations reconstituted in primary recipients of HOXB4- or neo-transducedbone marrow cells compared to normal (unmanipulated) mice.Except for the boxes representing the peripheral blood cells which were equivalent in numbers,the surface area of each box is drawn to scale to indicate the relative frequencies of the cellpopulations.109Previous studies have shown that, even after a single transplantation, therepopulating competence of the regenerated bone marrow cells is reduced -tenfold (Harrison, 1982; Harrison et al., 1990). The reasons for this change are notknown, although it has been hypothesized that the sustained proliferative stressimposed upon at least some of these cells during the early phase ofregeneration of the system may result in a decline in their probability of self-renewal in subsequent divisions. Consistent with these earlier studies, the poolof long-term repopulating stem cells (CRU) regenerated in recipients of neotransduced cells did not recover beyond a level equivalent to 3-6% of thatcharacteristic of normal mice despite the return to normal levels of bone marrowcellularity and clonogenic progenitors and frequencies.A different picture emerged in mice transplanted with marrowoverexpressing HOXB4. Here, CRU numbers recovered to a level that was 1.4-fold higher than the normal value or 47-fold higher than that observed inanimals transplanted with neo-transduced marrow. The enhanced proliferativecapacity of HOXB4-transduced CRU was further demonstrated by serialtransplantation studies in which as few as 5 CRU transplanted into secondaryrecipients were shown to be capable of regenerating a significant CRU pool notdemonstrably compromised in repopulating ability. In contrast serialtransplantation compromised even further the ability of nec-transduced and/ornon-transduced CRU to regenerate CRU in successive recipients.Expansion of day 12 CFU-S and in vitro clonogenic progenitors was alsodocumented in the mice transplanted with HQXB4-transduced marrow cells.Expansion of these later types of hematopoietic cells might simply be secondaryto the expansion of CRU. However, the fact that clonogenic progenitorstransduced with HOXB4 also replated much better in vitro than thosetransduced with nec strongly suggests that this gene can also directly influence110the proliferative capacity of later progenitors. The absence of any perturbation inthe proportions of different types of lineage-restricted clonogenic progenitorsproduced in vivo or the number of mature blood cells present in the circulationof mice repopulated with HOXB4-transduced cells strongly suggest that thisgene can influence stem cell self-renewal events in the absence of effects onlineage commitment or terminal differentiation. This is consistent with theconcept recently proposed by Fairbairn et al. (Fairbairn et aL, 1993) based onstudies with the FDCP-mix cell line that self-renewal and commitmentprocesses may not necessarily be linked at the molecular level. Together withthe results presented in chapter 3 which showed preferential expression ofHOXB4 in the most primitive bone marrow cell populations, the present datasuggest that the absolute level of HOXB4 can be a critical determinant of HSCproliferative ability. Interestingly, it was recently shown that inhibition of HOXB4expression using antisense oligonucleotides in peripheral blood progenitorscompromised the proliferation of these cells (Care et al., 1994; Giampaolo et al.,1994).In addition to clear effects on earlier hematopoietic cells in vivo,overexpression of HOXB4 had marked effects on cells cultured in vitro. Of these,the most striking effect was on the enhanced recovery of day 12 CFU-S afterseven days of in vitro culture (Figure 4.4). It has been previously reported thatthe CFU-S content of growth factor-stimulated post-5-FU treated bone marrowcells decreases dramatically with time using similar culture conditions (Bernadet al., 1994). The results obtained here with neo-transduced cells are consistentwith this report (i.e., there was a decrease of -2 log in the number of CFU-S atthe end of 7 days in culture). However, the CFU-S content of the culturesinitiated with HOXB4-transduced cells expanded by 2- to 5- fold during thissame period. Similar differences in the recovery of day 12 CFU-S after in vitro111culture were observed in comparisons of non 5-FU-treated bone marrowharvested from mice previously reconstituted with HOXB4- versus neotransduced marrows (data not shown). This difference in CFU-S content afterseven days of liquid culture may therefore be the result of HOXB4 “involvement”on various cellular mechanisms including cell death or proliferation of CFU-Sprecursors. In order to test a possible involvement of HOXB4 on cell death, twogrowth factor-dependent cell lines (FDC-P1 and BAF3) were transduced withHOXB4-neo (Figure 4.1C shows the Western of the FDC-P1) or neo viruses.Cell death assessement by trypan blue exclusion done every eight hours aftergrowth factor deprivation of log phase growing FDC-P1 or BAF3 cells failed toshow any effect of HOXB4 on apoptosis (data not shown, n=2 independentexperiments). Therefore the results presented in this Chapter are moresuggestive of HOXB4 playing a role in proliferation (cell division) than in theprevention of cell death but definitive proof to this statement remains to beshown. Nevertheless the magnitude of the differences observed in the “deltaCFU-S” assay suggest that this system could offer a powerful experimental toolfor the identification of HOXB4 target genes.Expansion of hematopoletic precursors without a concomitant increase inthe number of peripheral blood cells has not been previously observed whenthe effects of overexpression of various hematopoietic growth factors have beenstudied in a similar model (Chang and Johnson, 1991; Fraser et al., 1993;Hawley et al., 1992; Johnson et al., 1989; Tanaka et al., 1992; Wong et al.,1991). In most of these reports, it was found that overexpression of the cytokinesstudied resulted in an increase in the numbers of peripheral blood cells but thatthe content of bone marrow clonogenic progenitors was either unchanged or insome cases, diminished. Interestingly, overexpression of the non-clusteredhomeodomain-containing gene, TCL-3 (previously called HOX1 1, (Hawley et112al., 1994)) or HoxB8 (Perkins and Cory, 1993) in murine bone marrow cellswere also found to have proliferative effects. In both studies, generation of celllines from transduced bone marrow cells was observed with high frequency inthe presence of IL-3. In contrast, efforts to generate cell lines in similarconditions (i.e. high IL-3 concentration) were unsuccessful with HOXB4 (datanot shown). Interestingly and similar to our findings, mice transplanted withHoxB8-transduced marrow cells showed increased levels of bone marrow andsplenic clonogenic progenitors at three months post-transplantation. Asignificant proportion of mice transplanted with HoxB8-transduced cellsdeveloped leukemic transformation at —7 months post transplantation. Incontrast, no leukemic transformation was observed in the “HOXB4 mice” evenafter 1 year of observation and despite persistant high levels of expression ofHOXB4. Thus, although some similarities are observed between these threestudies, the results presented in this Chapter suggest fundamental differencesin HOXB4-mediated effects.Taken together, these results suggest that HOXB4 is a key regulator ofproliferation of HSC and that overexpression of this gene does not override theregulatory mechanisms involved in lineage determination or in the control ofend cell output. These findings demonstrate that it is possible to reverse thedecline of HSC that normally occurs during regeneration of the hematopoieticsystem after bone marrow transplantation resulting in a dramatic expansion ofgenetically modified HSC in vivo. Given the possibility of examining the functionof analogous populations of primitive human hematopoietic cells inimmunodeficient mice, it will be of interest to analyze such targets using thisapproach.113CHAPTER 5General Discussion and Conclusions5.1 Hox genes are expressed in normal early hematopoietic cellsThe nuclear factors involved in the regulation of proliferation and differentiationof primitive hematopoietic cells are still poorly understood. The goal pursued inthis thesis was to identify candidate factors and provide insight into theirpossible functional roles. The strategy taken was to focus on a particular classof transcription factors —the clustered homeobox genes— which as discussed inthe introduction, are central to differentiation and proliferation of embryoniccells. Considerable circumstantial evidence had implicated these genes inhematopoiesis notably findings of their expression in primary leukemic cellsand leukemic cell lines and of effects on proliferation and differentiation ofvarious hematopoietic cells following modulation of their expression.Capitalizing on the emergence of powerful reverse-transcriptase /polymerase-chain reaction strategies and on the recent availability of humanCD34 bone marrow subpopulations that are not only phenotypically pure butwhich also dramatically differ in their progenitor content, it was possible todocument the expression of more than 16 different Hox genes in earlypopulations of human bone marrow cells. These findings allowed theidentification of genes (e.g. HOXB3, HOXB4, etc.) which showed preferentialexpression in the earliest subpopulations studied; and of others—generallyfound at the 5’ region of the Hox cluster— that were equally expressed in allpopulations analyzed. Based on these observations, the hypothesis was putforward of the existence of a link between the position a Hox gene occupies on114the cluster and the function this gene has in hematopoiesis with 3’-positionedgenes postulated to have important roles in stem cell function and 5’-positionedgenes postulated to be involved in function of more mature cells.Another provocative feature of the expression pattern observed was theabsence of “upregulation” of any Hox gene with differentiation. In fact theexpression of each gene analyzed to date appears to decrease duringmaturation of hematopoietic cells from the most primitive where all 16 genesstudied were expressed (CD34++lineageCD38’°) to the more mature CD34cells where expression was barely detectable. Another intriguing finding wasthe absence of expression of the D cluster genes in these cells confirmingresults from studies of leukemic cell lines and raising the question of why thisparticular cluster is not expressed.5.2 HOXB4 overexpression causes expansion of earlyhematopoletic cellsFunctional studies presented in Chapter 4 provide striking new evidenceimplicating at least one 3’ Hox gene in stem cell expansion without detectableeffects on differentiation of later cells. This suggested that HOXB4overexpression did not override the regulation of the control of end-cell outputeven in the presence of large effects on earlier cells.What cellular mechanism was altered by HOXB4 overexpression? Onecan only speculate, at least at this time, four mechanisms to explain theobserved expansion of HSC described in Chapter 4. HOXB4 overexpressionmay inhibit cell death. Unfortunately very little is known about cell death of earlyhematopoietic cells in a BMT set up. Nevertheless, this possibility was exploredby overexpressing HOXB4 or neor in two growth factor-dependent cell lines.Upon growth factor deprivation, cells transduced with HOXB4 died just asquickly as the neo-controls thus suggesting that HOXB4 overexpression has115little or no effect on inhibition of apoptosis at least related to growth factorcontrol. Another possibility is that HOXB4 increased the recruitment of thenormally quiescent HSC into cycle (increased turn-over with attendant self-renewal divisions). This hypothesis which would predict a gradual CRUexpansion with time is currently being investigated. Interestingly clonogenicprogenitors obtained from mice transplanted with HOXB4-transduced cellsexpanded with time (see Table 4.2) but it remains to be shown if earlier cells willalso follow this trend. Alternatively, HOXB4 may increase the probability of self-renewal division of CRU cells during reconstitution. Effects observed afterHOXB4 overexpression may also result from improved seeding efficiencies ofHOXB4-transduced CRU cells at the time of transplantation. This alternative isinteresting since Hox genes are known to regulate adhesion molecules.However due to the magnitude of the effect reported (—50 fold more CRU inmice transplanted with HOXB4-transduced cells than in nec-controls) thishypothesis unlikely explains the phenomenon by itself.5.3 Hox genes: Master regulators of hematopoiesis?The “functional” studies previously reported (Care et al., 1994; Giampaolo et al.,1994; LIII et al., 1995; Perkins and Cory, 1993; Shen et al., 1992; Wu et al.,1992) combined with those described in Chapter 4 have all demonstrated thatseven out of the seven Hox genes tested in various systems produced profoundeffects on hematopoietic differentiation or proliferation. Considering that at least16 (see chapter 3, and likely >30) of these genes are expressed in primaryhematopoietic cells, it appears that Hex genes have the potential to regulate amultiplicity of targets in hematopoietic cells. This potential is even greater if oneconsiders the numerous alternate transcripts observed with some of thesegenes (HOXB3 for example has at least 5 different transcripts in K562 cells,116data not shown). Together these results point to the existence of a complex“Hox code” that may be deterministic for differentiation and proliferation ofhematopoietic cells.5.4 Future studiesConsistent with the expression study reported in Chapter 3, it will be necessaryto “test more exhaustively the hypothesis that there is a link between theposition of a particular Hox gene in the complex, its pattern of expression inimmature (3’ and 5’ Hox genes) versus more mature (5’ Hox genes) bonemarrow populations, and the effect of overexpression”. To test this hypothesis,HOXA1O (a 5’ gene also expressed in “mature” CD34 subpopulations, seeFigure 3.2) was recently overexpressed in murine bone marrow cells andpreliminary results from these studies also indicate effects on differentiation thatare dramatically different from those (no effect) observed with HOXB47.Finally HOXB3, which shows an even more restricted pattern of expressionin early hematopoietic cells than HOXB4, would be of interest to study in similarexperimental set ups. In an effort to pursue this goal and to obtain relevanthematopoietic HOXB3 transcripts, a phage cDNA library was constructed withmessenger RNA obtained from purified CD34 cells and a full-length (coding)novel HOXB3 transcript was isolated from this cDNA library (data not shown).The coding region of this cDNA was recently incorporated into a retroviral vectorand functional studies are underway.Hox genes found on the same paralog (see introduction, Figure 1 .4) mayhave functional redundancies. A dramatic example of this is the ability of themurine HoxA5 gene (which is highly similar to Drosophila’s Sex combs reduced7Thorsteinsdottir U., Sauvageau G., Hough M., et al. Manuscript in preparation.117(Scr)) to specifically activate Scr targets in a HoxA5- transgenic fruit fly (Jiaganget al., 1993). Since paralogous genes may also likely regulate similar targets inhematopoletic cells, one may predict that disruption of a specific Hox genewould not result in dramatic hematopoietic perturbations. Indeed, hematopoieticperturbations have never been reported with “knock out studies” involving morethan nine Hox genes including animals with genetic disruption of two Hoxgenes (HoxD3 ÷ HoxA3) (Condie and Capecchi, 1994; Krumlauf, 1994). Incollaboration with two other groups (Drs. H.J. Lawrence - C. Largman and Dr.M. Capecchi), we are currently studying the hematopoietic system of HoxA9“knock out” mice and, although no obvious hematopoietic defects were initiallydetected, detailed quantitative analysis of B, T and myeloid lineages aresuggestive of defective B lymphopoiesis in these mice (Lawrence et al., 1995).Therefore it will be important to test other available “knock out” mice (such asHoxB4, HoxB3 and HoxAlO) for other, perhaps subtle hematopoietic defect(s)that may have been overlooked. If viable, cluster and especially paralogdisruptions may generate more revealing results.Another important question that will need to be addressed is whether Hoxgenes are also involved in embryonic and I or fetal hematopoiesis. This issuecan be addressed by using the mouse embryonic stem (ES) cell which, undercontrolled conditions, can be induced to differentiate in vitro into primitive (i.e.yolk sac derived) and more definitive (i.e. fetal) hematopoietic cells. In acollaborative effort, Cheryl Helgason (a post doctoral fellow that has introducedin the laboratory the in vitro differentiation of ES cells) and myself, have recentlyanalyzed the effects of overexpressing HOXB4 in these cells and observedstriking proliferative effects on definitive multipotential progenitors whereas no118effect was observed on primitive hematopoietic cell differentiation8.This systemshould provide a powerful approach to the analysis of Hox gene function thatwill complement studies done in adult bone marrow as described in this thesis.In particular, it may facilitate detailed structure / function analysis of multiple Hoxgenes to identify critical domains that specifify proliferative versus differentiativeeffects.The recent findings that HOM-C genes and their mammalian Hoxhomologs may require accessory molecules such as extradenticule or PBX,respectively, (Chang et al., 1995; van Dijk and Murre, 1994) to bind andpossibly fully activate their targets (Sun et al., 1995) has raised questions aboutthe role these “accessory factors” may play in hematopoiesis. Is PBX requiredfor some (all?) Hox mediated effects in hematopoietic cells?. This question hasclear clinical relevance since it has been shown that the E2A-PBX fusionprotein involved in the t(1. 19) human pre-B leukemia interacts as well with theHox proteins as does PBX alone (Chang et al., 1995). Considering that Hoxmessenger RNA has been detected in fl human leukemias and that theoverexpression of certain Hox gene in murine bone marrow cells can lead toleukemic transformation (HoxB8, Perkin and Cory, 1993; HOXA1O, unpublishedobservations), it is possible that E2A-PBX requires certain members of the Hoxproteins to transform hematopoietic cells. Attempts at clarifying the necessicityof PBX-Hox interaction in hematopoietic cells can now be investigated byexploiting the recent generation of HOXB4 mutants that cannot interact with anyof the PBX proteins9.8Helgason C., Sauvageau G., Lawrence H.J., Largman C. and Humphries, R.K. (1995).Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cellsdifferentiated in vitro. Manuscript Submitted9Shen W.-F., Chang C.-P., Rozenfeld S., Sauvageau G., Humphries R.K., Lu M.,Lawrence H.J., Cleary M.L., Largman C. Hox homeoproteins exhibit selective complex stabilitieswith PBX and DNA, submitted.119In conclusion, the work presented in this thesis showed that multiple genesof the highly complex Hox family of transcription factor are expressed in earlyhematopoietic cells. This work also demonstrated that overexpression of theHOXB4 gene in bone marrow cells can dramatically influence expansion ofearly cells. Based on these results, a hypothesis was generated whichsuggested the existence of a relationship between the chromosomal position ofa particular Hox gene versus its function in primary hematopoiesis (see earlier).More studies will need to be done in order to gather further information aboutthe possible functions fulfilled by other Hox genes in early hematopoiesis.These should probably initially focus on determinating the function of 3’ versus5’ genes of the same cluster and also of paralogs (HOXA4 versus 84 and C4,etc.). Such studies should provide a better understanding of the possible“spectrum of Hox-mediated effects” and might allow the identification of anemerging functional pattern that may or may not be related to the chromosomalposition each gene occupy.Finally, there is accumulating evidence to suggest that molecules known toregulate Hox gene expression during embryogenesis are also active inhematopoietic cells. 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