Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX-ENL
1997; Springer Nature; Volume: 16; Issue: 14 Linguagem: Inglês
10.1093/emboj/16.14.4226
ISSN1460-2075
Autores Tópico(s)Retinoids in leukemia and cellular processes
ResumoArticle15 July 1997free access Immortalization and leukemic transformation of a myelomonocytic precursor by retrovirally transduced HRX–ENL Catherine Lavau Corresponding Author Catherine Lavau Systemix, Inc., 3155 Porter Drive, Palo Alto, CA, 94304 USA Search for more papers by this author Stephen J. Szilvassy Stephen J. Szilvassy Systemix, Inc., 3155 Porter Drive, Palo Alto, CA, 94304 USA Search for more papers by this author Robert Slany Robert Slany Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, 94305 USA Search for more papers by this author Michael L. Cleary Michael L. Cleary Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, 94305 USA Search for more papers by this author Catherine Lavau Corresponding Author Catherine Lavau Systemix, Inc., 3155 Porter Drive, Palo Alto, CA, 94304 USA Search for more papers by this author Stephen J. Szilvassy Stephen J. Szilvassy Systemix, Inc., 3155 Porter Drive, Palo Alto, CA, 94304 USA Search for more papers by this author Robert Slany Robert Slany Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, 94305 USA Search for more papers by this author Michael L. Cleary Michael L. Cleary Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, 94305 USA Search for more papers by this author Author Information Catherine Lavau 1, Stephen J. Szilvassy1,2, Robert Slany3 and Michael L. Cleary3 1Systemix, Inc., 3155 Porter Drive, Palo Alto, CA, 94304 USA 2Lucille P.Markey Cancer Center, Division of Hematology/Oncology, University of Kentucky, 800 Rose Street, Lexington, KY, 40536-0093 USA 3Department of Pathology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA, 94305 USA The EMBO Journal (1997)16:4226-4237https://doi.org/10.1093/emboj/16.14.4226 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A subset of chromosomal translocations in acute leukemias results in the fusion of the trithorax-related protein HRX with a variety of heterologous proteins. In particular, leukemias with the t(11;19)(q23;p13.3) translocation express HRX–ENL fusion proteins and display features which suggest the malignant transformation of myeloid and/or lymphoid progenitor(s). To characterize directly the potential transforming effects of HRX–ENL on primitive hematopoietic precursors, the fusion cDNA was transduced by retroviral gene transfer into cell populations enriched in hematopoietic stem cells. The infected cells had a dramatically enhanced potential to generate myeloid colonies with primitive morphology in vitro. Primary colonies could be replated for at least three generations in vitro and established primitive myelomonocytic cell lines upon transfer into suspension cultures supplemented with interleukin-3 and stem cell factor. Immortalized cells contained structurally intact HRX–ENL proviral DNA and expressed a low-level of HRX–ENL mRNA. In contrast, wild-type ENL or a deletion mutant of HRX–ENL lacking the ENL component did not demonstrate in vitro transforming capabilities. Immortalized cells or enriched primary hematopoietic stem cells transduced with HRX–ENL induced myeloid leukemias in syngeneic and SCID recipients. These studies demonstrate a direct role for HRX–ENL in the immortalization and leukemic transformation of a myeloid progenitor and support a gain-of-function mechanism for HRX–ENL-mediated leukemogenesis. Introduction Chromosomal translocations constitute important mechanisms for the activation of cellular proto-oncogenes with essential roles in leukemogenesis (Cleary, 1991; Rabbitts, 1991). A subset of acute leukemias carries translocations of chromosome band 11q23 with reciprocal partners located at 30 or more cytogenetically diverse loci (for a review, see Waring and Cleary, 1997). Chromosomal abnormalities of 11q23 are seen in B-lineage acute lymphoblastic leukemias (ALL) and myelomonocytic and monocytic subtypes of acute myeloid leukemias (AML). They result in structural interruption of the HRX gene (also called MLL, ALL1 or Htrx) which encodes a very large (430 kDa) protein with limited amino acid similarity to the Drosophila trithorax protein (Djabali et al., 1992; Gu et al., 1992; Tkachuk et al., 1992). Genetic analyses in flies and mice suggest that both trithorax and HRX are involved in the maintenance, but not initiation, of HOX gene expression during embryonic development (Breen and Harte, 1993; Kennison et al., 1993; Yu et al., 1995). Since HRX-associated leukemias often express markers of both lymphoid and myeloid lineages, it is possible that HRX translocations may occur at the level of a multipotent hematopoietic progenitor or hematopoietic stem cell (HSC), leading to the disruption of normal genetic programs for terminal differentiation. Chromosomal translocations involving HRX result in its in-frame fusion with a variety of heterologous proteins, several of which have now been cloned and characterized (Waring and Cleary, 1997). Some HRX partners are structurally related to each other, and many contain motifs previously associated with proteins involved in transcriptional regulation. One of the more common HRX fusion partners is ENL (Tkachuk et al., 1992), located at chromosome band 19p13.3. The ENL locus is involved in t(11;19) translocations in both B-lineage ALLs and myelomonocytic AMLs (Rubnitz et al., 1996). Leukemias bearing t(11;19) express HRX–ENL fusion proteins comprised of an amino-terminal portion of HRX, containing its DNA-binding A–T hook motifs, and the carboxy-terminal portion of ENL, which displays weak transcriptional activation potential (Rubnitz et al., 1994). ENL displays amino acid similarity to AF9, which is fused to HRX following t(9;11) translocations in another subset of AMLs (Iida et al., 1993; Nakamura et al., 1993). Both ENL and AF9 are distantly related to the yeast Anc1 protein (Welch and Drubin, 1994) which has been shown to be a component of two basal transcription complexes (TFIID and TFIIF) and the SWI/SNF complex (Henry et al., 1994; Cairns et al., 1996). It is not yet clear what biochemical role Anc1 plays in these transcription complexes or whether ENL or AF9 are components of the comparable mammalian complexes. Although available data suggest a role in transcriptional regulation, in vitro biological assays are necessary to establish a link between the transcriptional properties of ENL/AF9 and the oncogenic properties of their respective HRX fusion proteins. The genetic mechanisms by which mutations of HRX contribute to leukemogenesis are not yet clear. Consistently observed, in-frame fusions of the amino-terminal portions of HRX with heterologous proteins suggest an important role for 3′ portions of each fusion partner and support a potential gain-of-function mechanism. However, the diversity of observed HRX fusion partners, which lack any apparent unifying functional contribution, has led to counter-proposals for loss-of-function models. Furthermore, in a subset of myeloid leukemias, HRX is not fused to a heterologous protein but undergoes self-fusion, resulting in internal duplication of its amino-terminal portions (Schichman et al., 1994). Thus, a critical transforming event may be inactivating mutations of HRX resulting in its haplo-insufficiency. Another possibility is that translocations involving HRX contribute two oncogenic events by simultaneously creating both a gain-of-function mutation on the translocated allele as well as haplo-insufficiency for wild-type HRX (Yu et al., 1995). In an effort to address these mechanistic issues, we developed an in vitro model to characterize the effects of HRX–ENL on primitive murine hematopoietic cells by employing retroviral gene transfer. Following retroviral transduction, HRX–ENL increased the self-renewal and proliferative capacity of hematopoietic progenitors with in vitro clonogenic potential and resulted in the immortalization of early myelomonocytic cells. Hematopoietic cells transduced with HRX–ENL induced myeloid leukemias upon transplantation into syngeneic mice. In vitro transformation required fusion of HRX with ENL and was not observed with wild-type ENL or the 5′ portion of HRX alone. These data demonstrate a direct role for HRX–ENL in the immortalization and leukemic transformation of myeloid precursors, and support a simple gain-of-function mechanism for its oncogenic activity. Results Structure and expression of HRX–ENL retroviruses A cDNA encoding the HRX–ENL fusion transcript (Tkachuk et al., 1992) was inserted under the transcriptional control of the long terminal repeat (LTR) of the murine stem cell virus (MSCV) retroviral vector (Hawley et al., 1994) (Figure 1A). This vector was employed because it has previously has been used successfully with several genes to induce transformation of either myeloid or lymphoid cells (Hawley et al., 1995), suggesting that its LTR is capable of directing gene expression in multiple hematopoietic lineages. The MSCV/HRX–ENL vector also encodes a neomycin resistance gene (neo) under the control of the phosphoglycerate kinase (PGK) internal promoter and enables selection of infected cells on the basis of resistance to G418. High titer retroviral stocks for MSCV/HRX–ENL and MSCV/neo were produced by transient transfection into the ecotropic retroviral packaging cell line Bosc23 (Pear et al., 1993). Appropriate expression of HRX–ENL in transfected Bosc23 cells was confirmed by Western blot analysis (Figure 1B). Figure 1.Design and expression of MSCV constructs and experimental strategy for transduction of primitive hematopoietic cells. (A) Schematic illustrations of the retroviral vectors employed. (B) Western blot analysis of proteins expressed from the MSCV constructs shown in (A). Arrows indicate exogenously expressed proteins detected with the specific antibodies indicated beneath each panel. The anti-ENL antiserum (Butler et al., 1997) detects endogenous ENL proteins in Bosc23 cells whose level of expression is considerably lower than that observed in cells transfected with MSCV/ENL. (C) Experimental scheme employed for transduction of primitive hematopoietic cells. Download figure Download PowerPoint Murine hematopoietic cells transduced with HRX–ENL exhibit a growth advantage in vitro To determine if HRX–ENL could induce the leukemic transformation of primitive HSCs, we targeted its expression to two different populations of enriched murine bone marrow (BM) cells by retroviral infection (Figure 1C). In experiment 1, BM was harvested from mice 2 days after 5-fluorouracil (5-FU) treatment and enriched by depletion of mature cells expressing lineage-associated antigens, followed by fluorescence-activated cell sorting (FACS) to isolate a fraction of Thy-1loSca-1+H-2Khi cells shown previously to be highly enriched (one per 55 cells) in competitive long-term repopulating units (Szilvassy et al., 1996a). In experiment 2, BM was harvested from donor mice 5 days after 5-FU treatment, and primitive hematopoietic cells were enriched simply by immunomagnetic depletion of terminally differentiated (Lin+) cells. Enriched hematopoietic cells were infected with MSCV/HRX–ENL retrovirus by spinoculation and cultured in methylcellulose medium supplemented with interleukin (IL)-3, IL-6, granulocyte–macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF) with or without G418 selection. In the absence of G418, an average of 10% (experiment 1) and 2% (experiment 2) of the cells infected with either MSCV/HRX–ENL or MSCV/neo viruses formed colonies of mature myeloid cells in vitro. Culture in G418 did not significantly diminish the cloning efficiency of hematopoietic cells infected with MSCV/neo [80 and 100% of colony-forming cells (CFCs) were infected in experiments 1 and 2, respectively] but resulted in an ∼10-fold reduction in the number of colonies generated by both cell populations infected with MSCV/HRX–ENL. This difference in the efficiency of gene transfer into clonogenic progenitors probably reflected the lower titer of the MSCV/HRX–ENL viral stocks compared with the parental vector as measured on NIH3T3 cells (1–5×105versus 1–5×106G418R U/ml, respectively). G418-resistant colonies derived from progenitors infected with either vector were heterogeneous in size and exhibited a range of morphologies similar to colonies obtained from normal or mock-infected cells (data not shown). To determine if hematopoietic progenitors infected with HRX–ENL might display some perturbation in their normal self-renewal or differentiation potential not apparent in the primary colony assays, whole methylcellulose cultures were harvested 8–10 days after plating and a proportion of the cells was seeded into secondary methylcellulose cultures without G418. The total number of colonies generated in the secondary assays was similar for both HRX–ENL- and neo-infected cells: ∼30 per 104 cells plated for experiment 1, and ∼90 per 104 cells plated for experiment 2 (Figure 2). However, these secondary colonies displayed striking differences in their morphology, noticeable as early as 6 days after plating. Virtually all of the colonies derived from MSCV/neo-infected progenitors were very small (<30 cells) and diffuse, consistent with the limited self-renewal and proliferative potential of CFCs in normal BM. In contrast, secondary colonies derived from MSCV/HRX–ENL-infected cells were much larger (∼300 cells) and exhibited three distinct morphologies. Most (50–80%) were extremely compact and resembled colonies generated by primitive hematopoietic cells (Figure 3A). A smaller subset (20–40%) had a compact center with a diffuse halo of differentiating cells (Figure 3B) and a third minor subset (<15%) was comprised of large diffuse colonies (Figure 3C). Wright–Giemsa staining revealed that the very compact colonies consisted of immature myeloid cells while the colonies with a diffuse component also included differentiated macrophages (data not shown). Figure 2.Effect of HRX–ENL on the replating efficiency of hematopoietic progenitors. Each point represents the mean number of colonies generated per 104 cells seeded (±SEM) from three independent transduction experiments (one conducted on day 2 post-5-FU Thy-1loSca-1+H-2Khi BM cells, and two conducted on lineage-depleted day 5 post-5-FU BM cells as described in experiments 1 and 2, respectively, in Materials and methods). Download figure Download PowerPoint Figure 3.Morphology of secondary colonies generated by MSCV/HRX–ENL-infected hematopoietic cells. (A) Typical compact colony representing 50–80% of total HRX–ENL colonies. (B) Colonies with a dense center surrounded by a halo of migrating cells (20–40% of colonies). (C) Diffuse colonies of mobile differentiating cells ( 103 cells, secondary assays were harvested and tertiary assays initiated with MSCV/neo- or MSCV/HRX–ENL-transduced cells (104 cells/dish). Very few tertiary colonies were generated by MSCV/neo-infected cells, demonstrating that CFCs present in the secondary assays had exhausted virtually all proliferative capacity by the second generation. In contrast, progenitors expressing HRX–ENL exhibited a greatly enhanced proliferative potential in a third (1–7%) and fourth (10–19%) round of serial replating (Figure 2). The third and fourth generation HRX–ENL colonies conserved the distribution of morphologies noted in the secondary assays above. Furthermore, when 5–10 secondary colonies of each type were picked and replated according to morphology, all three colony types regenerated tertiary daughter colonies of all three morphologies at proportions equivalent to that produced by unselected secondary colonies (data not shown). It was noted, however, that the diffuse colonies had a low frequency of replating (only one of five colonies tested successfully replated), which correlates with the finding that these colonies consisted predominantly of macrophages. Taken together, these data demonstrate that HRX–ENL markedly enhances the self-renewal capacity and proliferative potential of clonogenic hematopoietic progenitors in vitro. Progenitors expressing HRX–ENL establish long–term myeloid progenitor cell lines To study further the properties of HRX–ENL-transduced progenitors in vitro, tertiary colonies described above were pooled and propagated in suspension cultures containing IL-3, IL-6 and SCF. Two cell lines were established by continuous passage of the expanding cells: line A from ∼250 pooled colonies derived from day 2 post-5-FU Thy-1loSca-1+H-2Khi cells, and line B from ∼100 colonies arising from lineage-depleted day 5 post-5-FU BM. After a few passages, IL-6 was omitted from the medium and the cells maintained in IL-3 and SCF for >7 months. The integration pattern of MSCV/HRX–ENL proviruses in each cell line was assessed by Southern blot analysis with an HRX probe. The presence of intact proviral DNA was confirmed in both cell lines by detection of two predicted bands of 5.8 and 3.0 kb in size following digestion with KpnI (which cuts once in each LTR and in the HRX–ENL cDNA) (Figure 4A). The number of proviral integrants in the infected cells was determined by Southern blot analysis of DNA digested with BamHI (which cuts once in the provirus to generate fragments whose size is dependent on the site of integration). A single fragment of ∼8.0 kb was found in line A, suggesting its derivation from a single retrovirally infected progenitor cell. In line B, a major band of ∼25 kb and a fainter band of ∼6 kb (not apparent in Figure 4A) were detected. Southern blot analysis of subclones of line B confirmed that the 6 kb fragment corresponds to an independent integration site and that this line is composed of at least two marked clones (data not shown). Southern blot analyses of proviral integrations were also performed on pooled and expanded tertiary colonies that originated from individually plucked secondary colonies. Unique hybridization patterns were observed in nine of 12 cultures (data not shown). HRX–ENL transcripts were detected in lines A and B by RT–PCR analysis employing oligonucleotide primers flanking the HRX–ENL fusion site (Figure 4B and data not shown). Thus, the lines were of monoclonal or oligoclonal origin and harbored transcriptionally active MSCV/HRX–ENL proviral genomes, confirming that HRX–ENL is directly responsible for immortalization of CFCs in vitro. Figure 4.Integration and expression of retrovirally transduced HRX–ENL sequences in progenitor lines A and B. (A) Southern blot analysis of genomic DNA digested with KpnI or BamHI and hybridized with a human HRX probe. KpnI cleaves once within each LTR and once in the HRX–ENL cDNA, thus generating two fragments of 3.0 and 5.8 kb which span the proviral genome. The 5.6 kb band also observed in genomic DNA from NIH3T3 cells represents the endogenous murine HRX gene which cross-hybridizes with the human HRX probe. BamHI cleaves once within the MSCV/HRX–ENL vector and allows detection of proviral DNA fragments whose size varies with the site of integration. The 9.0 kb BamHI fragment represents the endogenous HRX gene. (B) RT–PCR analysis for HRX–ENL transcripts. DNA-free RNA was converted to cDNA and amplified with primers flanking the fusion site of HRX–ENL. The predicted product of 373 bp is observed for MSCV/HRX–ENL-infected cells from line A and the t(11;19)-bearing control cell line HB1119, but not in negative control lanes in which reverse transcriptase (RT) was omitted from the amplification reaction. The lower band in HB1119 cells is a differentially spliced form of the HRX–ENL fusion transcript (Tkachuk et al., 1992). Download figure Download PowerPoint Both cell lines were composed predominantly of cells with morphologic features of immature myelomonocytic cells, but also contained some cells with more advanced stages of nuclear segmentation (Figure 5A and B). Flow cytometric analysis demonstrated that 100% of the cells were Mac-1+, Gr-1−, TER-119−, B220− and Thy-1− (Figure 5C). Both lines also expressed CD43 and c-kit, which are found on primitive hematopoietic precursors, but were Sca-1− (data not shown). Line A expressed slightly higher levels of Mac-1 and CD43 than line B (data not shown), suggesting that line A may be further engaged in the myeloid differentiation pathway (Moore et al., 1994). Like the tertiary colonies from which they were derived, both cell lines displayed a very high cloning efficiency in methylcellulose cultures supplemented with IL-3, IL-6, GM-CSF and SCF (80% for line A and 40% for line B). Most colonies had the typical compact appearance observed previously and were comprised of immature myeloid cells and monocytes/macrophages. Consistent with these findings, both lines expanded ∼25-fold after 5 days of culture in medium containing either IL-3, granulocyte colony-stimulating factor (G-CSF) or GM-CSF alone (Figure 6) and could be sustained for >20 days in these cytokines without SCF. The only significant difference noted between the two lines was the 2-fold lower proliferation of line B in G-CSF alone. Lower, but significant, proliferation of each line was also observed in IL-6 or SCF alone, and the two factors together had an additive effect on cell output (Figure 6). When both lines were stimulated with G-CSF, c-kit expression was lost while Mac-1 expression increased and terminally differentiated granulocytes were generated (Figure 5D and E). We were unable to demonstrate B lymphoid or erythroid differentiation potential (assessed by B220 or TER-119 expression, respectively) of HRX–ENL-infected progenitors upon culture with SyS-1 stromal cells in medium containing IL-7, or in liquid or methylcellulose cultures containing erythropoietin. Therefore, HRX–ENL-infected progenitors retained some capacity for terminal differentiation, but this was restricted to the myeloid lineage. Figure 5.Morphology and phenotype of cells immortalized by HRX–ENL. (A) and (B) Wright–Giemsa-stained cytospin preparation of lines A and B, respectively. Bar = 14 μm. (C) Flow cytometric analysis of surface antigen expression by line A. Black lines represent staining obtained with PE-conjugated antibodies specific for the indicated hematopoietic cell surface antigens. Gray lines represent the signal obtained with corresponding isotype control antibodies. (D) Wright–Giemsa-stained cytospin preparation and flow cytometric analysis demonstrating modulation of Mac-1 and c-kit expression of line A after culture in medium containing G-CSF. Download figure Download PowerPoint Figure 6.Proliferation of lines A and B in liquid cultures supplemented with recombinant hematopoietic growth factors. Cells (104 per well) from each line were cultured with the indicated cytokines and cell expansion measured after 5 days by adding Alamar blue substrate. Shown are the mean number of arbitrary fluorescence units (±SEM) obtained from triplicate wells from four independent experiments. Download figure Download PowerPoint Hematopoietic progenitor cells transduced with HRX–ENL induce acute myeloid leukemias in vivo To determine whether immortalized progenitor cells were capable of generating tumors in vivo, cells of line B were injected i.v. into sub-lethally irradiated syngeneic (B6.SJL) mice. Over an observation period of 7 months, all 10 mice injected with line B cells succumbed to AMLs (Figure 7). Histologic and cytologic analyses showed that >90% of BM cells had morphologic features of blasts similar to those displayed by cells of line B. Leukemic cells effaced the normal splenic architecture, and in the liver they were present as massive diffuse periportal and sinusoidal infiltrates. Southern blot analysis of DNA from the spleens of two animals revealed intact MSCV/HRX–ENL proviral DNA which confirmed that the tumor cells were derived from line B (data not shown). Notably, in two cases where explanted leukemia cells were analyzed, these remained dependent on IL-3 for in vitro growth. Similar results were obtained with line A. Following its injection into non-irradiated SCID mice (106 cells i.p. per recipient), nine of 10 recipients died 72–84 days later with AMLs (Figure 7). Figure 7.Development of leukemia in mice transplanted with virus-infected cell lines or sorted BM cells. A total of 106 cells from line A (●) or line B (○) were injected into 10 non-irradiated SCID or 10 sub-lethally irradiated syngeneic B6.SJL mice, respectively. For experiments with freshly infected BM cells, lethally irradiated B6.SJL mice were transplanted with 200 Thy-1loSca-1+H-2Khi day 2 post-5-FU BM cells (Ly-5.2+) transduced by MIN/HRX–ENL [17 mice (♦)] or MIN/neo (nine mice), together with 105 twice serially transplanted Ly-5.1 BM cells to provide radioprotection. Download figure Download PowerPoint Leukemic transformation of freshly isolated hematopoietic progenitor cells infected with MIN/HRX–ENL To determine whether expression of HRX–ENL in freshly isolated BM cells induced their leukemic transformation in vivo, lethally irradiated B6.SJL (Ly-5.1+) mice were transplanted with 200 Thy-1loSca-1+H-2Khi day 2 post-5-FU BM cells (Ly-5.2+) after infection with the MSCV-derived vector MIN/HRX–ENL (see Materials and methods). All mice were also co-transplanted with 105 twice serially transplanted Ly-5.1 BM cells to provide radioprotection (Szilvassy et al., 1990). Nine control mice injected with HSCs infected with MIN/neo exhibited a mean of 69% donor-derived (Ly-5.2+) peripheral blood cells but remained healthy up to 4 months after transplantation. In contrast, 17 mice injected with MIN/HRX–ENL-infected HSCs exhibited a mean of 78% donor blood cells, and eight animals (47%) developed myeloid leukemia with latencies of 73–118 days (Figure 7 and Table I). Elevated white blood cell counts and abnormal increases in the ratio of myeloid to lymphoid cells were detected in most animals before death or sacrifice. Histological analysis of the spleen, liver, kidney and BM of diseased animals revealed infiltration by large numbers of mitotically active, immature myeloid cells (Figure 8A). The leukemia cells present in peripheral blood and BM displayed a range of morphologic features from immature myelomonocytic cells to cells with more advanced stages of nuclear segmentation (Figure 8B). Southern blot analysis of KpnI-digested DNA isolated from the spleens of five animals showed intact MIN/HRX–ENL retroviral DNA, and BamHI digests demonstrated one or a few clones in each case (Figure 8C). Upon explantation, only one leukemia was observed to have acquired factor independence for in vitro growth. Two others were dependent upon IL-3 for optimal viability and proliferation. Of the nine animals which remained healthy even after 4 months, only two contained proviral DNA in peripheral blood cells, indicating that the absence of leukemias in most of these animals was likely to be due to poor efficiency of transduction of reconstituting HSCs by MIN/HRX–ENL. Figure 8.Characteristics of leukemias induced by HRX–ENL. (A) Histological analysis showing leukemia cells infiltrating sinusoids of liver. (B) Wright–Giemsa-stained preparation of peripheral blood showing leukemia cells displaying morphologic features ranging from blasts to early stage myelomonocytic cells with immature nuclear segmentation. (C) DNA isolated from spleens of moribund animals was subjected to Southern blot analysis employing a human HRX probe. KpnI digests (left panel) generated two fragments of 3.0 and 5.0 kb (arrows), confirming intact MIN/HRX–ENL proviral DNA in all cases. BamHI digests (right panel) showed one or two clonal bands in each case whose migration differed from the 9 kb fragment (dash) representative of the endogenous HRX gene. Download figure Download PowerPoint Table 1. Characteristics of leukemias induced in mice transplanted with HRX–ENL-infected hematopoietic cells Animal Latency (days) Terminal blood counts (×10−3/μl) Mac1+ cells in PB (%) Blasts in PB (%) Marrow blasts clonesa Tumor Secondary transplantation WBC RBC PLT 1 81 140 7 137 66 18 ++ 2 5 (36−44 days)/5 3b 116 11 9 625 nd nd nd nd 4 82 222 3 79 76 25 nd nd 5 117 72 7 149 nd 22 + 2 1 (98 days)/4 (>209 days) 6 119 3 2 36 nd 4 +++ 1 7c 73 25 7 268 nd 10 nd nd 8 109 55 6 109 nd 25 ++ 1 4 (37−103 days)/5 (>153 days) 16 104 192 2 55 86 26 ++ 1 1 (45 days)/3 (>158 days) Normald na 9 10 1048 11 0 − na a Bone marrow blasts were quantitated as: −, <10%; +, 10–19%; ++, 20–29%; and +++, 30–40%. b Blood smear and cell counts were performed 18 days before death. c Blood smear and cell counts were performed 11 days before death. d Mean of data obtained from three normal control mice; na, not applicable. nd, not determined. Fusion of HRX to ENL is necessary to induce the deregulated proliferation of hematopoietic progenitors To begin to address the mechanisms by which HRX–ENL contributes to the leukemic transformation of early hematopoietic cells, mutated forms of HRX–ENL were assessed for their ability to affect the proliferative capacity of progenito
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