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Previous Article | Next Article 
Molecular and Cellular Biology, November 2002, p. 7678-7687, Vol. 22, No. 21
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.21.7678-7688.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Pathology, Stanford University School of Medicine, Stanford, California 94305
Received 23 January 2002/ Returned for modification 28 May 2002/ Accepted 30 July 2002
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ABSTRACT |
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INTRODUCTION |
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Many of the genetic abnormalities in B cell precursor ALLs are recurrent chromosomal translocations that create fusion genes encoding chimeric kinases or transcription factors (23). An uncommon but well-studied chimera is E2a-Hlf, a basic-leucine zipper DNA-binding protein that contains the heterologous E2a transactivation domains as a consequence of t(17;19) chromosomal translocations in a subset of pediatric ALLs (11, 14). Gene transfer and candidate target gene studies suggest an oncogenic role for E2a-Hlf through induction of apoptotic resistance. E2a-Hlf prevents apoptosis after cytokine withdrawal in interleukin-3 (IL-3)-dependent pro-B-cell lines, possibly through effects on several subordinate pathways (13, 15, 16). One of its candidate target genes, SLUG, also mediates apoptotic resistance and shares similarity with Ces1, which functions downstream of Ces2 to modulate survival of a rare neuronal cell population in Caenorhabditis elegans (17). Since Hlf itself shares limited similarity with Ces2, E2a-Hlf may inappropriately activate a conserved signaling pathway that both worms and B-cell precursors use for modulation of cell survival. An alternative oncogenic role for E2a-Hlf is suggested by studies in transgenic murine models where it appears to antagonize lymphoid differentiation in the course of inducing primarily T-lineage malignancies (9, 33). Due to the limitations of previously employed experimental systems, it remains unclear which of the several roles ascribed to E2a-Hlf are more relevant for B cell precursor leukemogenesis and how E2a-Hlf may disrupt the normal program of lymphopoiesis.
In this report we evaluated the oncogenic contributions of E2a-Hlf employing an in vitro culture system that recapitulates the origin of B-cell precursor ALL from a bone marrow stromal microenvironment. Under these conditions, E2a-Hlf required coexpression with Bcl-2, a general suppressor of apoptosis, to immortalize bone marrow pre-B cells, which maintained their stromal and cytokine requirements for growth. Abrogation of stroma and cytokine dependence was associated with leukemic progression, suggesting a multistep model for induction of B-cell precursor ALL initiated by E2a-Hlf.
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MATERIALS AND METHODS |
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Retroviral stocks were produced by transfecting the Phoenix packaging cell line with 10 µg of purified plasmid DNA (27). Retrovirus-containing supernatants were collected 48 h later, filtered, and used for infection of primary bone marrow cells. Viral titers were determined by transfer of neomycin resistance to NIH 3T3 cells after infection with retroviral supernatants as described previously (21).
Isolation and transduction of B-cell progenitors. In vitro bone marrow cultures were performed on monolayers of the stromal cell line AC-6.21 (34). To allow selection for transduced bone marrow cells, AC-6.21 cells were converted to neomycin and puromycin (or neomycin and hygromycin) resistance by retroviral transduction with MSCV-neo and MSCV-puro (or MSCV-hygro) vectors, respectively. Stromal cells were maintained in RPMI 1640-5% fetal calf serum and 5 x 10-5 M ß-mercaptoethanol (R-5). On the day prior to bone marrow transduction, drug-resistant AC-6.21 cells were plated in six-well plates at a density of 5 x 105/well and then irradiated (3,000 rads) by using a gamma (137Cs) source. Primary bone marrow cells were isolated from 3.5-week-old BALB/c donor mice under sterile conditions. Bone marrow cells (4 x 105) were mixed with 1.0 ml of viral stock (ca. 107 PFU) supplemented with 4 µg of Polybrene/ml and spinoculated at 2,500 rpm for 2 h at 33°C. The spinoculated bone marrow cells were resuspended in R-5 medium, and equal numbers were plated into two replicate wells containing irradiated AC-6.21 stromal cells. Selection for drug-resistant colonies was initiated after 5 days. Cultures were fed weekly by carefully replacing half of the medium with fresh R-5 containing appropriate antibiotics (800 µg of G418/ml, and/or 1 µg of puromycin/ml, and/or 150 µg of hygromycin/ml). Growth of lymphoid cells overlying the stromal layer was assessed by using a manual hemocytometer after their removal by gentle pipetting.
Methylcellulose assays. Stromal contact and IL-7 requirements for growth were evaluated by using methylcellulose assays. Lymphoid cells were removed from the underlying stromal layer by gentle pipetting and then suspended (at 2.5, 5, and 7.5 x 104 cells/well) in methylcellulose medium with or without exogenous IL-7 (StemCell Technologies, Vancouver, British Columbia, Canada). Colonies were enumerated by light microscopy. Colonies that grew in the presence of supplemental IL-7 were transferred to liquid medium containing RPMI 1640-10% fetal calf serum supplemented with 1 ng of murine IL-7 (R&D Systems, Minneapolis, Minn.)/ml and 5 x 10-5 M ß-mercaptoethanol (R-10/7). Cytokine-dependent cells were maintained in R-10/7.
Leukemogenicity assays. Syngeneic, nonirradiated 4-week-old BALB/c mice were used to evaluate the leukemogenic potential of immortalized cells from in vitro bone marrow cultures. Mice were injected intravenous (i.v.) with 106 cultured lymphoid cells, and development of leukemia was monitored by periodic assessment of peripheral blood cell counts and morphology. Blood smears were fixed and stained with May-Grunwald and counterstained with Giemsa (Fluka Chemika/Sigma-Aldrich, St. Louis, Mo.). To study the leukemogenic potential in vivo of freshly isolated hematopoietic cells transduced with E2A-HLF retroviral constructs, lethally irradiated BALB/c mice (two equal doses of 495 rads of X-irradiation) were injected i.v. with 106 bone marrow cells (in 0.1 ml of R-5) subjected to spinoculation as described above. Leukemogenic potential was assessed periodically by morphological staining of blood cells obtained from tail bleeds. Moribund animals were euthanized by CO2 inhalation and tissues were removed for in vitro culture, Western blots, Southern blots, fluorescence-activated cell sorting (FACS) analysis or histologic examination (after fixation in buffered formalin and staining with hematoxylin and eosin). Leukemic cells were plated in vitro in R-10 medium with or without IL-7 to assess growth and cytokine requirements. Transplantability of leukemias was analyzed by injecting 106 nucleated cells from primary diseased mice i.v. into five nonirradiated secondary BALB/c recipient mice. Secondary leukemias were also evaluated for phenotypes and in vitro dependence on IL-7 for growth. All animals were bred and maintained in the Stanford University Research Animal Facility. Irradiated animals were maintained on a regimen of double antibiotic water solution (polymyxin-B sulfate, 850 U/ml; neomycin, 10% final concentration) throughout the duration of the experiments.
DNA and protein analysis.
DNA was isolated from fresh cells or tissues, digested with appropriate restriction enzymes, and analyzed by Southern blot hybridization by using standard methods. Probes for the mouse IgH-JH4 and IgL-J
5 have described previously (3). Protein extracts were prepared from cells or tissues by boiling in 1x sample buffer for 10 min. The extracts were then sonicated and protein concentration determined by UV spectroscopy (i.e., the optical density at 280 nm). Total cellular proteins (30 to 50 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then analyzed by Western blotting with monoclonal antibodies specific for E2a (18), Abl or p53 (clone AB-7; Calbiochem, Boston, Mass.), murine IL-7 (PeproTech, Rocky Hill, N.J.), human Bcl-2 (Dako, Carpinteria, Calif.), p19ARF (Novus Biologicals, Littleton, Colo.), or Mdm2 (clone C-18; Santa Cruz Biotechnology, Santa Cruz, Calif.) as previously described (32). Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.). Antibody specific for mutant p53 (clone PAB 240; Neomarkers, Fremont, Calif.) was used for immunoprecipitation.
Fluorescence-activated flow cytometric analysis. FACS analysis was performed as previously described (33) with antibodies purchased from Pharmingen Research Products (San Diego, Calif.). Specificities of antibodies were as follows: fluorescein isothiocyanate-conjugated S7 (anti-CD43), allophycocyanin-conjugated RA3-6B2 (anti-B220), phycoerythrin-conjugated M1/69 (anti-CD24/HSA), biotinylated 6C3/BP1 (anti-Ly51), phycoerythrin-conjugated DS-1 (anti-IgM, biotinylated RB6/8C5 (anti-Gr1), fluorescein isothiocyanate-conjugated M1/70 (anti-Mac1), and avidin-conjugated Texas red.
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RESULTS |
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genes (Fig. 3B and Table 1, BiEH.3-SD and BiEH.4-SD), suggesting an oligoclonal population of B lineage progenitors. In contrast, the cells in p190 primary cultures displayed one or two clonal IgH gene rearrangements, germ line Ig genes and single proviral integrations.
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The leukemogenic potentials of the stroma-independent (BiEH.1-SI, BiEH.2-SI, BiEH.3-SI, and BiEH.4-SI) and IL-7-independent (BiEH.5) cells were tested in syngeneic, nonirradiated recipients. BiEH.5 cells induced fatal leukemias with a latency of 2 to 3 weeks (Fig. 4 and 5). The pathological and cytologic features of the leukemias were comparable to those described above for p190-associated disease. In contrast, the stroma-independent, IL-7-dependent lines (BiEH.1-SI, BiEH.2-SI, BiEH.3-SI, and BiEH.4-SI) were nonleukemogenic under these conditions (Fig. 4 and 5B).
To address whether leukemogenicity required abrogation of the IL-7 cytokine requirement, stroma-independent cells were transduced with a retroviral construct expressing murine IL-7 under control of the MSCV LTR (Fig. 1A). After selection of stable transductants for resistance to puromycin, the resulting cells (BiEH.1-IL-7 and BiEH.2-IL-7) expressed exogenous IL-7 that was released into the culture medium (not shown). These lines displayed high clonogenic potentials in methylcellulose lacking added IL-7 (Fig. 4) and grew in liquid medium containing serum in contrast to their respective parental lines. Both lines induced rapidly fatal leukemias in recipient mice (Fig. 4). The disease latencies, pathology, and cytology were comparable to those observed in animals with BiEH.5 and p190 disease (Fig. 4 and data not shown). Taken together, our observations strongly suggested that abrogation of IL-7 dependence was a requirement for induction of aggressive leukemias by cells immortalized through the combined actions of E2A-HLF + BCL2.
Coexpression of IL-7 with E2a-Hlf is not sufficient to induce pre-B-cell leukemias. We investigated whether forced expression of IL-7 could substitute for Bcl-2 in collaborating with E2a-Hlf to immortalize pre-B-I cells and facilitate development of acute leukemias. Primary bone marrow cells were transduced with MSCV constructs expressing either IL-7 or IL-7-IRES-E2A-HLF (7iEH) or the control IL-7+BCL2 and plated on AC-6.21 stromal cells. Robust growth under these conditions was observed for cells transduced with these constructs. Phenotype analyses showed that the cultures were predominantly comprised of pre-B-I cells (Table 1) with some heterogeneity in cell compositions (Fig. 3D). Southern blot analyses showed multiple IgH gene rearrangements in the absence of IgL rearrangements consistent with an oligoclonal pre-B-I population (Fig. 3D and Table 1). Despite their vigorous in vitro growth, however, cells expressing IL-7 alone, IL-7+BCL2, or IL-7 + E2A-HLF were incapable of inducing leukemias in syngeneic, immunocompetent mice over an observation period in excess of 6 months. Thus, IL-7 could substitute for Bcl-2 to allow the in vitro growth of pre-B cells expressing E2a-Hlf, but the combined actions of IL-7 and E2a-Hlf were insufficient for leukemic conversion.
Cells expressing IL-7 + E2A-HLF preferred growth on stroma but could be adapted to growth in liquid culture without stroma. Pre-B cells expressing ectopic IL-7+BCL2, or IL-7 alone, in contrast, did not grow in the absence of stroma. In liquid culture, IL-7/E2A-HLF cells (7iEH-SI) maintained a pre-B-I phenotype. However, FACS analysis showed a more homogenous expression of surface antigens, and Southern blot analyses revealed a single IgH gene rearrangement (Fig. 3D), indicating that they had undergone a clonal selection in the process of adapting to growth in the absence of stroma. Injection of these cells into syngeneic, nonirradiated mice resulted in the rapid induction of fatal leukemias in all recipients within 3 weeks. Taken together, these results suggested that coexpression of IL-7 and E2a-Hlf was not sufficient for induction of pre-B-cell leukemias due to the requirement for yet an additional event that appeared to correlate with stroma independence.
Stromal independence is associated with p53 pathway alterations. We investigated whether the clonal selections occurring at different points in the evolution of E2a-Hlf-associated leukemias in vitro (summarized in Fig. 6) may involve inactivation of the p53 apoptosis and tumor surveillance pathway. Western blot analyses of Arf, Mdm2, and p53 expression were performed on various cell lines that differed in their stroma and cytokine requirements and leukemogenic potentials. Stroma-dependent cells immortalized by E2a-Hlf in combination with Bcl2 (BiEH.3 and BiEH.4) lacked expression of all three proteins above background levels. However, clonal sublines that had been selected for growth in the absence of stroma (BiEH.3-SI and BiEH.4-SI) expressed abundant Arf and Mdm2 (Fig. 7A, compare lanes 1 and 3 with lanes 2 and 4). Similarly, stroma-dependent cells immortalized by E2a-Hlf + IL-7 (7iEH-SD) lacked detectable Arf, Mdm2, or p53, whereas a stroma-independent subclone (7iEH-SI) expressed mutated p53 that reacts with a conformation-dependent monoclonal antibody (Fig. 7A, lanes 6 and 7, and Fig. 7B). Immortalized cells expressing abundant Arf and Mdm2 (e.g., BiEH.3-SI and BiEH.4-SI) were not leukemogenic if they retained a requirement for exogenous IL-7. Cells capable of inducing leukemias with short latencies in syngeneic hosts, however, harbored detectable alterations in the Arf/Mdm2/p53 pathway, evidenced by expression of high level Arf and/or Mdm2 and mutant p53 (7iEH-SI and BiEH.5) (Fig. 7). Leukemias arising de novo after transplantation of cells freshly transduced with E2a-Hlf + Bcl2 (see below) also expressed abundant Arf and Mdm2 (e.g., BiEH-L4 in Fig. 7A, lane 13). Cells immortalized by p190 expressed Arf and p53 (Fig. 7A, lanes 10 to 12), although p53 mutant status was not determined. Taken together, these observations indicated that alterations of the Arf/Mdm2/p53 pathway were a consistent feature of E2a-Hlf associated leukemogenesis and typically arose in vitro during selection for stroma-independent growth.
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DISCUSSION |
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E2a-Hlf collaborates with Bcl-2 to immortalize bone marrow B-cell progenitors. E2a-Hlf sustained the long-term in vitro growth of B-cell progenitors when coexpressed with Bcl-2. This was evident as developmental arrest and unlimited self-renewal of progenitors at the preB-I stage of differentiation comparable in phenotype to human leukemias bearing translocations of the HLF gene. The requirement for coexpressed Bcl-2 was unexpected, since E2a-Hlf itself has reported antiapoptotic properties (15). However, the survival of primary pre-B-I cells in vitro (the present study) and B cells in vivo (33) does not appear to be measurably enhanced by E2a-Hlf despite its ability to inhibit death associated with IL-3 deprivation of pro-B-cell lines in vitro (15). Thus, E2a-Hlf may be relatively selective in its antiapoptotic effects, which appear to be mediated at least in part through downstream perturbations of the IL-3 signaling pathway (13). In our cultures, IL-3 was unable to substitute for IL-7, the major cytokine associated with pre-B-I cell growth and survival in normal bone marrow microenvironments (1). Furthermore, increased apoptotic sensitivity, not resistance, of thymocytes has been reported in E2A-HLF transgenic mice (9), which develop profound thymic hypoplasias (9, 33).
In the present studies, coexpressed Bcl-2 was required for both long-term growth in vitro and induction of leukemias after transplantation of transduced cells. The requirement for Bcl-2 does not appear to reflect a need to suppress a tumor surveillance response to E2a-Hlf, since immortalized progenitors growing on stroma do not display activation of the p53 apoptosis pathway (see below). It is likely that the high apoptotic sensitivity of normal B-cell progenitors to even minimal alterations in their maturation programs constitutes a barrier to initiation of ALL by chimeric oncoproteins, as suggested by previous transgenic studies (3, 32). Coexpressed Bcl-2 may suppress apoptosis that is initiated by a developmental checkpoint that is triggered by E2a-Hlf under our experimental conditions. Abundant endogenous Bcl-2 levels in human cell lines harboring E2a-Hlf chromosomal translocations (K. S. Smith and M. L. Cleary, unpublished observations) raise the possibility of a similar Bcl-2 requirement in human leukemias, although the molecular mechanisms sustaining these Bcl-2 levels are not known.
B-cell progenitors immortalized by E2a-Hlf lack mutations in the Arf/Mdm2/p53 pathway, which subsequently arise during leukemic progression. Immortalization of murine B-cell progenitors by E2a-Hlf occurred in the absence of apparent mutations in the Arf/Mdm2/p53 pathway. This contrasts with murine fibroblast immortalization, which is invariably associated with perturbations of this pathway (20). Although Arf-deficient B-cell progenitors are immortal (28), our results clearly indicate that murine B-cell progenitors can be immortalized by an alternative route in the absence of apparent lesions in this pathway. Similar to Arf-null pre-B cells, primary B-cell progenitors immortalized by the combined actions of E2a-Hlf and Bcl-2 are nonleukemogenic and retained stromal and cytokine requirements for in vitro growth and survival. This resembles the supportive needs of normal B-cell progenitors, which differentiate and proliferate in close contact with bone marrow stromal cells (30). Our studies support the thesis that the primary effect of E2a-Hlf is to impair the differentiation of B-cell progenitors, arresting them in a proliferative state. Whereas normally this may result in programmed cell death, coexpression of Bcl-2 apparently prevents these arrested cells from undergoing apoptosis.
We were able to isolate subpopulations of immortalized cells that no longer required stromal contact by selective passage through methylcellulose containing high exogenous levels of IL-7. The resulting stroma-independent cells were clonal and invariably harbored alterations in the p53 pathway, as evidenced by mutations of p53 itself and/or increased Arf and Mdm2 expression, markers of downstream perturbations in the pathway (5, 31). This suggests a mechanism for limiting the growth of progenitors immortalized by E2a-Hlf that may be analogous to the tumor surveillance responses to other oncoproteins, such as c-Myc, whose forced expression induces apoptosis in low serum but not normal growth conditions (10). Thus, within the context of a stromal microenvironment, E2a-Hlf enhances the self-renewal of B-cell progenitors without initiating a p53-dependent surveillance response. However, inactivating alterations of the INK4A/ARF-MDM2-P53 axis were associated with leukemic progression in our model system and are commonly observed in pediatric ALLs (4, 26), including most t(17;19) cell lines and leukemias, which contain deletions or methylations that silence the INK4A/ARF gene (24). MDM2 overexpression is also observed in a subset of pediatric ALLs and has been linked to poor response to therapy and early relapse (36), a feature of leukemias expressing E2a-Hlf.
Leukemic progression of immortalized B-cell progenitors is associated with abrogation of stromal and cytokine requirements. The cytokine IL-7 is crucial for B-cell development, serving multiple roles that are not alleviated by forced expression of E2a-Hlf. This contrasts with the properties of cells transduced by p190Bcr-Abl, which lack similar stromal and cytokine requirements for growth in vitro. Bcr-Abl, a cytoplasmic kinase, is known to perturb multiple signaling pathways, including PI-3 kinase, ras and Jak/STAT pathways (37), which likely accounts for its potent ability to abrogate the cytokine requirements of primary B-cell progenitors. Furthermore, cells freshly transduced with p190 rapidly induced leukemias when transplanted into nonirradiated recipients, whereas BiEH-transduced cells induced leukemias only in irradiated syngeneic recipients. This, as well as the increased latency and reduced frequency, suggests that BiEH immortalized cells require a supportive microenvironmental niche to initiate a multistep neoplastic process in vivo. By comparison to p190, E2a-Hlf appears to mediate a more limited subset of the requirements for autonomous growth and leukemogenesis. As a consequence, the stromal microenvironment may serve a relatively greater role in nurturing the growth and survival of "preleukemic" B-cell precursors that express this fusion oncogene.
In spite of the need for IL-7 to sustain the growth of immortalized B-cell precursors in vitro, abrogation of IL-7 dependence was necessary for induction of leukemias by cells coexpressing E2a-Hlf and Bcl-2. Several observations support this conclusion. First, regardless of their method of establishment, cells that required exogenous IL-7 or stromal support for in vitro growth were consistently nonleukemogenic in syngeneic recipients. Second, immortalized cells adapted to grow without stroma were nonleukemogenic until programmed to express autocrine IL-7. Third, growth in RPMI medium lacking IL-7 was invariably associated with a rapidly leukemogenic phenotype. Fourth, leukemias that arose after long latency after transplantation of freshly transduced cells displayed cytokine-independent growth upon explantation and were rapidly leukemogenic in secondary recipients. Taken together with the lack of spontaneously arising IL-7-independent pro-B cells in vitro, these data strongly suggest that in our model system IL-7 dependence is a major rate-limiting step in leukemogenesis.
Our experimental model for B-cell precursor ALL reveals dichotomous roles for IL-7. It is essential for growth and survival of immortalized cells, but their dependence on exogenous IL-7 limits progression to a full leukemogenic phenotype. Thus, mutations that constitutively activate the IL-7 signal transduction pathway could provide a possible mechanism for satisfying both roles. To test this hypothesis, we coexpressed IL-7 and observed that it substitutes for Bcl-2 and collaborates with E2a-Hlf to immortalize B-cell precursors in vitro on stromal layers to fulfill both survival and cytokine requirements. Nevertheless, cells expressing E2a-Hlf and autocrine IL-7 underwent crisis and clonal outgrowth of a subpopulation with mutation of p53 upon withdrawal from stromal support. Notably, this was associated with progression to a leukemogenic phenotype in syngeneic, immunocompetent recipients. Thus, abrogation of IL-7 dependence is an important, albeit not sole, requirement for leukemogenic progression. Human cell lines representative of pediatric ALL, including three with the t(17;19) chromosomal translocation, generally lack cytokine requirements for in vitro growth. Although their reduced cytokine requirements may have been selected for during explantation, activating mutations in signal transduction pathways have been reported in a subset of primary ALL cells (29).
In summary, E2a-Hlf enhances the self-renewal of B-cell progenitors that retain their dependence on stromal and cytokine support in vitro and maintain an intact p53 pathway. This requires coexpression of Bcl-2 and is not sufficient for leukemogenesis. Our observations implicate perturbations of stromal and cytokine (IL-7) dependence pathways to collaborate with E2a-Hlf for progression to aggressive ALL from immortalized B-cell precursors. Mutations that disrupt these pathways have been reported previously in human B-cell progenitor leukemias, providing tentative support for the relevance of this multistep model for human leukemia pathogenesis.
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ACKNOWLEDGMENTS |
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These studies were supported by funds from the NIH (CA42971) and the Children's Health Initiative Innovations Fund.
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FOOTNOTES |
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