SH2-containing inositol 5'-phosphatase inhibits transformation of Abelson murine leukemia virus.

v-Abl protein tyrosine kinase encoded by Abelson murine leukemia virus (Ab-MLV) transforms pre-B cells. Transformation requires the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is antagonized by SH2-containing inositol 5'-phosphatase (SHIP), raising the possibility that v-Abl modulates PI3K signaling through SHIP. Consistent with this, we show that v-Abl expression reduces levels of full-length p145 SHIP in a v-Abl kinase activity-dependent fashion. This event requires signals from the Abl SH2 domain but not the carboxyl terminus. Forced expression of full-length SHIP significantly reduces Ab-MLV pre-B-cell transformation. Therefore, reduction of SHIP protein by v-Abl is a critical component in Ab-MLV transformation.

Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line.

The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.

Decreased virus population diversity in p53-null mice infected with weakly oncogenic Abelson virus.

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.

Absence of p53 complements defects in Abelson murine leukemia virus signaling.

The v-Abl protein encoded by Abelson murine leukemia virus (Ab-MLV) induces transformation of pre-B cells via a two-stage process. An initial proliferative phase during which cells with limited tumorigenic potential expand is followed by a crisis period marked by high levels of apoptosis and erratic growth. Transformants that survive this phase emerge as fully malignant cells and usually contain mutations that disable the p53 tumor suppressor pathway. Consistent with the importance of p53 in this process, pre-B cells from p53 null animals bypass crisis. Thus, the transformation process reflects a balance between signals from the v-Abl protein that drive transformation and those coming from the cellular response to inappropriate growth. One prediction of this hypothesis is that Ab-MLV mutants that are compromised in their ability to transform cells may be less equipped to overcome the effects of p53. To test this idea, we examined the ability of the P120/R273K mutant to transform pre-B cells from wild-type, p53 null, and Ink4a/Arf null mice. The SH2 domain of the v-Abl protein encoded by this mutant contains a substitution that affects the phosphotyrosine-binding pocket, and this mutant is compromised in its ability to transform NIH 3T3 and pre-B cells, especially at 39.5 degrees C. Our data reveal that loss of p53 or Ink4a/Arf locus products complements the transforming defect of the P120/R273K mutant, but it does not completely restore wild-type function. These results indicate that one important transforming function of v-Abl proteins is overcoming the effects of a functional p53 pathway.

A small molecule Abl kinase inhibitor induces differentiation of Abelson virus-transformed pre-B cell lines.

Abelson murine leukemia virus-transformed cell lines have provided a critical model system for studying the regulation of B cell development. However, transformation by v-Abl blocks B cell development, resulting in the arrest of these transformants in an early pre-B cell-like state. We report here that treatment of Abelson virus-transformed pre-B cell lines with the small molecule Abl kinase inhibitor (STI571) results in their differentiation to a late pre-B cell-like state characterized by induction of immunoglobulin (Ig) light chain gene rearrangement. DNA microarray analyses enabled us to identify two genes inhibited by v-Abl that encode the Igk 3' enhancer-binding transcription factors Spi-B and IRF-4. We show that enforced expression of these two factors is sufficient to induce germline Igk transcription in Abelson-transformed pro-B cell lines. This suggests a key role for these factors, and perhaps for c-Abl itself, in the regulated activation of Ig light chain gene rearrangement.

NF-kappaB1 can inhibit v-Abl-induced lymphoid transformation by functioning as a negative regulator of cyclin D1 expression.

Mounting evidence implicates deregulated Rel/NF-kappaB signaling as a common feature of lymphoid malignancies. Despite the fact that they promote the survival and proliferation of normal lymphocytes, the underlying mechanisms by which various Rel/NF-kappaB proteins with different transcriptional regulatory capacities might facilitate transformation remain to be established. Here we show that the proliferation and tumorigenicity of Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells are enhanced in the absence of NF-kappaB1 and that this coincides with elevated levels of cyclin D1. Support for a link between cyclin D1 expression and v-Abl transformation came from the finding that proliferation of transformed pre-B cells was reduced in the absence of cyclin D1, while enforced cyclin D1 expression increased the proliferation and tumorigenicity of wild-type transformants. A reduction in endogenous cyclin D1 levels that coincided with NF-kappaB1 transgene reversal of enhanced nfkb1(-/-) pre-B-cell transformation, coupled with NF-kappaB1 inhibition of v-Abl-induced kappaB-dependent murine cyclin D1 transcription, lends support to a model in which v-Abl-induced cyclin D1 transcription in transformed pre-B cells is controlled by Rel/NF-kappaB dimers with different activities.

Temperature-sensitive transformation by an Abelson virus mutant encoding an altered SH2 domain.

Abelson murine leukemia virus (Ab-MLV) encodes the v-Abl protein tyrosine kinase and induces transformation of immortalized fibroblast lines and pre-B cells. Temperature-sensitive mutations affecting the kinase domain of the protein have demonstrated that the kinase activity is absolutely required for transformation. Despite this requirement, mutations affecting other regions of v-Abl modulate transformation activity. The SH2 domain and the highly conserved FLVRES motif within it form a phosphotyrosine-binding pocket that is required for interactions between the kinase and cellular substrates. To understand the impact of SH2 alterations on Ab-MLV-mediated transformation, we studied the Ab-MLV mutant P120/R273K. This mutant encodes a v-Abl protein in which the beta B5 arginine at the base of the phosphotyrosine-binding pocket has been replaced by a lysine. Unexpectedly, infection of NIH 3T3 or pre-B cells with P120/R273K revealed a temperature-dependent transformation phenotype. At 34 degrees C, P120/R273K transformed about 10-fold fewer cells than wild-type virus of equivalent titer; at 39.5 degrees C, 300-fold fewer NIH 3T3 cells were transformed and pre-B cells were refractory to transformation. Temperature-dependent transformation was accompanied by decreased phosphorylation of Shc, a protein that interacts with the v-Abl SH2 and links the protein to Ras, and decreased induction of c-Myc expression. These data suggest that alteration of the FLVRES pocket affects the ability of v-Abl to interact with at least some of its substrates in a temperature-dependent fashion and identify a novel type of temperature-sensitive Abelson virus.

The absence of Msh2 alters abelson virus pre-B-cell transformation by influencing p53 mutation.

Defects in DNA mismatch repair predispose cells to the development of several types of malignant disease. The absence of Msh2 or Mlh1, two key molecules that mediate mismatch repair in eukaryotic cells, increases the frequency of mutation and also alters the response of some cells to apoptosis and cell cycle arrest. To understand the way these changes contribute to cancer predisposition, we examined the effects of defective mismatch repair on the multistep process of pre-B-cell transformation by Abelson murine leukemia virus. In this model, primary transformants undergo a prolonged apoptotic crisis followed by the emergence of fully transformed cell lines. The latter event is correlated to a loss of function of the p53 tumor suppressor protein and down-modulation of the p53 regulatory protein p19Arf. Analyses of primary transformants from Msh2 null mice and their wild-type littermates revealed that both types of cells undergo crisis. However, primary transformants from Msh2 null animals recover with accelerated kinetics, a phenomenon that is strongly correlated to the appearance of cells that have lost p53 function. Analysis of the kinetics with which p53 function is lost revealed that this change provides the dominant stimulus for emergence from crisis. Therefore, the absence of mismatch repair alters the molecular mechanisms involved in transformation by affecting a gene that controls apoptosis and cell cycle progression, rather than by affecting these processes directly.

Addition of constitutive c-myc expression to Abelson murine leukemia virus changes the phenotype of the cells transformed by the virus from pre-B-cell lymphomas to plasmacytomas.

Abelson murine leukemia virus (A-MuLV), a retrovirus that expresses the v-abl oncogene, characteristically induces pre-B-cell lymphomas following in vivo infection of BALB/c mice or in vitro infection of suspensions of fetal liver or bone marrow cells. ABL-MYC, a retrovirus that expresses both v-abl and c-myc, induces solely plasmacytomas in BALB/c mice. To investigate how the addition of overexpression of c-myc to that of v-abl accomplishes this dramatic change in the phenotype of the cells transformed by these closely related retroviruses, we utilized helper-free A-MuLV (psi 2) and ABL-MYC (psi 2) in vitro to infect suspensions of cells from different lymphoid tissues and purified immature and purified mature B cells. As expected, A-MuLV(psi 2) induced only pre-B-cell lymphomas in vivo and in vitro when immature B cells were present. ABL-MYC(psi 2), on the other hand, produced only plasmacytomas, even when purified immature B lymphocytes were infected in vitro. Although the A-MuLV(psi 2)-induced pre-B-cell lymphomas express easily detectable levels of c-myc mRNA, maturation into more-mature forms of B lymphocytes is blocked. The constitutively overexpressed c-myc in the ABL-MYC retrovirus abrogates this block, permits maturation of infected immature B cells, and yields transformed plasma cells.

Carboxyl-terminal determinants of Abelson protein important for lymphoma induction.

The carboxyl-terminal region of the Abelson protein is not absolutely required for Abelson virus transformation. However, Abelson virus strains encoding proteins missing portions of this region have a reduced ability to transform lymphoid cells in vitro and in vivo. One such strain, called P90A, is unique in that P90A-injected mice almost always develop tumors containing highly oncogenic variants that encode new forms of Abelson protein. In this work, we have examined the mechanism by which these variants are generated and used the variants to identify carboxyl-terminal protein sequences important for the induction of Abelson disease. Analysis of mice injected with helper-free P90A virus stocks demonstrates that the variants are generated during viral replication in vivo, probably as a consequence of error-prone reverse transcription. The sequence of the P90A viral genome reveals that a 19-base deletion is responsible for synthesis of the truncated Abelson protein. As a consequence of this mutation, 167 carboxyl-terminal amino acids normally found in the wild-type protein have been replaced by 33 amino acids derived from an alternative reading frame. Site-directed mutants show that the combination of the deletion and the P90A carboxyl terminus is required for the generation of variants. Thus, the particular structure of the P90A protein, not the specific residues lost or gained, alters the transforming potential of the Abelson protein. Finally, the sequence of the variants encoding smaller Abelson proteins reveals that as few as 452 v-abl-encoded amino acids are required for rapid induction of Abelson disease.

Chronic myeloproliferative disease induced by site-specific integration of Abelson murine leukemia virus-infected hemopoietic stem cells.

We recently showed that hemopoietic stem cells expressing the v-abl oncogene can cause leukemia when injected into lethally irradiated recipient mice. Progenitor cells expressing v-abl did not significantly contribute to disease development, and the leukemia was monoclonal in origin. By serially transplanting v-abl-transduced hemopoietic stem cells into normal, nonirradiated syngeneic recipients, we showed that multiple stem-cell clones do exist in some recipients. These cells fluctuated as normal stem cells do and could home to normal bone marrow. Based on the time course of disease, the recipients developed either an acute or a chronic phase of disorder. All recipients with the acute disease had stem-cell clones with random Abelson murine leukemia virus integration sites. All recipients with the chronic disorder had a specific Abelson murine leukemia virus integration site. We believe this abl-specific integration site, termed ASI, is important in abl-mediated stem-cell leukemogenesis.

The accelerating role of Abelson murine leukemia virus in murine plasmacytoma development: in vitro infection of spleen cells generates donor-type tumors after transfer to pristane-treated BALB/c mice.

The role of Abelson murine leukemia virus (A-MuLV) in the accelerated development of murine plasmacytomas (PCs) (Potter et al., 1973: Science, 132, 592-594) was studied in a new experimental system. Spleen cells from pristane-treated or untreated BALB/c mice carrying Robertsonian 6;15 fusion chromosomes were infected in vitro with helper-free A-MuLV overnight and subsequently transplanted into the peritoneal cavity of pristane-treated or untreated BALB/c mice. Donor-derived PCs developed in 4 out of 76 pristane-treated recipients [latent periods: 38-82 (mean 51) days] that had received spleen cells from pristane-treated donors, and also in 2 out of 41 pristane-treated recipients that had received untreated donor-derived spleen cells (latent periods: 65 and 120 days). Three of the PCs in the former and both PCs in the latter group were tested for integration and expression of the v-abl gene, with positive results. This indicates that the spleen contains PC-precursor cells that can be activated by A-MuLV even before the impact of pristane. All 6 donor-origin PCs carried a translocation involving chromosome 15, band D2/3. Four of these corresponded to a typical 12;15 translocation, one was a variant 6;15 translocation and the 6th may represent a previously unidentified translocation between chromosome 15 and the lambda gene-carrying chromosome 16. No PCs developed among 29 pristane-untreated recipients that had received pristane-treated donor-derived spleen cells. In addition to PCs, monocytic tumors developed in 37 (26%) of all recipients. Their development was independent of pristane treatment of recipients but was particularly frequent in those who had received spleen cells from pristane-treated donors.

Multiple steps are required for the induction of tumors by Abelson murine leukemia virus.

Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.

Distinct helper virus requirements for Abelson murine leukemia virus-induced pre-B- and T-cell lymphomas.

Abelson murine leukemia virus (A-MuLV) can induce pre-B- or T-cell lymphomas (thymomas) in mice depending on the route and time of injection. Previous studies have shown that the choice of the helper virus used to rescue A-MuLV greatly influences its ability to induce pre-B-cell lymphomas. In this study, we investigated the role of the helper virus in A-MuLV-induced thymomas. A-MuLV rescued with the helper Moloney MuLV, BALB/c endogenous N-tropic MuLV, and two chimeric MuLVs derived from these two parents were injected intrathymically in young adult NIH Swiss mice. All four A-MuLV pseudotypes were found to be equally efficient in the induction of thymomas, whereas drastic differences were observed in their pre-B-cell lymphomagenic potential. Thymoma induction by A-MuLV was independent of the replication potential of the helper virus in the thymus, and no helper proviral sequences could be detected in the majority of thymomas induced by A-MuLV rescued with parental BALB/c endogenous or chimeric MuLVs. In the thymomas in which helper proviruses were present, none of them were found integrated in the Ahi-1 region, a common proviral integration site found in A-MuLV-induced pre-B-cell lymphomas (Y. Poirer, C. Kozak, and P. Jolicoeur, J. Virol. 62:3985-3992, 1988). In addition, helper-free stocks of A-MuLV were found to be as lymphomagneic as other pseudotypes in inducing thymomas after intrathymic inoculation, in contrast to their inability to induce pre-B-cell lymphomas when injected intraperitoneally in newborn mice. Restriction enzyme analysis revealed one to three A-MuLV proviruses in each thymoma, indicating the oligoclonality of these tumors. Analysis of the immunoglobulin and T-cell receptor loci confirmed that the major population of cells of these primary thymomas belongs to the T-cell lineage. Together, these results indicate that the helper virus has no effect in the induction of A-MuLV-induced T-cell lymphomas, in contrast to its important role in the induction of A-MuLV-induced pre-B-cell lymphomas. Our data also revealed distinct biological requirements for transformation of these two target cells by v-abl.

Different genes control the susceptibility of mice to Moloney or Abelson murine leukemia viruses.

The susceptibility of mice to lymphoma induction by Moloney or Abelson murine leukemia virus has been compared in BALB/c, C57BL/6, and BALB/cXC57BL/6 recombinant inbred strains. BALB/c mice were found to be susceptible to lymphoma induction by either virus, and C57BL/6 mice were found to be relatively resistant to lymphoma induction by either virus. The genes that control these patterns of susceptibility to each virus are not the same because susceptibility to each virus segregated independently in CXB recombinant inbred strains. We also found, as reported by Cook (W. Cook, Proc. Natl. Acad. Sci. U.S.A. 79:2917-2921, 1982), when injected intrathymically that Abelson murine leukemia virus rapidly induced thymomas in weanling B6 mice. Examination of the cellular phenotypes of the tumors induced by Abelson murine leukemia virus or by Moloney murine leukemia virus indicated that different lymphocyte subpopulations were the targets for tumor induction by each virus.

The minimum transforming region of v-abl is the segment encoding protein-tyrosine kinase.

Only 1.2 kilobases (kb) at the 5' end of the 3.9-kb v-abl sequence in Abelson murine leukemia virus is required for fibroblast transformation. A precise delineation of this minimum transforming region was made by using small 5' or 3' deletions. Insertions of four amino acids, generated by putting synthetic DNA linkers into various restriction enzyme cleavage sites, abolished transforming activity, indicating that much of the internal sequence of the minimum transforming region plays a critical role in the transformation process. This 5' 1.2 kb of v-abl encodes protein-tyrosine kinase activity when expressed in Escherichia coli. Each of the mutations which caused a loss of transformation activity also resulted in a loss of protein-tyrosine kinase activity when expressed in E. coli. The minimum transforming region of v-abl contains amino acid homology to other protein-tyrosine kinase oncogenes, and a comparison with these oncogenes is presented.

Protein stabilization explains the gag requirement for transformation of lymphoid cells by Abelson murine leukemia virus.

The single protein encoded by Abelson murine leukemia virus is a fusion of sequence from the retroviral gag genes with the v-abl sequence. Deletion of most of the gag region from the transforming protein results in a virus capable of transforming fibroblasts but no longer capable of transforming lymphoid cells. Smaller deletions in gag reveal that p15 gag sequences are responsible for this effect, whereas deletion of p12 sequences had no effect on lymphoid transformation. In transformed fibroblasts, p15-deleted and normal proteins had similar activities and subcellular localization. When the p15-deleted genome was introduced into previously transformed lymphoid lines, its protein product exhibited a marked instability. The tyrosine-specific autophosphorylation activity per cell was less than 1/20th that of the nondeleted protein. Although pulse-Ia-beling showed that the p15-deleted protein was synthesized efficiently, immunoblotting demonstrated that its steady-state level was less than 1/10th that of the nondeleted Abelson protein. The specific instability of the p15-deleted protein in lymphoid cells explains the requirement of these sequences for lymphoid but not fibroblast transformation.