BCR-ABL Mediated Transformation Research Articles

The Role Of SOCS1 In BCR-ABL Mediated Transformation and Leukemogenesis

The BCR-ABL oncogene activates several signaling pathways, most notably by constitutive phosphorylation of the signal transducer and activator of transcription protein 5 (STAT5). After phosphorylation and nuclear translocation, STAT5 transcriptionally activates numerous genes responsible for proliferation, survival and differentiation of hematopoietic stem and progenitor cells. Among the STAT5 target genes are suppressor of cytokine signaling (SOCS) proteins. SOCS proteins inhibit JAK kinases by multiple mechanisms, thereby terminating cytokine signaling in a classical negative feedback loop. The SOCS family of proteins comprises eight members: cytokine-inducible SRC homology 2 (SH2) domain protein (CIS) and SOCS1–SOCS7. SOCS1 is frequently silenced by hypermethylation in multiple myeloma and inactivating mutations have been found in Hodgkin lymphoma with consecutive increase in JAK2 kinase activity. More recently, we identified SOCS1 as a “conditional oncogene” in the context of the FLT3-ITD oncogene (Reddy et al, Blood 2012): SOCS1 significantly enhanced FLT3-ITD-mediated myeloid transformation, both in vitro and in vivo. We hypothesized that this may be a more general mechanism of transformation and therefore analyzed the role of SOCS proteins in BCR-ABL mediated transformation and leukemogenesis.First, we investigated gene expression levels of SOCS proteins in BCR-ABL positive (versus BCR-ABL negative) cell lines and primary ALL long term-cultured cells. Upon treatment with the BCR-ABL inhibitor imatinib, mRNA expression levels of CIS and SOCS1-4 were reduced. SOCS5-7 did not exhibit any changes and were non-responsive to ABL-kinase inhibition. In lineage-depleted primary murine bone marrow retrovirally transduced with BCR-ABL, high induction of CIS and SOCS1-3 mRNA was detected, while SOCS4-7 showed only minor changes. When overexpressed in IL-3 dependent cell lines, SOCS1 led to a very rapid cell death within few days. Similar effects were demonstrated for CIS and SOCS2 overexpression, however, with a slower kinetics. In contrast, BCR-ABL transduced cells were insensitive to SOCS1 overexpression. In colony formation assays performed with primary hematopoietic cells, expression of SOCS1 led to a significant decrease of colony numbers. Interestingly, co-expression of SOCS1 and BCR-ABL (hereafter abbreviated as SOCS1/BCR-ABL) also lowered colony numbers compared to cells expressing BCR-ABL alone. However, when cells were subjected to interferon alpha or interferon gamma treatment, SOCS1/BCR-ABL positive cells displayed higher colony numbers and gained a growth advantage over BCR-ABL expressing cells, since anti-proliferative effects of the cytokines were inhibited by the presence of SOCS1. A careful analysis of the downstream signaling cascade of BCR-ABL and SOCS1/BCR-ABL expressing cells did not demonstrate any differences in the phosphorylation of AKT, ERK1/2 and STAT5. However, when BCR-ABL was inhibited by imatinib, STAT5 phosphorylation was significantly decreased in SOCS1/BCR-ABL transduced cells. Finally, the influence of SOCS1 in BCR-ABL mediated leukemia was investigated in a murine bone marrow transplantation model. BCR-ABL or SOCS1/BCR-ABL expressing cells led to disease formation with a chronic myeloid leukemia-like phenotype. Interestingly, the co-expression of SOCS1 and BCR-ABL prolonged disease latency, as opposed to the phenotype observed with FLT3-ITD (where SOCS1 co-expression shortened latency). In this setting SOCS1 acts as a tumor suppressor, protecting BCR-ABL transformed cells from rapid disease development, and a molecular analysis will be presented. Disclosures:No relevant conflicts of interest to declare.

Mtss1 Suppresses BCR-ABL Induced Cell Migration and Is Downregulated in CML Stem Cells.

Migration and adhesion properties of hematopoietic stem cells (HSC) are disrupted in chronic myeloid leukemia (CML). Egression of these cells from the bone marrow is associated with cytoskeletal changes including actin remodeling. In a microarray screen of differentially regulated genes in HSC from BCR-ABL positive transgenic mice, we found downregulation of multiple genes involved in actin-associated changes of cell structure, adhesion, and migration (i.e. intersectin-1, cortactin, Mtss1, synaptopodin, and Gem GTPase). Mtss1 was further studied since it has been described to be a binding partner of Rac, which is essential for BCR-ABL mediated transformation, and a potential tumor suppressor. Using quanitative RT-PCR, Mtss1 downregulation (6-fold) was confirmed in HSC and unfractionated bone marrow and spleen cells from SCLtTA/BCR-ABL transgenic mice after 3 weeks of BCR-ABL induction as well as in human BCR-ABL positive cell lines. Treatment of BCR-ABL positive (32D/BCR-ABL, K562, KYO-1) but not BCR-ABL negative (32D, U937) cell lines with 5μ M Imatinib led to upregulation of Mtss1 mRNA (5- to 10-fold) and protein, suggesting that downregulation of Mtss1 is dependent on BCR-ABL kinase activity. Retroviral transduction of Mtss1 into 32D/BCR-ABL cells almost completely inhibited BCR-ABL induced cell motility of individual cells seeded on murine bone marrow stromal cells in time-lapse video experiments over the course of two hours. Interestingly, we found that retroviral transduction of Mtss1 into 32D/BCR-ABL cells completely suppressed migration of these cells to extra-hematopoietic sites in vivo upon intravenous transplantation into syngeneic C3H mice. Moreover, when Mtss1-transduced cells were injected subcutaneously, the size of the tumors was significantly decreased as compared to empty vector-transduced 32D/BCR-ABL cells (p<0.05), confirming that Mtss1 may be a tumor suppressor.These results demonstrate that Mtss1 antagonizes BCR-ABL induced cell migration and is downregulated by BCR-ABL in CML stem cells, suggesting that downregulation of Mtss1 and other cytoskeletal adaptor proteins may be required for egression of CML stem cells from the bone marrow niche. Since the same Mtss1 protein domain is responsible for both Rac and actin binding, Mtss1 may interfere with Rac function and thereby inhibit the effects of Rac on migration and cytoskeletal dynamics of HSC.

Effect of Inhibition of Grb2 Expression on BCR-ABL Mediated Transformation of Primary Human Hematopoietic Cells.

The critical mechanisms underlying human hematopoietic stem cell (HSC) transformation by the BCR-ABL gene in chronic myeloid leukemia (CML) are still not well understood. Since treatment with imatinib fails to eliminate primitive CML hematopoietic cells, there is a pressing need to identify additional mechanisms that can be targeted to enhance elimination of CML stem cells. A tyrosine residue at position 177 (Y177) in the BCR-ABL protein binds the adapter protein Grb2 and appears to play an important role in BCR-ABL induced myeloid leukemogenesis in murine CML models. We have recently shown that a tyrosine to phenylalanine mutation of Y177 (Y177F) also results in significant reduction of abnormalities in proliferation and differentiation in BCR-ABL expressing human CD34+ cells and in BCR-ABL induced activation of Ras, Akt and STAT5 signaling (Cancer Res 2007 67:7045). Since Grb2 signaling is implicated in several other human malignancies and likely plays an important role in signaling downstream of BCR-ABL, we were interested in investigating the role of Grb2 in BCR-ABL-mediated transformation of primary human hematopoietic cells. Cord blood (CB) CD34+ cells were transduced with bicistronic retrovirus vectors coexpressing wild type or Y177F mutated BCR-ABL genes with the GFP gene. Coimmunoprecipitation studies confirmed the association of Grb2 with BCR-ABL and was abrogated by the Y177F mutation. We next investigated the effect of inhibition of Grb2 expression in BCR-ABL transduced human CD34+ cells. CB CD34+ cells were co-transduced with retroviral vectors coexpressing the BCR-ABL and GFP genes or control vectors expressing the GFP gene alone together with lentivirus vectors coexpressing Grb2 shRNA constructs and the dsRed gene or the dsRed gene alone. Cells expressing CD34+, GFP and dsRed were selected using flow cytometry. Western blot analysis indicated that Grb2 levels were reduced by 80.8±14.5% (n=3, P < 0.014); in CD34+ cells transduced with BCR-ABL and Grb2 shRNA compared with cells expressing BCR-ABL alone. Expression of Grb2 shRNA resulted in significant reduction in expansion of BCR-ABL expressing CD34+ cells compared with cells expressing BCR-ABL alone after 7 days of culture in serum free medium (SFM) with low concentrations of growth factor similar to those present in bone marrow stroma conditioned medium (BA alone 48.5±7.7; BA+Grb2 shRNA, 16.7±4.7, n=3). Co-expression of Grb2 shRNA also resulted in significant reduction in the total number of colonies generated by BCR-ABL expressing CD34+ cells in methylcellulose progenitor culture (BA, 123±31; BA+Grb2, 40±9; n=3, p<0.03), with reduction being seen mainly for erythroid colonies. Expression of Grb2 shRNA also reduced cell expansion from control CD34+ expressing GFP alone but the difference was not statistically significant. Similarly Grb2 inhibition did not result in a significant difference in the number of colonies generated from control CD34+ cells. Grb2 inhibition was associated with reduced levels of P-MAPK, but not P-AKT in BCR-ABL expressing CD34+ cells. Interestingly Grb2 inhibition also results in decreased levels of P-STAT5 consistent with the observed reduction in erythroid colonies. In conclusion RNAi mediated inhibition of Grb2 expression results in significant inhibition of BCR-ABL induced proliferation of human CD34+ cells proliferation, indicating an important role for Grb2 in BCR-ABL mediated transformation of CML cells. These observations support further evaluation of inhibition of Grb2 signaling in targeting of CML stem cells.

Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

Overexpression of the p65 Subunit of NF-kB and IkB Mediated Nuclear Sequestration of p53 as Common Events in Philadelphia Positive and Negative Chronic Myeloid Leukemia.

Introduction. Chronic Myeloid Leukemia (CML) is a myeloproliferative disorder caused by the Philadelphia translocation. Rare patients with a clinical presentation of CML are negative for the Ph chromosome. The pathogenesis of Ph negative CML is still unknown. Different reports have demonstrated that the transcription factor NF-kappaB is essential for Bcr-Abl mediated transformation. NF-kappaB is composed of two subunits (mainly p65 and p50) which are retained into the cytoplasm by the inhibitory protein IkappaB-alpha. Different stimuli trigger IkappaB degradation and nuclear translocation of NF-kappaB, where it mediates the transcription of different genes involved in cellular proliferation, transformation and resistance to apoptosis.Aim of the work. We have evaluated the role of NF-kappaB in primary chronic myeloid leukemia samples both positive and negative for the Philadelphia chromosome.Methods. Bone marrow samples of 10 chronic myeloproliferative disorders (6 Ph positive and 4 Ph negative CML), 1 CML blast phase, 2 Acute Myeloid Leukemia and 3 healthy donor have been collected at diagnosis. Each sample has been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of p65 and the regulatory protein IkappaB-alpha. DNA binding activity of NF-kappaB has been measured with an ELISA method (TransAM). To inhibit NF-kB, primary cells have been incubated with 1 microM MG-132, 90 microM Resveratrol and 5 microM Bay11-7082 or have been infected with a lentivirus construct containing dsRNA directed against the p65 subunit of NF-kB and a lentivirus expressing a mutated IKB which retains NF-kB inactive in the cytoplasm.Results. Ph+ and Ph- CML samples express both in the cytosol and in the nucleus higher levels of p65 respect to normal peripheral blood and normal bone marrow samples. In normal samples IkB-alpha is detectable only in the cytosol while in Ph+ and Ph- CML samples it is predominately expressed in the nucleus. DNA binding activity of NF-kappaB is not particularly increased in CML respect to normal bone marrow samples. Only CML blast phase and Acute Myeloid Leukemia show markedly increased DNA binding activity of NF-kappaB. Immunoprecipitates of nuclear and cytosolic IkappaB-alpha have been performed. The pro-apoptotic p53 co-immunoprecipitates with IkappaB-alpha. Treatment with NF-kappaB inhibitors MG-132, Resveratrol and Bay11-7082 disrupts p53-IkBalpha dimer rendering cells susceptible to the apoptotic stimulation induced by Doxorubicine. To confirm these data, primary CML samples have also been infected with a lentivirus construct containing dsRNA directed against p65 and a lentivirus expressing a mutated IKB which retains NF-kB inactive in the cytoplasm. In both cases cells becomes more sensitive to Doxorubicin-induced apoptosis, due to disruption of IkB - p53 dimer.Conclusion. Nuclear and cytosolic IkappaB-alpha mediated p53 sequestration is a common event in both Ph+ and Ph- CML, which may contribute to the pathogenesis of this disorder due to impairment of p53 pro-apoptotic functions. NF-kB inhibition may represent a powerful strategy to render CML cells more susceptible to apotosis.

Over-Expression of the P65 Subunit of NF-kB and Increased DNA Binding Activity of NF-kB Is a Common Event Both in Philadelphia Positive and Negative Chronic Myeloproliferative Disorders.

The chronic myeloproliferative diseases (CMPD) are clonal disorders characterized by increased proliferation of cells from one or more myeloid lineages. The most common CMPD is Chronic Myeloid Leukemia which is characterized by the Philadelphia t(9;22) chromosomal translocation. The pathogenesis of Philadelphia negative CMPD is poorly understood, although the activation of tyrosine kinases appears to be an essential feature. For example, a constitutively activated PDGF receptor tyrosine kinase (FIP1L1-PDGFRA) is involved in some cases of the hypereosinophilic syndrome (HES), which is a form of CMPD characterized by increased proliferation of eosinophils. Different reports have demonstrated that the transcription factor NF-kB is essential for Bcr-Abl mediated transformation. NF-kB is a transcription factor which is composed of two subunits (generally p65 and p50). NF-kB dimers are retained into the cytoplasm by the inhibitory protein IkB. Different stimuli trigger the Serine phosphorylation of IkB and its proteolitc degradation. Free NF-kB translocates into the nucleus where it mediates the transcription of different genes involved in cellular proliferation, transformation and in apoptosis resistance. The aim of this work is to evaluate whether NF-kB is active both in Ph positive and in Ph negative CMPD. Bone marrow samples of 8 myeloproliferative disorders (3 Philadelphia positive CML, 3 Ph negative CML-like, 1 HES, 1 Idiopathic Myelofibrosis) have been collected at the diagnosis. The t(9;22) positive K562 cell line, derived form a CML blast crysis, has been used as a positive cellular control of the following experiments. Each samples have been lysed to obtain cytosolic and nuclear extracts. Western blot have been performed to evaluate the expression of the p65 subunit of NF-kB, the regulatory protein IkB and the antiapoptotic protein Bcl-2, whose expression may be regulated by NF-kB. Subsequently the DNA binding activity of NF-kB have been measured with an ELISA method. Our data shows that in all samples p65 is over-expressed both in the cytosol and in the nucleus respect to normal peripheral blood and normal bone marrow samples. The antiapoptotic Bcl-2 is also detectable by western blot in all pathological samples. In normal samples IkB is detected only in the cytosol and not in the nucleus while in CMPD samples it is expressed predominately in the nucleus. Basal DNA binding activity of NF-kB is increased in all the nuclear samples but not in normal samples. To assess whether NF-kB is directly involved in the control of cell growth and apoptosis, we have developed a stable K562 cell line expressing the super repressor IkB. The SR-IkB is a mutated for of IkB which can not be degraded causing an cytosolic sequestration of NF-kB. This stable cell line has a marked reduction of cell growth and is more sensible to the apoptotic stimuli. The data described above suggest that NF-kB activation may be a common mechanism of transformation in both Ph positive and negative CMPD and that its inhibition may be a powerful targeted molecular therapy.

Inhibition of Phosphotyrosine Phosphatase-1B (PTP1B) Induces Resistance to the ABL Kinase Inhibitor Imatinib Mesylate (Gleevec®) in BCR-ABL Positive Leukemic Cells.

BCR-ABL chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) have recently received much attention as model diseases for the effectiveness of targeted drug therapy. The ABL kinase inhibitor imatinib mesylate has revolutionized treatment of CML. However, a serious drawback to this specific treatment is the emergence of drug resistance. Protein tyrosine phosphatase-1B (PTP1B) has been demonstrated to block the BCR-ABL mediated transformation of hematopoietic cells in vitro and in vivo. Therefore the impact of PTP1B function on imatinib-response of the BCR-ABL positive cell line K562 and an imatinib-resistant subclone of the cell line SupB15 was investigated.K562 cells were transfected with wild-type PTP1B (K562/PTP1B), a substrate-trapping dominant-negative mutant PTP1B (K562/D181A), or an empty vector (K562/mock), cultured in the presence or absence of imatinib (0.25–1.0 μM) and analyzed for proliferation, apoptosis and differentiation. K562/PTP1B and K562/D181A cells displayed 1.6-fold higher and 13-fold lower phosphatase activity than K562/mock cells, resp. In both K562/mock and K562/PTP1B cells, 0.5 μM imatinib induced growth arrest, an increase of cells in sub-G1 apoptotic phase (36.9±2.0% and 38.6±3.8%, resp.) and a decrease of cells in S/G2/M phases (18.9±1.1% and 23.7±2.7%, resp.) as determined by FACS analysis of propidium iodide stained cells. Imatinib-induced apoptosis was further confirmed by Annexin V staining and Western blot analysis showing increased active caspase-3 and -8 protein levels and induction of poly ADP-Ribose Polymerase (PARP) cleavage in both cells. Low doses of imatinib (0.25 μM) promoted erythroid differentiation of these cells as evaluated by enhanced expression of glycophorin A. Conversely, K562/D181A cells were significantly less sensitive to imatinib-induced growth arrest (sub-G1: 25±3.9 %, S/G2/M: 34.2±1.4 %) apoptosis, and promotion of erythroid differentiation. Inhibition of PTP1B phosphatase activity by the PTP1B inhibitor bis(N,N-Dimethylhydroxamido)hydroxooxovanadate (DHMV) decreased imatinib-induced apoptosis in wild-type K562 cells (46.7% vs. 28.3% annexin V positive cells; p<0,01). Moreover, the imatinib-resistant subclone of the ALL cell line SupB15, which had been generated in the presence of 1μM imatinib, showed significantly lower PTP1B activity (64.3±5.6%, p<0.05) as compared with imatinib-sensitive wild type cells. In conclusion, our data demonstrate that functional PTP1B is required for imatinib-induced apoptosis, growth arrest, and differentiation. Alterations of PTP1B gene expression and function might contribute to imatinib-resistence and should therefore be assessed in patients with BCR-ABL positive chronic and acute leukaemia who developed restance to imatinib.

The bcr-abl c-terminus is required for bcr-abl mediated apoptotic suppression and drug resistance but not proliferation or adhesion defects in haemopoietic progenitor cell line, fdcp-mix

The chimeric Bcr-Abl protein has constitutively activated Abl protein tyrosine kinase (PTK), a phenomenon deemed to be essential for the transforming ability of Bcr-Abl. The F- and G-actin binding region of the Bcr-Abl C-terminus is also thought to be important in Bcr-Abl mediated transformation. In order to investigate the importance of this domain a temperature-sensitive Bcr-Abl protein tyrosine kinase (tsBcr-Abl) and a tsBcr-Abl mutant deleted for 167 C-terminal amino acids were transfected into the mutipotent haemopoietic progenitor FDCP-Mix cell line. The expressed proteins have activated kinase function at 32°C but are inactive at 39°C. C-terminus truncation does not abrogate the temperature-sensitive PTK activity of the Bcr-Abl protein. Previous studies have shown that tsBcr-Abl transfected FDCP-Mix remain growth factor-dependent for survival and proliferation. However, at 32°C these cells display enhanced survival and proliferation in the absence or at low levels of growth factor (IL-3). In this investigation, viability assays and tritiated thymidine (3H) incorporation found that deletion of the C-terminus reversed both Bcr-Abl mediated apoptotic-suppression and resistance to cytotoxic drugs, cytosine arabinofuranoside and hydroxyurea, but had no effect on Bcr-Abl mediated proliferative synergy. Immunofluorescence studies found that full length Bcr-Abl localised to puntate areas within the cytoplasm. In contrast, C-terminus deleted Bcr-Abl displayed some diffuse, non-specific cytoplasmic staining while retaining a high level of nuclear staining similar to c-Abl. This may indicate an inability to bind to actin filaments. Furthermore, adhesion studies showed that both full-length and truncated Bcr-Abl mediated reduced adhesion to fibronectin, in the absence of IL-3. In conclusion, these results indicate that the Bcr-Abl C-Terminus has an important role in several aspects of Bcr-Abl mediated transformation of haemopoietic progenitor cells including apoptotic suppression and cytotoxic drug resistance.

Identification and characterization of two novel SH2 domain-containing proteins from a yeast two hybrid screen with the ABL tyrosine kinase.

To further our understanding of the molecular mechanism of Bcr-Abl mediated transformation, a yeast two hybrid screen was used to identify proteins binding to the Abl tyrosine kinase. Two partial cDNAs encoding novel SH2 domain-containing proteins were cloned and designated Shd and She. Both have homology to Shb, a previously reported SH2 domain-containing protein. Northern blot analysis showed that She is expressed in heart, lung, brain, and skeletal muscle, while expression of Shd is restricted to the brain. The deduced amino acid sequence of the full length mouse Shd cDNA contains an amino-terminal proline-rich region, and a carboxyterminal SH2 domain. A bacterially expressed Shd domain bound multiple tyrosine-phosphorylated proteins with relative molecular weights of 200, 170, 130, 100, 90, 78, 72 and 32 kDa from K562 cell lysates. Shd contains five YXXP motifs, a substrate sequence preferred by Abl tyrosine kinases. Shd was tyrosine phosphorylated in COS-7 cells co-transfected with Shd and c-Abl or Bcr-Abl. These results suggest that Shd may be a physiological substrate of c-Abl and may function as an adapter protein in the central nervous system.

Induction of Apoptosis by a Dominant Negative H-RAS Mutant (116Y) in K562 Cells

Recent extensive work on apoptosis has begun to reveal its molecular mechanisms. Several genes that regulate apoptosis have been identified. Among them, the BCL2 gene is considered to be an important gene that inhibits apoptosis. However, there must be other genes, yet to he identified, which suppress apoptosis. It has been suggested that the activation of RAS function by BCR-ABL fusion protein in chronic myelogenous leukemia may be an important mechanism in the BCR-ABL mediated transformation. Therefore, in this study we have investigated whether the suppression of endogenous H-RAS function inhibits the BCR-ABL mediated transforming activity in a K562 human chronic myelogenous leukemia cell line. The induced expression of a dominant negative v-H-RAS mutant (116Y) in K562 cells has resulted in cell death. The morphological characteristics and the detection of fragmented DNA by gel electrophoresis in the dead cells have revealed that this cell death is apoptosis. These results directly indicate that the RAS gene as well as the BCL2 gene has an ability to suppress apoptosis.