Tumorigenic activity of the BCR - ABL oncogenes is mediated byBCL 2 ( apoptosis / interleukin 3 / leukemia / Philadelphia chromosome / tumor development )

BCR-ABL is a chimeric oncogene generated by translocation of sequences from the c-abl protein-tyrosine kinase gene on chromosome 9 into the BCR gene on chromosome 22. Alternative chimeric proteins, p21OBCR-ABL and pl9OBCR-ABL, are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. Transformed murine hematopoietic cells can be used as a model of BCR-ABL function since these cells can be made growth factor independent and tumorigenic by the action of the BCR-ABL oncogene. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. Furthermore, BCRABL-expressing cells revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These results help to explain the ability of BCR-ABL oncogenes to synergize with c-myc in cell transformation. Hematopoietic growth factors are required to support the proliferation, survival, and differentiation of progenitor cells (1-3). Leukemic conversion of these cells to an autonomous growth state implies that specific genes must be activated to uncouple cellular proliferation/differentiation control and to generate intracellular signals that can substitute for growth factor requirements. A well-characterized example in the hematopoietic system involves the rearrangements of the BCR and ABL genes in Philadelphia chromosome-positive (Ph' ) chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (4-13). Depending on the precise breakpoint within the BCR gene, fusion proteins of 210 kDa (p210) or 190 kDa (p190) are produced (4, 6, 7, 9, 14-16). p210 and p190 BCR-ABL oncogenes contain identical ABL-derived sequences, respectively, but differ in the number of BCRencoded amino acid residues. The tyrosine kinase activity of the BCR-ABL proteins strongly correlates with their transforming potential in tissue culture and is higher for the 190-kDa form, which is characteristically associated with acute leukemias (12, 14, 17). Analysis of the mechanism of oncogene action can be studied in cell lines-often in NIH 3T3 fibroblasts. The latter are not transformed by either of the two BCR-ABL oncogenes or by v-abl itself (18). However, it has been shown that BCR-ABLP120 and v-abl convert the factor-dependent Ba/F3 cell line into factor independence and make it tumorigenic (19), but the mechanisms whereby this happens are unknown. Understanding these mechanisms requires identification of the downstream involvement of proteins that affect transcriptional control or cell survival. MYC is a candidate gene whose transcription is activated by some tyrosine kinase oncogenes as well as by growth factor stimulation (20, 21). Recent data have shown that MYC is essential for BCR-ABL transformation (22). However, an increase in MYC protein cannot be solely The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5287 responsible for the transforming effect of BCR-ABL oncogenes, because MYC overexpression does not substitute for the BCR-ABL oncogene transformation phenotype. Therefore, other components in addition to MYC must be required to reconstitute the BCR-ABL oncogene transformation signal. In the present study, we have examined the role of BCRABL oncogenes in the context of hematopoietic cell transformation using as a model system the Ba/F3 cell line (23). We investigated the possibility that BCR-ABL expression may function in Ba/F3 cells by inhibiting apoptosis and consequently prolonging cell survival. We show that the BCR-ABL oncogenes prevent apoptotic death in these cells by inducing a Bcl-2 expression pathway. We further show that Ba/F3 cells expressing BCR-ABL revert to factor dependence and nontumorigenicity after Bcl-2 expression is suppressed. These findings implicate the activation of Bcl-2 function as an important component in BCR-ABL-mediated transformation and can also explain the ability of BCR-ABL oncogenes to synergize with MYC in cell transformation. MATERIALS AND METHODS Cell Culture. Cell lines used include Ba/F3 (23), K562 (24), and DoHH2 (25). Ba/F3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 10% WEHI-3B-conditioned medium as a source of interleukin 3 (IL-3). DoHH2 and K562 cell lines were cultured in RPMI 1640 medium supplemented with 10% FCS and 5 x 10-5 M 2-mercaptoethanol. Plasmid Construction. The pAbc12 plasmid contains the mouse Bcl-2 cDNA in antisense orientation and the PGK-Hyg cassette into the pEF-BOS expression vector (26). The final product was confirmed by DNA sequence analysis. Cell Transfection. Ba/F3 cells were transfected by electroporation with 25 ,ug of KW3 expression plasmid and 25 ,ug of E1A2 plasmid along with 2 ,ug of MC1-neo expression vector. Cell lines were analyzed by Northern blotting for BCR-ABL expression and demonstrated resistance to IL-3 withdrawal. BCR-ABL-expressing Ba/F3 cells were transfected by electroporation (960 ,uF; 220 V) with 25 ,ug of the pAbc12 vector. Hygromycin-resistant cells were selected by using 600 ,ug-ml-1 of hygromycin B (Calbiochem) for 14 days. Clones were screened for high-level Bcl-2 expression by Northern blotting. The antisense Bcl-2 oligonucleotide used as a probe comprises the first 34 bases of the coding sequence of mouse Bcl-2 cDNA. RNA Analysis. Total cytoplasmic RNA (10 jig) was glyoxylated and fractioned in 1.4% agarose gels in 10 mM Na2HPO4 buffer (pH 7.0). After electrophoresis, the gel was blotted onto Abbreviations: CML, chronic myelogenous leukemia; FL, follicular lymphoma; IL-3, interleukin 3. *To whom reprint requests should be addressed at the present address: Departamento de Proliferaci6n y Diferenciaci6n Celular, Instituto de Microbiologia Bioguimica, Consejo Superior de Investigaciones Cientificas/Universidad de Salamanca, Avda del Campo Charro s/n, 37.007-Salamanca, Spain. 5288 Biochemistry: Sainchez-Garcia and Grutz Hybond-N (Amersham), UV-cross-linked, and hybridized to 32P-labeled probes. DNA Analysis. Low molecular weight DNA was isolated as follows. Cells were collected into 1.5 ml of culture medium and microcentrifuged for 5 min at 13,200 rpm (3000 x g), and the pellet was suspended in 300 ,ul of proteinase K buffer. After overnight incubation at 55°C, DNA was ethanol-precipitated, suspended in 200 ,A of TE buffer containing 50 jug-ml-' of RNase A, and incubated at 37°C for 2 h. DNA was extracted with phenol and chloroform and precipitated with ethanol. Aliquots of DNA (2 ,ug) were electrophoresed on 2% agarose gels, which were stained with ethidium bromide and photographed. Tumorigenicity Assay. To test the tumorigenicity of the various cell lines, 4to 6-week-old athymic (nude) male mice were injected subcutaneously on both flanks with 106 cells