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PubMed1996

Kinetic selectivity of complementary nucleic acids: bcr-abl-directed antisense RNA and ribozymes.

Efficacy and sequence specificity are two major requirements in the use of antisense nucleic acids and ribozymes. For long-chain complementary RNA sequences (>30 nt), effects in living cells are correlated with the association rate of the complementary RNA in vitro, but not with the stability of the formed double strand. Thus, sequence selectivity of complementary RNA has to be defined as fast versus slow annealing with the appropriate target or non-target sequences, respectively. In this work, we performed a systematic kinetic analysis to evaluate the selectivity of bcr-abl-directed antisense RNA and hammerhead ribozymes with a length of the complementary sequences of between 20 and 80 bases. By kinetic in vitro selection, we identified oligomeric as well as long-chain complementary RNA that annealed at least tenfold faster with the bcr-abl sequence in comparison with either of the wild-type sequences bcr or abl, respectively. In the presence of selected oligodeoxynucleotide sequences and RNase H, the bcr-abl transcript was specifically hydrolysed out of a mixture containing abl and bcr sequences as well. Hammerhead ribozymes were designed such that binding with their target was facilitated either via helix I or helix III-forming antisense arms but not both. Further, cleavage and binding occurred on opposite sides of the bcr-abl fusion point. Target selectivity was found for a ribozyme that annealed fast via abl sequences and cleaved within the bcr portion of bcr-abl RNA. Kinetic probing and calculations of the local folding potential indicate that the bcr-abl fusion point sequences are not easily accessible for complementary nucleic acids. This study supports the need for more detailed structural investigations of the bcr-abl fusion sequence and forms a more rational basis for the therapeutic use of nucleic acid inhibitors of the aberrant bcr-abl gene expression in Philadelphia chromosome-positive cells.

Base Sequence↗

PubMed1997

Regulation of Bcl-2 gene expression by BCR-ABL is mediated by Ras.

BCR-ABL is a chimaeric 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, p210(BCR-ABL) and p190(BCR-ABL), are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the aetiology of human leukemia remains to be defined. We have previously shown that the tumorigenic effect of BCR-ABL oncogenes is mediated by Bcl-2. In addition to Bcl-2, is a protein essential for transformation by BCR-ABL. However, it is not known how Bcl-2 and Ras fit together in cell transformation by BCR-ABL. The data presented here establish that Bcl-2 is a downstream target gene of the Ras signalling pathway in cells transformed by BCR-ABL, and that constitutive Ras activation results in constitutive expression of the gene. Conversely, a truncated form of the BCR-ABL, which lacks a critical BCR region required for activation of the Ras signalling pathway, failed to induce Bcl-2 expression. These results indicate that BCR-ABL prevents apoptosis by inducing Bcl-2 through a signalling pathway involving Ras and links constitutive Ras activation and Bcl-2 gene regulation. Hence, these results further imply that Ras is involved in both mitogenic signals and survival signals.

Animals↗

PubMed1997

Amplification of BCR protein associated with oncogenesis in human hepatocellular carcinoma.

The BCR gene is located on human chromosome 22. The normal cellular BCR gene encodes a 160,000-dalton phosphoprotein associated with a serine/threonine kinase activity. The BCR protein is involved in signal transduction. We investigated the expression of the BCR protein in hepatocellular carcinoma (HCC), surrounding noncancerous liver tissue, liver cirrhosis (LC), chronic hepatitis (CH), and normal liver with immunohistochemistry and a western blot analysis. BCR immunoreactivity was detected using a monoclonal antibody. In normal liver, and both CH and LC without association of HCC, the immunoreactivity of the BCR protein was minimal. In contrast, 73% (22 of 30) of noncancerous liver tissue adjacent to the HCC and 40% (12 of 30) of HCC expressed BCR protein; this difference was statistically significant (P < 0.01). The expression of the BCR protein expression correlated with the degree of histological differentiation of HCC (P < 0.05). In addition, the amplification of BCR protein in noncancerous cells was supported by the detection of specific protein using a western blot analysis. In two cases, the expression of BCR protein occurred only in overtly malignant HCC cells. As a result, the expression of the BCR protein may be associated with oncogenesis in human HCC.

Aged↗

PubMed2005

Dose-dependent effects of Bcr-Abl in cell line models of different stages of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is caused by Bcr-Abl, an activated tyrosine kinase. The amounts of Bcr-Abl mRNA and protein in cells from patients in blast crisis (BC) are higher than in those chronic phase (CP), indicating that their expression rises with disease progression. In order to study this phenomenon on cells with the same genetic background, we transfected the 32D cell line with the BCR-ABL transgene and selected clones with graded expression of Bcr-Abl within the range found in cells from CP to BC. In vitro, we found that Bcr-Abl exerted dose-dependent effects upon growth factor dependence, clonogenicity and migration. However, the relationship between Bcr-Abl expression and cellular adhesion to fibronectin was more complex: rather than a direct positive correlation, clones that expressed low, but not high, levels of Bcr-Abl were less adhesive than growth factor stimulated, parental BCR-ABL-negative 32D cells. This finding parallels the situation with normal and CML-CP progenitors where the latter exhibit defective adhesion to fibronectin. Treatment with the tyrosine kinase inhibitor imatinib mesylate reversed the adhesion deficient phenotype of clones expressing low levels of Bcr-Abl. When injected subcutaneously into syngeneic mice, cell lines expressing high levels of Bcr-Abl rapidly induced tumors, whereas low-expressing clones led to tumor formation only after a prolonged latency. These findings suggest that the level of Bcr-Abl may be essential in determining the phenotype of the leukemic clone at different stages of the disease.

Animals↗

PubMed2006

Sustained leukaemic phenotype after inactivation of BCR-ABLp190 in mice.

Pharmacological inactivation of cancer genes or products is being used as a strategy for therapy in oncology. To investigate the potential role of BCR-ABLp190 cessation in leukaemia development, we generated mice carrying a tetracycline-repressible BCR-ABLp190 transgene. These mice were morphologically normal at birth, and developed leukaemias. Disease was characterized by the presence of B-cell blasts co-expressing myeloid markers, reminiscent of the human counterpart. BCR-ABLp190 activation can initiate leukaemia in both young and adult mice. Transitory expression of BCR-ABLp190 is enough to develop leukaemia. Suppression of the BCR-ABLp190 transgene in leukaemic CombitTA-p190 mice did not rescue the malignant phenotype, indicating that BCR-ABLp190 is not required to maintain the disease in mice. Similar results were obtained by inactivation of BCR-ABLp190 with STI571 (Gleevec; Novartis, East Hanover, NJ, USA) in leukaemic CombitTA-p190 mice. However, gradual suppression of BCR-ABLp190 in leukaemic CombitTA-p190 mice identified a minimum level of BCR-ABLp190 expression necessary to revert the specific block in B-cell differentiation in the leukaemic cells. Overall, the findings indicate that BCR-ABLp190 appears to cause epigenetic and/or genetic changes in tumour-maintaining cells that render them insensitive to BCR-ABLp190 inactivation.

Animals↗

PubMed2002

Typical essential thrombocythaemia does not express bcr-abelson fusion transcript.

Essential thrombocythaemia (ET) is a chronic myeloproliferative disorder (MPD) characterized by an elevated platelet count and no identifiable underlying primary cause. According to the diagnostic criteria of the Polycythemia Vera Study Group (PVSG), ET lacks features diagnostic for other MPDs, including the Philadelphia chromosome (Ph) or bcr-abl rearrangement. Recently, some authors have reported bcr-abl transcript positivity in ET patients, but these findings remain controversial. The aim of this study was to investigate whether the bcr-abl transcript could be found in ET patients and to verify the hypothesis of a new ET variant. ET patients (n = 121) with a median age at diagnosis of 55 years were enrolled. The bcr-abl transcript status was examined by multiplex reverse transcription-polymerase chain reaction. Only two cases were positive for bcr-abl, one of which had the Ph at diagnosis. The positive bcr-abl transcript was associated, in both cases, with mild basophilia at diagnosis. After a median follow-up of 43 months (0-309 months), two patients in the bcr-abl-negative group developed Ph and bcr-abl-negative acute myeloid leukaemia (AML). In contrast, one of the two patients in the bcr-abl-positive group died from AML 13 years after diagnosis. In conclusion, our data on a large group of patients shows the rarity of the bcr-abl transcript in well-established ET. However, a subset of patients with apparent ET and basophilia may express the transcript and may constitute a novel entity intermediate between chronic myeloid leukaemia (CML) and typical ET. A prospective study is warranted in order to define better the clinical and biological characteristics of bcr-abl-expressing ET.

Adolescent↗

PubMed2000

Transformation of myeloid leukemia cells to cytokine independence by Bcr-Abl is suppressed by kinase-defective Hck.

Bcr-Abl is the constitutively active protein-tyrosine kinase expressed as a result of the Philadelphia translocation in chronic myelogenous leukemia. Bcr-Abl is coupled to many of the same signaling pathways normally regulated by hematopoietic cytokines. Recent work shows that Hck, a member of the Src tyrosine kinase family with myeloid-restricted expression, associates with and is activated by Bcr-Abl. Here we investigated the mechanism of Hck interaction with Bcr-Abl and the requirement for Hck activation in Bcr-Abl transformation signaling. Binding studies demonstrated that the Hck SH3 and SH2 domains are sufficient for interaction with Bcr-Abl in vitro. Hck binding localizes to the Abl SH2, SH3, and kinase domains as well as the distal portion of the C-terminal tail. To address the requirement for endogenous Src family kinase activation in Bcr-Abl signaling, a kinase-defective mutant of Hck was stably expressed in the cytokine-dependent myeloid leukemia cell line DAGM. Kinase-defective Hck dramatically suppressed Bcr-Abl-induced outgrowth of these cells in the absence of cytokine compared with a control cell line expressing beta-galactosidase. In contrast, kinase-defective Hck did not affect cell proliferation in response to interleukin-3, suggesting that the effect is specific for Bcr-Abl. These data show that Hck interacts with Bcr-Abl through a complex mechanism involving kinase-dependent and -independent components and that interaction with Hck or other Src family members is essential for transformation signaling by Bcr-Abl.

Animals↗

PubMed1993

BCR-ABL and v-abl oncogenes induce distinct patterns of thymic lymphoma involving different lymphocyte subsets.

The human BCR-ABL oncogenes encoded by the Philadelphia chromosome (Ph) affect the pathogenesis of diverse types of leukemia and yet are rarely associated with T-lymphoid leukemia. To determine whether BCR-ABL kinases are inefficient in transforming T lymphocytes, BCR-ABL-expressing retroviruses were injected intrathymically into mice. Thymomas that expressed BCR-ABL kinase developed after a relatively long latent period. In most thymomas, deletion of 3' proviral sequences resulted in loss of tk-neo and occasionally caused expression of kinase-active carboxy-terminally truncated BCR-ABL oncoprotein. In contrast, deletion of 3' proviral sequences was not observed in thymomas induced with Abelson murine leukemia virus (A-MuLV). BCR-ABL viruses induced distinct patterns of disease and involved different thymocyte subsets than A-MuLV and Moloney murine leukemia virus (Mo-MuLV). While Mo-MuLV only induced Thy-1+ thymomas, v-abl- and BCR-ABL-induced thymomas often contained mixed populations of B220+ and Thy-1+ lymphocytes in the same tumor. In most v-abl and BCR-ABL tumors, Thy-1+ lymphoid cells expressed CD8 and a continuum of CD4 ranging from negative to positive. Conversely, Mo-MuLV thymomas contained distinct populations of CD4+ cells that were either CD8+ or CD8-. A-MuLV-transformed T-lymphoid cells did not express the CD3/T-cell receptor complex, while BCR-ABL tumors were CD3+. Thus, BCR-ABL viruses preferentially induce somewhat more differentiated T lymphocytes than are transformed by A-MuLV. Furthermore, rare B220+ lymphocytes may represent preferred v-abl and BCR-ABL transformation targets in the thymus.

Animals↗

PubMed2001

Comparison of effects of the tyrosine kinase inhibitors AG957, AG490, and STI571 on BCR-ABL--expressing cells, demonstrating synergy between AG490 and STI571.

STI571 (formerly CGP57148) and AG957 are small molecule inhibitors of the protein tyrosine kinase (PTK) p145(abl) and its oncogenic derivative p210(bcr-abl). AG490 is an inhibitor of the PTK Janus kinase 2 (JAK2). No direct comparison of these inhibitors has previously been reported, so this study compared their effects on factor-dependent FDC-P1, 32D, and MO7e cells and their p210(bcr-abl)-expressing factor-independent derivatives. STI571 was a more potent inhibitor of (3)H-thymidine incorporation in p210(bcr-abl)-expressing cells than was AG957, and it showed superior discrimination between inhibitory effects on parental cell lines and effects on their p210(bcr-abl)-expressing derivatives. Assays performed with and without growth factor demonstrated that STI571 but not AG957 reversed the p210(bcr-abl)-driven factor independence of cell lines. p210(bcr-abl)-expressing cells were less sensitive to AG490 than to AG957 or STI571. However, for p210(bcr-abl)-expressing clones from all 3 cell lines, synergistic inhibition was demonstrated between STI571 and concentrations of AG490 with no independent inhibitory effect. Inhibition of nucleic acid synthesis with AG957 treatment was associated with reduced cell numbers, reduced viability, and small pyknotic apoptotic cells. At concentrations of STI571 that reversed the p210(bcr-abl) factor-independent phenotype, STI571 treatment and growth factor deprivation together were sufficient to induce apoptosis. This study concludes that, for the cell lines studied, (1) STI571 is a more potent and more selective inhibitor of a p210(bcr-abl)-dependent phenotype than AG957; (2) AG490 synergizes with STI571 to enhance its inhibitory effect on p210(bcr-abl)-driven proliferation; and (3) the combination of p210(bcr-abl)-tyrosine kinase inhibition and growth factor signal withdrawal can be sufficient to induce apoptotic death of transformed cells. (Blood. 2001;97:2008-2015)

Antineoplastic Agents↗

PubMed2001

Adhesion to fibronectin selectively protects Bcr-Abl+ cells from DNA damage-induced apoptosis.

The phenotype of Bcr-Abl-transformed cells is characterized by a growth factor-independent survival and a reduced susceptibility to apoptosis. Furthermore, Bcr-Abl kinase alters adhesion features by phosphorylating cytoskeletal and/or signaling proteins important for integrin function. Integrin-mediated adhesion to extracellular matrix molecules is critical for the regulation of growth and apoptosis. However, effects of integrin signaling on regulation of apoptosis in cells expressing Bcr-Abl are largely unknown. The influence of adhesion on survival and apoptosis in Bcr-Abl+ and Bcr-Abl- BaF3 cells was investigated. p185bcr-abl-transfected BaF3 cells preadhered to immobilized fibronectin had a significant survival advantage and reduced susceptibility to apoptosis following gamma-irradiation when compared with the same cells grown on laminin, on polylysin, or in suspension. Both inhibition of Bcr-Abl kinase by STI571 and inhibition of specific adhesion reversed the fibronectin-mediated antiapoptotic effect in BaF3p185. The DNA damage response of Bcr-Abl- BaF3 cells was not affected by adhesion to fibronectin. In contrast to parental BaF3 cells, BaF3p185 adherent to fibronectin did not release cytochrome c to the cytosol following irradiation. The fibronectin-mediated antiapoptotic mechanism in Bcr-Abl-active cells was not mediated by overexpression of Bcl-XL or Bcl-2 but required an active phosphatidylinositol 3-kinase (PI-3K). Kinase-active Bcr-Abl in combination with fibronectin-induced integrin signaling led to a hyperphosphorylation of AKT. Thus, cooperative activation of PI-3K/AKT by Bcr-Abl and integrins causes synergistic protection of Bcr-Abl+ cells from DNA damage-induced apoptosis.

Animals↗

PubMed2002

BCR-ABL-induced adhesion defects are tyrosine kinase-independent.

The t(9;22) chromosomal translocation results in expression of P210(BCR-ABL), a fusion protein necessary for the development of chronic myelogenous leukemia (CML). The constitutive activation of the P210(BCR-ABL) tyrosine kinase results in phosphorylation of multiple signaling pathways leading to the transformed phenotype. Additionally, extracellular interactions between P210(BCR-ABL)-expressing progenitor cells and bone marrow stroma may provide external signals that facilitate CML development. In contrast to the intracellular signaling pathways involved in CML, little is known about how P210(BCR-ABL) expression modifies cell-cell and cell-substratum interactions. To investigate the role of P210(BCR-ABL) in modulating cellular adhesion, we used a highly sensitive and quantitative cell detachment apparatus that measures the strength of association between a population of cells and an adhesive matrix. Our findings show that P210(BCR-ABL) expression increased adhesion nearly 2-fold between the myeloblastic cell line, 32D, and fibronectin compared to a control vector. We then investigated whether abnormal adhesion due to P210(BCR-ABL) expression was caused by its tyrosine kinase activity. A quantitative analysis of cell-fibronectin adhesion found that neither expression of a kinase-inactive P210(BCR-ABL) mutant in 32D cells or attenuation of kinase activity by STI571 (imatinib mesylate) in 32D cells transduced with wild-type P210(BCR-ABL) could correct the nearly 2-fold increase in cell-fibronectin adhesion. Similarly, STI571 treatment of Meg-01 cells, a P210(BCR-ABL)-expressing cell line derived from a patient in blast crisis, failed to inhibit adhesion to fibronectin. Together, our results indicate that changes in adhesion induced by P210(BCR-ABL) are independent of its tyrosine kinase activity.

3T3 Cells↗

PubMed2004

Serial monitoring of BCR-ABL transcripts in chronic myelogenous leukemia (CML) treated with imatinib mesylate.

Survival among chronic myelogenous leukemia (CML) patients can be linked to the reduction in leukemic cell burden. Treatment with imatinib mesylate results in a high frequency of complete cytogenetic response, which can be further stratified using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). We have serially monitored peripheral blood and bone marrow BCR-ABL transcripts using qRT-PCR in CML patients commencing imatinib therapy, and compared the results with bone marrow cytogenetics. Seventeen patients (aged 25-74 yr) with Philadelphia chromosome positive CML in first chronic phase were treated with imatinib targeting a dose of 400 mg/d. The median follow up is 30 mo (range 9-33 mo). Every third month the product of the BCR-ABL fusion gene was evaluated in both blood and bone marrow specimens by real-time RT-PCR using the TaqMan probe system. In 113 simultaneously obtained blood and bone marrow samples, the BCR-ABL transcript values agreed well with cytogenetic data. Blood and bone marrow specimens gave comparable values for BCR-ABL transcripts. Before start of imatinib therapy there was a considerable variation in BCR-ABL transcripts among the patients, ranging approximately one log (base 10). Similarly, patients with a complete cytogenetic response following imatinib therapy had variable BCR-ABL transcript levels, ranging at least three logs (base 10). The major decline in BCR-ABL transcripts occurred within 6 mo after start of imatinib therapy. The decline in BCR-ABL transcripts, following imatinib therapy, appears to level off at 12-15 mo. Two late responders were identified with a still decreasing level in BCR-ABL transcripts after 24 mo of treatment. It is concluded that BCR-ABL mRNA quantification in peripheral blood is suitable for routine monitoring of the response to treatment and long-term disease status in CML, especially in patients who have achieved a complete cytogenetic response. A plateau in BCR-ABL transcripts seems to have been reached after 12-15 mo of imatinib treatment; however, some "late responders" are seen.

Adult↗

PubMed1992

Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: a Cancer and Leukemia Group B Study (8762).

The Philadelphia (Ph1) chromosome, or its molecular counterpart, the BCR-ABL fusion gene, is a rare but important prognostic indicator in childhood acute lymphoblastic leukemia (ALL), but its impact on adult ALL has not been well ascertained. A prospective study of the BCR-ABL fusion gene was begun on patients entered on clinical trials conducted by the Cancer and Leukemia Group B (CALGB). All patients received intensive, multiagent chemotherapy that included daunorubicin. Over 2 years, 56 patients were studied for molecular evidence of a BCR-ABL gene using Southern blot and pulsed-field gel hybridization analysis. Results were compared with cytogenetic detection of a Ph1 chromosome, and clinical features were compared for the BCR-ABL-positive and -negative groups. Molecular methods detected the BCR-ABL gene in 30% of cases compared with cytogenetic detection of the Ph1 chromosome in only 23%. The majority of cases (76%) showed the p190 gene subtype similar to pediatric ALL; the BCR-ABL-positive cases displayed a more homogeneous immunophenotype than the BCR-ABL-negative cases and were predominantly CALLA positive (86%) and B-cell surface antigen positive (82%). The rate of achieving complete remission was similar in the BCR-ABL-positive and -negative groups (71% and 77%, respectively, P = .72). There were more early relapses in the BCR-ABL-positive group, resulting in a shorter remission duration that was especially marked in the CALLA-positive and B-cell antigen-positive populations. These preliminary data suggest that the impact of the BCR-ABL gene on clinical outcome in ALL may be on maintenance of complete remission (CR) rather than achievement of CR when aggressive, multiagent chemotherapy is used. This study identifies the BCR-ABL gene as an important factor in adult ALL and demonstrates the utility of molecular methods for its accurate diagnosis.

Adolescent↗

PubMed1988

bcr genes and transcripts.

Human chronic myelogenous leukemia (CML) is a clonal hematologic disorder. CML is characterized by the t(9:22) chromosome translocation which results in translocation of the oncogene abl from chromosome 9 into the breakpoint cluster region (bcr) gene on chromosome 22. We cloned and characterized the cDNA of the normal bcr gene. The bcr gene codes for a protein of 1271 amino acids. The open reading frame is preceded by a region high in GC. At the 5' of this region we identified several GC motifs which are probably involved in the initiation of bcr transcription. bcr transcripts of 7.0 and 4.5 kb are expressed in all cell types examined. These transcripts share all cDNA sequences analysed, including the 5' untranslated region. The latter as well as 902 or 927 amino acids are included within the CML-specific bcr-abl mRNA transcribed from the chimeric bcr-abl gene on chromosome 22. In addition to the complete bcr gene, the human genome contains 3 bcr-related genes containing the last seven exons of the intact bcr gene. One of these genes was analysed in detail and showed high sequence homology with the latter. The three bcr-related genes were probably derived from the intact gene by subsequent steps of duplication.

Base Sequence↗

PubMed2006

Deletion of any part of the BCR or ABL gene on the derivative chromosome 9 is a poor prognostic marker in chronic myelogenous leukemia.

To evaluate the prognostic significance of submicroscopic deletions of the ABL or BCR gene associated with t(9;22) in chronic myelogenous leukemia (CML), we investigated the incidence of an ABL or BCR deletion on derivative chromosome 9 using fluorescence in situ hybridization (FISH). FISH was performed using the LSI BCR/ABL dual-fusion translocation probe on bone marrow cells of 86 patients with CML. Of 86 patients, ABL deletion was detected in 13 (15.1%) patients and BCR deletion in 8 patients (9.3%). Patients with ABL deletion showed shorter event-free survival time (EFS) than those without ABL deletion (P = 0.020). Patients with BCR deletion showed significantly short overall survival time (OS; P = 0.039). Patients with ABL and/or BCR deletion (14/86 patients, 16.3%) showed significantly short OS and EFS (median OS, 43.0 months; median EFS, 40.0 months), compared to the patients without any BCR or ABL gene deletions (median OS, 94.0 months; median EFS, 90.0 months; P = 0.041 for OS, P = 0.008 for EFS). All the patients with BCR deletion, except for one, had a concomitant ABL deletion, suggesting that BCR deletion occurs in conjunction with ABL deletion. In patients with ABL deletion only, BCR/ABL rearrangement with b2a2 mRNA type tended to be more frequent than in patients without any deletion of the two genes (P = 0.073). Deletion of any of the BCR or ABL genes on derivative chromosome 9 was associated with both short OS and EFS. We conclude that deletion of not only the ABL gene, but also of the BCR gene, is a poor prognostic marker that indicates rapid disease progression in CML.

Acute Disease↗

PubMed1993

BCR-ABL-induced oncogenesis is mediated by direct interaction with the SH2 domain of the GRB-2 adaptor protein.

BCR-ABL is a chimeric oncoprotein that exhibits deregulated tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph1)-positive human leukemias. Sequences within the first exon of BCR are required to activate the transforming potential of BCR-ABL. The SH2/SH3 domain-containing GRB-2 protein links tyrosine kinases to Ras signaling. We demonstrate that BCR-ABL exists in a complex with GRB-2 in vivo. Binding of GRB-2 to BCR-ABL is mediated by the direct interaction of the GRB-2 SH2 domain with a phosphorylated tyrosine, Y177, within the BCR first exon. The BCR-ABL-GRB-2 interaction is required for activation of the Ras signaling pathway. Mutation of Y177 to phenylalanine (Y177F) abolishes GRB-2 binding and abrogates BCR-ABL-induced Ras activation. The BCR-ABL (Y177F) mutant is unable to transform primary bone marrow cultures and is impaired in its ability to transform Rat1 fibroblasts. These findings implicate activation of Ras function as an important component in BCR-ABL-mediated transformation and demonstrate that GRB-2 not only functions in normal development and mitogenesis but also plays a role in oncogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Bcr - Abl-mediated resistance to apoptosis is independent of PI 3-kinase activity.

The Bcr - Abl tyrosine kinase is responsible for the oncogenic phenotype observed in Philadelphia chromosome-positive leukemia and induces resistance to apoptotic cell death in a variety of cell types. Recent evidence supports the hypothesis that these two properties of Bcr - Abl are derived from cooperative but distinct signaling pathways. Phosphatidylinositol 3-kinase (PI3K), which has been suggested to associate with and become activated by Bcr - Abl, has been shown to be required for Bcr - Abl-mediated cell growth. Also, PI3K has been implicated in resistance to apoptosis induced by some growth factors. We therefore examined the role of PI 3-kinase in the anti-apoptotic effect of Bcr - Abl. First, we confirmed that expression of p185(bcr - abl) in HL-60 cells, which renders these cells resistant to apoptosis, induces tyrosine phosphorylation of the p85 subunit of PI3K. Consistent with this result, we observed a 20-fold increase in PI3K activity upon immunoprecipitation of tyrosine-phosphorylated proteins from cells expressing Bcr - Abl versus control cells. Nevertheless, treatment of HL-60.p185(bcr - abl) cells with wortmannin, a potent inhibitor of PI3K, eliminated PI3K activity but did not interfere with the resistance of these cells to apoptosis. Similar results were obtained with the CML line K562 and with the BaF3.p185 (bcr - abl) line. We conclude that while PI3K participates in the anti-apoptotic response mediated by some growth factors and also seems to be important for the growth of Bcr-Abl-positive cells, it does not play any role in Bcr - Abl-mediated resistance to apoptosis.

Journal Article↗

PubMed1988

Variable Philadelphia breakpoints and potential lineage restriction of bcr rearrangement in acute lymphoblastic leukemia.

Philadelphia (Ph1) chromosome breakpoints in acute lymphoblastic leukemia (ALL) are of two kinds: those within the breakpoint cluster region (bcr+), as in chronic myeloid leukemia (CML), and those outside it (bcr-). These encode different c-abl messenger RNAs (mRNAs), p210 and p190, respectively. It has been suggested that one class of Ph+ ALL (bcr+) may be a variant of CML arising in a multipotent stem cell, the other (bcr-) de novo ALL initiated in a lymphoid-committed progenitor. Thirty-two cases of ALL (12 Ph1+, ten chromosomally normal, and ten non-mitotic cases) were investigated for bcr involvement. Breakpoints were found within five Ph1+ and in one normal case. There was no difference in clinical features, common ALL antigen (CALLA) positivity, cytogenetics, or response to treatment between the 6 bcr+ and 7 Ph1+ bcr- patients. Myeloid antigen expression was found in 2 bcr+ cases. Bcr rearrangement appeared to be restricted to the lymphoblastic component of marrow or blood in at least four bcr+ cases. In one case, separated myeloid and lymphoid cell fractions were both bcr+. Potential heterogeneity of the Ph1+ target cell, as seen in this study, may be more important in determining disease outcome than the precise location of the Ph breakpoint.

Genes, Immunoglobulin↗