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PubMed2002

Jak2 is involved in c-Myc induction by Bcr-Abl.

We have previously shown that the Jak2 tyrosine kinase is activated in Bcr-Abl positive cell lines and blood cells from CML blast crisis patients by tyrosine phosphorylation. We are searching for downstream targets of Jak2 in Bcr-Abl positive cells. It is known that c-Myc expression is required for the oncogenic effects of Bcr-Abl, and that over-expression of c-Myc complements the transformation defect of the Bcr-Abl SH2 deletion mutant. Moreover, the Bcr-Abl SH2 deletion mutant and an Abl C-terminal deletion mutant are deficient in activating c-Myc expression. Since the Jak2 binds to the C-terminal domain of Bcr-Abl and optimal Jak2 activation requires the SH2 domain, we tested whether Jak2 was involved in c-Myc protein induction by Bcr-Abl. We treated the 32Dp210 Bcr-Abl cells with the Jak2 specific tyrosine kinase inhibitor, AG490, and found that this drug, like the Abl tyrosine kinase inhibitor STI-571, inhibited c-Myc protein induction by Bcr-Abl. Treatment of 32Dp210 Bcr-Abl cells with AG490 also inhibited c-MYC RNA expression. It is also known that c-Myc protein is a labile protein that is increased in amounts in response to various growth factors by a mechanism not involving new Myc protein formation. Treatment of 32Dp210 Bcr-Abl cells with both the proteasome inhibitor MG132 and AG490 blocked the reduction of the c-Myc protein observed by AG490 alone. An adaptor protein SH2-Bbeta is involved in the enhancement of the tyrosine kinase activity of Jak2 following ligand/receptor interaction. In this regard we showed that the Jak2/Bcr-Abl complex contains SH2-Bbeta. Expression of the SH2-Bbeta R555E mutant in 32Dp210 Bcr-Abl cells reduced c-Myc expression about 40% compared to a vector control. Interestingly, we found the reduction of the c-Myc protein in several clones of dominant-negative (DN) Jak2 expressing K562 cells correlated very well with the reduction of tumor growth of these cells in nude mice as compared to vector transfected K562 cells. Both STI-571 and AG490 also induced apoptosis in 32Dp210 cells. Of interest, IL-3 containing medium reversed the STI-571 induced apoptosis of 32Dp210 cells but did not reverse the induction of apoptosis by AG490, which strongly supports the specificity of the inhibitory effects of AG490 on the Jak2 tyrosine kinase. In summary, our findings indicate that Jak2 mediates the increase in c-Myc expression that is induced by Bcr-Abl. Our results indicate that activated Jak2 not only mediates an increase of c-MYC RNA expression but also interferes with proteasome-dependent degradation of c-Myc protein.

Adaptor Proteins, Signal Transducing↗

PubMed1998

The involvement of Bcr in leukemias with the Philadelphia chromosome.

In this review, the role of Bcr sequences within Bcr-Abl and the role of the Bcr protein itself in leukemias with the Philadelphia chromosome are discussed. An overview of the oncogenic effects of Bcr-Abl is presented together with Bcr-Abl's effects on several signal transduction pathways. Bcr sequences within Bcr-Abl are known to have an important function in transducing Bcr-Abl's oncogenic signal, yet the role of the Bcr protein encoded by the normal allele is not known. However, several recent findings suggest that Bcr is a conditional negative regulator of Bcr-Abl. From these findings, my colleagues and I have developed a molecular model of the early stage (benign phase) of chronic myelogenous leukemia in which the Bcr protein antagonizes the oncogenic effects of Bcr-Abl.

Animals↗

PubMed1991

Long-range mapping of the normal BCR gene.

Pulsed field gel electrophoresis was used to construct a long-range map of the normal BCR gene. A single BssHII restriction fragment encompasses all the known exons of the BCR gene (except a small 5' part of exon one). MIuI has one restriction site within the first intron of the BCR gene and another 250 kb downstream. This MIuI fragment contains most of the BCR gene coding sequences apart from the first exon and contains more sequences downstream of the BCR gene than the BssHII fragment. The NarI restriction sites are very close to the BssHII sites in the BCR gene, but they differ in the ABL gene, so that NarI digests could theoretically provide additional information in chronic myeloid leukaemia (CML) patients. This map was used to confirm BCR gene involvement in two CML patients in whom results of conventional Southern blotting of DNA were ambiguous. It was also used in a third patient to demonstrate the presence of a breakpoint apparently outside the BCR gene. Preliminary evidence from the use of PFGE confirms the presence of three BCR-related genes homologous to 3' sequences in the classical BCR gene (BCR-1). These BCR-related genes are located at a considerable distance from BCR-1.

Chromosome Mapping↗

PubMed2004

Coexistence of phosphotyrosine-dependent and -independent interactions between Cbl and Bcr-Abl.

Cbl is one of the major tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells. A direct association between the SH2 domain of Bcr-Abl and tyrosine-phosphorylated Cbl has been demonstrated. The purpose of this study was to determine if and how unphosphorylated Cbl and Bcr-Abl may associate. Interactions between Cbl and Bcr-Abl were investigated in yeast two- and three-hybrid systems, gel overlay assays, and immunoprecipitates from mammalian cells expressing wild-type and the Y177F mutant of Bcr-Abl. No direct interaction between Bcr-Abl and unphosphorylated Cbl was observed. Bcr-Abl did, however, associate with Grb2, an adaptor protein that binds tyrosine 177 of Bcr-Abl. Additionally, Grb2 interacted with Cbl. In a yeast three-hybrid assay, Grb2 mediated an interaction between Cbl and Bcr-Abl that was dependent on a functional Grb2 binding site. This interaction was confirmed in vitro using purified proteins. In cells expressing Bcr-Abl with a mutation in the Grb2 binding site, binding of Cbl to Bcr-Abl was significantly reduced, but Cbl tyrosine phosphorylation was maintained. Imatinib treatment of these cells further reduced but did not abrogate Cbl binding, reflecting residual kinase activity. Multiple phosphotyrosine-dependent and -independent interactions stabilize the interaction between Cbl and Abl. Grb2 or another, yet unidentified, protein may mediate an initial interaction between Cbl and Bcr-Abl that is independent of Cbl tyrosine phosphorylation. Following this initial interaction, Cbl can then become tyrosine phosphorylated and interact with the SH2 domain of Bcr-Abl, further stabilizing the complex.

Adaptor Proteins, Signal Transducing↗

PubMed2003

The c-Myc Oncoprotein Interacts with Bcr.

Bcr is a multifunctional protein that is the fusion partner for Abl (p210 Bcr-Abl) in Philadelphia chromosome positive leukemias. We have identified c-Myc as a binding partner for Bcr in both yeast and mammalian cells. We are also able to observe interactions between natively expressed c-Myc and Bcr in leukemic cell lines. Although Bcr and Max have overlapping binding sites on c-Myc, Bcr cannot interact with Max, or with the c-Myc.Max heterodimer. Bcr expression blocks activation of c-Myc-responsive genes, as well as the transformed phenotype induced by coexpression of c-Myc and H-Ras, and this finding suggests that one function of Bcr is to limit the activity of c-Myc. However, Bcr does not block c-Myc function by preventing its nuclear localization. Interestingly, increased Bcr dosage in COS-7 and K-562 cells correlates with a reduction in c-Myc protein levels, suggesting that Bcr may in fact be limiting c-Myc activity by regulating its stability. These data indicate that Bcr is a novel regulator of c-Myc function whose disrupted expression may contribute to the high level of c-Myc protein that is observed in Bcr-Abl transformed cells.

Animals↗

PubMed2006

The Bcr-Abl mutations T315I and Y253H do not confer a growth advantage in the absence of imatinib.

Mutations in the Bcr-Abl kinase domain are a frequent cause of imatinib resistance in patients with advanced CML or Ph+ ALL. The impact of these mutations on the overall oncogenic potential of Bcr-Abl and on the clinical course of the disease in the absence of imatinib is presently unclear. In this study, we analyzed the effects of the Bcr-Abl P-loop mutation Y253H and the highly imatinib resistant T315I mutation on kinase activity in vitro and transforming efficiency of Bcr-Abl in vitro and in vivo. Immunoprecipitated Bcr-AblY253H and Bcr-AblT315I proteins displayed similar kinase activities and substrate phosphorylation patterns as Bcr-Abl wildtype. We directly compared the proliferative capacity of mutant to wildtype Bcr-Abl in primary BM cells in vitro and in a murine transplantation model of CML by using a competitive repopulation assay. The results implicate that in the absence of imatinib, there is no growth advantage for cells carrying Bcr-AblT315I or Bcr-AblY253H compared to Bcr-Ablwt, whereas imatinib treatment clearly selects for leukemic cells expressing mutant Bcr-Abl both in vitro and in vivo. Thus, the analysed Bcr-Abl mutants confer imatinib resistance, but do not induce a growth advantage in the absence of imatinib.

Amino Acid Substitution↗

PubMed1998

Bcl-2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL.

Bcr - Abl is the molecule responsible for both the transformation phenotype and the resistance to chemotherapeutic drugs found in chronic myelogenous leukemia (CML) cells. Wild-type HL-60, a transformed pro-myelocytic cell line, is very susceptible to apoptosis-inducing agents. We show here that expression of Bcr - Abl in HL-60 cells rendered them extremely resistant to apoptosis induced by a wide variety of agents. The anti-apoptotic effect of Bcr - Abl was found to be independent of the phase of the cell cycle. Treatment with antisense oligonucleotides directed to bcr decreased the expression of the ectopic bcr - abl and restored susceptibility to apoptosis. Double mutations affecting the autophosphorylation site and the phosphotyrosine-binding motif (FLVRES) have been previously shown to impair the transforming activity of Bcr - Abl in fibroblasts and hematopoietic cells, however HL-60 cells expressing this double mutant molecule exhibited the same level of resistance to apoptosis as those expressing the wild-type Bcr - Abl. Interestingly, wild type and mutant Bcr - Abl induced in HL-60 cells a dramatic down regulation of Bcl-2 and increased the levels of Bcl-xL. The level of Bax did not change in response to the presence of Bcr - Abl. Antisense oligonucleotides targeted to bcl-x downregulated the expression of Bcl-x, and increased the susceptibility of HL-60. Bcr - Abl cells to staurosporine. Importantly, HL-60 cells overexpressing Bcl-xL showed higher expression of Bcl-xL but lower resistance to apoptosis when compared to HL-60. Bcr - Abl cells. The results described here show that Bcr - Abl is a powerful mammalian anti-apoptotic molecule and can act independently of Bcl-2. Bcl-xL, however, seems to participate in part in Bcr - Abl-mediated resistance to apoptosis in HL-60 cells.

Apoptosis↗

PubMed2003

The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain.

The protein kinase Bcr is a negative regulator of cell proliferation and oncogenic transformation. We identified Bcr as a ligand for the PDZ domain of the cell junction and Ras-interacting protein AF-6. The Bcr kinase phosphorylates AF-6, which subsequently allows efficient binding of Bcr to AF-6, showing that the Bcr kinase is a regulator of the PDZ domain-ligand interaction. Bcr and AF-6 colocalize in epithelial cells at the plasma membrane. In addition, Bcr, AF-6, and Ras form a trimeric complex. Bcr increases the affinity of AF-6 to Ras, and a mutant of AF-6 that lacks a specific phosphorylation site for Bcr shows a reduced binding to Ras. Wild-type Bcr, but not Bcr mutants defective in binding to AF-6, interferes with the Ras-dependent stimulation of the Raf/MEK/ERK pathway. Since AF-6 binds to Bcr via its PDZ domain and to Ras via its Ras-binding domain, we propose that AF-6 functions as a scaffold-like protein that links Bcr and Ras to cellular junctions. We suggest that this trimeric complex is involved in downregulation of Ras-mediated signaling at sites of cell-cell contact to maintain cells in a nonproliferating state.

Amino Acid Sequence↗

PubMed1996

A novel BCR-ABL fusion gene (e6a2) in a patient with Philadelphia chromosome-negative chronic myelogenous leukemia.

A novel variant of the chimeric BCR-ABL mRNA transcript was detected in a patient with Philadelphia chromosome-negative (Ph-) chronic myelogenous leukemia (CML) by multiplex reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis of the fusion region of the amplified cDNA fragment showed an in-frame joining of exon e6 of the BCR gene and exon a2 of the ABL gene, giving rise to an e6a2 BCR-ABL transcript. This finding was confirmed by Southern blot analysis using a specific probe corresponding to intron 6 of the BCR gene, whereas conventional Southern blot for rearrangement of the major breakpoint cluster region (M-bcr) was negative. Western blot studies detected a BCR-ABL protein slightly larger than p185 BCR-ABL. Metaphase fluorescence in situ hybridization showed an insertion of ABL material into the BCR region without reciprocal BCR translocation. The findings in this case show that atypical BCR-ABL transcripts are detectable even in Ph- CML patients without M-bcr-rearrangements. Multiplex PCR using primers that allow for amplification of all known BCR-ABL transcripts is an appropriate method to exclude these rare variants.

Adult↗

PubMed1991

Characterization of the BCR promoter in Philadelphia chromosome-positive and -negative cell lines.

The t(9;22) Philadelphia chromosome translocation fuses 5' regulatory and coding sequences of the BCR gene to the c-ABL proto-oncogene. This results in the formation of hybrid BCR-ABL mRNAs and proteins. The shift in ABL transcriptional control to the BCR promoter may play a role in cellular transformation mediated by this rearrangement. We have functionally localized the BCR promoter to a region 1 kb 5' of BCR exon 1 coding sequences by using a chloramphenicol acetyltransferase reporter gene assay. Nucleotide sequence analysis of this region revealed many consensus binding sequences for transcription factor SP1 as well as two potential CCAAT box binding factor sites and one putative helix-loop-helix transcription factor binding site. No TATA-like or "initiator" element sequences were found. Because of low steady-state levels of BCR mRNA and the high GC content (78%) of the promoter region, definitive mapping of transcription start sites required artificial amplification of BCR promoter-directed transcripts. Overexpression from the BCR promoter in a COS cell system was effective in demonstrating multiple transcription initiation sites. In order to assess the effects of chromosomal translocation on the transcriptional control of the BCR gene, we determined S1 nuclease protection patterns of poly(A)+ RNA from tumor cell lines. No differences were observed in the locations and levels of BCR transcription initiation sites between those lines that harbored the t(9;22) translocation and those that did not. This demonstrates that BCR promoter function remains intact in spite of genomic rearrangement. The BCR promoter is structurally similar to the ABL promoters. Together, this suggests that the structural fusion of BCR-ABL and not its transcriptional deregulation is primarily responsible for the transforming effect of the t(9;22) translocation.

Amino Acid Sequence↗

PubMed1996

Comparison of bcr-abl protein expression and Philadelphia chromosome analyses in chronic myelogenous leukemia patients.

The Philadelphia chromosome (Ph) is found in most chronic myelogenous leukemia (CML) patients. The bcr-abl oncoprotein (P210 or P185), the product of the fused bcr-abl gene produced by the Ph, is known to be the major factor in initiation and maintenance of the leukemic state in these types of leukemias. The authors have devised a Western blot test to detect and quantitate the bcr-abl oncoprotein in blood and bone marrow cells. The authors analyzed 1,155 peripheral blood samples from CML patients, for which there were same day Ph data, to determine if there was a correlation between bcr-abl protein levels and the percentage of Ph. A ratio of bcr-abl protein (P210 or P185) to normal abl protein (P145), the latter is ubiquitously expressed in all blood cells, has been established as a criterion for quantitating bcr-abl levels. The bcr-abl/abl protein ratio from 399 patient who were 100% Ph-positive had a mean of 2.47 (standard error [SE] of +/- 0.05); no false negatives were detected. A decreasing ratio was found in patients with decreasing percentages of Ph. The relationship between the ratio of bcr-abl/abl proteins to the percentage of Ph-positive cells was nearly linear in 392 patients with Ph percentages between 5% to 95% (r = 0.97, P < .001). For patients in remission with no detectable Ph, the bcr-abl/abl ratio had a mean of 0.01 (SE = 0 +/- 0.00). The level of bcr-abl expression in samples from peripheral blood and bone marrow also were compared. The results indicated that the amount of bcr-abl protein within peripheral blood is usually similar to that in marrow. In conclusion, quantitation of the bcr-abl oncoprotein in peripheral blood cells or marrow cells as measured by a Western blot assay provides reliable data for both diagnosis and monitoring patients with pH-chromosome positive leukemia.

Blotting, Western↗

PubMed2002

Specific inhibition of bcr-abl gene expression by small interfering RNA.

Small interfering RNAs (siRNAs) were designed to target the bcr-abl oncogene, which causes chronic myeloid leukemia (CML) and bcr-abl-positive acute lymphoblastic leukemia (ALL). Chemically synthesized anti-bcr-abl siRNAs were selected using reporter gene constructs and were found to reduce bcr-abl mRNA up to 87% in bcr-abl-positive cell lines and in primary cells from CML patients. This mRNA reduction was specific for bcr-abl because c-abl and c-bcr mRNA levels remained unaffected. Furthermore, protein expression of BCR-ABL and of laminA/C was reduced by specific siRNAs up to 80% in bcr-abl-positive and normal CD34(+) cells, respectively. Finally, anti-bcr-abl siRNA inhibited BCR-ABL-dependent, but not cytokine-dependent, proliferation in a bcr-abl-positive cell line. These data demonstrate that siRNA can specifically and efficiently interfere with the expression of an oncogenic fusion gene in hematopoietic cells.

Animals↗

PubMed2000

Bcr-Abl kinase down-regulates cyclin-dependent kinase inhibitor p27 in human and murine cell lines.

Chronic myeloid leukemia (CML) is a malignant stem cell disease characterized by an expansion of myeloid progenitor cells expressing the constitutively activated Bcr-Abl kinase. This oncogenic event causes a deregulation of apoptosis and cell cycle progression. Although the molecular mechanisms protecting from apoptosis in CML cells are well characterized, the cell cycle regulatory event is poorly understood. An inhibitor of the cyclin-dependent kinases, p27, plays a central role in the regulation of growth factor dependent proliferation of hematopoietic cells. Therefore, we have analyzed the influence of Bcr-Abl in the regulation of p27 expression in various hematopoietic cell systems. An active Bcr-Abl kinase causes down-regulation of p27 expression in murine Ba/F3 cells and human M07 cells. Bcr-Abl blocks up-regulation of p27 after growth factor withdrawal and serum reduction. In addition, p27 induction by transforming growth factor-beta (TGF-beta) is completely blocked in Bcr-Abl positive M07/p210 cells. This deregulation is directly mediated by the activity of the Bcr-Abl kinase. A Bcr-Abl kinase inhibitor completely abolishes p27 down-regulation by Bcr-Abl in both Ba/F3 cells transfected either with a constitutively active Bcr-Abl or with a temperature sensitive mutant. The down-regulation of p27 by Bcr-Abl depends on proteasomal degradation and can be blocked by lactacystin. Overexpression of wild-type p27 partially antagonizes Bcr-Abl-induced proliferation in Ba/F3 cells. We conclude that Bcr-Abl promotes cell cycle progression and activation of cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27. (Blood. 2000;96:1933-1939)

Animals↗

PubMed2001

Nonrandom distribution of interspersed repeat elements in the BCR and ABL1 genes and its relation to breakpoint cluster regions.

The Philadelphia translocation, t(9;22)(q34;q11), is the microscopically visible product of recombination between two genes, ABL1 on chromosome 9 and BCR on chromosome 22, and gives rise to a functional hybrid BCR-ABL1 gene with demonstrated leukemogenic properties. Breakpoints in BCR occur mostly within one of two regions: a 5 kb major breakpoint cluster region (M-Bcr) and a larger 35 kb minor breakpoint cluster region (m-Bcr) towards the 3' end of the first BCR intron. By contrast, breakpoints in ABL1 are reported to occur more widely across a >200 kb region which spans the large first and second introns. The mechanisms that determine preferential breakage sites in BCR, and which cause recombination between BCR and ABL1, are presently unknown. In some cases, Alu repeats have been identified at or near sequenced breakpoint sites in both genes, providing indications, albeit controversial, that they may be relevant. For the present study, we carried out a detailed analysis of genomic BCR and ABL1 sequences to identify, classify, and locate interspersed repeat sequences and to relate their distribution to precisely mapped BCR-ABL1 recombination sites. Our findings confirm that Alu are the most abundant class of repeat in both genes, but that they occupy fewer sites than previously estimated and that they are distributed nonrandomly. r-Scan statistics were applied to provide a measure of repeat distribution and to evaluate extremes in repeat spacing. A significant lack of Alu elements was observed across the major and minor breakpoint cluster regions of BCR and across a 25-kb region showing a high frequency of breakage in ABL1. These findings counter the suggestion that occurrence of Alu at BCR-ABL1 recombination sites is likely by chance because of the high density of Alu in these two genes. Instead, as yet unidentified DNA conformation or nucleotide characteristics peculiar to the preferentially recombining regions, including those Alu elements present within them, more likely influence their fragility.

Alu Elements↗

PubMed2003

Effects of the tyrosine kinase inhibitor imatinib mesylate on a Bcr-Abl-positive cell line: suppression of autonomous cell growth but no effect on decreased adhesive property and morphological changes.

Expression of the Bcr-Abl oncoprotein alters various aspects of hematopoietic cells. We investigated the effects of a Bcr-Abl tyrosine kinase inhibitor, imatinib mesylate, on the proliferation, adhesive properties, and morphology of a Bcr-Abl-transferred cell line, TF-1 Bcr-Abl, in comparison with parental TF-1. First, the factor-independent growth of TF-1 Bcr-Abl was inhibited in the presence of imatinib mesylate, but this inhibition was overcome by addition of exogenous granulocyte-macrophage colony-stimulating factor. Imatinib mesylate remarkably reduced tyrosine phosphorylation of Bcr-Abl, Cbl, and Crkl in a time-dependent manner, and their complex formation also was affected. Imatinib mesylate inhibited activation of Stat5 rather than the MEK-ERK1/2 pathway. TF-1 Bcr-Abl cells exhibited a round shape, unlike TF-1, and the adhesive property to fibronectin was much lower than that of TF-1. Although the Bcr-Abl oncoprotein may be involved negatively in cell adhesion, the decreased adhesion and altered morphology of TF-1 Bcr-Abl cells were minimally affected by imatinib mesylate and seemed independent of Bcr-Abl kinase activity. The present data indicated that the Bcr-Abl-specific kinase inhibitor cannot control Bcr-Abl-induced cell alterations other than autonomous growth.

Benzamides↗

PubMed2001

Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette.

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycline added to the medium (1 microg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 microg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin- bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.

Animals↗

PubMed2002

BCR-ABL alters the proliferation and differentiation response of multipotent hematopoietic cells to stem cell factor.

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.

Antineoplastic Agents↗

PubMed1997

Heterogenous expression of bcr-abl fusion mRNA in a patient with Philadelphia-chromosome-positive acute lymphoblastic leukaemia.

We performed molecular and cytogenetic analysis on a 56-year-old woman with Philadelphia chromosome (Ph1)-positive acute lymphoblastic leukaemia (ALL) having two types of major and minor bcr-abl (M-bcr-abl, m-bcr-abl/fusion mRNA at diagnosis. In the course of her disease, unexpected heterogenous bcr-abl fusion mRNA was detected by sequential analysis using the reverse transcription and polymerase chain reaction (RT-PCR). The RT-PCR analysis showed both M-bcr-abl (b2-a2 type) and m-bcr-abl at diagnosis. Although b2-a2 type M-bcr-abl disappeared after complete remission (CR) was achieved with intensive induction chemotherapy, expression of both m-bcr-abl and a new type of M-bcr-abl mRNA (b1-a2 type), which may have been produced through splicing out of exon b2, was detected in the early stage of CR. The leukaemic cells contained these heterogenous bcr-abl mRNAs in both the CR and relapsed state, and showed more stable expression of the m-bcr-abl type mRNA than that of the b2-a2 type. These findings of heterogenous bcr-abl mRNA due to alternative or missplicing during the disease course in the present ALL case may provide important evidence of disease progression.

Alternative Splicing↗