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At least 577 records · Page 32Linked to original sources
PubMed2000

Detailed mapping of methylcytosine positions at the CpG island surrounding the Pa promoter at the bcr-abl locus in CML patients and in two cell lines, K562 and BV173.

Chronic myelogenous leukemia (CML) is associated with a translocation of the protooncogene c-abl from chromosome 9 to chromosome 22, where it fuses to proximal exons of the bcr gene. The expression of the hybrid gene bcr-abl is regulated by the bcr promoter and results in a translation product with high tyrosine kinase activity. In most CML cases, one of two abl promoters (Pa) is nested within the bcr-abl transcription unit, but appears to be usually silent. Recently, de novo methylation of the Pa region and its correlation with disease progression were reported. As these previous studies were limited to the use of methylation-sensitive restriction endonucleases, our aim here was to obtain a complete map of methylcytosines and its variants in CML patients and in model cell lines. To achieve this, bisulfite conversion of cytosines (but not methylcytosines) to uracils in genomic DNA was employed. After modification, the region of interest was PCR-amplified and the products were cloned and sequenced. The results show methylation at a high level and in a homogenous pattern in the BV173 cell line, corresponding to the translocated abl alleles. Variant methylation observed in K562 cells correlates with multiple bcr-abl loci and an intact chromosome 9. Patients that were methylation-positive in restriction analysis showed sporadic and heterogenous occurrence of methylcytosines in bisulfite modification assays. Corresponding results were obtained using a quantitative Southern analysis of the extent of methylation. We conclude that restriction analysis combined with PCR is able to find rare cases of hypermethylation, e. g., for diagnostic purposes, but does not reflect the dominating level of methylation in Ph-positive cells.

Base Sequence↗
PubMed2002

Lymphoid preponderance and the absence of basophilia and splenomegaly are frequent in m-bcr-positive chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) with a minor bcr-abl transcript is a rare entity. We describe a 66-year-old female who was diagnosed with CML in the chronic phase. Molecular analysis of her Philadelphia chromosome using the reverse transcriptase polymerase chain reaction and subsequent sequencing revealed a minor bcr-abl transcript. Monocytosis resembling chronic myelomonocytic leukemia was observed without splenomegaly and basophilia. Her clinical course was indolent and maintained the chronic phase of CML for nearly 3 years under hydroxyurea treatment. A review of the 23 cases of m-bcr CML including this case showed the presence of monocytosis and the absence of basophilia and splenomegaly in 55.0%, 55.0%, and 70.0% of patients, respectively. The absence of basophilia was a significant finding in patients without monocytosis ( P=0.01). Although the hematological features or clinical outcomes were variable in m-bcr CML cases, all three cases at the onset of the blastic phase showed lymphoid crisis, implying an increased lymphoid leukemogenicity of minor bcr-abl transcripts.

Aged↗
PubMed2026

Late acquisition of BCR::ABL1 during clonal evolution of SAMD9-associated MDS with phenotypic shift from AML to B-ALL.

We describe a unique case of SAMD9-associated myelodysplastic syndrome (MDS) with monosomy 7 that evolved over 16 years into BCR::ABL1-positive acute myeloid leukemia (AML) and subsequently manifested as B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis at AML diagnosis revealed a germline SAMD9 mutation together with somatic RUNX1 and PPM1D mutations, supporting stepwise clonal evolution, with BCR::ABL1 emerging as a late leukemogenic event. The dominant leukemic population at AML onset showed myeloid morphology and immunophenotype, whereas a minor CD19+CD10+ population was already detectable. Following venetoclax and azacitidine therapy, the dominant leukemic phenotype shifted to B-ALL while retaining BCR::ABL1 positivity. Detection of the Philadelphia chromosome in mature neutrophils at both AML onset and ALL relapse supported multilineage involvement of a multipotent BCR::ABL1-positive clone. Ponatinib achieved disease control. This case highlights late acquisition of BCR::ABL1 during SAMD9-associated clonal evolution and therapy-driven phenotypic shift within a shared Ph-positive leukemic stem-cell hierarchy.

Humans↗
PubMed1995

Alternative signals to RAS for hematopoietic transformation by the BCR-ABL oncogene.

Biological function of the BCR-ABL oncogene is dependent on its activated tyrosine kinase. Mutations that inactivate the SRC homology 2 (SH2) domain, the GRB2-binding site in BCR, or the major autophosphorylation site of the kinase domain selectively disrupt downstream signaling but not tyrosine kinase activity. Despite a loss of fibroblast transformation activity, all three mutants retain the ability to render hematopoietic cell lines growth factor independent and transform primary bone marrow cells in vitro. In vivo tests of malignant potential reveal a most critical role for signals dependent on the BCR-ABL SH2 domain. The efficiency of both fibroblast and hematopoietic transformation by BCR-ABL is strongly affected by increased dosage of the SHC adapter protein, which can connect tyrosine kinase signals to RAS. The BCR-ABL oncogene activates multiple alternative pathways to RAS for hematopoietic transformation.

Animals↗
PubMed1996

The effect of bcr-abl antisense oligonucleotide on DNA synthesis and apoptosis in K562 chronic myeloid leukemia cells.

Mutations in oncogenes have traditionally been viewed as inducing malignancy by causing excessive cell division. However, an additional possible tumorigenic mechanism is inhibition of normally occurring apoptosis. We have studied the mechanism of action of bcr-abl in chronic myeloid leukemia (CML) by inhibiting its expression using antisense oligonucleotides. K562 cells, derived initially from a patient with CML, were incubated with 16 microM 3',5'-capped bcr-abl antisense phosphodiester 18mer targeting the bcr-abl junctional sequence. Antisense reduced cell number by day 5 by 44% +/- 2.5% S.E. compared to nonsense or no-oligomer controls. Compared to nonsense oligomer, antisense oligomer reduced [3H]thymidine incorporation by only 13% +/- 1%. By the more reliable bromodeoxyuridine incorporation method, antisense had no inhibiting effect on DNA synthesis. In contrast to its minimal effect on DNA synthesis, antisense had a large effect on apoptosis. At day 4, after 3 days of oligomer treatment, antisense increased the proportion of cells with less than 2 N DNA 2.5 +/- 0.3-fold compared to nonsense, as revealed by analysis of DNA distribution following propidium iodide-staining. After 3 days of oligomer treatment and 24 h of serum deprivation, antisense increased the proportion of cells with less than 2 N DNA even more, over 3.1 +/- 1.1-fold compared to nonsense. Because CML cells are resistant to the induction of apoptosis (as judged by DNA laddering on electrophoresis, which requires double-stranded breaks), we also assayed the binding of terminal deoxynucleotidyl transferase (TdT), which requires only single-stranded DNA breaks. Antisense treatment for 3 days increased TdT binding at day 4 by 16.4 +/- 8.7-fold. We conclude that, in CML, bcr-abl may lead to the accumulation of myeloid cells to a greater extent by inhibiting apoptosis than by increasing cell division. This bcr-abl induced inhibition of apoptosis may thwart chemotherapy and foster the accumulation of further mutations leading to the development of the blastic phase of the disease.

Apoptosis↗
PubMed1989

The variable hematologic expression of the BCR-ABL genomic mutation and its possible determinants.

The Philadelphia (Ph) chromosome usually results from the t(9;22), which causes the physical association of the BCR1 and ABL genes and their function as a single new gene. This precise genomic mutation probably has a significant role in the development of leukemia in humans, but that leukemia may take several forms: chronic myeloid leukemia (CML), acute myeloid leukemia, acute lymphocytic leukemia, and essential thrombocythemia; CML also transforms to a lymphoid or myeloid acute phase. Two models are considered with regard to determinants of this variable hematologic expression of BCR-ABL. The first is variation in the breakpoint site of BCR1. Two breakpoint sites, M-BCR and m-BCR, are known, and their occurrence shows a nonrandom association with the different forms of leukemia. The precise position of the breakpoint within M-BCR may also be important. The second model concerns the role of other genes in determining the leukemic form shown by BCR-ABL. Results are reviewed of a patient who entered blast crisis CML and whose leukemic clones involved ten genetic loci with known leukemic associations. Many of these were probably genetic variants that allowed leukemic proliferations following the initiation of blast crisis. The multiplicity of these genes may obscure the prime determinant of blast crisis, which is unknown at the present time.

Gene Expression Regulation, Leukemic↗
PubMed2005

Cytogenetic characterization of a BCR-ABL transduced mouse cell line.

Most patients with Philadelphia (Ph)-positive acute lymphoblastic leukemia (ALL) show evidence of secondary chromosome aberrations that may influence the course of disease and response to treatment. To better understand how these secondary chromosomal aberrations occur and to investigate whether the p185/p190 BCR-ABL fusion protein may directly induce an increased chromosomal instability and subsequently the appearance of clonal chromosome aberrations, three BRC-ABL (p185/ p190)-transduced mouse pre-B cell lines were analyzed by spectral karyotyping and fluorescence in situ hybridization. The human wild-type BCR-ABL gene was expressed at a level comparable with that in human Ph-positive leukemias at diagnosis. All BCR-ABL-transduced cell lines acquired similar clonal chromosomal aberrations. Trisomy 5 was always present, followed by loss of the Y chromosome, trisomy of chromosomes 12 and 18, and an unbalanced translocation between chromosomes X and 12. Thus, ectopic p185/p190 BCR-ABL expression, such as p210 BCR-ABL, PML-RARA, or C-MYC transduction, may induce an increased chromosomal instability leading to clonal karyotypic evolution, which may mimic secondary chromosome aberrations in human Ph-positive ALL.

Animals↗
PubMed2005

Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl.

The Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50 <30 nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants. Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107 prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolonged survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and Ph+ ALL.

Animals↗
PubMed2004

Imatinib mesylate (STI571) prevents the mutator phenotype of Bcr-Abl in hematopoietic cell lines.

Progression of CML from chronic phase to blast crisis is accompanied by accumulating genetic alterations. To analyze whether this abnormality can be prevented by inhibition of Bcr-Abl, we measured the frequency of spontaneous and irradiation-induced HPRT mutations in cells treated with or without imatinib mesylate (Gleevec, STI571). Imatinib treatment of cells expressing Bcr-Abl reversed the mutation frequency to a value comparable to that of Bcr-Abl negative cells. Experiments with a Bcr-Abl deletion mutant indicate that in addition to the kinase activity, protein-protein interactions are required for induction of the mutator phenotype by Bcr-Abl.

Animals↗
PubMed2005

RNA interference targeting of Bcr-Abl increases chronic myeloid leukemia cell killing by 17-allylamino-17-demethoxygeldanamycin.

17-Allylamino-17-demethoxygeldanamycin (17-AAG) induces degradation of Hsp90 client proteins, including Bcr-Abl, however, its clinical use as an anti-tumor agent may be limited by toxicity and modest efficacy. We reasoned that Bcr-Abl targeting by RNA interference (RNAi) might selectively increase the activity of 17-AAG against Bcr-Abl+ leukemia cells. 17-AAG in combination with targeting small interfering RNAs (siRNAs) reduced Bcr-Abl protein levels, triggered increases in markers of apoptosis and decreased cell viability more effectively than did control siRNA and 17-AAG together, or Bcr-Abl targeting siRNA alone. Combination targeting strategies such as this may therefore achieve enhanced therapeutic potency.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

Detection of BCR-ABL transcripts in chronic myeloid leukaemia by nested PCR.

Polymerase chain reaction (PCR) is a powerful and rapid method for specifically detecting BCR-ABL rearrangement by amplification of the complementary DNA (cDNA) produced by reverse transcription of BCR-ABL mRNA. We studied 29 patients for detecting the presence of BCR-ABL transcripts before and after bone marrow transplantation (BMT). Our sample was composed of two different groups of patients: one group (n = 18) was studied by serial follow-ups before and after BMT; a second group (n = 11) was studied several years after BMT. Detection of BCR-ABL was carried out with different primer sets at different periods of the clinical outcome of chronic myeloid leukaemia (CML). A comparison of PCR data and clinical-haematological conditions showed clear differences between patients. In the first group, eight patients showed a positive correlation between a favourable clinical outcome and molecular remission. Conversely, in the second group, six patients were BCR-ABL positive between 20 and 117 months after BMT, while only two of these patients showed signs of clinical relapse. Among all patients whose isoforms were known at some time during the course of CML, the more frequent isoform was b3a2. These results were compared to previous findings in the literature on diagnosis, outcome and prognosis of CML.

Bone Marrow Transplantation↗
PubMed1997

Interaction of BCR-ABL with the retinoblastoma protein in Philadelphia chromosome-positive cell lines.

The tyrosine kinase activity of BCR-ABL fusion proteins plays an important role in the pathogenesis of leukemia that is for the Philadelphia chromosome (Ph1). Because nuclear c-ABL is regulated during the cell cycle through a specific interaction with the retinoblastoma protein (pRB), the possible interaction of BCR-ABL with pRB in Ph1-positive cell lines was investigated. P145 c-ABL as well as P190 and P210 BCR-ABL proteins interacted with pRB. Furthermore, c-ABL and BCR-ABL associated with both phosphorylated and nonphosphorylated forms of pRB. These findings suggest that BCR-ABL interferes with pRB function and thereby regulates cell growth.

Animals↗
PubMed1997

The chimeric BCR-ABL gene.

The 1982 discovery that in chronic myeloid leukaemia (CML) the ABL proto-oncogene is translocated to the BCR gene located on chromosome 22 initiated many studies on the structural organization and function of these genes. The nucleotide sequence of the entire BCR and major parts of the ABL gene has now been determined. However, the actual cause of the fusion of BCR with ABL remains essentially unknown. Mouse models have been helpful to unravel the normal cellular function of BCR and ABL, as well the activity of BCR-ABL, although a single mechanism explaining the transforming activity of the latter has not been discovered. The cause of progression of the disease remains unknown, and no single genetic abnormality has been linked to the blast phase of CML. Much has been learned concerning the molecular biology of CML, but answers to the fundamental questions above may be expected in the coming years in parallel to increasing knowledge of genome structure, signal transduction and cell cycle control.

Animals↗
PubMed2003

Autoinhibition of Bcr-Abl through its SH3 domain.

Bcr-Abl is a dysregulated tyrosine kinase whose mechanism of activation is unclear. Here, we demonstrate that, like c-Abl, Bcr-Abl is negatively regulated through its SH3 domain. Kinase activity, transformation, and leukemogenesis by Bcr-Abl are greatly impaired by mutations of the Bcr coiled-coil domain that disrupt oligomerization, but restored by an SH3 point mutation that blocks ligand binding or a complementary mutation at the intramolecular SH3 binding site defined in c-Abl. Phosphorylation of tyrosines in the activation loop of the catalytic domain and the linker between the SH2 and catalytic domains (SH2-CD linker) is dependent on oligomerization and required for leukemogenesis. These results suggest that Bcr-Abl has a monomeric, unphosphorylated state with the SH3 domain engaged intramolecularly to Pro1124 in the SH2-CD linker, the form that is sensitive to the inhibitor imatinib (STI-571). The sole function of the coiled-coil domain is to disrupt the autoinhibited conformation through oligomerization and intermolecular autophosphorylation.

3T3 Cells↗
PubMed2006

Allosteric inhibitors of Bcr-abl-dependent cell proliferation.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-abl, a 210-kDa fusion protein with deregulated tyrosine kinase activity. Encouraged by the clinical validation of Bcr-abl as the target for the treatment of CML by imatinib, we sought to identify pharmacological agents that could target this kinase by a distinct mechanism. We report the discovery of a new class of Bcr-abl inhibitors using an unbiased differential cytotoxicity screen of a combinatorial kinase-directed heterocycle library. Compounds in this class (exemplified by GNF-2) show exclusive antiproliferative activity toward Bcr-abl-transformed cells, with potencies similar to imatinib, while showing no inhibition of the kinase activity of full-length or catalytic domain of c-abl. We propose that this new class of compounds inhibits Bcr-abl kinase activity through an allosteric non-ATP competitive mechanism.

Animals↗
PubMed2004

Requirement of Src kinases Lyn, Hck and Fgr for BCR-ABL1-induced B-lymphoblastic leukemia but not chronic myeloid leukemia.

The Abl kinase inhibitor imatinib mesylate is the preferred treatment for Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML) in chronic phase but is much less effective in CML blast crisis or Ph(+) B-cell acute lymphoblastic leukemia (B-ALL). Here, we show that Bcr-Abl activated the Src kinases Lyn, Hck and Fgr in B-lymphoid cells. BCR-ABL1 retrovirus-transduced marrow from mice lacking all three Src kinases efficiently induced CML but not B-ALL in recipients. The kinase inhibitor CGP76030 impaired the proliferation of B-lymphoid cells expressing Bcr-Abl in vitro and prolonged survival of mice with B-ALL but not CML. The combination of CGP76030 and imatinib was superior to imatinib alone in this regard. The biochemical target of CGP76030 in leukemia cells was Src kinases, not Bcr-Abl. These results implicate Src family kinases as therapeutic targets in Ph(+) B-ALL and suggest that simultaneous inhibition of Src and Bcr-Abl kinases may benefit individuals with Ph(+) acute leukemia.

Animals↗
PubMed1996

Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.

The bcr-abl oncogene, present in 95% of patients with chronic myelogenous leukemia (CML), has been implicated as the cause of this disease. A compound, designed to inhibit the Abl protein tyrosine kinase, was evaluated for its effects on cells containing the Bcr-Abl fusion protein. Cellular proliferation and tumor formation by Bcr-Abl-expressing cells were specifically inhibited by this compound. In colony-forming assays of peripheral blood or bone marrow from patients with CML, there was a 92-98% decrease in the number of bcr-abl colonies formed but no inhibition of normal colony formation. This compound may be useful in the treatment of bcr-abl-positive leukemias.

Animals↗
PubMed1997

Effect of bcr-abl oligodeoxynucleotides on the clonogenic growth of chronic myelogenous leukaemia cells.

We studied the effect of phosphorothioate oligodeoxynucleotides ([S]ODNs) complementary to the bcr-abl junction on cells taken at diagnosis from 41 patients with Philadelphia-positive chronic myelogenous leukaemia (CML). Experiments included the evaluation of the anti-leukaemic effect of 16- and 26-mer antisense [S]ODNs on both mononuclear and CD34+ cells, evaluation of incubation time and correlation of colony growth inhibition with the down-regulation of p210(bcr-abl). At the same time, the uptake of [S]ODNs by mononuclear and purified CD34+ cell populations and the cross-hybridization of 26- and 16-mer [S]ODNs with the complementary sequences were evaluated. After incubation for 120 h with 26-mer antisense [S]ODNs on mononuclear cells, overall mean colony recovery was 41.9% of the untreated control samples; in particular, a significant reduction in colony formation was observed in 22 of the 35 cases tested. The effect of 26-mer ODNs on CD34+ cells was comparable to that observed on mononuclear cells in terms of colony inhibition; however, a higher proportion of cases showed a significant inhibition of colony formation. In comparison with the 26-mer antisense [S]ODNs, the anti-leukaemic effect of the 16-mer antisense [S]ODNs was less evident on mononuclear cells and comparable on CD34+ cells; however, a more specific effect was evident on both target cells. Hybridization experiments confirmed a partial cross-reactivity when the 26-mer ODNs were hybridized with their complementary sequence; this did not occur when 16-mer ODNs were similarly tested. Experiments aimed at evaluating the effect of the incubation time showed a significant increase in anti-leukaemic effect after a 120 h incubation period compared to that measured after a 24 h incubation period; this was parallelled by a progressive increase in the intracellular concentrations of [S]ODNs from day 1 to day 5. The accumulation of [S]ODNs correlated with a marked down-regulation of p210(bcr-abl) levels which was first detectable after 72 h of treatment. The down-regulation of p210(bcr-abl) levels following treatment with [S]ODNs showed a correlation between the effect of antisense [S]ODNs on leukaemic colony formation and protein expression. These studies confirm that, under optimal conditions of target cell culture and ODN size, antisense [S]ODNs complementary to the bcr-abl junction have specific anti-leukaemic effects.

Antigens, CD↗