Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “BCR”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed1999

Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay.

Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular permeabilization and fixation, the mRNA BCR-ABL was reverse transcribed and amplified by PCR using digoxigenin-labelled dUTP. The reaction was revealed with the anti-digoxigenin FITC antibody. On the fluorescent microscope, a strong positive green fluorescence signal was observed in 98-99% cells in Ph1-positive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2 +/- 1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in situ RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), and two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematological remission after alpha interferon (three patients), hydroxyurea (one patient) autologous bone marrow transplantation (BMT) (one patient) and allogeneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4% (range 1.8-3.2). All six patients had normal karyotype and negative two-step RT-PCR results. In the five other patients, two were treated by hydroxyurea alone or autologous BMT, and 11 and 13% of the cells were strongly positive; three were treated with interferon and 14-62% of the cells were positive, generally weakly. All five patients had persistence of Ph1 (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in situ RT-PCR can specifically identify cells with BCR-ABL transcript and its results are concordant with those of karyotype and RT-PCR. Because of its limited sensitivity and specificity, however, it appears to have limited value in the analysis of MRD. On the other hand, it can evaluate the presence and intensity of BCR-ABL fusion transcript at the single cell level, and this could be useful in treatment monitoring.

Bone Marrow Transplantation↗
PubMed2002

Bcr-abl-positive cells secrete angiogenic factors including matrix metalloproteinases and stimulate angiogenesis in vivo in Matrigel implants.

To further elucidate the role of angiogenesis in the pathogenesis of chronic myelogenous leukemia (CML) we evaluated the effects of the bcr-abl translocation on the secretion of the angiogenic factors VEGF, FGF-2, HGF, IL-8 and matrix metalloproteinases (MMPs) as well as on the angiogenic potential in vivo of bcr-abl+ cells. First, we examined murine FL5.12 cells transfected with the bcr-abl constructs p185, p210 and p230 and found that the transfected cells secreted as much as four-fold more VEGF (p185 > p210 >p230) than wild-type (wt) cells, as well as MMP-9 and MMP-2. When Matrigel fragments containing these bcr-abl+ cells were implanted subcutaneously in SCID or Balb-C mice they became significantly more vascularized and hemoglobinized than implants containing normal or wt cells (p185 > p210 > p230). Similarly, we found that myeloblasts expanded from bone marrow (BM) CD34+ cells derived from Philadelphia-positive CML patients secreted up to 10 times more VEGF, FGF-2, HGF and IL-8 compared to myeloblasts derived from normal donors' BM CD34+ cells and that BM mononuclear cells (MNC) isolated from CML patients induced vascularization of Matrigel implants in mice. Moreover, we found that peripheral blood MNC expressed MMP-2 and membrane-type (MT)1-MMP in about 50% of CML patients studied, and MMP-9 in all of them. Furthermore, VEGF stimulated the secretion of MMP-9 in these primary CML cells. We conclude that stimulation of angiogenesis by angiogenic factors, including MMPs, could play an important role in the pathogenesis of CML, suggesting that therapies targeting the newly formed endothelium could be developed for CML.

Adult↗
PubMed2004

Efficacy of dual-specific Bcr-Abl and Src-family kinase inhibitors in cells sensitive and resistant to imatinib mesylate.

Monotherapy of chronic myeloid leukemia (CML) with imatinib mesylate has been cast into shadow by the evolution of clinical resistance during therapy. Resistance to imatinib can arise by multiple mechanisms including amplification or mutation of Bcr-Abl, and continuity of imatinib therapy is probably a poor option for either of these patient groups. Recently, however, a structurally distinct new class of drugs, the pyrido[2,3-d]pyrimidines, has been described, and these compounds are predicted to make different molecular contacts in the Abl kinase domain. These drugs potently target both the Bcr-Abl and Src-family kinase activities, both of which are thought to be relevant to survival of the leukemic cell. We asked whether these drugs could selectively induce cell death in murine cell line models of CML cells sensitive and resistant to imatinib by different mechanisms. We show that whereas the pyrido[2,3-d] pyrimidines are indeed highly potent in suppressing proliferation of Bcr-Abl-overexpressing imatinib-resistant cells, they are almost completely ineffective against cells expressing the T315I mutant. This implies that despite structural differences from imatinib, these drugs are unlikely to be useful in patients expressing this mutant Bcr-Abl protein, but may be effective in cases where selection of cells overexpressing the oncoprotein leads to refractoriness to imatinib.

Animals↗
PubMed2006

BCR-ABL activity and its response to drugs can be determined in CD34+ CML stem cells by CrkL phosphorylation status using flow cytometry.

In chronic myeloid leukaemia, CD34(+) stem/progenitor cells appear resistant to imatinib mesylate (IM) in vitro and in vivo. To investigate the underlying mechanism(s) of IM resistance, it is essential to quantify Bcr-Abl kinase status at the stem cell level. We developed a flow cytometry method to measure CrkL phosphorylation (P-CrkL) in samples with <10(4) cells. The method was first validated in wild-type (K562) and mutant (BAF3) BCR-ABL(+) as well as BCR-ABL(-) (HL60) cell lines. In response to increasing IM concentration, there was a linear reduction in P-CrkL, which was Bcr-Abl specific and correlated with known resistance. The results were comparable to those from Western blotting. The method also proved to be reproducible with small samples of normal and Ph(+) CD34(+) cells and was able to discriminate between Ph(-), sensitive and resistant Ph(+) cells. This assay should now enable investigators to unravel the mechanism(s) of IM resistance in stem cells.

Adaptor Proteins, Signal Transducing↗
PubMed2006

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

Amino Acid Sequence↗
PubMed2007

FIP1L1-PDGFRA in chronic eosinophilic leukemia and BCR-ABL1 in chronic myeloid leukemia affect different leukemic cells.

We investigated genetically affected leukemic cells in FIP1L1-PDGFRA+ chronic eosinophilic leukemia (CEL) and in BCR-ABL1+ chronic myeloid leukemia (CML), two myeloproliferative disorders responsive to imatinib. Fluorescence in situ hybridization specific for BCR-ABL1 and for FIP1L1-PDGFRA was combined with cytomorphology or with lineage-restricted monoclonal antibodies and applied in CML and CEL, respectively. In CEL the amount of FIP1L1-PDGFRA+ cells among CD34+ and CD133+ cells, B and T lymphocytes, and megakaryocytes were within normal ranges. Positivity was found in eosinophils, granulo-monocytes and varying percentages of erythrocytes. In vitro assays with imatinib showed reduced survival of peripheral blood mononuclear cells but no reduction in colony-forming unit growth medium (CFU-GM) growth. In CML the BCR-ABL1 fusion gene was detected in CD34+/CD133+ cells, granulo-monocytes, eosinophils, erythrocytes, megakaryocytes and B-lymphocytes. Growth of both peripheral blood mononuclear cells and CFU-GM was inhibited by imatinib. This study provided evidence for marked differences in the leukemic masses which are targeted by imatinib in CEL or CML, as harboring FIP1L1-PDGFRA or BCR-ABL1.

AC133 Antigen↗
PubMed2002

Studying the pathogenesis of BCR-ABL+ leukemia in mice.

Animal models of BCR-ABL+ leukemias have provided important new knowledge about the molecular pathophysiology of these diseases, and answered questions that are difficult or impossible to address using BCR-ABL-expressing cell lines or primary Ph+ leukemia samples from patients. The power of mouse models lies in their ability to recapitulate precisely the phenotypes of BCR-ABL+ leukemias in vivo, but this comes at the price of significant complexity. Here I review recent studies of leukemias induced in mice by BCR-ABL with an emphasis on the intricate nature of these diseases and the need for careful pathological and molecular analysis.

Animals↗
PubMed1997

An antisense Bcr-Abl phosphodiester-tailed methylphosphonate oligonucleotide reduces the growth of chronic myeloid leukaemia patient cells by a non-antisense mechanism.

The specificity of antisense oligonucleotides targeted to the mRNA breakpoint region of the Bcr-Abl oncogene, found in leukaemic cells from patients with chronic myeloid leukaemia, remains controversial due to non-specific effects. To prevent protein binding of oligonucleotides we designed and tested a methylphosphonate oligonucleotide with an attached 3' soluble phosphodiester tail. Growth of chronic myeloid leukaemia (CML) cell lines BV173, KCL-22 and cells of CML patients tested was inhibited by the b2a2 type antisense Bcr-Abl oligonucleotide and not with controls. Also the growth of control CD34+ cells of two healthy donors, control cell lines and cells from AML patients was only moderately affected or not affected. Bcr-Abl protein studies in combination with growth-determination experiments indicated that the antisense methylphosphonate Bcr-Abl oligonucleotide tested is a potent inhibitor of the growth of CML cells but works in a non-antisense manner.

Apoptosis↗
PubMed1998

BCR-ABL-positive progenitors in chronic myeloid leukaemia patients in complete cytogenetic remission after treatment with interferon-alpha.

To determine the source of residual disease detected in patients with chronic myeloid leukaemia (CML) in complete cytogenetic remission (n=8) after treatment with interferon-alpha (IFN-alpha), we have tested CFU-GM colonies grown from bone marrow mononuclear cells or from plastic-adherent (Pdelta) cells for BCR-ABL mRNA using a nested multiplex RT-PCR. We compared our results with those obtained by analysis of colonies from newly diagnosed patients (n=4) and patients achieving no cytogenetic response (n=1) or incomplete cytogenetic response to treatment with IFN-alpha (n=5). A total of 1239 informative colonies were analysed. A small proportion of BCR-ABL-positive colonies was detected in all eight patients in complete cytogenetic remission, suggesting the persistence of leukaemia that could potentially lead to relapse. The overall proportion of BCR-ABL-positive colonies in patients achieving a cytogenetic response to IFN-alpha correlated with the levels of BCR-ABL transcripts detected in the peripheral blood by competitive RT-PCR (P=0.004). We conclude that residual disease detected in the peripheral blood of complete cytogenetic responders to IFN-alpha is at least partly derived from clonogenic myeloid cells. It is probable that the leukaemia clone in CML is only very rarely or never entirely eradicated by treatment with IFN-alpha.

Adult↗
PubMed1998

Expression of p210 and p190 BCR-ABL due to alternative splicing in chronic myelogenous leukaemia.

The hallmark of chronic myelogenous leukaemia (CML) is the presence of the Philadelphia chromosome and its resultant fusion message, BCR-ABL, and fusion protein, p210. Patients with CML in blast crisis, or with Philadelphia positive acute lymphoblastic leukaemia (ALL), can have a smaller BCR-ABL fusion transcript possessing only the first exon of BCR fused to ABL. This smaller transcript encodes a 190 kD protein which is more strongly transforming than the p210 protein derived from the larger CML-associated transcript. We performed RT-PCR on samples from CML patients in chronic phase to determine the frequency and mechanism of p190 and p210 co-expression and to see if this correlated with clinical indices. We examined the peripheral blood or marrow of 67 patients with CML and found that 35 of them expressed both transcripts whereas the remainder expressed the p210-encoding transcript exclusively. Additional PCR products of an intermediate size were also frequently detected and have been isolated and sequenced. Data from two of these products indicate that they are the result of alternative splicing and include variable combinations of BCR exons. We believe that the expression of the p190-encoding transcript in the chronic phase of CML is also due to alternative splicing. A comparison of patients co-expressing the p190- and p210-encoding transcripts with those patients who expressed only the p210-encoding transcript detected significantly higher white blood cell (WBC) counts and blast cell counts at time of testing as well as significantly higher white blood cell counts at diagnosis.

Adolescent↗
PubMed2000

Ontogeny of natural killer cells and T cells by analysis of BCR-ABL rearrangement from patients with chronic myelogenous leukaemia.

Chronic myelogenous leukaemia (CML) is a haematological malignant disorder characterized by the Philadelphia chromosome (Ph) and BCR-ABL gene rearrangement. This abnormal fusion gene can be considered to serve as a marker for the transformed cell clone in CML and is found in all cells arising from the same malignant precursor cell. It has been detected in CML cells of the myeloid, monocytic, erythroid and B-lymphocytic lineages. However, it is still arguable as to whether T lymphocytes or natural killer (NK) cells carry this marker. Answering this question would clarify the ontogenic relationship between NK cells and T cells. We examined 12 CML patients and studied the expression of BCR-ABL rearrangement by fluorescence in situ hybridization (FISH) in both NK cells and T cells sorted by flow cytometry. The purity of T cells was 95.6-99.8% and that of NK cells was 95.3-99.3% after sorting. Neither NK cells nor T cells showed any positive BCR-ABL signal with the exception of one patient who recovered from a lymphoid blastic crisis. We speculate that T cells and NK cells originate from BCR-ABL-negative stem cells.

Adult↗
PubMed2000

Novel type of BCR-ABL transcript in a chronic myelogenous leukaemia patient relapsed after bone marrow transplantation.

We identified a novel BCR-ABL transcript in a chronic myelogenous leukaemia (CML) patient who relapsed after bone marrow transplantation (BMT), containing a fusion between part of BCR exon 3, 44 nucleotides derived from ABL intron 1b and ABL exon 2. The breakpoints were located within BCR exon 3 on chromosome 22 and within the ABL intron 1b on chromosome 9, and the transcript derives from a splicing of ABL exon 2 to a putative splicing acceptor site 44 nucleotides downstream to the breakpoint on chromosome 9. The patient's clinical course strengthens the idea that short forms of BCR-ABL transcripts are associated with a more aggressive disease.

Adult↗
PubMed2002

Blast crisis of Philadelphia chromosome-positive chronic myeloid leukaemia carrying micro-bcr breakpoint (e19a2 and e191a).

We describe two Philadelphia chromosome-positive chronic myeloid leukaemia (Ph-positive CML) patients carrying micro-bcr (micro-bcr) breakpoint, who developed blast crisis within 5 years of diagnosis. Reverse transcription polymerase chain reaction analysis of bone marrow cells using primers specific for the p210BCR-ABL fusion transcript showed aberrant large-sized bands in both cases (986 bp in patient 1 and 1031 bp in patient 2). Sequencing analysis of these products revealed BCR-ABL fusion transcripts with e19a2 in patient 1 and e191a in patient 2. These findings suggest that CML carrying micro-bcr breakpoint may exhibit a similar clinical course to classical CML, and that it may not be a mild form of the disease.

Aged↗
PubMed1998

Detecting residual bcr-abl transcripts in chronic myeloid leukaemia patients using coupled reverse transcriptase-polymerase chain reaction with rTth DNA polymerase.

The bcr-abl fusion gene is the hallmark of chronic myeloid leukaemia (CML) and presumably the cause of its development. Accordingly, long-term disappearance of the bcr-abl gene after intensive therapy suggests that a patient is probably cured of CML. The diagnostic protocol based on coupling of two enzymatic reactions, reverse transcription (RT) and nested polymerase chain reaction (nPCR), for the detection of bcr-abl transcripts in peripheral blood provides a powerful tool for minimal residual CML detection. We have developed a new detection protocol using rTth DNA polymerase as the only enzyme catalysing both reactions for simplifying CML diagnosis. We demonstrate its efficacy investigating residual leukaemic cells in the peripheral blood of 10 patients. This assay offers several advantages over the use of conventional RT-PCR, being more sensitive, faster, less prone to false positives since no opening of the tube is required between the two reactions and requires no special oils or waxes. Our simple assay for bcr-abl chimeric transcripts detection is a practical addition to the diagnostic evaluation of the patient with chronic myeloid leukaemia.

DNA-Directed DNA Polymerase↗
PubMed2001

Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.

BACKGROUND: BCR-ABL is a constitutively activated tyrosine kinase that causes chronic myeloid leukemia (CML). Since tyrosine kinase activity is essential to the transforming function of BCR-ABL, an inhibitor of the kinase could be an effective treatment for CML. METHODS: We conducted a phase 1, dose-escalating trial of STI571 (formerly known as CGP 57148B), a specific inhibitor of the BCR-ABL tyrosine kinase. STI571 was administered orally to 83 patients with CML in the chronic phase in whom treatment with interferon alfa had failed. Patients were successively assigned to 1 of 14 doses ranging from 25 to 1000 mg per day. RESULTS: Adverse effects of STI571 were minimal; the most common were nausea, myalgias, edema, and diarrhea. A maximal tolerated dose was not identified. Complete hematologic responses were observed in 53 of 54 patients treated with daily doses of 300 mg or more and typically occurred in the first four weeks of therapy. Of the 54 patients treated with doses of 300 mg or more, cytogenetic responses occurred in 29, including 17 (31 percent of the 54 patients who received this dose) with major responses (0 to 35 percent of cells in metaphase positive for the Philadelphia chromosome); 7 of these patients had complete cytogenetic remissions. CONCLUSIONS: STI571 is well tolerated and has significant antileukemic activity in patients with CML in whom treatment with interferon alfa had failed. Our results provide evidence of the essential role of BCR-ABL tyrosine kinase activity in CML and demonstrate the potential for the development of anticancer drugs based on the specific molecular abnormality present in a human cancer.

Adult↗
PubMed1994

Progressive de novo DNA methylation at the bcr-abl locus in the course of chronic myelogenous leukemia.

De novo methylation of CpG islands is a rare event in mammalian cells. It has been observed in the course of developmental processes, such as X chromosome inactivation and genomic imprinting. The methylation of DNA, an important factor in the epigenetic control of gene expression, may also be involved in tumorigenesis. After the t(9;22) chromosomal translocation and generation of the Philadelphia chromosome, the initiating event in chronic myelogenous leukemia (CML), most of the abl coding sequence is fused to the 5' region of the bcr gene. Expression of the hybrid bcr-abl gene is, therefore, regulated by the bcr promoter. In most cases of CML, one of the two abl promoters (Pa) is nested within the bcr-abl transcriptional unit and should be able to transcribe the type Ia 6-kb normal abl mRNA from the Philadelphia chromosome. However, we have found that the 6-kb transcript is present only in CML cell lines containing a normal abl allele and that the apparent inactivation of the nested Pa promoter is associated with allele-specific methylation. Furthermore, we have noticed that the Pa promoter is contained within a CpG island and undergoes progressive de novo methylation in the course of the disease. This is attested to by the fact that DNA samples from CML patients that are methylation-free at the time of diagnosis invariably become methylated in advanced CML. Since tumor progression in CML cannot always be inferred from the clinical presentation, assessment of de novo CpG methylation may prove to be of critical value in management of the disease. It could herald blastic transformation at a stage when bone marrow transplantation, the only potentially curative therapeutic procedure in CML, is still effective.

Animals↗
PubMed1999

The BCR-ABL oncoprotein potentially interacts with the xeroderma pigmentosum group B protein.

The previously uncharacterized CDC24 homology domain of BCR, which is missing in the P185 BCR-ABL oncogene of Philadelphia chromosome (Ph1)-positive acute lymphocytic leukemia but is retained in P210 BCR-ABL of chronic myelogeneous leukemia, was found to bind to the xeroderma pigmentosum group B protein (XPB). The binding appeared to be required for XPB to be tyrosine-phosphorylated by BCR-ABL. The interaction not only reduced both the ATPase and the helicase activities of XPB purified in the baculovirus system but also impaired XPB-mediated cross-complementation of the repair deficiency in rodent UV-sensitive mutants of group 3. The persistent dysfunction of XPB may in part underlie genomic instability in blastic crisis.

Adenosine Triphosphatases↗
PubMed2001

p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).

p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).

Animals↗