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At least 829 records · Page 46Linked to original sources
PubMed2000

Down-regulation of bcr-abl and bcl-x(L) expression in a leukemia cell line and its doxorubicin-resistant variant by topoisomerase II inhibitors.

K562 cells are usually resistant to apoptosis induction, probably because of the expression of bcr-abl, the hybrid gene characteristic of the Philadelphia chromosome (t 9;22). However, we have previously shown that amsacrine and, to a lesser extent, doxorubicin, could induce apoptosis in the doxorubicin-resistant variant of this cell line. In order to elucidate the role of bcr-abl in triggering apoptosis, we investigated the effect of the topoisomerase II inhibitors doxorubicin, amsacrine, and etoposide on the expression of several genes that may be related to apoptosis induction in both cell lines. This was done using a technique of reverse transcription-polymerase chain reaction coupled with HPLC of the amplified fragments to obtain semiquantitative evaluations. We showed that amsacrine, at pharmacologically relevant concentrations, was able to decrease the expression of bcr-abl down to 20% of the basal value in the doxorubicin-resistant variant only, whereas doxorubicin and etoposide were unable to do so. No effect of these drugs was seen on the expression of the normal abl gene. In addition, there was an effect of amsacrine on the expression of bcl-x(L) in the resistant cell line only, but at concentrations higher than the IC(50) of this drug. Our results emphasize the role of bcr-abl in protecting cells from apoptosis and the possible involvement of specific topoisomerase II inhibitors in overcoming resistance to apoptosis.

Amsacrine↗
PubMed2003

BCR-ABL binds to IRS-1 and IRS-1 phosphorylation is inhibited by imatinib in K562 cells.

In the present study we used K562 cells to demonstrate that insulin receptor substrate 1 (IRS-1) is expressed and constitutively phosphorylated in BCR-ABL(+) cells. We observed association between BCR-ABL/IRS-1, IRS-1/phosphoinositide 3'-kinase (PI3-kinase), and IRS-1/Grb2 in the K562 cell line. Our findings demonstrate that imatinib treatment resulted in marked attenuation of BCR-ABL/IRS-1 association and of IRS-1-stimulated PI3-kinase activity in K562 cells. We concluded that the IRS-1 protein is involved in the signalling pathway of the BCR-ABL tyrosine kinase.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Inhibition of protein kinase C delta has negative effect on anchorage-independent growth of BCR-ABL-transformed Rat1 cells.

The BCR-ABL oncoprotein transmits transformation signals mainly through pathways involving Ras, Myc and PI3 kinase. Here we report that inhibition of protein kinase C (PKC) delta had negative influence on anchorage-independent growth of Rat1 cells transformed by BCR-ABL. The effect was observed with delta isoform-specific inhibitor rottlerin, but not with Go6976 that inhibits only conventional isoforms. The kinase activity of delta isoform was found to be roughly two-fold higher in BCR-ABL-expressing Rat1 cells than that in mock. Although overexpression of wild type PKC delta did not enhance soft agar colony number by BCR-ABL-transformed Rat1 cells, that of dominant-negative delta isoform reduced it by approximately 40%.

Animals↗
PubMed2004

p190(BCR-ABL) rearrangement as a secondary change in a case of acute myelo-monocytic leukemia with inv(16)(p13q22).

The simultaneous occurrence of two specific primary chromosomal changes in hematological malignancies is rare. We report on a patient with acute myelo-monocytic leukemia and both inv(16)(p13q22) and t(9;22)(q34;q11) with a p190(BCR-ABL) rearrangement. The t(9;22)(q34;q11) translocation appears to be a secondary change. Similar secondary BCR-ABL rearrangements have already been described and, in most cases, the chimeric protein was of the p190(BCR-ABL) type as in our case. A complete remission was obtained by conventional chemotherapy followed with imatinib mesylate maintenance therapy. At relapse, the BCR-ABL transcripts were undetectable, which suggests that imatinib mesylate could be an effective adjuvant treatment in acute leukemia with a secondary t(9;22)(q34;q11).

Adult↗
PubMed1998

Detection of BCR-ABL transcripts in acute lymphoblastic leukemia in Indian patients.

Thirty-three patients with acute lymphoblastic leukemia (ALL) from India were studied for the presence of BCR-ABL chimeric transcripts, by a seminested cDNA-PCR. We report the presence of BCR-ABL chimeric transcripts in 4/17 (24%) children (under 15 years) and 3/16 (19%) adults (15-50 years). This is in sharp contrast to the published literature from the West where the presence of BCR-ABL has been reported in only 2-5% children and 35% adults. Whether the presence of BCR-ABL fusion mRNA, which is generally an attribute of ALL in adults and of poorer prognosis, may contribute to chemo-incurability in young Indian patients, remains to be seen, as a larger number of patients are studied for treatment outcome and survival on uniform therapy protocols.

Adolescent↗
PubMed1997

Regulation of the oncogenic activity of BCR-ABL by a tightly bound substrate protein RIN1.

RIN1 was originally identified by its ability to physically bind to and interfere with activated Ras in yeast. Paradoxically, RIN1 potentiates the oncogenic activity of the BCR-ABL tyrosine kinase in hematopoietic cells and dramatically accelerates BCR-ABL-induced leukemias in mice. RIN1 rescues BCR-ABL mutants for transformation in a manner distinguishable from the cell cycle regulators c-Myc and cyclin D1 and the Ras connector Shc. These biological effects require tyrosine phosphorylation of RIN1 and binding of RIN1 to the Abl-SH2 and SH3 domains. RIN1 is tyrosine phosphorylated and is associated with BCR-ABL in human and murine leukemic cells. RIN1 exemplifies a new class of effector molecules dependent on the concerted action of the SH3, SH2, and catalytic domains of a cytoplasmic tyrosine kinase.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Characterization of the different BCR-ABL transcripts with a single multiplex RT-PCR.

The diagnosis of chronic myeloid leukemia is based on detection of the Philadelphia (Ph) chromosome or the BCR-ABL gene. The junction present in the transcript may vary according to the reciprocal translocation t(9;22)(q34;11). Identification of the transcript (p190, p210 or p230) does not reveal the type of junction but this information is very important for classification of patients in clinical trials. Most identification kits do not explore p230 transcripts and are unable to determine exotic breakpoints. We have developed a clinical molecular diagnosis assay, able to identify all of the BCR-ABL transcripts and, by single assay, to characterize all of the possible transcript junctions. This technique is based on RT-PCR and PCR-capillary electrophoresis. For each patient sample, we performed RT-PCR with three different BCR primers each coupled to a specific different fluorochrome and a unique reverse ABL primer. Depending on the transcript, only one BCR primer was used for each RT-PCR. After capillary electrophoresis and fluorescence determination, we were able to identify both the transcript and its junction at the same time.

DNA Primers↗
PubMed2005

Orientation and oligomerization specificity of the Bcr coiled-coil oligomerization domain.

The Bcr oligomerization domain, from the Bcr-Abl oncoprotein, is an attractive therapeutic target for treating leukemias because it is required for cellular transformation. The domain homodimerizes via an antiparallel coiled coil with an adjacent short, helical swap domain. Inspection of the coiled-coil sequence does not reveal obvious determinants of helix-orientation specificity, raising the possibility that the antiparallel orientation preference and/or the dimeric oligomerization state are due to interactions of the swap domains. To better understand how structural specificity is encoded in Bcr, coiled-coil constructs containing either an N- or C-terminal cysteine were synthesized without the swap domain. When cross-linked to adopt exclusively parallel or antiparallel orientations, these showed similar circular dichroism spectra. Both constructs formed coiled-coil dimers, but the antiparallel construct was approximately 16 degrees C more stable than the parallel to thermal denaturation. Equilibrium disulfide-exchange studies confirmed that the isolated coiled-coil homodimer shows a very strong preference for the antiparallel orientation. We conclude that the orientation and oligomerization preferences of Bcr are not caused by the presence of the swap domains, but rather are directly encoded in the coiled-coil sequence. We further explored possible determinants of structural specificity by mutating residues in the d position of the coiled-coil core. Some of the mutations caused a change in orientation specificity, and all of the mutations led to the formation of higher-order oligomers. In the absence of the swap domain, these residues play an important role in disfavoring alternate states and are especially important for encoding dimeric oligomerization specificity.

Amino Acid Sequence↗
PubMed2000

Purging in BCR-ABL-positive acute lymphoblastic leukemia using immunomagnetic beads: comparison of residual leukemia and purging efficiency in bone marrow vs peripheral blood stem cells by semiquantitative polymerase chain reaction.

Twenty autologous bone marrow (BM) and 25 peripheral blood stem cell (PBSC) grafts were collected from a total of 40 consecutive patients with BCR-ABL+ acute lymphoblastic leukemia (ALL) in first (n = 37) or second (n = 3) complete morphological remission and subsequently purged with a cocktail of anti-CD19, -CD10, AB4 MoAbs and immunomagnetic beads (IMB). Residual BCR-ABL-positive cells before purging were detected in 19 of 20 BM grafts at a median of 4 (range 0-6) logs and in 17 of 25 evaluable PBSC grafts at a median of 1 (range 0-3) log above the limit of detection assessed by a semiquantitative limiting log10-dilution RT-PCR (P < 0.0001). IMB purging depleted a median of 2.5 (range 1-4) log of residual BCR-ABL+ cells from BM and a median of 1 (range 0-2) log from PBSC grafts, achieving RT-PCR negativity in 1/20 BM and 12/25 PBSC grafts after purging. Cell recoveries were 62% and 86% (P < 0.0001) of MNC and 74% and 97% (P = 0.065) of CD34+ cells after BM and PBSC purging, respectively. BM purging was superior using the triple MoAb cocktail which depleted 2.64 +/- 0.4 log (n = 14) compared to 1.6 +/- 0.4 log (n = 5) using the MoAb cocktail not including AB4 (P = 0. 02). We conclude that unpurged BM grafts contain 2-3 log more residual BCR-ABL+ cells than unpurged PBSC grafts and that purging efficacy is superior in BM compared to PBSC grafts, but median titers in purged BM grafts still exceed those in purged PBSC grafts. Bone Marrow Transplantation (2000) 25, 97-104.

Adolescent↗
PubMed1998

High rate of chromosome abnormalities detected by fluorescence in situ hybridization using BCR and ABL probes in adult acute lymphoblastic leukemia.

The value of dual-color fluorescence in situ hybridization (FISH) with BCR and ABL probes for the detection of the Philadelphia (Ph) translocation and of other alterations involving ABL and/or BCR was evaluated in adult acute lymphoblastic leukemia (ALL). One hundred and four patients were studied prospectively using interphase nuclei FISH, chromosome analysis (CA), and PCR assays for the chimeric BRC/ABL transcript. FISH detected a Ph translocation in 24 cases (23.1%), as was confirmed by CA and/or PCR. FISH revealed a false positive diagnosis of a Ph translocation in four cases (5% false positive rate). Among 54 cases with combined FISH, CA and PCR assays, FISH failed to establish a correct diagnosis in 3.7%, PCR in 5.6%, and CA in 7.4%. The combination of two screening methods led to discrepant results in 9.3% (FISH + PCR), 11.1% (FISH + CA), or 13% (CA + PCR) of the cases. In seven of 80 (8.8%) Ph-negative patients, gain of BCR and/or ABL was identified. Overall, FISH detected alterations of the BCR and/or ABL genes with an incidence of 29.8% of the current study. Due to the possibility of false positive diagnosis of a Ph translocation using dual-color FISH the combination with chromosome and/or RT-PCR analyses is recommended in adult ALL patients.

Adolescent↗
PubMed2000

The existence of lymphoid lineage restricted Philadelphia chromosome-positive acute lymphoblastic leukemia with heterogeneous bcr-abl rearrangement.

Analysis of lineage involvement was performed in 17 Philadelphia chromosome-positive acute lymphoblastic leukemia patients with no history of chronic myeloproliferative disorder. The percentage of blastic cells as defined by flow cytometry matched that of the Ph-positive cells in 14 out of 17 patients. The bcr-abl rearrangement was investigated by fluorescent in situ hybridization in morphologically identified blastic cells, myeloid elements, lymphocytes and erythroblasts using a combined light and fluorescent microscopical imaging. Lymphoid lineage restriction could be determined in all but three of the patients. These 14 patients exhibited heterogeneity in terms of m-bcr and M-bcr types of translocation as revealed by reverse transcription polymerase chain reaction. The three patients with multilineage involvement and M-bcr type of translocation reverted to chronic phase and the percentage of Ph-positive cells remained high. Thus, we could identify an uncommitted stem cell origin among Ph-positive ALLs only in those patients whose disease subsequently proved to be a lymphoid blastic crisis with clinically silent chronic phase.

Adult↗
PubMed2000

Increase in mutant frequencies in mice expressing the BCR-ABL activated tyrosine kinase.

The acquisition of the Philadelphia (Ph) chromosome (or BCR-ABL translocation) represents a detrimental pathophysiological event in humans. The activated tyrosine kinases, which are produced by this translocation, are associated with fatal hematological malignancies. The initial molecular dissection of BCR-ABL has linked the expression of this constitutively activated kinase with enhanced genomic instability. We directly evaluated the consequence of BCR-ABL expression on genomic instability using the Big Blue in vivo mutagenesis mouse system. We report that the expression of BCR-ABL in both spleens and kidneys confers a mutator phenotype represented by a statistically significant elevation in mutant frequencies.

Animals↗
PubMed2004

JAK/STAT, Raf/MEK/ERK, PI3K/Akt and BCR-ABL in cell cycle progression and leukemogenesis.

The roles of the JAK/STAT, Raf/MEK/ERK and PI3K/Akt signal transduction pathways and the BCR-ABL oncoprotein in leukemogenesis and their importance in the regulation of cell cycle progression and apoptosis are discussed in this review. These pathways have evolved regulatory proteins, which serve to limit their proliferative and antiapoptotic effects. Small molecular weight cell membrane-permeable drugs that target these pathways have been developed for leukemia therapy. One such example is imatinib mesylate, which targets the BCR-ABL kinase as well as a few structurally related kinases. This drug has proven to be effective in the treatment of CML patients. However, leukemic cells have evolved mechanisms to become resistant to this drug. A means to combat drug resistance is to target other prominent signaling components involved in the pathway or to inhibit BCR-ABL by other mechanisms. Treatment of imatinib-resistant leukemia cells with drugs that target Ras (farnysyl transferase inhibitors) or with the protein destabilizer geldanamycin has proven to be a means to inhibit the growth of resistant cells. This review will tie together three important signal transduction pathways involved in the regulation of hematopoietic cell growth and indicate how their expression is dysregulated by the BCR-ABL oncoprotein.

Animals↗
PubMed2005

Direct transcriptional regulation of Bim by FoxO3a mediates STI571-induced apoptosis in Bcr-Abl-expressing cells.

In this study, we have used the human BV173 and the mouse BaF3/Bcr-Abl-expressing cell lines as model systems to investigate the molecular mechanisms whereby STI571 and FoxO3a regulate Bim expression and apoptosis. FoxO3a lies downstream of Bcr-Abl signalling and is constitutively phosphorylated in the Bcr-Abl-positive BV173 and BaF3/Bcr-Abl cells. Inhibition of Bcr-Abl kinase by STI571 results in FoxO3a activation, induction of Bim expression and apoptosis. Using reporter gene assays, we demonstrate that STI571 and FoxO3a activate Bim transcription through a FoxO-binding site (FHRE) located within the promoter. This was verified by DNA pull-down and chromatin immunoprecipitation analyses. We find that conditional activation of FoxO3a leads to induction of Bim expression and apoptosis. Conversely, silencing of FoxO3a in Bcr-Abl-expressing cells abolishes STI571-mediated Bim induction and apoptosis. Together, the results presented clearly confirm FoxO3a as a key regulator of apoptosis induced by STI571, and show that Bim is a direct transcriptional target of FoxO3a that mediates the STI571-induced apoptosis. Thus, STI571 induces an accumulation of FoxO3a activity which in turn binds directly to an FHRE in the promoter to activate Bim expression and apoptosis.

Animals↗
PubMed2000

Three-dimensional spectral precision distance microscopy of chromatin nanostructures after triple-colour DNA labelling: a study of the BCR region on chromosome 22 and the Philadelphia chromosome.

Topological analysis of the three-dimensional (3D) chromatin nanostructure and its function in intact cell nuclei implies the use of high resolution far field light microscopy, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that, in practice, under biologically relevant conditions, the spatial resolution of CLSM is limited to about 300 nm in the lateral direction and about 700 nm in the axial direction. To overcome this shortcoming, the use of a recently developed light microscopical approach, spectral precision distance microscopy (SPDM) is established. This approach is based on the precise localization of small labelling sites of a given target in spectrally differential images. By means of quantitative image analysis, the bary centres (intensity weighted centroid analogous to the centre of mass) of these independently registered labelling sites can be used as point markers for distance and angle measurements after appropriate calibration of optical aberrations (here, polychromatic shifts). In combination with specific labelling of very small chromatin target sites with dyes of different spectral signatures by fluorescence in situ hybridization (FISH), SPDM presently allows us to analyse the nuclear topology in three-dimensionally conserved nuclei with a 'resolution equivalent', many times smaller than the conventional optical resolution. Chronic myelogeneous leukaemia (CML) is genetically characterized by the fusion of parts of the BCR and ABL genes on chromosomes 22 and 9, respectively. In most cases, the fusion leads to a translocation t(9; 22) producing the Philadelphia chromosome. SPDM was applied to analyse the 3D chromatin structure of the BCR region on the intact chromosome 22 and the BCR-ABL fusion gene on the Philadelphia chromosome (Ph) by using a new triple-colour FISH protocol: two different DNA probes were used to detect the BCR region and the third DNA probe was used to identify the location of the ABL gene. Consistent 3D distance measurements down to values considerably smaller than 100 nm were performed. The angle distributions between the three labelled sites on the Philadelphia chromosome territory were compared to two state-of-the-art computer models of nuclear chromatin structure. Significant differences between measured and simulated angle distributions were obtained, indicating a complex and non-random angle distribution.

Bone Marrow Cells↗
PubMed2006

Loss of p53 impedes the antileukemic response to BCR-ABL inhibition.

Targeted cancer therapies exploit the continued dependence of cancer cells on oncogenic mutations. Such agents can have remarkable activity against some cancers, although antitumor responses are often heterogeneous, and resistance remains a clinical problem. To gain insight into factors that influence the action of a prototypical targeted drug, we studied the action of imatinib (STI-571, Gleevec) against murine cells and leukemias expressing BCR-ABL, an imatinib target and the initiating oncogene for human chronic myelogenous leukemia (CML). We show that the tumor suppressor p53 is selectively activated by imatinib in BCR-ABL-expressing cells as a result of BCR-ABL kinase inhibition. Inactivation of p53, which can accompany disease progression in human CML, impedes the response to imatinib in vitro and in vivo without preventing BCR-ABL kinase inhibition. Concordantly, p53 mutations are associated with progression to imatinib resistance in some human CMLs. Our results identify p53 as a determinant of the response to oncogene inhibition and suggest one way in which resistance to targeted therapy can emerge during the course of tumor evolution.

Animals↗
PubMed1993

Induction of protein-tyrosine-phosphatase activity by interleukin 6 in M1 myeloblastic cells and analysis of possible counteractions by the BCR-ABL oncogene.

Interleukin 6 (IL-6) induces in M1 myeloblastic cells growth arrest and terminal differentiation toward monocytes. It is reported here that IL-6 reduced by 5- to 20-fold the tyrosine phosphorylation of cellular proteins in these cells. The same-fold reduction was also observed in M1 cells that were transfected with the BCR-ABL deregulated protein kinase. In these stable clones, the levels of tyrosine phosphorylation of cellular proteins were 30- to 100-fold higher than in the parental cells. IL-6 did not reduce the expression levels or the inherent tyrosine kinase activity of BCR-ABL p210. By measuring the protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in crude cell lysates, we found that protein dephosphorylation resulted, at least partially, from induction of PTPase activity by IL-6. The induction of PTPase in the BCR-ABL-transfected clones was not sufficient to confer the minimal protein phosphorylation levels characteristic of IL-6-treated cells. Yet, the transfected M1 clones showed normal growth and differentiation responses to IL-6. None of the gene responses to IL-6 including suppression in the levels of c-myc, c-myb, and cyclin A mRNA; junB and c-jun mRNA induction; and dephosphorylation of retinoblastoma protein were rescued by the BCR-ABL oncogene. The functional relevance of PTPase induction by IL-6 is discussed.

Cyclins↗
PubMed2005

Solution structure of AF-6 PDZ domain and its interaction with the C-terminal peptides from Neurexin and Bcr.

AF-6 is a key molecule essential for structure organization of cell-cell junction of polarized epithelia. It belongs to a novel cell-cell adhesion system. The AF-6 PDZ domain mediates interactions by binding to a specific amino acid sequence in target proteins. Here we report the solution structure of the AF-6 PDZ domain determined by NMR. Previously, the AF-6 PDZ domain was considered to be a class II PDZ domain. However we found that a unique hydrophilic amino acid, Gln70, at position alphaB1 makes the alphaB/betaB groove of the AF-6 PDZ domain significantly different from that of the canonical class II PDZ domain. The AF-6 PDZ domain does not have the second hydrophobic binding pocket, and the N-terminal end of alphaB is closer to betaB. Using BIACORE and NMR chemical shift perturbation experiments, we have studied the binding characteristics of the PDZ domain to the C-terminal peptide of Neurexin, KKNKDKEYYV, and that of Bcr, KRQSILFSTEV. The C-terminal peptide of Neurexin is a class II ligand, whereas that of Bcr is a class I ligand. The dissociation constants of these ligands were 4.08 x 10(-7) and 2.23 x 10(-6) m, respectively. Each of the four C-terminal positions in Neurexin and Bcr may contribute to the interaction. The three-dimensional models of the AF-6 PDZ-Neurexin C-terminal peptide complex and the AF-6 PDZ-Bcr C-terminal peptide complex were built up by molecular dynamics simulations. Unlike the canonical class II PDZ domain, Ala74 at alphaB5 rather than the residue at alphaB1 makes direct hydrophobic contact with the side chain of Tyr at the -2 position of the ligand.

Amino Acid Sequence↗