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PubMed2000

Consequences of BCR-ABL expression within the hematopoietic stem cell in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) was the first human malignancy shown to be associated with a specific cytogenetic lesion, the Philadelphia chromosomal translocation. Forty years on, many biological and biochemical properties have been ascribed to its molecular product, the BCR-ABL tyrosine kinase fusion protein. However, it has been difficult to establish their precise contribution to the deregulation of normal survival, proliferative and differentiative control in chronic phase CML and the degree to which the involvement of stem cells extends beyond their role as the aetiological target. This review will focus on our current understanding of the pathogenesis of CML from the perspective of stem cell involvement, and how the biological and biochemical properties ascribed to BCR-ABL from studies of in vitro transformation and in vivo leukemogenesis systems relate to the abnormalities manifest in the human disease.

Animals↗

PubMed2002

Intracellular antibody capture technology: application to selection of intracellular antibodies recognising the BCR-ABL oncogenic protein.

The expression of antibodies inside cells to ablate protein function has the potential for disease therapy and for target validation in functional genomics. However, due to inefficient expression or folding, only a few antibodies or antibody fragments, usually as single-chain Fv antibody fragments (scFv), bind their antigens in an intracellular environment. We have established a genetic-selection technology (intracellular antibody capture, IAC) to facilitate the isolation of functional intracellular scFv from a diverse repertoire. This approach comprises an in vitro library screen with scFv-expressing bacteriophage, employing bacterially expressed antigen, followed by a yeast in vivo antibody-antigen interaction screen of the sub-library of in vitro scFv antigen-binders. Accordingly, we have isolated panels of scFv that bind intracellularly to the BCR or the ABL parts of the BCR-ABL oncogenic protein. Sequence analysis of the intracellular antibody scFv panels revealed a sequence conservation indicating an intracellular antibody consensus for both VH and VL, which could form the basis for the de novo synthesis of intracellular antibody libraries to be used with intracellular antibody-capture technology.

Amino Acid Sequence↗

PubMed2002

Detection of the BCR-ABL gene by interphase fluorescence in situ hybridization (iFISH) in chronic myelogenous leukemia patients after hemopoietic stem cell transplantation: the feasibility of iFISH monitoring of therapeutic response in peripheral blood.

The detection of the Philadelphia (Ph) translocation has been accomplished primarily by cytogenetic analysis and reverse transcriptase polymerase chain reaction (RT-PCR). RT-PCR is highly sensitive (1/10(4)-10(6)) but not quantitatively reliable and is thus unsuitable for the monitoring of Ph-positive cells during therapy. Interphase fluorescence in situ hybridization (iFISH) allows analysis of a large number of cells (> 500) in a timely and efficiently quantitative manner. We obtained 118 peripheral blood (PB) and 127 bone marrow (BM) samples from 75 adult chronic myelogenous leukemia (CML) patients undergoing stem cell transplantation. We simultaneously performed nested RT-PCR and iFISH for all samples. False-positive cells were detected in 2.48% +/- 0.93% (mean +/- SD) of PB samples and 2.75% +/- 0.83% of BM samples. The iFISH results for PB and BM ranged from 1.4% to 92.8% and 1.0% to 93.8%, respectively. Correlation analysis of iFISH results for PB versus BM samples showed a strong relation (r = .993). A significant correlation (P < .05) was also found between iFISH and first-round RT-PCR. The sensitivity of BCR-ABL iFISH was similar to that of first-round RT-PCR, and iFISH results for PB and BM were also well correlated. Thus, iFISH analysis of PB and/or BM samples may be more clinically reliable than RT-PCR in the quantitative monitoring of BCR-ABL fusion in CML after transplantation.

Blood Cells↗

PubMed1988

Multilineage cell involvement in Ph1-negative, bcr-negative chronic myeloid leukemia.

We report a case of Ph1-negative, bcr-negative CML-BC, in which the primary leukemic cells displayed T-related antigens (CD7, CD4) in addition to HLA-DR and CD25 determinants. No B-lymphoid, myeloid and megakaryoblastic surface antigens were detected. In spite of this phenotype, DNA analysis revealed a germ-line configuration of the T-cell receptor beta chain gene region. Moreover, in-vitro culture studies demonstrated a proliferative response of the blast cell population to natural and recombinant myeloid-related factors, while no proliferative signal was observed in the presence of IL-2. The myeloid lineage was further demonstrated by the expression of myeloid-associated antigens on cultured blast cells, which still retained the CD7 antigen. Finally, cytogenetic analysis revealed a monosomy 7 which is usually associated with a stem cell leukemia. These results support the hypothesis that Ph1-negative, bcr-negative CML is characterized by the involvement of a multipotent stem cell capable of multilineage expression and indicate that differentiative and proliferative assays provide a further tool towards a more precise recognition of hematological disorders of uncertain origin.

Aged↗

PubMed1992

Relationship of the type of bcr-abl hybrid mRNA to clinical course and transforming activity in Philadelphia-positive chronic myelogenous leukemia.

We studied the type of bcr-abl mRNA for 34 patients with chronic myelogenous leukemia and analyzed for correlations among the mRNA type, the clinical outcome and the transforming activity using the tumorigenicity assay. There was no difference in the distribution of the mRNA-types (b2-a2 and b3-a2) between clinical phases. We found no correlation between the two types of bcr-abl mRNA and the chronic phase duration or survival. The DNA from 12 of 20 chronic phase patients and all five blastic phase patients showed transforming activity. Although there was no difference in the positive rate of transforming activity among the two mRNA-type groups, the blastic phase patients showed a tendency to have higher transforming activity.

3T3 Cells↗

PubMed1995

Further evidence for the lack of correlation between the breakpoint site within M-BCR and CML prognosis and for the occasional involvement of p53 in transformation.

The actual significance of the type of BCR-ABL rearrangement in chronic myeloid leukemia (CML) prognosis remains controversial. Also, the molecular events that lead to CML progression are largely unknown. We analyzed the M-BCR breakpoint position in 64 CML patients by Southern blot and correlated the molecular findings with the cytogenetic, hematologic, and clinical data. No statistically significant differences were found with respect to the clinical and hematologic data presented at diagnosis or in the median duration of chronic phase (CP) and survival between the groups of patients with 5' and 3' breakpoints. We also studied by PCR-SSCP and direct sequencing the p53 gene in patients with specimens available in both chronic phase and blast crisis. We identified p53 mutations in 17% of the blast crisis samples analyzed, whereas no abnormalities were found in CP. This finding suggests that only in a minor fraction of cases are lesions in the p53 gene involved in transformation. Given the present findings, along with previous reports, we believe that a novel mechanism to explain the heterogeneity of CML should be postulated and actively pursued, as should the identification of secondary molecular events more consistently involved in progression.

Adolescent↗

PubMed1995

Possible significance of Ph, zinc and BCR-ABL chimaerism in the pathogenesis of chronic myeloid leukaemia.

Available evidence suggests a double-pathway two-staged genetic alteration in the pathogenesis of Chronic Myeloid Leukaemia (CML). The regular Ph' defect results in BCR-ABL gene chimaerism on the one hand and suppressed synthesis of the protein responsible for Zn absorption on the other. The resulting Zn deficiency leads, through its metalloenzymes, to a low NAP activity and depressed DNA & RNA polymerase activities: the latter necessitates an adaptive mechanism to sustain cell division despite low zinc. This adaptation is in the form of another gene alteration; a point mutation in the BCR-ABL chimaeric gene, now an oncogene, whose onco-proteins are zinc-independent and stimulate cell division more efficiently (though abnormally also) than the polymerases while defying the usual mechanisms regulating DNA synthesis and cell division. Thus it seems possible that assisted transcellular zinc transport could prevent development of CML in Ph'-positive individuals and the enhanced (abnormal) cellular proliferation might be specifically inhibited.

Fusion Proteins, bcr-abl↗

PubMed2005

Deletions of the 3'BCR and 5'ABL regions in patients with Philadelphia-positive chronic myeloid leukemia: a one-step process occurring in about 10% of the cases without any evidence of genetic instability in the target cells.

Deletions of the 5'ABL region adjacent to the t(9;22)(q34;q11) have recently been reported in 8-32.7% of patients with chronic myeloid leukemia (CML). The deletions were visualized with fluorescence in situ hybridization using, in the majority of the cases, the Vysis LSI BCR/ABL ES (extra signal) probe. In our series, 10 of 99 CML patients (10.1%) were characterized by a 5'ABL deletion. We show that 3'BCR losses are observed in nearly all the cases with 5'ABL deletions. Moreover, the different genetic events (Philadelphia chromosome formation; 5'ABL and 3'BCR deletions) occur simultaneously in a one-step process without any evidence for genetic instability in the target bone marrow cells.

Female↗

PubMed2006

Persistent Cdk2 inactivation drives growth arrest of BCR-ABL-expressing cells in response to dual inhibitor of SRC and ABL kinases SKI606.

Complementary inhibition of tyrosine and SRC kinases implement dual SRC/ABL inhibitor effects in chronic myeloid leukemia (CML). Here, we show that one such inhibitor, SKI-606, induces persistent Cdk2 inactivation leading to growth arrest of BCR-ABL-expressing cells either IM-sensitive or driven to IM-resistance by other events than gene overexpression and point mutations. Inhibition of Akt serine/threonine kinase, a phosphatidylinositol 3 kinase (PI-3k) target that integrates p210 TK signaling with membrane-associated SRC kinases, is a central component of restored expression and subcellular redistribution of Cdk2 regulatory signals (p21 and p27 and Cdc25A phosphatase) in response to SKI-606. The putative roles of growth factor (namely IL-3) autocrine loop in BCR-ABL-expressing progenitor progression towards a drug-resistant phenotype are discussed.

Aniline Compounds↗

PubMed2007

BCR-ABL mutant kinetics in CML patients treated with dasatinib.

Dasatinib is efficient in vitro against most of CML cells harboring ABL kinase domain mutations and induces high rates of response in imatinib-resistant CML patients. Here, we monitored the mutated BCR-ABL transcripts during the follow-up of 12 CML patients treated with dasatinib. We identified four groups of patients based on their sensitivity to dasatinib. Clinical responses were correlated to the in vitro sensitivity of BCR-ABL mutants to dasatinib, however, some discrepancies were observed in a subfraction of CML patients, suggesting subtle differences between in vitro observations and clinical entities and/or the onset of other mechanisms responsible for dasatinib resistance.

Adult↗

PubMed2004

Endoplasmic reticulum stress initiates apoptotic death induced by STI571 inhibition of p210 bcr-abl tyrosine kinase.

The endoplasmic reticulum (ER) is the site where proteins destined to either secretion or different subcellular compartments assemble and the major storage of intracellular Ca(2+). The ER stress resulting from a variety of toxic insults leads to apoptosis. Here, we showed that the apoptotic death triggered by STI571, an inhibitor of the p210 bcr-abl tyrosine kinase, in murine myeloid progenitors transducing the p210 bcr-abl tyrosine kinase of Chronic Myeloid Leukemia (CML) proceeds from ER stress. The Bcl-2 dowmodulation and inactivation induced by the binding to its antagonist: Bad, the release of caspase 12 from the ER membranes in its active form and of Ca(2+) from the ER pool addressed towards ER a sensor of STI571-induced pro-apoptotic signal.

Animals↗

PubMed2002

Identical abnormality of the short arm of chromosome 18 in two Philadelphia-positive chronic myelocytic leukemia patients with erythroblastic transformation, resulting in duplication of BCR-ABL1 fusion.

Two patients with Ph-positive chronic myelocytic leukemia in erythroblastic transformation and rearrangement of the short arm of chromosome 18 are reported. Fluorescence in situ hybridization studies showed that the 18p rearrangement resulted from translocation of the main part of chromosome 22 long arm to 18p, including BCR-ABL1 fusion. The 18p abnormality resulted, thus, in loss of 18p and duplication of BCR-ABL1 in both patients. The possible relation to the erythroblastic type of blastic phase is briefly discussed. In addition an apparently intact germline ABL1 gene was duplicated and inserted into chromosome 6 at band p21 in one of these patients.

Aged↗

PubMed1999

Insertion of the 5' part of BCR within the ABL gene at 9q34 in a Philadelphia-negative chronic myeloid leukemia.

We report a chronic myeloid leukemia patient without evidence of a Philadelphia (Ph) chromosome in whom RT-PCR analysis performed in blast crisis demonstrated the existence of both common b3a2 and b2a2 BCR/ABL fusion transcripts. In situ hybridization studies with BCR- and ABL-specific probes showed location of the BCR/ABL fusion gene on chromosome 9, band q34, instead of at chromosome 22q11, and that it resulted from an insertion of the 5' side of BCR within the ABL gene on chromosome 9. The vast majority of cells showed a BCR/ABL fusion gene on both chromosomes 9, which is equivalent to a double Ph chromosome, thus reinforcing the notion that the critical event in CML is the formation of a functional BCR/ABL fusion gene.

Chromosomes, Human, Pair 9↗

PubMed2000

Transposition of duplicated chromosomal segment involving fused BCR-ABL gene or ABL oncogene alone in chronic myelocytic leukemia and Ph chromosome-positive acute leukemia with complex karyotypes.

Thirty-six patients with chronic myelocytic leukemia (CML) in the blastic phase were examined by fluorescence in situ hybridization to clarify the mechanisms of progression of the disease. Two of 19 CML patients in the blastic phase (10.5%) had an extra fused BCR-ABL gene on structurally complex chromosome aberrations in addition to the Ph chromosome. Another patient had an extra ABL oncogene on the end of a deleted chromosome, resulting in three copies of the ABL oncogene. These three patients showed additional chromosome aberrations, such as der(12), der(15), and der(18), which differ from the standard karyotypic evolution in the blastic phase. Amplification of the fused BCR-ABL gene or the ABL oncogene seemed to be induced by transposition. These segmental transpositions suggest that these regions have high genetic instability possibly leading to blastic transformation.

Chromosome Aberrations↗

PubMed2000

Bcr-Abl and inhibition of apoptosis in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) cells are highly resistant to apoptosis induced by chemotherapeutic drugs. The observation that production of Bcr-Abl is the initiating event in CML has focussed attention on the survival signals triggered by this oncogene. A number of signal transducers and transcription factors have been associated with the antiapoptotic phenotype of CML cells, some of which lead to the expression and/or activation of members of the Bcl-2 family of apoptosis modulators, such as Bcl-xL and Bad. In this article, recent advances in understanding the antiapoptotic pathways triggered by Bcr-Abl in CML cells, are discussed.

Apoptosis↗

PubMedDate not supplied

Kinetics of BCR-ABL fusion transcript levels in chronic myeloid leukemia patients treated with STI571 measured by quantitative real-time polymerase chain reaction.

The activated tyrosine kinase, which arises as a result of the balanced t(9,22) translocation in chronic myeloid leukemia (CML), is thought to be essential for the development of the leukemic phenotype. Recently, designer drugs have been introduced which specifically inhibit such specific kinases. Among these, STI571 (Glivec) has entered clinical trials and shown promising activities in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) as evidenced by significant hematological and cytogenetic responses in CML patients. To evaluate the effect of STI571 at the molecular level we have employed quantitative real-time PCR (RQ-PCR) to measure the amount of BCR-ABL fusion transcript in a series of 19 patients treated with STI571, either in CP(11) or in (AP)(8) of the disease for 3--9 months (median 6 months). Employing this method, which is able to detect at least one BCR-ABL+ cell in 500,000, in serial blood and bone marrow specimens we found decreases in transcript levels in 10/11 CP patients, but only in 1/8 of the AP patients. When present such decreases were gradual and became evident only after 3 months of STI571 treatment, and their kinetics in blood closely mirrored those seen in parallel marrow samples. Moreover, decreases were between 10- and 100-fold in 11/13 patients, with only two patients reaching residual disease levels below 10(-2) (a 900-fold decrease). Thus, no patient reached PCR negativity. We conclude that the RQ-PCR method is a highly suitable tool for following the effect of STI571 in CML and that further validation of the method, performed in a prospective manner, will contribute significantly to the elucidation of the proper role of STI571 in CML.

Adult↗

PubMed1997

Analysis of the BCR-ABL protein in Philadelphia chromosome-positive adult acute lymphocytic leukemia.

Philadelphia chromosome (Ph)-positive acute lymphocytic leukemia (ALL) constitutes 15-35% of all ALL in adults. Its detection is prognostically significant. The Ph abnormality is usually detected through standard cytogenetic analysis but 20-30% of patients have insufficient metaphases (IM) with such analysis. To detect the BCR-ABL oncoprotein in peripheral blood specimen of patients with ALL at the time of diagnosis and at follow-up, a new sensitive technique of enhanced chemiluminescence Western blot (ECL-WB) analysis was investigated. Among 41 patients with newly diagnosed ALL, nine were Ph positive by cytogenetic studies; they were also BCR-ABL positive according to ECL-WB. Eight had p190 disease, and one had p210 disease. Among the 16 patients with IM, none demonstrated the oncoprotein through ECL-WB or through simultaneous Southern blot (SB) for p210 rearrangement. Follow-up studies were available for seven patients: four had detectable protein and three of them relapsed 4-20 weeks later; three had undetectable protein and one of them (who had low level protein at the time of diagnosis) relapsed 11 weeks later. Although none of the patients with IM at diagnosis had detectable protein according to ECL-WB, this was probably due to the small number of patients studied. One patient with IM studied at follow-up demonstrated the protein by ECL-WB. In summary, we describe a technique that is useful in the detection of p190/p210 ALL at diagnosis. It is less time consuming, and more cost effective than standard chromosome banding techniques. It may also detect the oncoprotein in cases with IM. Although a larger number of patients should be studied to prove its clinical usefulness, this technique may also be of value for monitoring residual disease at follow-up.

Blotting, Western↗

PubMed2003

In BCR-ABL-positive cells, STAT-5 tyrosine-phosphorylation integrates signals induced by imatinib mesylate and Ara-C.

In BCR-ABL-positive cells, the transcription factor STAT-5 is constitutively activated by tyrosine phosphorylation. STAT-5 activation results in upregulation of bcl-X(L) and increased resistance to induction of apoptosis. Here, we investigated the effects of imatinib mesylate and cytosine arabinoside (Ara-C) on STAT-5 tyrosine-phosphorylation, cellular proliferation and induction of apoptosis in cell lines and primary hematopoietic cells. Imatinib mesylate treatment strongly suppressed STAT-5 tyrosine-phosphorylation in K562 and primary CML blasts. In contrast to JAK-2 and PI-3-kinase inhibition, exposure of K562 cells to imatinib mesylate resulted in obvious suppression of proliferation. Reduced cell growth was due to specific induction of caspase activation followed by apoptotic cell death. In addition, we investigated the effects of Ara-C on STAT-5 tyrosine-phosphorylation. Exposure to Ara-C resulted in significant downregulation of STAT-5 tyrosine-phosphorylation and inhibition of DNA binding. Treatment of K562 cells with Ara-C in combination with imatinib mesylate revealed synergistic effects at the level of STAT-5 tyrosine-phosphorylation and DNA binding, Hck tyrosine-phosphorylation, cell growth and induction of apoptosis. Overall, in this report we demonstrate that STAT-5 tyrosine-phosphorylation is a specific target of imatinib mesylate and Ara-C. Our results suggest that, in combination therapy, inhibition of STAT-5 tyrosine-phosphorylation may be responsible for synergistic or additive effects on BCR-ABL-positive cells.

Antineoplastic Agents↗