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PubMed1994

No evidence for genomic imprinting of the human BCR gene.

Chronic myeloid leukemias and 5% to 20% of acute lymphoid leukemias are characterized by the Philadelphia chromosome, a reciprocal chromosomal translocation, t(9;22)(q34;q11), generating BCR-ABL and ABL-BCR fusion genes. Cytogenetic studies have recently shown a preferential involvement of the paternally derived chromosome 9 and the maternally derived chromosome 22 in this translocation, indicating that imprinting might be involved in the formation or selection of the translocation. In this study, we have identified a BamHI polymorphism in the coding region of BCR exon 1, allowing us to investigate whether both BCR alleles are transcribed. By using a reverse transcriptase-polymerase chain reaction assay, we show that both BCR alleles are expressed in the peripheral blood cells of normal individuals.

Alleles↗
PubMed1993

Detection and direct sequence identification of BCR-ABL mRNA in Ph+ chronic myeloid leukemia.

The reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR-ABL mRNA is increasingly used to diagnose and monitor patients with Ph+ chronic myeloid leukemia (CML). We investigated an alternative approach to detect BCR-ABL mRNA in CML in order to overcome some of the potential drawbacks of RT-PCR. Nucleic acid sequence based amplification (NASBA) is a homogeneous, isothermal, in vitro process that provides the direct amplification of RNA. Peripheral blood from seven patients with Ph+ CML and Ph+ EM-2 cells were investigated by NASBA and RT-PCR. A nested set of four primers flanking the BCR-ABL junction was used in two serial NASBA reactions performed for 2 hours. The two methods were fully concordant for detection of transcripts with bcr3-abl2 and bcr2-abl2 junctions. Ethidium bromide fluorescence with NASBA indicated in repeated experiments that similar quantities of total RNA from patient material contained different amounts of BCR-ABL mRNA. The data suggest that direct amplification of RNA is suitable for identifying and monitoring patients with Ph+ CML and may provide a means to quantify BCR-ABL mRNA levels.

Base Sequence↗
PubMed1995

Inhibition of chronic myelogenous leukemia cells harboring a BCR-ABL B3A2 junction by antisense oligonucleotides targeted at the B2A2 junction.

Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder characterized by the BCR-ABL hybrid gene. Two types of hybrid BCR-ABL mRNA have been found, B2A2 and B3A2. As the BCR-ABL rearrangement is specific to leukemic cells, selective inhibition of leukemic cell growth by BCR-ABL antisense oligonucleotides (ASO) has been reported in vitro for CML patients and cell lines. However, controversial results have been obtained from preclinical studies using anti-BCR-ABL ASO, as nonspecific inhibition of leukemic cell growth was evidenced in some cases. B3 exon secondary structure was deduced from its sequence and found to be a loop. According to this predictive structure of exon B3, a 56-mer antisense oligonucleotide targeting the polypurine bases from the B2A2 junction was devised which would inhibit proliferation (MTT assay) of B3A2 junction cell lines (K562 and a murine cell line Ba/F3 transfected with the B3A2 junctional sequence). This ASO had a hairpin-like secondary structure and was found to be much more resistant to the action of nucleases than control 18-mer standard oligonucleotides. Hybridization to its target mRNA occurs via formation of a triplex structure. A concentration of 5 microM of specific 56-mer B2A2 ASO was necessary to demonstrate 50% optical density (OD) reduction for K562 cell line and Ba/F3 transformed by B3A2 cDNA. Sense and non-sense 56-mer sequence or 18-mer linear ASO showed no effect for these concentrations. Western blot showed a partial inhibition of P210 protein; expression of P145abl remains unchanged. The 56-mer ASO also inhibited the proliferation of B2A2 junction cell line BV173 at the same concentration and showed no effect on the HL60 cell line used as control.

Base Sequence↗
PubMed1995

Detection of bcr-abl mRNA in single progenitor colonies from patients with chronic myeloid leukemia by PCR: comparison with cytogenetics and PCR from uncultured cells.

Bone marrow and/or peripheral blood of patients with chronic myeloid leukemia (CML) was investigated by the following three parameters: Ph' chromosome, bcr-abl expression in fresh blood and/or bone marrow, and bcr-abl expression in single hematopoietic progenitor colonies generated from blood and/or bone marrow. Expression of bcr-abl was proven by a reverse "nested primer" polymerase chain reaction (PCR) that is able to detect 1 pg of hybrid mRNA. We performed 108 investigations on 68 patients containing all three parameters: 12 on untreated patients, seven after interferon-alpha (IFN-alpha), seven after low-dose cytosine arabinoside (Ara-C), 22 after cyclic high-dose hydroxyurea (HU), 49 after allogeneic BMT, five before and three after stem cell mobilization, and three after autologous stem cell transplantation (ASCT). In 53 cases (49%), cytogenetics and PCR gave identical results. In 40 cases (37%), PCR from single colonies gave additional information compared to cytogenetics (e.g., mosaic in colonies when all metaphases were positive or negative). Most interesting were the results of one patient after IFN, one patient after ASCT, and 10 patients after BMT (14 investigations = 13%), showing only Ph'-negative mitoses accompanied by a negative nested primer PCR from fresh blood/bone marrow but single bcr-abl-positive progenitor colonies. False-positive results could be widely excluded by repeated insertion of negative controls into the experiments. One explanation for these results could be that CML, progenitors survive in the patient's body by being inactive and not proliferating. These cells express no or very little RNA and bcr-abl is not detectable by reverse PCR. When stimulated ex vivo in a colony assay by external growth factors, cells proliferate and produce detectable amounts of hybrid mRNA. The value of these observations is not clear. A follow-up of the patients will show if such sleeping progenitors can be activated in vivo. Concluding our observations, we can say that in special cases (therapy follow-up, detection of minimal residual disease) it could be useful to perform a PCR analysis of single progenitors in parallel with the routine investigations.

Base Sequence↗
PubMed1996

Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia.

Using chronic myelogenous leukemia (CML) as a model, we tested the hypothesis that cytokine-independent growth of leukemia cells results from aberrant activation of cytokine signaling pathways. The STAT5 (signal transducer and activator of transcription) protein, which is activated transiently in normal myeloid cells by cytokines such as GM-CSF (granulocyte-macrophage colony stimulating factor), was constitutively activated in cell lines derived from CML patients, even in the absence of GM-CSF. STAT5 was also activated in primary mouse bone marrow cells acutely transformed by the CML-specific BCR-ABL oncogene, but not by the serine kinase oncogene v-MOS. Reconstitution experiments in non-hematopoietic cells show that STAT5 activation by BCR-ABL occurs independent of cytokines. Results using BCR-ABL mutants which specifically uncouple connections to known signal transduction pathways show that STAT5 activation is kinase dependent and correlates directly with ability to confer cytokine independent growth in hematopoietic cells. BCR-ABL also activates JAK kinases, which may provide a mechanism for STAT activation. These findings are consistent with a role for STAT5 in hematopoietic transformation by BCR-ABL.

Animals↗
PubMed1996

Different methylation patterns of the M-BCR gene in myeloblastic and lymphoblastic crisis of chronic myelocytic leukemia.

OBJECTIVE: To investigate the relationship between methylation status of M-bcr gene and transformation of chronic myelocytic leukemia (CML) from chronic to blastic phase. MATERIAL AND METHODS: The methylation patterns of M-bcr in 23 patients with Ph' positive CML were studied. DNAs extracted from mononuclear cells of both chronic and blastic phases (20 cases) or blastic phases only (3 cases), were doubly digested with restriction enzymes HpaII and BglII, hybridized with a 5'M-bcr probe labeled with 32p-deoxycytidine triphosphate, and autoradiographed. RESULTS: In all the patients with myeloblastic crisis, DNAs from both chronic and blastic cells of each patient showed identical methylation patterns. There was substantial heterogeneity in methylation patterns in the patients with lymphoblastic crisis. All the lymphoblastic patterns were distinct from the chronic patterns as well as the patterns shown in myeloblastic crisis. Moreover, in four out of six patients with lymphoblastic crisis, the chronic patterns were different from those in cases with myeloblastic crisis. CONCLUSIONS: The methylation status of M-bcr was stable during evolution of CML from chronic to myeloblastic phase. Analysis of M-bcr methylation status may be of clinical use in distinguishing lymphoblastic from myeloblastic crisis and predicting the cell lineage of crisis when the disease is still in its chronic phase.

Blast Crisis↗
PubMed1997

HLA-DR1-restricted bcr-abl (b3a2)-specific CD4+ T lymphocytes respond to dendritic cells pulsed with b3a2 peptide and antigen-presenting cells exposed to b3a2 containing cell lysates.

Chronic myeloid leukemia (CML) is characterized by a specific translocation of the c-abl oncogene on chromosome 9 to the break point cluster region (bcr) on chromosome 22, t(9;22) (q34;q11). This translocation results in the expression of a 210-kD bcr-abl protein fusion gene product. The juxtaposition of the bcr and abl genes produces a novel junctional amino acid sequence, which may be presented by antigen-presenting cells and recognized specifically by human T lymphocytes. We have generated a CD4+ T lymphocyte line (NG-1) which recognizes the peptide epitope (GFKQSSKALQR) in association with HLA-DRbeta1*0101-02. A comparison of antigen-presenting cells showed that CMRF-44+ blood dendritic cell presented a 12mer b3a2 peptide effectively. The b3a2 peptide was able to generate specific primary T-lymphocyte responses in other HLA-DR1 donors. We also show that bcr-abl, b3a2 peptide-specific T-lymphocyte lines proliferate in response to bcr-abl b3a2 containing cell lysates (K562 or CML PBMC derived) but not control (including b2a2 CML PBMC) lysates.

Amino Acid Sequence↗
PubMed1989

Distribution of breakpoint within the breakpoint cluster region (bcr) in chronic myelogenous leukemia with a complex Philadelphia chromosome translocation.

We analyzed, by Southern blot hybridization, the site of breakpoint within the breakpoint cluster region (bcr) in six patients with a complex Philadelphia chromosome (Ph) translocation and in 23 unselected patients with a standard Ph. The breakpoint was found within the 5.8 kb bcr in all 29 patients. When the bcr was subdivided into four parts, fragments I-IV, based on the restriction enzyme sites, among the six patients with a complex Ph, two had a breakpoint at fragment I, three at fragment II, and one at fragment III. This distribution of breakpoints in patients with a complex Ph did not differ significantly from that in patients with a standard Ph. A deletion of an allele within the bcr was found in three patients (50%) with a complex Ph and in three (13%) with a standard Ph. The internal bcr deletion may be more common in patients with a complex Ph.

Alleles↗
PubMed2005

AMN107, a novel aminopyrimidine inhibitor of p190 Bcr-Abl activation and of in vitro proliferation of Philadelphia-positive acute lymphoblastic leukemia cells.

BACKGROUND: Previous studies have shown that patients with Bcr-Abl-positive acute lymphoblastic leukemia (ALL) either have primary disease that is refractory to imatinib mesylate or develop disease recurrence after an initial response. METHODS: The authors investigated the effects of a newly designed Bcr-Abl inhibitor, AMN107, by comparing its in vitro inhibitory potency on p190 Bcr-Abl ALL cell lines with that of imatinib. RESULTS: In two Philadelphia (Ph)-positive ALL cell lines, AMN107 was found to be 30-40 times more potent than imatinib in inhibiting cellular proliferation. AMN107 was also more effective than imatinib in inhibiting phosphorylation of p190 Bcr-Abl tyrosine kinase in cell lines and primary ALL cells. The inhibition of cellular proliferation was associated with the induction of apoptosis in only one of the cell lines. No activity was observed in cell lines lacking the BCR-ABL genotype. CONCLUSIONS: The results of the current study suggest the superior potency of AMN107 compared with imatinib in Ph-positive ALL and support clinical trials of AMN107 in patients with Ph-positive ALL.

Antineoplastic Agents↗
PubMed1987

Absence of the hybrid bcr-abl mRNA in Ph1-positive B lymphoblastoid cell lines established from a patient with chronic myelogenous leukemia.

The expression of c-abl, c-sis, c-myc and N-ras oncogenes was examined in 2 lymphoblastoid cell lines, one with Ph1 (PB-1049) and the other without Ph1 (LN-1049), both established from a patient with chronic myelogenous leukemia (CML), and in a Ph1-positive cell line (PB-1049-T) derived from a tumor formed after transplantation of PB-1049 cells in a nude mouse with reference to their tumorigenic potential in nude mice. The normal transcripts of c-abl were detected in all 3 lymphoblastoid cell lines. Although in situ hybridization of v-abl proved, and restriction endonuclease analyses of the bcr region strongly indicated the occurrence of bcr-abl rearrangement in PB-1049 and PB-1049-T, we could not obtain any evidence for the expression of the hybrid bcr-abl mRNA. These results indicate that the Ph1 translocation does not ensure the production of the hybrid bcr-abl mRNA, and that the expression of hybrid bcr-abl gene is not essential for the maintenance of tumorigenicity of these cell lines. Expression of c-sis was not detected in any of the cell lines examined, whereas the expression of c-myc was uniformly higher in the 3 cell lines than in normal control cells. The levels of N-ras expression varied considerably, probably in parallel with the changes in tumorigenicity of the cell lines. N-ras expression in the PB-1049 and PB-1049-T cell lines was higher than that in the LN-1049 line when they retained tumorigenic potential, but it fell to the level of LN-1049 with loss or decline of tumorigenicity.

Fusion Proteins, bcr-abl↗
PubMed1987

cDNA sequence for human bcr, the gene that translocates to the abl oncogene in chronic myeloid leukaemia.

The hallmark of human chronic myeloid leukaemia is a 9;22 chromosome translocation that fuses most of the c-abl oncogene to the 5' portion of the breakpoint cluster region (bcr) gene, such that a hybrid bcr-abl mRNA and polypeptide are generated. To clarify further the nature of this translocation, we have analysed the structure of normal human bcr mRNA by isolating large cDNA clones that collectively span the entire coding region and extend 2.6 kb upstream of those previously described. The 3150-bp nucleotide sequence reported here includes 534 bp of a GC-rich 5' non-coding segment and indicates, in conjunction with published sequences, that the bcr polypeptide comprises 1271 amino acid residues. The predicted polypeptide is unrelated to serine or tyrosine kinases, or indeed to any previously published sequence; its structure provides no evidence of a transmembrane region. Since probes from throughout the 4.8-kb cloned region hybridized to both the 4.5 and 6.7 kb normal bcr transcripts, both RNAs contain most or all of that region.

Amino Acid Sequence↗
PubMed1996

Deletion of the ABL SH3 domain reactivates de-oligomerized BCR-ABL for growth factor independence.

Biological activities of BCR-ABL, an activated tyrosine kinase oncogene responsible for pathogenesis of human leukemias, can be completely inactivated by a deletion of the BCR aminoterminal sequence with tetramerizing property (BCR-ABL delta 1-40). We attempted several ways to restore the ability to induce growth factor independence to the de-oligomerized BCR-ABL delta 1-40 and found that an additional deletion of the ABL SH3 domain could. In BCR-ABL delta 1-40 reactivated by the SH3 deletion, transphosphoryation of other cellular proteins like p62 or SHC in vivo and autophosphorylation with recruitment of GRB-2 were also recovered.

Adaptor Proteins, Signal Transducing↗
PubMed1988

The karyotype of Philadelphia chromosome-negative, bcr rearrangement-positive chronic myeloid leukemia.

Philadelphia (Ph) chromosome negative chronic myeloid leukemia (CML) can be distinguished from clinically similar disorders on the basis of the presence of rearrangement of the breakpoint cluster region (bcr) of chromosome 22. We have identified six patients with Ph-negative CML, each with bcr rearrangement. Apparently normal karyotypes were observed in two cases, and a third contained a rearrangement that did not appear to involve chromosomes 9 or 22. The other three cases had translocations involving chromosome band 9q34 but no case contained the common derivative chromosome 9pter----9q34::22q11----22qter. One case appeared to contain either a deletion of an unrearranged bcr locus in approximately 50% of cells or duplication of rearranged bcr, both 5' and 3' of the chromosome 22 breakpoint. Considerable complexity exists in the types of genetic changes that can juxtapose bcr and the c-abl oncogene in CML. Based on the molecular and cytogenetic analyses of these and other cases described in the literature, we conclude that most cases of true Ph-negative CML arise from submicroscopic genetic exchanges rather than masking of simple t(9;22)(q34;q11) translocations by secondary rearrangements.

Adult↗
PubMed2006

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells.

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

Aminolevulinic Acid↗
PubMed2005

Treatment of elderly patients with acute lymphoblastic leukemia--evidence for a benefit of imatinib in BCR-ABL positive patients.

All patients with acute lymphoblastic leukemia (ALL) over age 60 years seen at Princess Margaret Hospital over an 11-year period were analysed retrospectively. Of 53 patients, 45 received multiagent induction chemotherapy using a variety of regimens. There were 13 BCR-ABL positive patients, 9 of who received imatinib mesylate, either during induction or post-remission therapy. The overall complete remission (CR) rate of all 45-induction patients was 56%, with a 27% induction-related mortality rate. The CR rate was not influenced by induction regimen, age, initial WBC, LDH or BCR-ABL status. The median overall survival of the induction patients was 9 months, while the median progression-free survival (PFS) of the patients achieving CR was 10 months. The estimated overall survival (OS) at 3 years was 18.4% (95% CI: 9.8-34.3%). Age and initial WBC did not significantly predict for OS when evaluating the entire group of induction patients. However, there was a strong trend for BCR-ABL status to favorably predict for PFS, and for OS when only patients treated after July 2000 (when imatinib became available) were evaluated. The results indicate that ALL remains a poor prognosis disease in elderly patients, and that aggressive induction regimens designed for younger patients are very toxic for these patients. These data suggest that BCR-ABL+ ALL is becoming a relatively more favorable prognosis disease in the elderly, likely due to the influence of imatinib therapy. Further regimens should explore the use of less aggressive regimens in elderly patients and should evaluate the optimal way of combining imatinib with conventional agents in BCR-ABL+ patients.

Aged↗
PubMedDate not supplied

Effects of transfection of p210bcr-abl and bcr-v-abl into the factor-dependent human leukemia cell line HSM-911.

In order to clarify the action of the bcr-abl, a growth factor dependent human leukemic cell line (HSM-911) was transfected with p210bcr-abl or bcr-v-abl by electroporation. The cells transfected with bcr-v-abl, but not the cells transfected with p210bcr-abl, became growth factor independent. Some clones of the cells transfected with p210bcr-abl demonstrated cellular maturation (nuclear segmentation, becoming positive for naphthol ASD chloroacetate esterase, the disappearance of CD34 expression and the appearance of glycophorin A and CD10 expression). Moreover, these clones transfected with p210bcr-abl demonstrated apoptosis (increased expression of Fas and DNA ladder formation suggesting apoptotic DNA fragmentation). These findings demonstrated the different actions of p210 bcr-abl and bcr-v-abl, the former of which gave the cells the characteristics of maturation like the cells from chronic myelogenous leukemia, and the latter of which rendered the cells grow autonomously.

Apoptosis↗
PubMed2002

Comprehensive comparison of FISH, RT-PCR, and RQ-PCR for monitoring the BCR-ABL gene after hematopoietic stem cell transplantation in CML.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with fluorescence in situ hybridization (FISH) and real-time quantitative RT-PCR (RQ-PCR) for minimal residual disease (MRD) monitoring in 266 post-transplant bone marrow samples from 78 patients with chronic myelogenous leukemia (CML). The sensitivities of FISH to BCR-ABL positive samples determined by first-round (1st) RT-PCR, second-round (2nd) RT-PCR, and RQ-PCR were 64.2%, 25.8%, and 20.7%, respectively. The BCR-ABL/ABL ratio by RQ-PCR had a mean of 0.000 13 in the 1st RT-PCR-negative samples and 1.42 in the 1st RT-PCR-positive samples (P<0.001), and means of 0.000 39 and 0.51 in the 2nd RT-PCR-negative and -positive samples (P< 0.001). The mean ratios of BCR-ABL/ABL by RQ-PCR were significantly different in N/N (1st/2nd RT-PCR) or N/P and P/P (P<0.001), but not in N/N and N/P, which showed that the discriminative power of RQ-PCR is confined to the 1st RT-PCR level. In this respect, monitoring of the 1st RT-PCR might be useful for estimating normalized BCR-ABL levels after transplantation. Nested RT-PCR was of limited use, as RQ-PCR quantified the BCR-ABL transcripts in 60 (91%) of 66 samples determined to be negative by 2nd RT-PCR. FISH was significantly correlated with RQ-PCR in FISH-positive samples (n=24, r=0.79, P=0.001). An increase of FISH preceded that of RQ-PCR in a few cases with molecular relapse. By analyzing a large number of samples post-transplant, we found that RQ-PCR might be the most useful assay for MRD monitoring; however, FISH and RT-PCR were found to be useful complementary tools.

Bone Marrow↗
PubMed2006

Requirement for CD44 in homing and engraftment of BCR-ABL-expressing leukemic stem cells.

In individuals with chronic myeloid leukemia (CML) treated by autologous hematopoietic stem cell (HSC) transplantation, malignant progenitors in the graft contribute to leukemic relapse, but the mechanisms of homing and engraftment of leukemic CML stem cells are unknown. Here we show that CD44 expression is increased on mouse stem-progenitor cells expressing BCR-ABL and that CD44 contributes functional E-selectin ligands. In a mouse retroviral transplantation model of CML, BCR-ABL1-transduced progenitors from CD44-mutant donors are defective in homing to recipient marrow, resulting in decreased engraftment and impaired induction of CML-like myeloproliferative disease. By contrast, CD44-deficient stem cells transduced with empty retrovirus engraft as efficiently as do wild-type HSCs. CD44 is dispensable for induction of acute B-lymphoblastic leukemia by BCR-ABL, indicating that CD44 is specifically required on leukemic cells that initiate CML. The requirement for donor CD44 is bypassed by direct intrafemoral injection of BCR-ABL1-transduced CD44-deficient stem cells or by coexpression of human CD44. Antibody to CD44 attenuates induction of CML-like leukemia in recipients. These results show that BCR-ABL-expressing leukemic stem cells depend to a greater extent on CD44 for homing and engraftment than do normal HSCs, and argue that CD44 blockade may be beneficial in autologous transplantation in CML.

Animals↗