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PubMed2003

BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome.

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BcR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C(16)- ceramide predominated, was observed 6 h after BcR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BcR triggering. The formation of these very long-chain ceramide species, in which C(24)-ceramide predominated, required the activation of effector caspases. BcR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BcR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BcR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta-lactone, an irreversible inhibitor of the proteasome, prevented BcR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BcR cross-linking-induced activation of caspases. The formation of C(16)-ceramide in response to BcR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.

Apoptosis↗

PubMed1996

Negative selection of human germinal center B cells by prolonged BCR cross-linking.

The antigen receptors on T and B lymphocytes can transduce both agonist and antagonist signals leading either to activation/survival or anergy/death. The outcome of B lymphocyte antigen receptor (BCR) triggering depends upon multiple parameters which include (a) antigen concentration and valency, (b) duration of BCR occupancy, (c) receptor affinity, and (d) B cell differentiation stages. Herein, using anti-immunoglobulin kappa and lambda light chain antibodies, we analyzed the response of human naive, germinal center (GC) or memory B cells to BCR cross-linking regardless of heavy chain Ig isotype or intrinsic BCR specificity. We show that after CD40-activation, anti-BCR (kappa + gamma) can elicit an intracellular calcium flux on both GC and non-GC cells. However, prolonged BCR cross-linking induces death of CD40-activated GC B cells but enhances proliferation of naive or memory cells. Anti-kappa antibody only kills kappa + GC B cells without affecting surrounding gamma + GC B cells, thus demonstrating that BCR-mediated killing of GC B lymphocytes is a direct effect that does not involve a paracrine mechanism. BCR-mediated killing of CD40-activated GC B cells could be partially antagonized by the addition of IL-4. Moreover, in the presence of IL-4, prestimulation through CD40 could prevent subsequent anti-Ig-mediated cell death, suggesting a specific role of this combination in selection of GC B cells. This report provides evidence that in human, susceptibility to BCR killing is regulated along peripheral B cell differentiation pathway.

Antibodies, Anti-Idiotypic↗

PubMed2002

Depletion of Lyn kinase from the BCR complex and inhibition of B cell activation by excess CD21 ligation.

The human and murine CD21 gene products have been functionally linked to B cell activation by the co-ligation of the BCR and the CD21/CD19/CD81 complexes. Binding of low levels of antigen complexed to the complement ligand(s) for CD21 enhances B cell activation compared to the stimulation caused by antigen alone. Mice lacking functional CD21 predispose to autoimmune responses suggesting that this receptor may also play a negative role: thus in the presence of excess complement-bearing immune complexes, B cell antigen-specific activation may be inhibited. This possibility was investigated using intracellular calcium elicitation analyses to follow BCR-mediated activation. Ligation of the BCR and limiting quantities of the CD21 receptor demonstrated the expected enhanced cellular response compared to BCR ligation alone: CD21 ligation alone demonstrated no alteration in calcium flux. However, co-ligation of the BCR with excess CD21 binding resulted in the elimination of the calcium response, suggesting that CD21 ligation was down-modulating the BCR response. Immunoprecipitation of kinases associated with the BCR and CD21/CD19/CD81 complexes demonstrated that Lyn is preferentially depleted from the BCR complex following excess binding of CD21. Localization of other kinases integral for B cell activation is not altered. These data suggest that excess CD21 ligand binding can negatively impact B cell activation by sequestering Lyn kinase away from the BCR complex.

Animals↗

PubMed2002

In vitro resistance of leukaemic blasts to prednisolone in bcr-abl positive childhood acute lymphoblastic leukaemia.

BACKGROUND & OBJECTIVES: Although the outcome of children with acute lymphoblastic leukaemia (ALL) has improved dramatically over the last decade, some children still fare poorly and relapses are seen. The sensitivity of leukaemic cells to corticosteroids has emerged as an important prognostic factor in ALL. The t(9,22) translocation, resulting in the bcr-abl fusion gene, is a non-random translocation found in B-lineage acute lymphoblastic leukaemia. It is also known to be an independent poor prognostic factor for long-term disease free survival. We studied the association between the presence of bcr-abl fusion gene and in vitro prednisolone resistance in children with B-lineage acute lymphoblastic leukaemia at diagnosis. METHODS: A total of 23 children (aged 1-16 yr, median age: 12 yr) with B-lineage acute lymphoblastic leukaemia at diagnosis were included in the study. The presence of bcr-abl fusion gene was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and the in-vitro resistance to prednisolone was measured by short term colorimetric methyl thiazol tetrazolium (MTT) assay. RESULTS: A median LD50 (lethal dose for 50% cells) for prednisolone in bcr-abl positive children (n=7) was 1.6 mg/ml (range: 0.25-5.0 mg/ml) and that of bcr-abl negative children (n=16) was 0.35 mg/ml (range 0.62-1.0 mg/ml). The median LD50 for prednisolone differed significantly between the bcr-abl positive and negative groups of children with acute lymphoblastic leukaemia (P<0.005). INTERPRETATION & CONCLUSION: This is probably the first report to show that leukaemic blasts of bcr-abl positive children with ALL are about four-fold resistant to prednisolone as compared to blasts from bcr-abl negative children. This suggests that one of the reasons for the poor prognosis of bcr-abl positive ALL could be a lower steroid sensitivity.

Adolescent↗

PubMed1988

Fusion of the bcr and the c-abl genes in Ph'-positive acute lymphocytic leukemia with no rearrangement in the breakpoint cluster region.

Two types of Philadelphia (Ph') chromosome positive acute lymphoblastic leukemias (ALL) have been described. One shows rearrangements within the 5.8 kb breakpoint cluster region (bcr), which forms the mid-portion of the bcr gene, on chromosome 22, while the other carries rearrangements involving a more proximal region on chromosome 22. To understand the nature of the breakpoints on chromosome 22 in bcr rearrangement negative, Ph'-positive ALLs, we have cloned and sequenced the cDNA of the c-abl oncogene in such ALL cells. The 5' ends of the cDNA clones correspond to the normal sequences of the bcr gene first exon with two of the clones extending beyond the GCCATGG consensus sequence for the initiation of translation. The bcr sequence stops at nucleotide 1813 of the coding sequence of the bcr gene, while the c-abl sequence starts at the beginning of the second c-abl exon (nucleotide 227). Thus the joining point between bcr and c-abl is at the boundary between two exons, suggesting intronic fusion and the occurrence of a splicing event. Our current observations indicate that the Ph' translocation in bcr negative ALL involves bcr gene sequences, albeit only a proximal portion of those involved in CML. These genomic differences may be important factors in the pathogenesis of the distinct phenotypes of ALL and CML.

Base Sequence↗

PubMed1995

Correlation between the proportion of Philadelphia chromosome-positive metaphase cells and levels of BCR-ABL mRNA in chronic myeloid leukaemia.

We have sought to define the relationship between the proportion of marrow metaphases showing the Philadelphia chromosome (Ph) and levels of BCR-ABL mRNA assessed by quantitative polymerase chain reaction (PCR) in patients with chronic myeloid leukaemia (CML). From a total of 141 patients, 164 PCR assays were performed on peripheral blood samples taken within 2 weeks of a bone marrow specimen analysed by cytogenetics. BCR-ABL mRNA was quantified in all 106 PCR-positive samples by competitive PCR; results ranged from < 10 to 3.4 x 10(6) transcripts/micrograms RNA. Twenty-one chronic-phase patients had a median of 5.0 x 10(5) BCR-ABL transcripts/micrograms RNA; no difference in levels of the fusion mRNA was found between 15 Ph-positive and six Ph-negative, BCR-ABL-positive patients. Ph-positive metaphases were not detected in any individual who was PCR negative (n = 58) and in only a single patient who was PCR positive with < 10(3) BCR-ABL transcripts/micrograms RNA (n = 44). Conversely, of 41 samples from patients in haematological remission who had > 10(3) BCR-ABL transcripts/micrograms RNA, 30 had at least one Ph-positive metaphase. The highest level of BCR-ABL transcripts at which Ph-positive metaphases were not detected was 1.5 x 10(4). For the 46 patients who had at least one Ph-positive metaphase, a good correlation (Spearman coefficient = 0.83, P < 0.0001) was found between the percentage of Ph-positive metaphases and BCR-ABL transcript levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Marrow↗

PubMed1993

Influence of M-BCR breakpoint sites on the duration of chronic phase in 100 patients with chronic myelocytic leukemia.

Rearrangements of the bcr (M-BCR) gene were studied in 100 patients with chronic myelocytic leukemia (CML). To determine the significance of a chimeric gene expression in the progression of CML, we analyzed 43 patients for bcr-ABL chimeric mRNA expression. Both DNA and RNA analyses revealed a possible influence of breakpoint sites in the bcr region on the duration of the chronic phase. Patients with the breakpoint located at about the 1-kb region between BamHI and HindIII in bcr exon 3 (region C2) had a significantly shorter chronic phase (31 months) (p = 0.028) than patients in whom the breakpoint was located in other regions. When the bcr locus was divided into 5' and 3' regions as for the BamHI cleavage site located near the 5' region of bcr exon 3, the chronic phase duration in patients with the 5' site (HindIII-BamHI) and 3' site (BamHI-EcoRI site) was 75 and 38 months, respectively. However, the difference was not statistically significant (p = 0.128). These results suggest that only the breakpoint site at C2 on the bcr locus, rather than breakpoint sites in other regions, has an important role in the progression of CML.

Chromosome Mapping↗

PubMed2001

The role of Bcr-Abl in chronic myeloid leukemia and stem cell biology.

Hematopoietic stem cells, the critical target cells in chronic myeloid leukemia (CML), are difficult to study owing to their rarity. An in vitro differentiation model was used to examine the role of the Bcr-Abl tyrosine kinase in the pathogenesis of CML. A tetracycline-regulated promoter was developed to drive Bcr-Abl expression in differentiating embryonal stem cells. Enforced Bcr-Abl expression was sufficient to increase the number of multilineage progenitors. Myeloid progenitors were expanded in particular and erythroid development was suppressed, consistent with the phenotype of CML. A complementary approach was used to investigate the effect of specifically blocking Bcr-Abl-deregulated tyrosine kinase activity in transformed cells. For this purpose a fusion "escort/phosphatase" was created that binds to Bcr-Abl and effectively inhibits kinase activity in vitro and in vivo. Loss of function was demonstrated by growth inhibition of bcr-abl-transformed fibroblasts, reacquisition of growth factor dependence by Bcr-Abl-expressing hematopoietic cells, and normalization of erythroid differentiation in a human CML line. These studies reaffirm the central role of stem cell biology in understanding the causes and possible treatments for CML, and suggest that direct targeting and inhibition of Bcr-Abl kinase activity is a valid therapeutic approach to treating this disease.

Animals↗

PubMed2000

Cytogenetic response to autografting in chronic myelogenous leukemia correlates with the amount of BCR-ABL positive cells in the graft.

OBJECTIVE: An important step in successful autografting of patients with chronic myelogenous leukemia is the delivery of a leukemia-free graft. We conducted this study to determine whether the cytogenetic response after autografting was correlated with the number of BCR ABL-positive cells present within the stem cell grafts. MATERIALS AND METHODS: By BCR-ABL mRNA quantification, we studied the serial pheresis products from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplantation. We correlated the residual disease within the graft reinfused with the cytogenetic response following transplantation, taking into consideration those responses that lasted 12 months or more. RESULTS: Thirty-two patients received a graft with 0-35% Ph-metaphases and 19 received a graft with BCR-ABL/ABL ratio < or =0.01. After a median of 27 months (range, 12-50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months, and 14 of them (78%) received a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.0003-0.01). Twenty-two patients experienced short-lived responses or had >35% Ph-positive cells in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio < or =0.01 (range, 0.001-0.01). Therefore, we found a strong association between a BCR-ABL/ABL ratio less than or =0.01 and the achievement of complete or major cytogenetic remission after autografting (chi(2) test, p = 0.0001). Patients reinfused with grafts contaminated at low levels with leukemic cells also showed a longer duration of the response (log-rank test, p = 0.0009). Eleven patients were reinfused with the lowest level of contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients experienced prolonged neutropenia or thrombocytopenia following stem cell reinfusion and nine of them had long-lasting complete or major cytogenetic responses after transplant. CONCLUSION: This study demonstrates that the number of BCR-ABL positive cells present in a stem cell graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting. [corrected]

Adult↗

PubMed2001

Induction of apoptosis in chronic myelogenous leukemia cells through nuclear entrapment of BCR-ABL tyrosine kinase.

The chimeric BCR-ABL oncoprotein is the molecular hallmark of chronic myelogenous leukemia (CML). BCR-ABL contains nuclear import and export signals but it is localized only in the cytoplasm where it activates mitogenic and anti-apoptotic pathways. We have found that inhibition of the BCR-ABL tyrosine kinase, either by mutation or by the drug STI571, can stimulate its nuclear entry. By combining STI571 with leptomycin B (LMB) to block nuclear export, we trapped BCR-ABL in the nucleus and the nuclear BCR-ABL tyrosine kinase activates apoptosis. As a result, the combined treatment with STI571 and LMB causes the irreversible and complete killing of BCR-ABL transformed cells, whereas the effect of either drug alone is fully reversible. The combined treatment with STI571 and LMB also preferentially eliminates mouse bone marrow cells that express BCR-ABL. These results indicate that nuclear entrapment of BCR-ABL can be used as a therapeutic strategy to selectively kill chronic myelogenous leukemia cells.

Active Transport, Cell Nucleus↗

PubMed2000

Quantification of BCR-ABL transcripts in CML patients in cytogenetic remission after interferon-alpha-based therapy.

We measured using a competitive quantitative polymerase chain reaction-capillary electrophoresis (PCR-CE)-based assay, the levels of bcr-abl transcripts in 44 patients with chronic myeloid leukemia (CML) after interferon-alpha (IFN-alpha) therapy, who achieved a major (10 patients, MCR group) or complete (34 patients, CCR group) cytogenetic response. All 34 CCR patients had molecular evidence of residual disease detected in bone marrow samples at the time of best karyotypic response. The median number of bcr-abl transcripts of 34 evaluable patients in the CCR group at the time of complete cytogenetic remission was 4/microg RNA (range 3-4600), while the median number of bcr-abl transcripts of 10 patients in the MCR group at the time of best cytogenetic response was 4490/microg RNA (range 600-23 900) (P = 0.000024). In nine CCR and five MCR patients we were able to quantify the amount of bcr-abl transcript both at diagnosis and after interferon therapy: no statistical difference (P = 0.18) was found between the two groups at diagnosis (median bcr-abl transcripts/microg RNA was 30 000 vs. 39 650, respectively). During IFN-alpha therapy, the two groups were evaluable at the time of major karyotypic conversion: at this point, there was a statistical difference of expression of bcr-abl transcript between the CCR group (17 patients) (median 2700; range 76-40 000) and the MCR group (10 patients) (median 4490; range 600-23 900), respectively (P = 0.046). No differences of bcr-abl amount of transcript were found in patients with CCR obtained either by IFN-alpha therapy alone (20 patients) vs. IFN-alpha plus ABMT (13 patients) (P = 0.47). We firstly demonstrated that although the CCR and MCR groups were clinically, cytogenetically and molecularly indistinguishable at diagnosis, the two groups could be recognized successfully during interferon therapy based on the level of bcr-abl transcript.

Adolescent↗

PubMed2002

Purging of chronic myelogenous leukemia cells by retrovirally expressed anti-bcr-abl ribozymes with specific cellular compartmentalization.

In patients with chronic myelogenous leukemia (CML), abnormal expansion of myeloid cells is maintained by expression of the p210(bcr-abl) fusion protein. Thus, this protein and its mRNA represent primary targets to inhibit proliferation of these cells. Here we describe the properties of a ribozyme against the bcr-abl mRNA, expressed as a fusion transcript with the human U1 small nuclear RNA or the adenovirus VA1 RNA and delivered to the cells through retroviral vectors. These fusion ribozymes are specifically localized in the nucleus or in the cytoplasm, respectively. Transduction of 32D-LG7 myeloid cells, whose growth is IL-3 independent thanks to deregulated bcr-abl expression, imposed strong negative selective pressure on cell growth and induced restoration of an IL-3-dependent phenotype. Although expressed at a level similar to that of the U1-fusion ribozyme, the cytoplasmic VA1 ribozyme was a more powerful inhibitor of p210(bcr-abl) gene expression. In cells transduced with the vector expressing this ribozyme, the levels of the bcr-abl transcript were reduced up to 10(4)-fold, the p210(bcr-abl) protein became undetectable, and the cells underwent massive apoptosis when cultured in the absence of IL-3. Transduction of primary hematopoietic cells obtained from bone marrow of patients with CML resulted in remarkable reduction of bcr-abl mRNA levels, starting a few days after transduction. These results show the feasibility and efficacy of vector-expressed anti-bcr-abl ribozymes for purging of CML cells.

Animals↗

PubMed2005

Stable RNA interference (RNAi) as an option for anti-bcr-abl therapy.

RNA interference (RNAi) has recently been used for sequence-specific gene silencing of disease-related genes including oncogenes in hematopoietic cells. To characterize its potential therapeutic value, we analyzed different modes to activate RNAi as well as some pharmacokinetic aspects of gene silencing in bcr-abl+ cells. Using lentiviral gene transfer of transcription cassettes for anti-bcr-abl shRNAs and red fluorescence protein (RFP) as a quantitative reporter, we demonstrate that stable but not transient RNAi can efficiently deplete bcr-abl+ K562 and murine TonB cells from suspension cultures. Importantly, depletion of bcr-abl+ cells depends on the dose of lentivirus used for transduction and correlates with the RFP-expression level of transduced target cells: RFP-high K562 cells are eradicated, whereas RFP-low or -intermediate cells may recover after prolonged cell culture. Interestingly, these cells still show reduced bcr-abl mRNA levels, aberrant proliferation kinetics, and enhanced sensitivity to the Bcr-Abl-kinase inhibitor STI571. Quantitative PCR from genomic DNA suggests that more than three lentiviral integrations are required for effective depletion of K562 cells. Finally, we demonstrate that lentivirus-mediated anti-bcr-abl RNAi can inhibit colony formation of primary CD34+ cells from chronic myeloid leukemia patients. These data demonstrate dose-dependent gene silencing by lentivirus-mediated RNAi in bcr-abl+ cells and suggest that stable RNAi may indeed be therapeutically useful in primary hematopoietic cells.

Animals↗

PubMed1998

Arginine butyrate downregulates p210 bcr-abl expression and induces apoptosis in chronic myelogenous leukemia cells.

Downregulation of bcr-abl expression in the chronic myelogenous leukemia cell line K562 using antisense oligonucleotides has been shown to enhance the sensitivity of the cells to apoptotic stimuli, suggesting that p210 bcr-abl, like bcl-2 functions as an anti-apoptosis factor (McGahon A et al, Blood 1994, 83: 1179). In these experiments, the inhibition of p210 bcr-abl expression alone was not sufficient to induce apoptosis. We demonstrated that exposure to low doses (0.5 mM) of a butyric acid analog, arginine butyrate, was capable of inducing apoptosis in selected leukemia cell lines, including K562 cells, and in fresh leukemia cells from patients with chronic myelogenous leukemia. To further explore the mechanisms of this effect, we examined expression of p210 bcr-abl after butyrate exposure and found a dose-related inhibition of p210 bcr-abl protein without concordant change in other phosphoproteins, including the JAK-1 kinase. Further analysis revealed that the inhibition of bcr-abl expression occurs due to transcriptional regulation of the bcr-abl gene by arginine butyrate. These results suggest that arginine butyrate and other butyrate analogs alone or in combination may be useful in the therapy of patients with chronic myelogenous leukemia or bcr-abl expressing acute leukemias.

Antineoplastic Agents↗

PubMed2002

Transcription-mediated amplification and hybridisation protection assay to determine BCR-ABL transcript levels in patients with chronic myeloid leukaemia.

Detection of BCR-ABL transcripts in chronic myeloid leukaemia (CML) is used to confirm the diagnosis and to monitor residual disease. Quantitative techniques are required to predict response to therapy or early relapse. We have evaluated an assay in which transcription-mediated amplification (TMA) of BCR-ABL and ABL transcripts is achieved using reverse transcriptase and RNA polymerase. The products are quantified in the hybridisation protection assay (HPA) using acridinium ester-labelled DNA probes and chemiluminescence. The method is a single tube procedure which uses small amounts of RNA (<500 ng/triplicate analysis), is technically simple (requiring just two waterbaths and a luminometer), rapid (total assay time <4 h) and sensitive (capable of detecting one BCR-ABL-positive K562 cell in the presence of 10(4)-10(5) BCR-ABL-negative cells). BCR-ABL signals from patient RNA samples were quantified relative to known amounts of K562 RNA and normalised to levels of ABL. BCR-ABL/ABL ratios ranged from 0.15 to 1.59 (median 0.65) in RNA from diagnostic blood or bone marrow of 18 CML patients and were < or =0.0001 in 20 normal controls. Sequential samples analysed from six CML patients post-allogeneic bone marrow transplantation who relapsed and received donor lymphocyte infusions showed BCR-ABL/ABL ratios which reflected patient status or treatment. A BCR-ABL/ABL ratio of 0.01 served as a useful arbitrary indicator value, with results above and below this value generally correlating with relapse or remission, respectively.

Acridines↗

PubMed2003

Phosphorylation of tyrosine 393 in the kinase domain of Bcr-Abl influences the sensitivity towards imatinib in vivo.

The Bcr-Abl fusion protein arising through the t(9;22)(q34;q11) reciprocal translocation is the causative agent in chronic myeloid leukemia and a subset of acute lymphocytic leukemia. Imatinib mesylate is a specific inhibitor of the Bcr-Abl kinase and has shown promising results in clinical studies. The structural relation between the Bcr-Abl oncogene and the tyrosine kinase inhibitor imatinib has recently been elucidated by an elegant crystal structure analysis, emphasizing the importance of dephosphorylated tyrosine 393 (Tyr393) in Bcr-Abl for access of the inhibitor to the kinase domain. By mutating this tyrosine to phenylalanine and thereby mimicking a constitutively dephosphorylated state, we now show that Ba/F3 cells transformed by this mutant demonstrate an increased sensitivity towards imatinib in vivo. This effect is not due to an impaired kinase activity of Bcr-Abl Y393F, since a synthetic substrate is phosphorylated with similar kinetics. Treatment of Ba/F3 cells transfected with Bcr-Abl wild type with a phosphatase inhibitor diminished the effect of imatinib, but did not influence the growth of Ba/F3 cells transfected with Bcr-AblY393F. The results support the findings of the crystal structure and indicate that Tyr393 indeed plays a significant role for the sensitivity of Bcr-Abl towards imatinib in vivo. These data implicate the regulation of Tyr393 phosphorylation as a potential mechanism of imatinib resistance.

Antineoplastic Agents↗

PubMed2006

Transient or long-term silencing of BCR-ABL alone induces cell cycle and proliferation arrest, apoptosis and differentiation.

BCR-ABL fusion protein, a t(9;22) translocation product is indispensable for generation, maintenance and progression of chronic myeloid leukemia. RNA interference is an approach to silence gene at post-transcriptional level. We show that dsRNA targeted against the translocation region leads to more than 90% inhibition of BCR-ABL mRNA and protein expression levels using K562 as a model. Lack of BCR-ABL leads to cell cycle arrest in G1 phase as observed by decrease in cyclin D1 and increase in p21 and p27 cdk inhibitors mRNA. Apoptosis resistance imparted by BCR-ABL is lost in these cells in caspase-dependent or independent manner. Decrease in Bcl-XL is observed along with decrease in mitochondrial membrane integrity. Transient removal of BCR-ABL expression has a profound effect on proliferation and clonogenic capacity also confirmed by long-term silencing using lentiviral vectors. Interestingly, low level of BCR-ABL message leads to enhanced erythroid differentiation and reduced expression of megakaryocytic markers. Importantly, in six CML patient samples studied, silencing BCR-ABL in the lineage depleted enriched stem cell population leads to a decrease in colony-forming capacity. Thus, long-term silencing of BCR-ABL might prove to be a promising alternative approach in CML patients especially for those who do not respond to any other drug treatment.

Apoptosis↗

PubMed2000

Bcr-Abl has a greater intrinsic capacity than v-Abl to induce the neoplastic expansion of myeloid cells.

The Bcr-Abl/p210 fusion protein plays a primary role in the pathogenesis of chronic myelogenous leukemia (CML). Abelson murine leukemia virus, which encodes v-Abl/p160, induces a pre-B cell leukemia/lymphoma in mice. It has been unclear whether the apparent specificity of these two abl oncogenes for myeloid versus lymphoid neoplasms is due to specific intrinsic properties of these Abl oncoproteins, or due to the properties of the target cells expressing them. We have recently shown that expression of Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder in mice resembling human CML. In this study, we compared Bcr-Abl/p210 and v-Abl/p160 in this mouse CML model. We found that early in the course of disease, both Bcr-Abl/p210 and v-Abl/p160 expanded early immature hematopoietic cells. Later Bcr-Abl/p210 selectively expanded myeloid cells while v-Abl/p160 primarily induced the rapid in vivo expansion of B lymphoblastic cells, along with a minor population of myeloid cells. In vitro, Bcr-Abl/p210 induced more growth of myeloid colonies from 5-fluorouracil treated bone marrow than v-Abl/p160. These results, obtained under equal bone marrow transduction/transplantation conditions, indicate that Bcr-Abl/p210 has a greater intrinsic capacity than v-Abl/p160 to induce the neoplastic growth of myeloid cells. In addition, we found that cultured cells expressing Bcr-Abl/p210 had more activated STAT5 than cells that expressed v-Abl/p160. This suggests that activation of STAT5 might be one part of the mechanism of abl oncogene disease specificity.

Animals↗