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PubMed1993

Structural organization of BCR-ABL gene in chronic phase and blast transformation in chronic myeloid leukemia patients.

We studied 36 DNA samples of 18 patients affected with chronic myeloid leukemia (CML) for the presence of mutations in the first exon of the BCR gene was divided into four regions amplified by polymerase chain reaction (PCR). By single strand conformation polymorphism analysis (SSCP) and direct sequencing of amplified fragments, we found different banding profiles in 9 out of 18 patients in the PCR fragment spanning nucleotide 506-826. In one patient, sequence analysis revealed the presence of a point mutation at nucleotide 669 (A-T; Gln-Leu). No difference was found between DNA samples collected during the chronic phase and the blastic transformation. No different mobility shifts of single stranded PCR products were found in the other amplified fragments. The activation of BCR-ABL involves direct interaction between BCR first exon sequences and the tyrosine kinase regulatory domains of ABL. In the first BCR exon, and around the mutated sequences two SH-2-binding sites, are retained. These domains are essential for BCR-ABL-mediated transformation. Our results demonstrate the presence of point mutation in this regulatory region, which may suggest a role for the altered BCR sequence in activation of the BCR-ABL oncogene.

Base Sequence↗
PubMed1993

The relationship between the type of bcr-abl hybrid messenger RNA and thrombopoiesis in Philadelphia-positive chronic myelogenous leukemia.

Formation of the Philadelphia (Ph1) chromosome, which contains the hybrid bcr-abl gene, is thought to be the initial event in chronic myelogenous leukemia (CML). The positions of the breakpoint within the breakpoint cluster region (bcr) on the bcr-abl gene in 22 chronic-phase cases of Ph1-positive CML were determined using conventional Southern blots, and the splicing pattern were also determined the species of the fused bcr-abl mRNA in 79 CML cases using the polymerase chain-reaction procedure (RT-PCR). The location of the breakpoint within the bcr locus was assigned to one of five zones. Breakpoints in zones 1 and 2 were grouped as 5', and those in zones 3, 4 and 5 as 3'. Nine patients had 5' breakpoints and 13 patients had 3' breakpoints. The platelet counts of 3' patients were significantly higher than those of 5' patients (1395 vs 274 x 10(9)/L; p < 0.03). The megakaryocyte counts from bone marrow histological sections in 3' patients (n = 12) and 5' patients (n = 7) were 63.4/mm2 and 19.5/mm2, with a significant difference at p < 0.006. The mean number of megakaryocyte progenitor cells assayed by in vitro cloning was 128.3/2 x 10(5) bone marrow cells for 3' patients (n = 7) compared with 46.3 for 5' patients (n = 4). Using the RT-PCR technique, the bcr exon 2/abl exon 2 fused mRNA (b2-a2) was detected in 18 patients, the bcr exon 3/abl exon 2 fused mRNA (b3-a2) was detected in 45 patients, and both types of mRNA were detected in 16 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1997

Sequential interphase FISH analysis of m-BCR/ABL chimeric gene-positive cells in Ph-positive acute myeloid leukemia.

We report a case of Philadelphia (Ph)-positive AML in which interphase fluorescence in situ hybridization (FISH) analysis was performed from diagnosis throughout the course of therapy using major (M-) breakpoint cluster region (BCR)/minor (m-) BCR and ABL cosmid probes. We also investigated the existence of the M-BCR or m-BCR at the RNA or DNA level by the reverse transcriptase polymerase chain reaction and Southern blot analysis, respectively. Complete remission with a normal karyotype was achieved after several regimens of chemotherapy and peripheral blood stem cell transplantation (PBSCT), but relapse occurred and his cells became 100% Ph-positive. We detected the m-BCR/ABL fusion gene by interphase FISH analysis using an m-BCR/ABL translocation probe, and found that FISH analysis was useful for classifying the BCR, identifying minimal residual disease, and for predicting imminent relapse after chemotherapy and PBSCT.

Acute Disease↗
PubMed2001

Requirement for Mdm2 in the survival effects of Bcr-Abl and interleukin 3 in hematopoietic cells.

The p53/Mdm2 pathway plays an important role in the induction of cell cycle arrest or apoptosis in response to genotoxic stress. Both the oncogene Bcr-Abl and physiological growth factors such as interleukin (IL)-3 can modulate the outcome of cellular exposure to DNA damage. To determine whether Bcr-Abl and growth factors can affect the p53/Mdm2 pathway, we studied the expression of Mdm2 in the IL-3-dependent pre-B cell line BaF3 and its bcr-abl-transfected derivative BaF3p185 after IL-3 deprivation or treatment with the c-Abl tyrosine kinase inhibitor STI571. We found that both growth factor withdrawal and inhibition of Bcr-Abl kinase lead to a down-regulation of Mdm2 preceding the induction of apoptosis. Apoptotic cell death induced by STI571 is partially dependent on p53. The early decrease of Mdm2 protein was not attributable to transcriptional regulation or to caspase-mediated cleavage. On the other hand, it could be completely blocked by the proteasomal inhibitor lactacystin. Targeted down-regulation of Mdm2 protein by antisense oligodeoxynucleotides overcame the survival effects of IL-3 and Bcr-Abl and resulted in accelerated apoptosis. Taken together, survival signals provided either by physiological growth factors or by oncogenic Bcr-Abl can positively regulate Mdm2, whereas Mdm2 ablation can reduce cell survival. These findings imply that, similarly to physiological growth factors such as IL-3, Bcr-Abl can promote cell survival through modulating the p53-Mdm2 pathway.

Apoptosis↗
PubMed2006

[Detection of phosphotyrosine in bcr-abl-positive cells with PY20 antibody and its clinical applications].

OBJECTIVE: To explore the specificity of anti-phosphotyrosine monoclonal antibody PY20 in bcr-abl+ cells and its possible clinical applications. METHODS: Bcr-abl cell lines( K562, MEG-01) and bcr-abl- cells lines( Jurkat, MCF-7 )were stained with PY20. Phosphotyrosine protein of K562 and MEG-01 cells was detected by flow cytometry before and after treatment with imatinib. Phosphotyrosine protein in bone marrow cells from 49 patients with chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia(Ph(+) -ALL), Ph- ALL, acute myeloid leukemia (AML-M1, M2, M3, M5, FAB classification), chronic lymphocytic leukemia (CML) and 3 normal donor. Positive cells over 5% of total cells was considered positive cases for phosphotyrosine protein. The level of tyrosine phosphorylation was determined by median fluorescence intensity (MFI). RESULTS: Bcr-abl cell lines and marrow cells from 10 CML patients and 8 ALL patients were all PY20-positive, while bcr-abl- cell lines and marrow cells from 18 leukemia patients and 3 normal donor were all PY20-negative. MFI decreased remarkably after blocked by imatinib in K562 cells and MEG-01 cells. The positive cell percent of marrow cells from 10 newly diagnosed CML patients and 9 imatinib-sensitive CML patients was (54.20 +/- 19.82)% and (14.84 +/- 6.17)% (P < 0.05), while that of 2 cases of imatinib-resistant was 64.3% and 57.2%. There was significant difference of MFI between imatinib-resistant patients and imatinib-sensitive patients (99.42 +/- 4.87 vs 46.41 +/- 4.67, P < 0.01). CONCLUSION: PY20 monoclonal antibody is highly specific for bcr-abl+ cells. It might be useful in rapid detection of bcr-abl+ cells and sensitivity to imatinib of CML patients.

Antibodies, Monoclonal↗
PubMed1995

The SH2 domain of ABL is not required for factor-independent growth induced by BCR-ABL in a murine myeloid cell line.

Chronic myelogenous leukemia (CML) is characterized by the presence of a specific chromosomal translocation between the long arms of chromosomes 9 and 22 that results in the fusion of BCR encoded sequences upstream of exon 2 of c-ABL. This fusion gene produces a 210-kDa chimeric BCR-ABL protein that has elevated tyrosine kinase activity. Several substrates of this activated tyrosine kinase have been reported. However, their necessity for the transforming functions of BCR-ABL has not been determined. A specific deletion of the SH2 domain of ABL was created to determine whether this mutation would alter the ability of BCR-ABL to induce factor-independent growth of a murine myeloid cell line and to determine whether the SH2 domain mediates the interaction of BCR-ABL with any of its substates. Our results indicate that the SH2 domain of BCR-ABL is not required for the induction of growth factor independence and is not required for the association of BCR-ABL with rasGAP or SHC. However, myeloid cells expressing this mutant lack the tyrosine phosphorylation of a 62-kDa rasGAP associated protein.

Animals↗
PubMed1994

Variable transcription of BCR-ABL by Ph+ cells arising from hematopoietic progenitors in chronic myeloid leukemia.

Recent studies suggest that the BCR-ABL gene plays a critical role in the pathogenesis of Ph+ chronic myeloid leukemia (CML). We investigated the hematopoietic colonies derived from the marrows of 12 patients with Ph+ CML in chronic phase by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of BCR-ABL mRNA and by cytogenetics. Colonies were individually harvested and each colony divided into two portions, one for cytogenetics and the other for isolation of total RNA for PCR of BCR-ABL transcripts and for an RNA internal control. We found that 23% +/- 18% (mean +/- SD, range 0% to 60%) of Ph+ colonies did not transcribe the aberrant gene. In each case when BCR-ABL transcription was not detected, normal ABL mRNA was present. The data suggest that hitherto unknown mechanisms may regulate BCR-ABL expression in some Ph+ cells and indicate that caution should be exercised in the interpretation of results using RT-PCR analysis of hematopoietic colonies from clinical specimens and from experiments with antisense oligonucleotides directed at the BCR-ABL gene. These data also raise the notion of a transitional Ph+ precursor cell in which BCR-ABL may become upregulated and lead to a fully expressed phenotype. We conclude that further studies correlating the frequency of Ph+ PCR- progenitors with prognostic clinical variables are warranted.

Adult↗
PubMed1993

Frequent detection of BCR-ABL specific mRNA in patients with chronic myeloid leukemia (CML) following allogeneic and syngeneic bone marrow transplantation (BMT).

We performed a two-step polymerase chain reaction (PCR) to detect bcr-abl-specific mRNA in 440 peripheral blood and/or bone marrow samples of 30 chronic myeloid leukemia (CML) patients (mean 15, range 2-50 samples) following non T-cell-depleted allogeneic (n = 28) or syngeneic (n = 2) bone marrow transplantation (BMT). Median follow-up after BMT is 40 months (range 2-116 months), the median observation time 29 months (range 2-40 months). In 15 patients (50%), bcr-abl-specific mRNA could be detected following BMT. Bcr-abl positivity was rare in patients who were in hematological remission for at least 40 months (2/11). In five patients, PCR positivity was observed only once; all five patients are in complete hematological remission. Ten patients showed bcr-abl specific mRNA in two or more consecutive samples. Hematological relapse occurred in five of the latter patients. Bcr-abl positivity preceded hematological relapse in all cases. Bcr-abl positivity was detected more frequently in patients without graft-versus-host disease (GVHD) (11/15), than in patients with GVHD (4/15) (p < 0.02). Our data indicate that transient bcr-abl positivity is not usually followed by hematological relapse, while patients, who are positive in serial samples have a high risk of relapse.

Adult↗
PubMed1993

Molecular characterization of a variant Ph1 translocation t(9;22;11) (q34;q11;q13) in chronic myelogenous leukemia (CML) reveals the translocation of the 3'-part of BCR gene to the chromosome band 11q13.

We performed cloning and sequence analysis of translocation junctions at 11q- and 22q- (Ph1) chromosomes and the corresponding germline DNAs of a variant Ph1-positive CML with t(9;22;11)(q34;q11;q13). Southern blot analysis using probes for different regions of bcr mapped the translocation break near the 5'-side of bcr exon 4. Cloning, Southern blot analysis and restriction map analysis of both bcr fragments showed that the part of bcr 3'- to the translocation break moved to 11q13. Sequence analysis of the translocation junction on the Ph1 chromosome showed that the translocation break occurred 63 bp upstream of exon 4. Compared to the germline sequence, bcr sequence from the translocated partners showed deletion of seven basepairs at the site of translocation. A probe derived from the 5'-region of the clone isolated from the 11q- chromosome identified clonal rearrangements in the leukemic DNA. Restriction map and sequence analysis showed that this clone consisted of the 3'-half of the glutathione S-transferase Pi (GST-Pi) gene and the 3'-part of bcr. We identified two point mutations in the GST-Pi allele involved in translocation. Northern blot analysis showed that the GST-Pi gene was expressed in the leukemic cells at blast crisis but not at chronic phase; however, no fusion mRNA between GST-Pi and bcr was identified. We did not find any sequence homology between 11q13 DNA and 22q11 DNA around the translocation breakpoints; however, sequences homologous to ALU repeats were identified close to the sites of translocation breaks at 22q11 and 11q13. This study supports our hypothesis that variant Ph1 translocations may occur as primary cytogenetic changes similar to the classical Ph1 translocations.

Base Sequence↗
PubMed1993

Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line.

The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

Base Sequence↗
PubMed1993

Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation.

We have developed a competitive polymerase chain reaction (PCR) titration assay that estimates the number of BCR-ABL transcripts in chronic myeloid leukemia patients to monitor minimal residual disease after bone marrow transplantation (BMT). The assay gave reproducible results and allowed differences in BCR-ABL message levels of half an order of magnitude to be distinguished. Of 91 patients studied by nonquantitative PCR, 28 who had a positive PCR result on at least one occasion posttransplant were analyzed by competitive PCR. Seventeen patients had no evidence in their marrow of cytogenetic relapse during the period of observation; BCR-ABL transcript numbers in these cases ranged from approximately 10 to 800/micrograms RNA. Ten of the 11 patients who relapsed cytogenetically were studied when Philadelphia-positive metaphases were first detected in their marrow; transcript numbers ranged from 1,600 to 7 x 10(5)/micrograms RNA. Patients in hematologic relapse had between 9 x 10(4) and 10(6) BCR-ABL transcripts/micrograms RNA. Patients who progressed from cytogenetic remission to cytogenetic relapse and then to hematologic relapse had increasing numbers of BCR-ABL transcripts in their blood. Three patients had clear evidence of rising numbers of BCR-ABL transcripts before routine detection of cytogenetic relapse. Conversely patients without cytogenetic relapse generally had low or falling numbers of transcripts. We conclude that serial monitoring of residual disease post-BMT by estimating the number of BCR-ABL transcripts provides more information than conventional cytogenetics or nonquantitative PCR and may identify patients in need of therapeutic intervention before the onset of overt relapse.

Base Sequence↗
PubMed1996

Clinical features and outcome of children with first marrow relapse of acute lymphoblastic leukemia expressing BCR-ABL fusion transcripts. BFM Relapse Study Group.

Although the Philadelphia chromosome (Ph1) has been identified as an adverse prognostic factor in acute lymphoblastic leukemia (ALL), little is known about the incidence and clinical course of relapsed Ph1-positive ALL in children. The incidence was determined by screening of 170 consecutive children with first bone marrow relapse of ALL using the reverse transcriptase-polymerase chain reaction (RT-PCR) and comparison, with cytogenetic analysis. Among these 170 children, 20 (12%) were found to be BCR-ABL-positive, representing a rate that is about three times higher than that reported for newly diagnosed ALL. Ten of the cases were identified by RT-PCR only. In none of the 21 patients with T-cell immunophenotypes could an expression of the BCR-ABL mRNA be detected. BCR-ABL positivity was associated with a significantly shorter duration of first remission (P = .0086) and higher white blood cell (P = .0157) and blast cell counts (P = .0304) at relapse diagnosis. All patients were treated according to the ALL-REZ BFM 87 and 90 relapse trials of the BFM Relapse Study Group. The intensive multiagent chemotherapy induced a second complete remission in only 60% of children with BCR-ABL-positive ALL compared with in 91% of those without BCR-ABL expression (P = .0023). The prognosis of BCR-ABL-positive ALL in children is poor, with a probability of event-free survival at 2 years of 8% versus 50% in those without BCR-ABL mRNA or cytogenetic analysis should become part of the routine diagnostic panel for children with newly diagnosed ALL and is fundamental for children presenting with an early bone marrow relapse.

Adolescent↗
PubMed1996

Kinetics of increasing BCR-ABL transcript numbers in chronic myeloid leukemia patients who relapse after bone marrow transplantation.

We prospectively studied 98 chronic myeloid leukemia (CML) patients after bone marrow transplantation by competitive polymerase chain reaction to detect and quantify leukemia-specific BCR-ABL mRNA. Of 69 patients who had persistently undetectable, decreasing, or low BCR-ABL levels ( < 50 transcripts/microgram RNA) on sequential analysis, one (1%) subsequently relapsed. Of 29 patients who had increasing or persistently high BCR-ABL (> 50 transcripts/microgram RNA) on sequential analysis, 21 (72%) have relapsed (P < .00001). In 19 patients studied sequentially, a constant logarithmic increase in the number of BCR-ABL transcripts was found before relapse, indicating a constant BCR-ABL transcript doubling time. The doubling time for patients relapsing cytogenetically or into chronic phase (median, 24.7 days) was significantly longer than that of patients relapsing into advanced phases (median, 14.7 days; P = .005). Eight patients were treated for relapse by donor leukocyte transfusions. The doubling time of responders was significantly longer than that of nonresponders (P = .017). We conclude that quantification of BCR-ABL transcripts after bone marrow transplantation (BMT) is valuable in predicting relapse: a more rapid BCR-ABL transcript doubling time before relapse might indicate a more aggressive disease.

Biomarkers, Tumor↗
PubMed1996

Investigating and improving the specificity of ribozymes directed against the bcr-abl translocation.

Chronic myelogenous leukaemia (CML) is associated with a translocation between the ABL and BCR genes on chromosomes 9 and 22, t(9;22). The resulting transcription and translation products, bcr-abl mRNA and p210bcr-abl, are unique to the malignant cells and as such are ideal targets for specific chemicals or drugs. We have designed hammerhead ribozymes to cleave the two predominant forms of bcr-abl mRNA, b2a2 and b3a2. Synthetic bcr-abl RNA substrates were cleaved by the ribozymes in vitro, but so was a wild-type abl RNA sequence. bcr RNA was not cleaved in vitro and mutant ribozymes showed no cleavage activity. Ribozymes designed to cleave 9 nucleotides (nt) from either of the fusion points were non-specific for the bcr-abl substrate, but a ribozyme designed to cleave 3 nt upstream of the b3a2 fusion point was specific for b3a2 RNA. However, this ribozyme was less efficient than the others. The shortening of one of the ribozymes arms from 10 nt to 4 nt resulted in a ribozyme that was more specific without losing any efficiency. We conclude that it is possible to specifically cleave bcr-abl RNA in vitro by using hammerhead ribozymes.

Base Sequence↗
PubMed1996

BCR-ABL expression in different subpopulations of functionally characterized Ph+ CD34+ cells from patients with chronic myeloid leukemia.

In patients with chronic myeloid leukemia (CML), the leukemic (BCR-ABL+/Ph+) clone typically includes cells belonging to all of the myeloid lineages and frequently some B cells. From such observations it has been inferred that the initial BCR-ABL gene rearrangement event occurs in a pluripotent hematopoietic stem cell and that the clone subsequently generated is maintained by a subpopulation of neoplastic, BCR-ABL-expressing cells that retain at least some of the defining properties of normal hematopoietic stem cells. To test this hypothesis directly, we isolated various subpopulations of CD34+ cells from fresh or cryopreserved samples of peripheral blood from 5 CML patients with high white blood cell counts, 4 of which were selected because of their exclusive content of Ph+ progenitors (both colony-forming cells and long-term culture-initiating cells [LTC-IC]). Cells in each of the CD34+ subpopulations isolated were examined for the presence of BCR-ABL mRNA using a reverse transcriptase-polymerase chain reaction technique that reproducibly gave a positive signal from single K562 cells. BCR-ABL mRNA was detected in 117 of 147 samples (80%) in which actin mRNA was demonstrable. This included 60% to 90% of a large number of individually analyzed CD34+ cells including 46 single CD34+CD71-CD38- cells and 27 single CD34+CD71+CD38+ cells from 3 patients. In 2 of these cases, the same populations also contained a very high frequency of Ph+ LTC-IC. Our findings demonstrate BCR-ABL gene expression in neoplastic cells with functional as well as surface marker characteristics of very primitive normal hematopoietic cells. This implicates the BCR-ABL gene product directly in the acquisition by these cells of properties that alter their interactions with the microenvironment and deregulate their proliferation control.

ADP-ribosyl Cyclase↗
PubMed1998

Inhibition of BCR-ABL expression with antisense oligodeoxynucleotides restores beta1 integrin-mediated adhesion and proliferation inhibition in chronic myelogenous leukemia hematopoietic progenitors.

Chronic myelogenous leukemia (CML) is characterized by the continuous proliferation and abnormal circulation of malignant hematopoietic progenitors. This may be related to the unresponsiveness of CML progenitors to beta1 integrin adhesion receptor-mediated inhibition of progenitor proliferation by the marrow microenvironment. In hematopoietic cell lines, the BCR-ABL oncogene product, p210(BCR-ABL), interacts with a variety of cytoskeletal elements important for normal integrin signaling. We studied the role of p210(BCR-ABL) in abnormal integrin function in CML by evaluating the effect of inhibition of BCR-ABL expression with antisense oligodeoxynucleotides (AS-ODNs) on integrin-mediated adhesion and proliferation inhibition of malignant primary progenitors from CML marrow. Preincubation of CML CD34(+)HLA-DR+ (DR+) cells with breakpoint-specific AS-ODNs significantly increased adhesion of CML progenitors to stroma and fibronectin (FN). Pretreatment with breakpoint-specific ODNs also resulted in significant inhibition of CML progenitor proliferation after ligand or antibody-mediated beta1 integrin engagement. Breakpoint-specific ODNs were significantly more effective in restoring CML progenitor adhesion and proliferation inhibition than control ODNs. BCR-ABL mRNA and p210(BCR-ABL) levels in CML CD34(+) cells were significantly reduced after incubation with breakpoint-specific AS-ODN. These studies indicate a role for BCR-ABL in abnormal circulation and defective integrin-dependent microenvironmental regulation of proliferation of CML hematopoietic progenitors.

Cell Adhesion↗
PubMed2002

Reduced focal adhesion kinase and paxillin phosphorylation in BCR-ABL-transfected cells.

BACKGROUND: BCR-ABL formation is critical to oncogenic transformation in chronic myelogenous leukemia and has been implicated as a key event leading to alterations in cytoskeletal structures and adhesion in the leukemic cells. The authors therefore investigated the effect of p210(BCR-ABL) on actin polymerization as well as on the expression and phosphorylation state of the adhesion proteins paxillin and focal adhesion kinase (FAK). METHODS: Transfection with BCR-ABL constructs abrogated the ability of NIH 3T3 fibroblasts to adhere and the cells underwent striking morphologic changes. RESULTS: Scanning electron microscopy revealed that the cells lost their elongated appearance and became rounded. This alteration was associated with significantly reduced actin polymerization. In addition, steady-state levels of paxillin and FAK protein were increased. However, while the overall level of phosphotyrosines was also increased, the amount of tyrosine phosphorylated paxillin and FAK was reduced in the BCR-ABL-transfected cells as compared to the parental cells. Culture on extracellular fibronectin matrix partially reversed the morphologic changes and resulted in a return, albeit incomplete, of filamentous actin in BCR-ABL-transfected 3T3 fibroblasts. In addition, phosphorylation of paxillin and FAK in the BCR-ABL-transfected NIH 3T3 cells was restored. CONCLUSIONS: The authors conclude that, in the current system, transfection of BCR-ABL attenuates FAK and paxillin phosphorylation and reduces actin polymerization, events accompanied by significant alterations in cellular morphology. The observation that exposure of the cells to fibronectin partially reverses all these changes suggests that the focal adhesion proteins and actin structures nevertheless remain responsive to signaling from the outside.

3T3 Cells↗
PubMed2004

Inhibition of bcr-abl and/or c-abl gene expression by small interfering, double-stranded RNAs: cross-talk with cell proliferation factors and other oncogenes.

BACKGROUND: Short, 21-mer, double-stranded/small interfering RNAs (ds/siRNAs) were designed to target bcr-abl mRNA in chronic myelogenous leukemia (CML) with a potential also to target c-abl mRNA. METHODS: ds/siRNAs were transfected into bcr-abl-positive K-562 cells (derived from blast-crisis) or bcr-abl-negative/c-abl-positive Jurkat cells (derived from acute lymphoblastic leukemia) using lipofectamine. ds/siRNAs intracellular uptake was detected by fluorescent confocal microscopy using fluorescein-labeled ds/siRNAs. The treatment was performed over 6 days with repetitive siRNA transfections. Efficiency of the siRNAs was determined 24 hours after single siRNA transfection and 6 days after repetitive siRNA transfections. RESULTS: Two of the designed ds/siRNAs decreased the target mRNA levels markedly (determined by reverse transcriptase-polymerase chain reaction analysis) and bcr-abl/c-abl oncoproteins (determined by flow cytometry using Fluor-488-labeled, anti-c-abl antibody as well as by Western blot analysis). These sequences also inhibited protein tyrosine kinase activity significantly and suppressed cell proliferation. One of the three selected ds/siRNAs expressed only slight effects on the bcr-abl/c-abl mRNA in K-562 cells (but not on the oncoprotein level), on protein tyrosine kinase activity, and on cell proliferation. The combination of the three ds/siRNA constructs provoked stronger decreases in bcr-abl/c-abl mRNAs and their respective oncoproteins and produced the strongest suppression of cell proliferation. CONCLUSIONS: The cross-talk between siRNA interference of bcr-abl oncogene and the expression of several apoptotic/antiapoptotic factors, cell proliferation factors, and other oncogenes exists and it was determined by microarray analysis in K-562 cells that were treated over 6 days.

Base Sequence↗