Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The t(9;22) translocation resulting in the fusion of BCR and ABL genes is pathognomonic in chronic myeloid leukemia (CML) and may be investigated at the molecular level using fluorescence in situ hybridization (FISH). Two-color BCR-ABL probes visualizing one fusion signal (1F FISH) have high false positive rates (FPR) and false negative rates (FNR). The FPR is a result of the random spatial association of probe signals within normal interphase cells so that some cells appear to contain the BCR-ABL fusion gene. The FNR of 1F FISH probes depends on the distance between the BCR and ABL probes hybridized to the BCR-ABL fusion gene (< or =368 kb); the "gap" between the signals causing the cell to be interpreted as normal. To overcome these difficulties, a two-color probe was used, employing four yeast artificial chromosome (YAC) sequences that span the breakpoint regions of the BCR and ABL genes and that visualize the two fusion signals BCR-ABL and ABL-BCR in CML cells (2F FISH). The FNR for the 2F FISH probes was assessed on clonal Ph+ granulocyte-macrophage-colony-forming cell (CFU-GM) derived colonies and was reduced to 0.4% (2/450), compared with an FNR of 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells for the 2F FISH was 0. 19 +/- 0.12% (3/1,700), whereas the FPR using 1F FISH was 4.5 +/- 2.3% (63/1,294). The 2F FISH can thus be used to evaluate very small frequencies of BCR-ABL-positive and -negative interphase cells and may be of use in the clinical monitoring of CML.
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PMID:A two-color BCR-ABL probe that greatly reduces the false positive and false negative rates for fluorescence in situ hybridization in chronic myeloid leukemia. 973 13

Approximately 2-5% of children with newly diagnosed acute lymphoblastic leukemia (ALL) have a Philadelphia (Ph) chromosome detectable on cytogenetic analysis, which is associated with a poor prognosis. In rare ALL cases the Ph chromosome may appear in leukemic cells during the course of the disease. We report here the case of a 5.5-year-old male patient with T-ALL who was found to have the b2a2 BCR-ABL mRNA transcript by reverse transcriptase-polymerase chain reaction (RT-PCR) at first marrow relapse. At the time of initial diagnosis, no BCR-ABL transcripts had been detected by PCR in the patient's blood and marrow samples. Further studies were performed using a competitive PCR titration assay and the fluorescence in situ hybridization (FISH) method to monitor the leukemic clone. Progression of the disease was associated with a higher BCR-ABL transcript level and an increasing proportion of BCR-ABL-positive cells. Metaphase FISH analysis identified the presence of the BCR-ABL fusion gene on a normal chromosome 22. This study shows that a late-appearing Ph translocation in ALL may be cytogenetically invisible. Quantitative RT-PCR and FISH techniques are appropriate and efficient methods for detecting these rare ALL variants expressing the BCR-ABL fusion gene and for estimating the level of residual disease following treatment.
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PMID:Molecular detection of a late-appearing BCR-ABL gene in a child with T-cell acute lymphoblastic leukemia. 976 Jan 54

The number of genetic lesions necessary to generate leukemia in humans is unknown, but it is possible that certain specific abnormalities, eg, fusion genes, known to be associated with acute and chronic leukemia are produced relatively frequently in human cells but require other events to occur before the leukemia becomes manifest. We investigated this possibility by studying peripheral blood leukocytes from normal individuals and various hematopoietic cell lines for the presence and expression of the p210 and the p190 types of the BCR-ABL gene associated with chronic myeloid leukemia (CML) and acute lymphoblastic leukemia. We used two-step reverse transcriptase-polymerase chain reaction (RT-PCR) assays in which batches of 10(8) cells per sample were tested in 40 replicate reactions. We estimate that this assay is 1.5 logs more sensitive than the two-step RT-PCR assays that we use routinely to assess minimal residual disease. BCR-ABL fusion gene transcripts of various configurations were found in circulating leukocytes from 12 of the 16 healthy adults analyzed. Transcripts with an e1a2 junction (p190 BCR-ABL) were present in 11 and p210-type transcripts with b2a2 and/or b3a2 junctions were detected in 4 individuals. The same RT-PCR assays in non-CML cell lines showed the presence of classical or aberrant p210-type mRNA in 3 of 7 lines and of p190-type transcripts in all 7 lines of hematopoietic origin (HL60, KG1, U937, Kasumi, Jurkat, JVM13, and JVM25), whereas the NIH3T3 murine fibroblast line was reproducibly negative for these fusion genes. These findings confirm and extend previous reports on the detection of leukemia-associated genes in normal leukocytes and suggest that certain fusion genes are generated relatively frequently in hematopoietic cells, but only infrequently do the cells acquire the additional changes necessary to produce leukemia in humans. Although there is only a small probability that such innocent BCR-ABL-carrying leukocytes are detected by conventional RT-PCR assays, they may be the source of some sporadically positive tests in leukemia patients in long-term remission.
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PMID:The presence of typical and atypical BCR-ABL fusion genes in leukocytes of normal individuals: biologic significance and implications for the assessment of minimal residual disease. 978 74

The hallmark of chronic myelogenous leukaemia (CML) is the presence of the Philadelphia chromosome and its resultant fusion message, BCR-ABL, and fusion protein, p210. Patients with CML in blast crisis, or with Philadelphia positive acute lymphoblastic leukaemia (ALL), can have a smaller BCR-ABL fusion transcript possessing only the first exon of BCR fused to ABL. This smaller transcript encodes a 190 kD protein which is more strongly transforming than the p210 protein derived from the larger CML-associated transcript. We performed RT-PCR on samples from CML patients in chronic phase to determine the frequency and mechanism of p190 and p210 co-expression and to see if this correlated with clinical indices. We examined the peripheral blood or marrow of 67 patients with CML and found that 35 of them expressed both transcripts whereas the remainder expressed the p210-encoding transcript exclusively. Additional PCR products of an intermediate size were also frequently detected and have been isolated and sequenced. Data from two of these products indicate that they are the result of alternative splicing and include variable combinations of BCR exons. We believe that the expression of the p190-encoding transcript in the chronic phase of CML is also due to alternative splicing. A comparison of patients co-expressing the p190- and p210-encoding transcripts with those patients who expressed only the p210-encoding transcript detected significantly higher white blood cell (WBC) counts and blast cell counts at time of testing as well as significantly higher white blood cell counts at diagnosis.
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PMID:Expression of p210 and p190 BCR-ABL due to alternative splicing in chronic myelogenous leukaemia. 985 21

Interferon-alpha (IFN-alpha) can be considered as treatment of choice for patients with chronic myeloid leukaemia (CML) in chronic phase. With this treatment major cytogenetic responses can be achieved in 30% to 50% of patients. Regular monitoring of cytogenetic response is essential for the therapeutic management of these patients. As conventional cytogenetics is not always successful, especially under IFN-alpha treatment, molecular cytogenetic methods have been established for the examination of interphase nuclei for the presence of the BCR-ABL fusion gene, the molecular counterpart of the Philadelphia chromosome. To demonstrate the value of these new methods we have analysed interphase nuclei from sequentially cultured bone marrow cells from 14 CML patients who were treated with IFN-alpha and whose bone marrow was investigated regularly during therapy. Dual-colour FISH with a breakpoint spanning BCR-YAC and a flanking cosmid from the ABL region was applied. When compared with conventional cytogenetics the results achieved by FISH were favourable. The most evident advantage of FISH analysis is that in case of failure of conventional cytogenetics a reliable determination of the remission status can be done. Together with other recent studies our results illustrate the advantages and limitations of the interphase FISH method for monitoring CML patients.
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PMID:Monitoring of remission status by fluorescence in situ hybridisation in chronic myeloid leukaemia patients treated with interferon-alpha. 986 21

The Philadelphia (Ph) chromosome, the main product of the (9;22)(q34;q11) translocation, is the cytogenetic hallmark of chronic myeloid leukemia (CML), a clonal myeloproliferative disorder of the hematopoietic stem cell; the Ph chromosome is also found in a sizeable portion of acute lymphoblastic leukemia (ALL) patients and in a small number of acute myeloid leukemia (AML) cases. At the molecular level, the t(9;22) leads to the fusion of the BCR gene (on chromosome 22) to the ABL gene (translocated from chromosome 9); this fusion gene BCR-ABL with its elevated tyrosine kinase activity must to be central to the pathogenesis of these disorders. Three different breakpoint cluster regions are discerned within the BCR gene on chromosome 22: M-bcr, m-bcr, and mu-bcr. Ph + leukemia cell lines are important tools in this research area. More than 20 ALL-and more than 40 CML-derived Ph + leukemia cell lines have been described. Furthermore, three Ph + B-lymphoblastoid cell lines, established from patients with Ph + ALL or CML, are available. Molecular analysis has documented BCR-ABL fusion genes in three apparently Ph chromosome-negative cell lines, all three derived from CML. Nearly all Ph + ALL cell lines have the m-bcr e1-a2 fusion gene (only two ALL cell lines have a b3-a2 fusion) whereas all CML cell lines, but one carry the M-bcr b2-a2, b3-a2 or both hybrids. The mu-bcr e19-a2 has been detected in one CML cell line. Four cell lines display a three-way translocation involving chromosomes 9, 22 and a third chromosome. Additional Ph chromosomes (up to five) have been found in four Ph + ALL cell lines and in 18 CML cell lines; though in some cell lines the extra Ph chromosome(s) might be caused by the polyploidy (tri- and tetraploidy) of the cells. Another modus to acquire additional copies of the BCR-ABL fusion gene is the formation of tandem repeats of the BCR-ABL hybrid as seen in CML cell line K-562. Both mechanisms, selective multiplication of the der(22) chromosome and tandem replication of the fusion gene BCR-ABL, presumably lead to enhanced levels of the fusion protein and its tyrosine kinase activity (genetic dosage effect). The availability of a panel of Ph + cell lines as highly informative leukemia models offers the unique opportunity to analyze the pathobiology of these malignancies and the role of the Ph chromosome in leukemogenesis.
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PMID:Leukemia cell lines: in vitro models for the study of Philadelphia chromosome-positive leukemia. 1007 Oct 72

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia chromosome resulting from the translocation t(9-22) producing the chimeric 190 and 210 kDa BCR-ABL fusion proteins. Evolution of the CML to the more agressive acute myelogenous leukemia (AML) is accompanied by increased cellular proliferation and genomic instability at the cytogenetic level. We hypothezised that genomic instability at the nucleotide level and spontaneous error in DNA replication may also contribute to the evolution of CML to AML. Murine Ba/F3 cell line was transfected with the p190 and p210-encoding BCR-ABL oncogenes, and spontaneous mutation frequency at the Na-K-ATPase and the hypoxanthine guanine phosphoribosyl transferase (HPRT) loci were measured. A significant 3-5-fold increase in mutation frequency for the transfected cells relative to the untransfected control cells was found. Furthermore, we observed that BCR-ABL transfection induced an overexpression of DNA polymerase beta, the most inaccurate of the mammalian DNA polymerases, as well as an increase in its activity, suggesting that inaccuracy of DNA replication may account for the observed mutator phenotype. These data suggest that the Philadelphia abnormality confers a mutator phenotype and may have implications for the potential role of DNA polymerase beta in this process.
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PMID:Mutator phenotype of BCR--ABL transfected Ba/F3 cell lines and its association with enhanced expression of DNA polymerase beta. 1034 41

Quantitative competitive RT-PCR techniques have been developed to detect BCR-ABL fusion transcripts in CML but they are hardly reproducible. In this work, we have developed BCR-ABL quantification by real time RT-PCR using the ABI PRISM 7700 (Perkin Elmer), a new technique which allows simple and rapid quantification of a target sequence during the extension phase of PCR amplifications. A fluorogenic probe labeled with both a reporter dye at the 5' end and a quencher-dye at the 3' end hybridizes to the target sequence on the third exon of the ABL gene. The exonuclease activity of the Taq DNA polymerase cleaves the probe and releases the reporter dye, resulting in an increase in the fluorescence signal. The absolute copy number of the target sequence (BCR-ABL) or a control gene (ABL) in an unknown sample can then be calculated using a calibration curve prepared from a set of BCR-ABL RNA standards, and results are expressed as a BCR-ABL/ABL ratio. In our hands, the sensitivity of a serial dilution of total RNA from a positive cell line (K562) in a negative cell line (HL60) was 10(-4). Fifteen CML patients in cytogenetic CR, including 11 allografted patients, two autografted patients and two patients treated by IFN were studied sequentially by this new real time quantitative RT-PCR technique in parallel with conventional qualitative two round nested RT-PCR. The two autografted patients showed high BCR-ABL/ABL ratio in all samples. The two patients treated by IFN showed a progressive decrease in the ratio. In the 11 allografted patients, four were sequentially studied 2 years or more after allo-BMT, and all ratios were below 10(-4). The four patients remained in clinical and cytogenetic CR. The seven other allografted patients were studied immediately after the procedure. Three of them showed a progressive decrease in the BCR-ABL/ABL ratio which reached 10(-4) 7 months after allo-BMT. The three patients remained in hematologic and cytogenetic CR. The remaining four allografted patients had progressive increase of BCR-ABL ratio; three developed cytogenetic relapse 9, 11, 28 months after allo-BMT, and the last patient remained in cytogenetic CR in the bone marrow but developed granulocytic sarcoma. Results of real-time quantitative RT-PCR were in agreement with those of qualitative two round nested PCR. However, evolution changes in the results of real-time quantitative RT-PCR often preceded those of the conventional technique: a decrease of the BCR-ABL/ABL ratio preceded progression from first round to second round positivity and then negativity with the classical technique; conversely, an increase in the ratio preceded evolution with the classical technique. Thus, real-time quantitative RT-PCR may show better correlation with clinical and cytogenetic evolution than conventional qualitative techniques and may help in making early therapeutic decisions in CML, especially after molecular relapse.
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PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using a 'real time' quantitative RT-PCR assay. 1036 Mar 86

Methods of minimal residual disease (MRD) detection in chronic myelogenous leukemia (CML) include chromosome analysis, reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) techniques. We report a novel method to detect intracellular BCR-ABL messenger on single cells using in situ RT-PCR, which can be performed on blood and marrow slides, without extraction of the nucleic acid. After cellular permeabilization and fixation, the mRNA BCR-ABL was reverse transcribed and amplified by PCR using digoxigenin-labelled dUTP. The reaction was revealed with the anti-digoxigenin FITC antibody. On the fluorescent microscope, a strong positive green fluorescence signal was observed in 98-99% cells in Ph1-positive cell lines. A faint signal was detected in 1.5% and 2% of negative cell lines. Likewise, a faint signal was found in 1.6-2.8% of the cells in five normal controls (mean 2.2 +/- 1.1%). The positive threshold for in situ RT-PCR was therefore determined as mean + 2 s.d. = 4.4% cells. We used in situ RT-PCR by comparison to cytogenetics (at least 30 mitoses examined), and two-step RT-PCR (10(-6) sensitivity in our hands) in bone marrow samples from 13 CML patients: two patients at diagnosis and 11 patients in hematological remission after alpha interferon (three patients), hydroxyurea (one patient) autologous bone marrow transplantation (BMT) (one patient) and allogeneic BMT (six patients). In the two diagnostic patients, 90 and 95% cells were respectively strongly positive by in situ RT-PCR. In the six patients treated by allogeneic BMT, the median percentage of positive cells was 2.4% (range 1.8-3.2). All six patients had normal karyotype and negative two-step RT-PCR results. In the five other patients, two were treated by hydroxyurea alone or autologous BMT, and 11 and 13% of the cells were strongly positive; three were treated with interferon and 14-62% of the cells were positive, generally weakly. All five patients had persistence of Ph1 (in 9-56% mitoses), and positive RT-PCR results after one round. In conclusion, in situ RT-PCR can specifically identify cells with BCR-ABL transcript and its results are concordant with those of karyotype and RT-PCR. Because of its limited sensitivity and specificity, however, it appears to have limited value in the analysis of MRD. On the other hand, it can evaluate the presence and intensity of BCR-ABL fusion transcript at the single cell level, and this could be useful in treatment monitoring.
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PMID:Detection of BCR-ABL transcripts in chronic myeloid leukemia (CML) using an in situ RT-PCR assay. 1037 89

Chronic myelogenous leukemia (CML) is characterized by a balanced translocation that leads to the formation of the the BCR-ABL fusion gene. Although autografts can prolong the life of CML patients, patients relapse owing to malignant cells that persist in the graft and the host. This review discusses various experimental strategies that target the BCR-ABL gene or gene products that are downstream of it. Various strategies have been adopted to block BCR-ABL at the gene, mRNA and protein level. One promising strategy involves the cotransduction of a patient's hematopoietic stem cells (HSCs) with anti-BCR-ABL antisense sequences and a drug resistance gene. This might allow for the elimination of any residual disease in the graft or host by chemotherapy while rendering any drug-resistant, malignant CML HSCs functionally normal.
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PMID:Gene therapy for chronic myelogenous leukemia. 1043 Nov 69


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