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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chronic myelogenous leukemia is a disease of the pluripotent stem cell that involves the myeloid and, to a varying degree, the lymphoid compartment. We studied the involvement of B cells in chronic myelogenous leukemia at diagnosis and during treatment. B lymphocytes were immortalized by infection with Epstein-Barr virus. B-lymphoid cell lines could be established from 25 patients suffering from Philadelphia-chromosome (Ph1)-positive chronic myelogenous leukemia. The cell lines were tested for expression of the typical 210-kDa fusion protein, p210, using Western-blot analysis, and/or for mRNA expression of bcr-abl fusion genes, using reverse transcriptase polymerase chain reaction analysis. At diagnosis, mosaicism of B cells was demonstrated in every patient. During treatment with interferon alpha, p210-expressing B-lymphoid cell lines could not be established from 8 of 8 patients. Following discontinuation of IFN-alpha therapy, p210-positive cell lines were found early, even before cytogenetic recurrence. Resistance to IFN-alpha therapy and progression of the disease were both associated with the appearance of p210-positive cell lines. Cell lines established from 3 healthy individuals and from patients suffering from Ph1-negative diseases did not show p210 expression in Western blots. Our data suggest that B lymphocytes are involved early in the disease, and that B-cell mosaicism may be a sensitive marker for resistance to IFN-alpha therapy and disease progression.
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PMID:Mosaicicm in bcr-abl protein expression in B cells in chronic myelogenous leukemia. 893 37

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

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.
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PMID:2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous leukemia cells. 983 40

Improvement in diagnostic cytogenetic techniques has led to the recognition of an increasing number of leukemia-associated chromosomal translocations and inversions. These genetic lesions frequently are associated with the disruption of putative transcription factors and the production of hybrid transcripts that are implicated in leukemogenesis. Epidemiologic evidence suggests that some, but not all, individuals with a history of gamma-irradiation exposure are at increased risk of developing chronic myeloid leukemia (CML). CML is characterized by the Philadelphia chromosome and transcription of the resulting hybrid BCR-ABL gene. Utilizing the leukemia-associated BCR-ABL p210 transcript as a marker, we sought differences in the induction of illegitimate genetic recombination following high-dose gamma-irradiation of karyotypically normal lymphoblastoid cell lines (LCL) derived from individuals with and without a history of myeloid leukemias. Six LCL [4 leukemia patient derived [2 acute myeloid leukemia and 2 CML] and 2 from normal individuals were analyzed with reverse transcriptase polymerase chain reaction for BCR-ABL under stringent conditions following exposure to 0, 50, or 100 Gy of LET gamma-irradiation delivered via a Varian linear accelerator at 4 MV. Transcripts identical to disease-associated b2a2 and b3a2 transcripts were detected both spontaneously (background illegitimate genetic recombination) and following gamma-irradiation. Background BCR-ABL positivity was demonstrable in 4 of the 6 LCL, with no significant difference in detection between leukemic- and nonleukemic-derived LCL. Overall, increasing gamma-irradiation dose resulted in an increased frequency of BCR-ABL transcript detection (0 Gy vs 50 Gy vs 100 Gy,p = 0.0023, Chi-square test). Within the leukemic- but not the nonleukemic-derived LCL there was significantly greater BCR-ABL positivity after gamma-irradiation compared to unirradiated equivalents. Furthermore, the BCR-ABL positivity of both the AML- and CML-derived LCL after gamma-irradiation was significantly greater than that of the nonleukemic-derived LCL after gamma-irradiation. We speculate that this difference in the detection of illegitimate after gamma-irradiation recombination may be due to aberrant DNA double strand break repair mechanisms in individuals predisposed to the development of myeloid leukemias.
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PMID:Leukemia patient-derived lymphoblastoid cell lines exhibit increased induction of leukemia-associated transcripts following high-dose irradiation. 1048 Apr 30

We studied lineage-specific chimerism and minimal residual disease (MRD) in sequential posttransplant samples from 55 patients who underwent unmanipulated (n = 44) or partially T-cell-depleted (n = 11) allogeneic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Chimerism was assessed by polymerase chain reaction (VNTR [variable number of tandem repeats]-PCR) analysis in highly purified CD19+, CD3+, CD15+, and CD56+ cell fractions, whereas MRD was investigated in whole blood by reverse transcriptase-PCR (RT-PCR) of both p210(BCR-ABL) and p190(BCR-ABL) hybrid transcripts. Of 55 patients, 14 (including 6 T-cell-depleted patients) had cytogenetic relapse at 5-80 months and progressed to hematologic relapse, while 41 patients remained in prolonged cytogenetic remission 12-107 months post-BMT. Before leukemia recurrence, patients in the relapse group showed a consistent evolution pattern sequentially featured by persistent p210(BCR-ABL) positivity, increasing mixed chimerism (MC) in myeloid cells, p190(BCR-ABL) positivity, and, finally, cytogenetic relapse. Myeloid MC preceded cytogenetic relapse by 2-12 months, whereas p190(BCR/ABL) was detected 1-6 months prior to cytogenetic relapse in 11 patients and concomitant with cytogenetic relapse in 3 patients. In the remission group, all patients invariably tested negative for p190(BCR-ABL); 10 patients tested positive for p210(BCR-ABL) at variable time-points but showed persistent full donor chimerism (DC), whereas 31 patients tested p210(BCR-ABL) negative and displayed full DC or transient MC due to the persistence of recipient T cells. Two patients in the relapse group were successfully reinduced into molecular remission with donor lymphocyte infusion. Sequential molecular analysis after such treatment showed the inverse pattern to that observed prior to relapse, ie, progressive disappearance of p190(BCR-ABL) transcripts, conversion of myeloid chimerism to donor type, and, finally, p210(BCR-ABL) negativity. We conclude that lineage-specific chimerism and p190(BCR-ABL) messenger RNA (mRNA) analyses contribute a better characterization of CML evolution after BMT and enable early identification of patients at the highest risk of relapse. (Blood. 2000;95:2659-2665)
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PMID:Molecular analysis of lineage-specific chimerism and minimal residual disease by RT-PCR of p210(BCR-ABL) and p190(BCR-ABL) after allogeneic bone marrow transplantation for chronic myeloid leukemia: increasing mixed myeloid chimerism and p190(BCR-ABL) detection precede cytogenetic relapse. 1075 48

The BCR/ABL fusion gene is pathognomonic for chronic myelogenous leukaemia (CML). We have previously reported alternative splicing of BCR/ABL, as indicated by the detection of both p190- and p210-encoding transcripts, in about 60% of CML patient samples. These exon-skipping events involved the joining of ABL exon 2 to variable upstream BCR exons. Similarly, ABL exon 2 is alternatively spliced to either of two upstream ABL exons (1a or 1b) in c-ABL. We have constructed BCR and BCR/ABL minigenes to study this phenomenon in more detail. These constructs were transfected into various cell types and splicing was assessed by reverse transcriptase PCR. Whereas the basic BCR minigene expressed exon-inclusive transcripts only, insertion of genomic DNA spanning ABL exon 2 induced exon-skipping but only when expressed in the CML cell lines K562 and EM3. In this study we localized the required sequence element to ABL exon 2 itself. These results mimic the splicing phenotype displayed by most CML patients. We propose a model where a trans-factor present in some CML cells interacts with ABL exon 2 pre-mRNA to promote skipping of upstream BCR exons.
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PMID:Exon-skipping in BCR/ABL is induced by ABL exon 2. 1079 14

The Philadelphia (Ph) chromosome is observed in approximately 1% of patients with acute myeloblastic leukaemia (AML), especially subtypes M1 and M2 in the French-American-British classification. We describe here a cytogenetic and molecular investigation of a rare case with Ph-positive AML M6 (erythroleukaemia). A 63-yr-old woman was diagnosed as having erythroleukaemia. Leukaemic cells were positive for CD4 and CD7 as well as CD13, CD33, CD34 and HLA-DR. They were analyzed by G-banding, fluorescence in situ hybridization (FISH), Southern blot and reverse transcriptase polymerase chain reaction analyses. The karyotypes at diagnosis were as follows: 61, XX, -X, -1, -2, -3, -4, -5, -7, t(9;22)(q34;q11)x 2, -15, -16, -17, -18, + 19, +21, +22 [3]/61, idem, -22, +der(22)t(9;22) [36]. FISH with BCR/ABL probes showed that 39% and 57% of interphase nuclei had double and triple BCR/ABL fusion signals, respectively. Chromosome analysis in complete remission showed a normal karyotype in all 20 metaphases, confirming the diagnosis as Ph positive-acute leukaemia. FISH at relapse showed that 92% of interphase nuclei had triple fusion signals. Rearrangement of major breakpoint cluster region (M-bcr) in the BCR gene and coexpression of p210-type (b2a2) and p190-type (e1a2) BCR/ABL fusion transcripts due to alternative splicing were also detected. We conclude that clonal evolution from double to triple Ph chromosomes may be implicated in the disease progression. Considering other two reported cases, Ph-positive erythroleukaemia appears to be correlated with coexpression of myeloid/T-lymphoid markers and hyperdiploidy with double or triple Ph chromosomes, although breakpoints in the BCR gene are heterogenous.
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PMID:Triple Philadelphia chromosomes with major-bcr rearrangement in hypotriploid erythroleukaemia. 1100 54

We designed a novel multiplex in-cell reverse transcription-polymerase chain reaction method for the simultaneous detection and differentiation of p190 and p210 BCR-ABL mRNAs within single cells from the human chronic myeloid leukemia and Philadelphia positive acute lymphoblastic leukemia. Human K562 chronic myeloid leukemia and SUP B-15 Ph+ acute lymphoblastic leukemia cell lines were used as positive controls for p210 and p190 BCR-ABL mRNAs, respectively. HL60 cell line was used as a negative control. After the leukemia cells were fixed and permeabilized, without extracting nucleic acids, the mRNAs were reverse transcribed to cDNAs, and the cDNAs were amplified by multiplex polymerase chain reaction with fluorescent primers specific for p190 and p210 BCR-ABL mRNAs. After transfer onto glass slides by cytospin, the amplified cells were detected by fluorescence microscopy. Fluorescence microscopy after propidium iodide or 4',6-diamidino-2-phenylindone counterstaining showed that the positive K562 cells exhibited a yellow-green fluorescent cytoplasm around a red nucleus, and that the positive SUP B-15 cells exhibited an orange cytoplasm around a blue nucleus. Only the red or blue nucleus was visible in respective negative HL60 cells. The specificity of amplification was confirmed by the absence of a signal when control experiments were performed either with RNase digestion of mRNA or without reverse transcriptase/Taq polymerase. We conclude that the multiplex in-cell reverse transcription-polymerase chain reaction method is capable of simultaneously detecting and differentiating the p210 and p190 BCR-ABL mRNAs of chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia cells, and that it may be useful in quantitatively monitoring the minimal residual disease during therapy.
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PMID:Multiplex in-cell reverse transcription-polymerase chain reaction for the simultaneous detection of p210 and p190 BCR-ABL mRNAs in chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia cell lines. 1109 54

There are two major forms of the BCR/ABL fusion gene, involving ABL exon 2, but including different exons of BCR gene. The transcripts b2a2 or b3a2 code for a p210 protein. Another fusion gene leads to the expression of an e1a2 transcript, which codes for a p190 protein. Another, less common fusion gene is c3a2[e19a2], which encodes a p230 protein. The incidence of one or the other rearrangement in chronic myeloid leukaemia (CML) patients varies in different reported series. This study was designed to determine the frequency of coexpresion of the p210, p190 and p230 transcripts in 250 Mexican patients with CML. We performed nested and multiplex reverse transcriptase polymerase chain reaction (RT-PCR) on bone marrow samples from adult patients and found that all cases were positive for some type of BCR/ABL rearrangement. In 226 (90.4%) patients it was p210, while the remaining 9.6% showed coexpression or one of the transcripts of p190/p210/p230. In 7% of patients with p210 expression there are both isoforms (b3a2/b2a2), presumably the result of alternative splicing. The rate of coexpression of the p190/p210 transcripts was 5%, which is much lower than in other reports. This may be due to the technical factors. These patients had high platelet counts, marked splenomegaly and chromosomal abnormalities in addition to Ph'. Other types of coexpression seen were p210/p230 and p190/p210/p230, in patients with high-risk clinical factors. Our study confirms the occurrence of coexpression of different BCR/ABL transcripts, although the rate (9.6%) was much lower than has been reported in other populations. This may reflect either the sensitivity of the detection techniques used or the possibility of genetic differences between the populations studied. Coexpression may be due to alternative splicing or to phenotypic variation, with clinical courses different from classical CML.
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PMID:BCR/ABL p210, p190 and p230 fusion genes in 250 Mexican patients with chronic myeloid leukaemia (CML). 1206 77

Chronic myelogenous leukemia is characterized by the presence of the reciprocal t(9;22)(q34;q11) in which c-abl located on chromosome 9, and the bcr locus located on chromosome 22, are disrupted and translocated creating a novel bcr-abl fusion gene residing on the derivative chromosome 22. In most cases, the breakpoint in abl occurs within intron 1. Depending on the breakpoint in bcr, exon 2 of abl (a2) joins with exons 1 (e1), 13 (b2), or 14 (b3), or rarely to exon 19 (e19) of bcr resulting in chimeric proteins of p190, p210 and p230, respectively. Currently, several multiplex real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays for detecting bcr-abl are available to assess the levels of the three common fusion transcripts, b2a2, b3a2 and e1a2. Although these assays circumvent the requirement for individual fusion sequence quantitative polymerase chain reaction-based assays, they do not identify the specific fusion transcript. Knowledge of the latter is useful to rule out false-positive results and to compare clones before and after therapy. We designed a novel multiplex real-time RT-PCR assay to detect bcr-abl that allows accurate quantification and determination of the specific fusion transcript. In this assay, abl primer labeled at its 5' end with the fluorescent dye NED (Applied Biosystems) is incorporated into the bcr-abl fusion product during amplification. The NED fluorescent dye in abl primer, without interfering with fluorescent TaqMan probe signal, allows subsequent identification of the fusion transcript by semiautomated high-resolution capillary electrophoresis and GeneScan analysis.
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PMID:TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type. 1465 55


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