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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Partially purified platelet DNA polymerase (PDP) was able to synthesize DNA transcripts of platelet polyadenylated RNA. PDP was elevated in the earliest stages of CML and PV. In PV, successful chemotherapy resulted in rapid return of PDP to normal levels while in CML this was not the case. An hypothesis is presented proposing that PDP contributes to oncogenesis by regulating the expression of oncogenes.
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PMID:Platelet DNA polymerase levels in CML: implications for oncogenesis. 620 35

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase which is only found in immature cells of lymphoid lineage (pre-T/pre-B). Because of this restricted distribution of TdT, biochemical and immunofluorescence techniques have been employed to determine the distribution of TdT phenotypes in human leukemias and lymphomas, showing high levels of TdT in approximately 95% of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL), approximately 50% of patients with acute undifferentiated leukemia (AUL), approximately 10 of patients with acute nonlymphoblastic leukemia (ANLL), and approximately 30% of patients with chronic myeloid leukemia (CML) and other myeloproliferative (MPS) or myelodysplastic (MDS) syndromes in blast crisis. High levels of TdT activity are associated with a clinical response to remission inducing therapy with vincristine and prednisone in a high proportion of patients (50%-90%), irrespective of clinical and morphologic diagnosis. Preliminary studies furthermore suggest that TdT might serve as a sensitive indicator of subclinical disease in ALL in complete remission.
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PMID:Methods and clinical relevance of terminal deoxynucleotidyl transferase determination in leukemic cells. 694 40

Terminal deoxynucleotidyl transferase is a unique DNA polymerase that can carry out DNA synthesis on an initiator molecule in the absence of a template. The usefulness of this enzyme as a biological marker for following patients during treatment and remission has been suggested. The potential usefulness of this enzyme in predicting the onset of relapse before any morphological indications has been demonstrated in chronic myelogenous leukemia patients in blast phase of the disease. In order to be able to detect low levels of TdT activity especially during remission phase, we have used cell separation techniques which can enrich cell populations containing TdT activity. A number of cell separation techniques have been developed to separate different cell types. We have used the techniques of unit gravity sedimentation and free flow electrophoresis to achieve enrichment of TdT positive cell populations. Our results show that up to 20 fold enrichment of TdT activity in normal human bone marrow can be accomplished by using cell separation techniques. With the use of free flow electrophoresis, we have achieved enrichment of TdT positive cell populations from normal human bone marrow, cells from patients with acute lymphoblastic leukemia and chronic myelogenous leukemia in blast phase of the disease. No TdT positive cells were detected in patients with acute myelogenous leukemia. These cell separation techniques should prove to be useful in early detection of relapse in patients in remission.
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PMID:Enrichment of terminal deoxynucleotidyl transferase activity by cell separation. 698 Dec 92

We have developed an in vitro assay to assess and predict the potential efficacy of in vivo interferon-alpha (IFN-alpha) treatment (5 x 10(6) units/m2 per day) for patients with chronic myelogenous leukemia (CML). Although determining the numbers and affinities of IFN-alpha receptors on CML cells has been developed as a method for predicting treatment response to IFN-alpha, it fails to predict response in CML. Previously, we and others observed that mitogens, toxins and lectins that bind to cell-surface receptors are endocytosed, escaping endosomes in order to act directly on cellular targets. Therefore, we tested the ability of low concentrations of IFN-alpha (5-10 units) to act directly on DNA polymerase (Pol) in purified chromatin nucleoprotein complexes (NPC). NPC were prepared by a methodology that uses direct treatment of leukocyte nuclei with MspI to generate six NPC-containing fractions (S1, M1, S2, M2, 0.1K and R). We found three general categories of in vitro DNA synthesis response for the six different NPC fractions isolated from the white blood cells of patients with CML (n = 19) before their treatment with IFN-alpha. IFN-alpha induced either stimulation, inhibition or had no apparent effect on Pol activity in the six different NPC fractions in a blind assay. In most of the NPC fractions isolated from the leukocytes of patients with progressive CML and in those from CML patients who failed to show a clinical response to IFN-alpha, this cytokine stimulated or had no effect on Pol activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Interferon-alpha directly inhibits DNA polymerase activity in isolated chromatin nucleoprotein complexes: correlation with IFN-alpha treatment outcome in patients with chronic myelogenous leukemia. 754 66

The induced expression of multiple drug resistance (MDR)-associated genes as a direct response of tumor cells to antineoplastic drugs could be an important factor influencing the success of cancer chemotherapy. We investigated the effects of such compounds on mdr1/P-glycoprotein (P-gp) gene expression and drug sensitivities in the T-lymphoblastoid human cell line CCRF-CEM and MDR sublines. Thereby, we observed that actinomycin D or adriamycin administered at sublethal concentrations induced increases of mdr1 mRNA levels and resistance within 72 h. Furthermore, on leukemia cell samples collected before and after chemotherapy we checked by a complementary DNA polymerase chain reaction (cDNA-PCR) approach for similar alterations in the relative expression levels of the MDR-associated genes (a) mdr1/P-gp (b) mrp (MDR related protein), and (c) the topoisomerase II isoforms alpha and beta. We found a concomitant increase in mdr1 and mrp gene expression combined with a decreased expression of topoisomerase II alpha in the course of the second relapse of an acute lymphoblastic leukemia (ALL). This points to the emergence of at least three different MDR mechanisms in this type of leukemia unresponsive to chemotherapy. A chronic myeloid leukemia (CML) in blast crisis, however, showed combined increases in mdr1 (about 20-fold) and mrp (about four fold) gene expression after intense but unsuccessful chemotherapy over a 6-month period. Our results indicate the occurrence of induced resistance in vitro and in vivo and suggest a contribution of the newly identified ATP-binding cassette (ABC) transporter MRP in MDR.
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PMID:Drug-induced changes in the expression of MDR-associated genes: investigations on cultured cell lines and chemotherapeutically treated leukemias. 791 48

The purpose of this study was twofold: (1) to develop an optimized, reliable method for the flow cytometric analysis of the intranuclear DNA polymerase, terminal deoxynucleotidyl transferase (TdT) in acute myeloid leukemia, and (2) to establish the usefulness of a novel, fluorescein-isothiocyanate conjugated monoclonal anti-TdT antibody (HT-6) in double-fluorescence staining for surface antigens in the characterization of leukemic cells. Inclusion of an aldehyde blocking buffer in the staining protocol reduced background fluorescence sufficiently to allow for the detection of the low-level fluorescent TdT+ myeloblasts. When admixed to normal peripheral blood mononuclear cells, 0.4-0.5% of HLA-DR+ or myeloid surface antigen+, TdT+ double-stained myeloblasts could be reliably detected above background levels. Flow cytometric TdT measurements using the HT-6 antibody in 55 patients with TdT+ acute lymphocytic or myelocytic leukemia or blast crisis of chronic myelogenous leukemia were equal or superior to the results obtained with a mixture of monoclonal anti-TdT antibodies (anti-HTDT-Mix) and comparable to those obtained by the conventional slide method employing polyclonal rabbit anti-human TdT antiserum. This flow cytometric TdT determination in combination with surface antigen staining using a novel anti-TdT monoclonal antibody (HT-6) allows for the recognition of minimal leukemic blast cells during clinical remission in acute myeloid leukemia.
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PMID:Detection of terminal transferase in acute myeloid leukemia by flow cytometry. 792 95

We studied the proliferative activity of leukemic cells obtained from the peripheral blood and bone marrow of 34 patients; 30 with acute leukemia and 4 with chronic myelogenous leukemia in blastic crisis. Flow cytometry was performed using monoclonal antibody against DNA polymerase alpha. Since fresh and frozen cells showed virtually identical DNA polymerase alpha-positive populations and flow cytometric histograms, 52 cryopreserved samples (25 from peripheral blood and 27 from bone marrow) were used in this study. The DNA polymerase alpha-positive population ranged from 20.4% to 84.7% in peripheral blood, and from 6.5% to 92.1% in bone marrow. A positive correlation (r = 0.76, P < 0.01) was found between DNA polymerase alpha-positive populations in peripheral blood and bone marrow from the same patient. This suggests that the DNA polymerase alpha-positive population in the bone marrow can be estimated from that in peripheral blood. No relationship was observed between the positive population and the response to chemotherapy. Statistical analyses for all cases showed no relationship between the DNA polymerase alpha-positive population and either the tumor cell count or time to reach a nadir. However, a negative correlation was observed between the positive population in bone marrow samples and the time to reach a nadir (r = -0.64, P < 0.05) in those patients who achieved a complete response. In addition, in the cases of acute non-lymphocytic leukemia who did not respond to chemotherapy, a positive correlation was observed between the tumor cell count in bone marrow and the DNA polymerase alpha-positive population (r = 0.93, P < 0.01). Thus, the method described here provides a simple and time-efficient means of detecting the proliferative activity of leukemic cells, which is a useful parameter in the treatment of leukemia.
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PMID:Flow cytometric detection of proliferative cells in leukemias. 814 1

The bcr-abl fusion gene is the hallmark of chronic myeloid leukaemia (CML) and presumably the cause of its development. Accordingly, long-term disappearance of the bcr-abl gene after intensive therapy suggests that a patient is probably cured of CML. The diagnostic protocol based on coupling of two enzymatic reactions, reverse transcription (RT) and nested polymerase chain reaction (nPCR), for the detection of bcr-abl transcripts in peripheral blood provides a powerful tool for minimal residual CML detection. We have developed a new detection protocol using rTth DNA polymerase as the only enzyme catalysing both reactions for simplifying CML diagnosis. We demonstrate its efficacy investigating residual leukaemic cells in the peripheral blood of 10 patients. This assay offers several advantages over the use of conventional RT-PCR, being more sensitive, faster, less prone to false positives since no opening of the tube is required between the two reactions and requires no special oils or waxes. Our simple assay for bcr-abl chimeric transcripts detection is a practical addition to the diagnostic evaluation of the patient with chronic myeloid leukaemia.
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PMID:Detecting residual bcr-abl transcripts in chronic myeloid leukaemia patients using coupled reverse transcriptase-polymerase chain reaction with rTth DNA polymerase. 977 68

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


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