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)

Examples of collateral sensitivity, even in experimental tumor systems, remain few. Preliminary data from this laboratory indicated that certain tumor cells expressed increased sensitivity to cisplatin after exposure in vitro to x-irradiation. To further clarify whether the type of fractionated radiation procedure used clinically can induce hypersensitivities to certain antitumor drugs we have pre-exposed the human ovarian carcinoma cell line JA-T/P derived from a tumor from an untreated patient to fractionated x-irradiation (total dose 50 Gy) in vitro. The resultant subline JA-T/DXR-10 expressed collateral sensitivity to cisplatin (CDDP), methotrexate (MTX) and fluorouracil (5-FU), but not to acute x-irradiation. Hypersensitivity to CDDP was associated with decreased activity of DNA polymerase beta (3.5-fold, P less than .01), but unaltered glutathione metabolism. Pre-incubation with cyclosporin A or with 3-aminobenzamide significantly enhanced (twofold, P less than .01) CDDP-induced cytotoxicity in JA-T/P cells, but not in the DXR-10 subline. Consistent with MTX hypersensitivity dihydrofolate reductase activity was significantly decreased (2.9-fold, P less than .01). Despite collateral sensitivity to 5-FU, however, thymidylate synthase activity was increased (twofold, P less than .05) suggesting alternative mechanisms for 5-FU-induced cytotoxicity in these JA-T/DXR-10 cells. These data demonstrate that DNA repair and associated reduced folate metabolism can be modified not only by drugs but also by fractionated x-irradiation.
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PMID:Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro. 155 59

The proliferative activity of various parts of normal and malignant endometrium was evaluated using an immunohistochemical approach and flow cytometry (FCM). The two monoclonal antibodies, Ki-67 and anti-DNA polymerase alpha antibody (anti-poly alpha antibody) were used to detect the proliferative activity of cells, and the percentage of the Ki-67 and anti-poly alpha positive cells were measured. Proliferative indices (PI; percentage of S and G2M phase) and DNA ploidy were measured by FCM. Normal endometrial specimens from 29 patients with benign diseases were used and three different parts (fundus, middle, and low part of the uterus) were examined. In the proliferative phase of normal endometrium, there was no significant difference in the proliferative activity in the three parts. In 20 patients with endometrial carcinomas with myometrial invasion, tissues were taken from the myometrial invasive site and the central part of the tumor tissue. In the cases of endometrial carcinoma, the myometrial invasive site had a higher proliferative activity than central part of the tissue. The proliferative activity measured by the immunohistochemistry was correlated with the histological grade of malignancy, but it was not consistent with PI by FCM. This suggests that the proliferative activity measured by the immunohistochemistry is independent of flow cytometric PI.
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PMID:Proliferative activity in normal endometrium and endometrial carcinoma measured by immunohistochemistry using Ki-67 and anti-DNA polymerase alpha antibody, and by flow cytometry. 157 57

We have investigated the species-specific replication of polyomavirus DNA in the cell-free system that was established previously (Y. Murakami, T. Eki, M. Yamada, C. Prives, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 83:6347-6351, 1986). Extracts from various species of cells supported polyomavirus DNA replication in a species-specific manner that was consistent with the host range specificity of polyomavirus; extracts prepared from mouse and hamster cells were active, whereas extracts prepared from human, monkey, and insect cells were inactive. The addition of DNA polymerase alpha-primase purified from mouse cells induced the replication of polyomavirus DNA in a cell-free system containing polyomavirus large tumor antigen and nonpermissive cell extracts, such as human and insect cell extracts. Isolated mouse DNA primase alone also induced polyomavirus DNA replication in human cell extracts but not in insect cell extracts, indicating that mouse DNA primase plays the principal role in determining permissiveness for polyomavirus DNA replication.
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PMID:Mouse DNA primase plays the principal role in determination of permissiveness for polyomavirus DNA replication. 165 10

Proliferating cell nuclear antigen (PCNA) is a 36-kDa DNA polymerase-delta auxiliary protein which accumulates in the nucleus during S phase of the cell cycle. Immunohistochemical labeling indices (LI) of PCNA and Ki-67 were compared using an avidin-biotin complex method on frozen sections of 27 nervous system tumors. 3 normal cerebral cortices, and 3 peripheral nerves. In glial tumors, PCNA and Ki-67 LI increased with increasing tumor grade (Daumas-Duport system). In 5 low-grade glial tumors, PCNA and Ki-67 LI were less than or equal to 1%, except for one optic nerve glioma (Ki-67 LI = 6%). In 7 grade 3 astrocytomas and 1 mixed glioma, PCNA LI were less than or equal to 1-1.5%, while Ki-67 LI were 2%-10%. In 7 grade 4 astrocytomas and 1 metastatic carcinoma, PCNA LI ranged from 6%-15% while Ki-67 LI ranged from 17%-30%. In 5 of 6 schwannomas, focally high PCNA LI (4%-65%) were noted, despite low LI with Ki-67 (less than or equal to 1.6%). Scattered normal schwann cell nuclei also stained with PCNA, but normal cerebral cortex did not.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Proliferating cell nuclear antigen and Ki-67 immunohistochemistry in brain tumors: a comparative study. 167 78

Biochemical assays for ras mutations are capable of detecting a mutant allele only if it is present in at least 5% of cells tested. Further, ras mutation assays which utilize the polymerase chain reaction (PCR) are unable to distinguish a ras mutation in a small population of cells from mutations resulting from Taq DNA polymerase base misincorporation. We used a standard restriction fragment length polymorphism assay of PCR-amplified c-Ki-ras to detect codon 12 mutations in tumor cells and found a cumulative error frequency for Taq DNA polymerase of one codon 12 mutation per 2 X 10(4) molecules of total amplification product. The Taq polymerase-induced mutations were found to be multiple base transitions and represented a constant proportion of the amplification product at each step of the PCR. The ability to detect the in vitro generated mutation was dependent on the number of thermal cycles and the sensitivity of the detection assay. With these considerations in mind, we developed a two-step RFLP assay in which the thermal cycle number was kept low and molecules containing mutations at codon 12 were selectively amplified in the second step. We were able to detect a ras mutation occurring in 1 per 1000 cells (a two log improvement over standard RFLP methods) without detecting mutations resulting from Taq DNA polymerase infidelity.
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PMID:A method to detect ras point mutations in small subpopulations of cells. 167 14

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
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PMID:Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses. 169 11

DNA polymerase alpha is a DNA replicating enzyme expressed in all proliferating cells. This nuclear antigen in paraformaldehyde-postfixed frozen sections of normal, benign, and malignant tissues was identified by the peroxidase-antiperoxidase technique with the use of a mouse monoclonal antibody against bovine/human DNA polymerase alpha. The nuclei of normal proliferating cells were positive. Malignant tumors (n = 95) showed a higher proportion of positive nuclei than did low-grade malignant tumors (n = 7) or benign lesions (n = 67). The number of positive nuclei in squamous cell carcinomas (n = 19) was higher than in adenocarcinomas (n = 45). Eight (18%) adenocarcinomas and all five renal cell carcinomas had less than 10% positive cells, whereas in benign tissues, such as pituitary adenomas, a thymoma, reactive lymphoid lesions, and some benign mammary nodules, more than 10% of nuclei were labeled. In addition, foci of proliferating cells were clearly recognized. DNA polymerase alpha is, therefore, an excellent marker of proliferative activity that provides an approach to analyzing tumor cell heterogeneity not only in fully developed neoplasms, but also in their precursor lesions.
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PMID:DNA polymerase alpha. An immunohistochemical marker for proliferating cells in normal and neoplastic human tissues. 169 87

The mechanism of tumor cell killing by HO-221, a novel benzoylphenylurea derivative that shows broad-spectrum antitumor activities, was studied. HO-221 strongly inhibited the activity of mammalian DNA polymerase alpha but not that of DNA polymerases beta or gamma. The inhibition was equivalent to that induced by aphidicolin and ara-CTP, which were selective inhibitors of the enzyme. Furthermore, the inhibition by HO-221 of DNA polymerase alpha was found to be non-competitive with respect to dCTP as a substrate, unlike that induced by aphidicolin and ara-CTP. The inhibition was reduced the addition of an excess of DNA polymerase alpha but not by excess amounts of activated DNA as a template primer. These results suggest that HO-221 inhibits the activity of DNA polymerase alpha by direct interaction with the enzyme in contrast to the impairment of template activity through intercalation into DNA induced by anthracycline compounds. On the other hand, HO-221 showed almost no effect on RNA polymerase activity, the reverse transcriptase activity of avian myeloblastosis virus or protein synthesis in a cell-free system. The flow-cytometry analysis revealed that HO-221 accumulated HL-60 cells in G1-S phases at a low concentration but increased the number of cells in the G1 phase at a higher concentration, stopping cell-cycle progression. The results suggest a correlation between cell-cycle progression and inhibition by HO-221 of DNA polymerase alpha, which plays a role in DNA replication during the S phase in living cells.
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PMID:Mechanism of tumor cell killing by HO-221, a novel antitumor compound. 170 66

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Effects of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5'-triphosphate. 170 52

A new class of compounds, 9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] [(RS)-FPMP] derivatives of purines, is described that has selective activity against a broad spectrum of retroviruses [including human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2)] but not other RNA or DNA viruses. This activity spectrum is completely different from that of the parental compounds, 9-[(2S)-3-hydroxy-2-phosphonylmethoxypropyl] [(S)-HPMP] derivatives of purines, which are active against a broad range of DNA viruses. The racemic (RS)-FPMP derivatives of adenine and 2,6-diaminopurine, termed (RS)-FPMPA and (RS)-FPMPDAP, respectively, are markedly more selective as in vitro antiretroviral agents than their 9-(2-phosphonylmethoxyethyl) (PME) counterparts, PMEA and PMEDAP. Also, (RS)-FPMPA and (RS)-FPMPDAP have a substantially higher therapeutic index in mice in inhibiting Moloney murine sarcoma virus-induced tumor formation and associated death and are markedly less inhibitory to human bone marrow cells than PMEA and PMEDAP. The diphosphate derivative of (RS)-FPMPA [(RS)-FPMPApp] is a potent and selective inhibitor of HIV-1 reverse transcriptase but not of HSV-1 DNA polymerase or DNA polymerase alpha. (RS)-FPMPApp, akin to PMEA diphosphate (PMEApp), acts as a DNA chain terminator. The DNA chain-terminating properties of PMEApp and (RS)-FPMPApp seem to be a prerequisite for acyclic nucleoside phosphonates to exhibit antiretrovirus (i.e., anti-HIV) activity.
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PMID:9-[(2RS)-3-fluoro-2-phosphonylmethoxypropyl] derivatives of purines: a class of highly selective antiretroviral agents in vitro and in vivo. 171 Dec 14


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