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)

Human lung cancers of distinct histology exhibit different responses to radiation therapy in vivo. For examination of the basis of this phenomenon, the radiation survival curves and levels of relevant enzymes were determined in 16 lung cancer cell lines derived from tumors of different histology. These included lines from 5 adenocarcinomas, 7 small cell tumors, 3 variant small cell tumors, and 1 large cell tumor. These findings were compared to those obtained with the use of a normal skin fibroblast cell line. Whether cloned in liquid culture or soft agarose, cell lines had similar radiation survival curves. These curves were consistent with the apparent in vivo radiation responsiveness of the tumors. Although considerable heterogeneity in radiation survival curves was observed among the cell lines, cells from large cell lines and small variant lines had pronounced shoulders and extrapolation numbers (n) from 5.6 to 14. In contrast, cells from small cell lines and adenocarcinoma cell lines were more "sensitive" (-n values of 1-3.3). In these cell lines, levels of DNA polymerase beta, glutathione (GSH), GSH transferase, GSH reductase (NAD(P)H), gamma-glutamyltransferase did not correlate with radiation parameters of sensitivity. DNA polymerase beta and GSH levels were, however, higher than those in a line of normal skin fibroblasts. These cell lines may be useful in identifying the basis of the variable responsiveness of human lung cancer cells to ionizing radiation.
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PMID:Heterogeneity in the radiation survival curves and biochemical properties of human lung cancer cell lines. 614 44

6-(p-n-Butylanilino)uracil and N2-(p-butylphenyl)guanine inhibited the activity of DNA polymerase alpha from calf thymus but had no effect on other eukaryotic polymerases (DNA polymerases beta and gamma) or Escherichia coli DNA polymerase I. Inhibition was competitive with deoxyguanosine 5'-triphosphate and did not occur in the reaction of DNA polymerase alpha with a template that did not contain cytosine residues. The results support a mechanism which involves hydrogen bonding of inhibitors with cytosines in the DNA template and binding with an inhibitor specific site on the enzyme. A screen of inhibitor effects on normal and cancer cell growth in culture showed that cells were not uniformly sensitive to these compounds, a mouse lymphoma line being least sensitive and a human lung cancer line being most sensitive. It is suggested that these inhibitors may be useful to probe possible structural differences among DNA polymerases alpha.
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PMID:Inhibition of calf thymus DNA polymerase alpha and of normal and cancer cell growth by butylanilinouracil and butylphenylguanine. 675 79

The development of non-P-glycoprotein-mediated multi-drug resistance is a frequent event among lung-cancer cell lines. In an attempt to understand the underlying mechanisms of this phenotype, we have selected a multi-drug-resistant subline (POGB/DX) in vitro for doxorubicin resistance. The original cell line (POGB) was established in vitro from a non-treated patient with a small-cell lung cancer. POGB/DX cells were cross-resistant to other drugs, associated with MDR phenotype. In contrast, they were not resistant to taxol, camptothecin or melphalan, but were instead hypersensitive to 5-fluorouracil. Although expression of the mdr-1 gene was not detected in POGB/DX cells, cellular pharmacokinetics showed a reduced drug accumulation and altered intracellular localization in the POGB/DX cell line. This defect in drug accumulation was associated with overexpression and amplification of the MRP gene. Interestingly, verapamil, a known modulator of P-glycoprotein function, was able to reverse drug resistance and to increase drug accumulation. In Northern-blot analysis no differences in expression of topoisomerase I and II (alpha and beta), DNA polymerase beta, or HSP70 and HSP60 genes were observed between POGB and POGB/DX. Coupled to lack of changes in expression of known resistance factors, overexpression of MRP and modulation by verapamil strongly support a role for this gene product in the development of drug resistance in this SCLC cell system. This study provides evidence that (a) altered cellular pharmacokinetics is related to MRP expression; (b) MRP-mediated phenotype is characterized by a specific pattern of cross-resistance, which does not involve taxol; and (c) verapamil may be effective in modulating the function of the MRP gene product.
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PMID:MRP gene overexpression in a human doxorubicin-resistant SCLC cell line: alterations in cellular pharmacokinetics and in pattern of cross-resistance. 760 72

A retroviral vector system was developed to transduce a K-ras antisense construct efficiently into human cancer cells. A 2-kb fragment of K-ras gene DNA in antisense orientation was linked to a beta-actin promoter and inserted into retroviral vector LNSX in two different orientations. The constructs were transfected into amphotropic packaging cell line GP+envAm12 followed by alternating transduction between the ecotropic packaging cell line psi-2 and GP+envAm12. Titers up to 9.7 x 10(7) colony-forming units (cfu)/ml were achieved without detectable replication-competent virus. The human large cell lung carcinoma cell line H460a, which has a homozygous codon 61 K-ras mutation, was transduced with an efficiency of 95% after five to seven repeated transductions. DNA polymerase chain reaction (PCR) and genomic DNA Southern blot analysis showed that the retroviral construct was integrated into the genome of H460a cells. K-ras antisense RNA expression was detected in the cells by Northern analysis, slot blot hybridization, and reverse transcriptase-PCR. Translation of the mutated K-ras p21 protein RNA was specifically inhibited, whereas expression of other p21 species was unchanged. Proliferation of H460a cells was suppressed 10-fold following transduction by the antisense construct. Colony formation in soft agarose and tumorigenicity in an orthotopic lung cancer model in nu/nu mice were dramatically reduced in H460a cells expressing antisense K-ras. We conclude that an antisense construct for K-ras can be expressed effectively in a retroviral vector that can efficiently transduce human cancer cells.
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PMID:Retroviral vector-mediated transduction of K-ras antisense RNA into human lung cancer cells inhibits expression of the malignant phenotype. 839 92

The pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and dosage and administration of gemcitabine are reviewed. Gemcitabine is a deoxycytidine-analogue antimetabolite with activity against some solid tumors. Gemcitabine is phosphorylated intracellularly to difluorodeoxycytidine triphosphate, which terminates DNA-chain elongation and competitively inhibits DNA polymerase and ribonucleotide reductase. After i.v. administration, gemcitabine is rapidly distributed into total body water. The drug is deaminated in the plasma to inactive difluorodeoxyuridine; both gemcitabine and difluorodeoxyuridine are primarily renally eliminated. In clinical studies, gemcitabine reduced pain and improved function in patients with advanced pancreatic cancer. Gemcitabine has shown some activity against non-small-cell lung cancer, particularly when combined with cisplatin or ifosfamide. The agent has also shown modest activity against advanced ovarian and breast cancer. Adverse effects include dose-limiting myelosuppression, flu-like symptoms, nausea, vomiting, and rash. Gemcitabine has FDA-approved labeling for use in the treatment of locally advanced and metastatic pancreatic cancer. The recommended dosage for this indication is 1000 mg/m2 (as the hydrochloride salt) i.v. given over 30 minutes weekly for seven weeks, followed after one week of rest by 1000 mg/ m2 i.v. given over 30 minutes weekly for three weeks every four weeks. Gemcitabine palliates symptoms in patients with advanced or metastatic pancreatic cancer. More study is needed to determine gemcitabine's role in the treatment of non-small-cell lung cancer, ovarian cancer, and breast cancer.
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PMID:Gemcitabine: a cytidine analogue active against solid tumors. 911 4

In situ PCR is a new technique for the localization of low copy number sequences. We report here a method for the in situ visualization of a point mutation in K-ras codon 12 by indirect in situ PCR. Twenty-five primers were examined to select mutant-specific primers. Harvested cell lines were fixed and suspended in PCR mixture. Forty cycles of PCR in cell suspension was performed in a thermal cycler using a hot start method. Cells were cytocentrifuged onto slides, and post-fixation was performed. The specimens on the slides were then hybridized with a digoxigenin-labeled probe, followed by color reaction. Both Calu-1 (mutated: TGT) and NCI-H460 (wild type: GGT) cells had strong hybridization signals in the nuclei with general primers. But with mutant-specific primers, only Calu-1 cells had hybridization signals. No signal was observed without primers or Taq DNA polymerase. Southern blotting of the same preparation confirmed desired amplification. We also applied direct in situ PCR, but this method failed to detect the point mutation. We conclude that our indirect in situ PCR method shows the feasibility of in situ identification of single cells carrying point mutations.
Lung Cancer 1997 Jul
PMID:Detection of K-ras point mutation by in situ PCR in cell suspensions: comparison of the indirect and direct methods. 923 54

DNA polymerase beta (pol beta) provides most of the gap-filling synthesis at apurinic/apyrimidine sites of damaged DNA in the base excision repair pathway. A truncated form of the pol beta protein is expressed in colon and breast cancers. However, the role of the pol beta gene in lung cancer is not known. Thus, we investigated a possible occurrence of pol beta variants in primary lung tumors. The entire cDNA of pol beta obtained by RT-PCR amplification was analyzed for nucleotide sequencing in lung tumor and matched normal lung tissue of the same patient. Three types of variants were detected in squamous, non-small, or large cell carcinomas. The most common variant was a deletion of 87 bp from pol beta cDNA at a site corresponding to exon 11. In addition, a variant exhibiting deletions of 87 and 140 bp together with an insertion of 105 bp was identified in three lung tumors. This is the first report of the occurrence of pol beta variants, possibly splicing variants, in lung cancer. A truncated pol beta protein resulting from variant forms of the gene may impact the function of the enzyme and increase susceptibility to carcinogenesis.
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PMID:Variant forms of DNA polymerase beta in primary lung carcinomas. 1043 53

Telomerase is a ribonucleoprotein DNA polymerase that maintains the telomeric region of chromosomes lost during successive rounds of cell division. We used the telomeric repeat amplification protocol (TRAP) assay to examine telomerase activity in bronchial lavage (BL) samples from individuals undergoing diagnosis of lung cancer. Telomerase activity was detected in 17 (47%) of 36 samples examined. In particular, 16 (70%) of 23 BL specimens obtained from lung cancer patients showed detectable telomerase activity, while only 1 of 13 (8%) specimens obtained from patients without lung cancer demonstrated activity (P=0.00038). Moreover, 9 (90%) of 10 BL specimens, which were cytologically positive for lung cancer, were also positive for telomerase activity, while 7 (54%) of 13 cytologically negative BL specimens for lung cancer showed detectable telomerase activity. Detection of telomerase activity combined with cytology were able to identify 17 (74%) of 23 lung cancer cases whereas cytology alone identified 10 (43%) of 23 such cases (P=0.035). Our findings indicate that telomerase is a specific marker for malignant lung disease and a potential complementary tool to cytology in the diagnosis of certain lung cancer cases.
Lung Cancer 2000 Apr
PMID:Evaluation of telomerase activity in bronchial lavage as a potential diagnostic marker for malignant lung disease. 1070 7

The main mechanism of action of the anticancer drug gemcitabine is assumed to be incorporation of its triphosphate (dFdCTP) into DNA, resulting in inhibition of DNA polymerization, inhibition of DNA synthesis and repair. Another mechanism is inhibition of ribonucleotide reductase leading to imbalance in the deoxyribonucleotide (dNTP) pools. One assay to measure dNTP pools is based on oligonucleotide elongation mediated by DNA polymerase. Since the latter may be affected by dFdCTP, we studied the effect of 0.1-600 pmol dFdCTP on this assay; 10 pmol and more dFdCTP significantly increased the average dpm of the blank (absence of other dNTP) and that of the calibration line of dATP (1.4-1.6-fold); 0.1 pmol and more increased that of the standard dGTP curve significantly (1.1-1.8-fold); 10-75 pmol decreased that of dCTP while 75 and 100 pmol significantly increased that of dCTP (1.3-fold); 50 pmol significantly increased that of dTTP (1.3-1.5-fold). For dATP, dGTP and dTTP, a saturation was reached at 100 pmol dFdCTP, but not yet for dCTP. To minimize these effects, we added an excess of 200 pmol dFdCTP to all samples and calibration lines when measuring dNTP levels of gemcitabine treated samples. In this way the effects of gemcitabine on dNTP levels were studied in human A2780 ovarian, HT29 colon, K562 myelogenous leukemia, H322 non-small cell lung cancer cell lines and the murine lung cancer cell line Lewis Lung. In all cell lines, intrinsic dTTP pools (3-77 pmol/106 cells) were the highest, followed by dATP (1.5-31), dCTP (0.7-27) and (nd-14) dGTP. Exposure to 1 and 10 microM gemcitabine for 4-h concentration dependently decreased dATP 3-10-fold and dGTP to undetectable levels, but dCTP at most 3-fold, while dTTP increased. In conclusion, dFdCTP affects dNTP measurements with the DNA polymerase elongation assay, but its effect could be controlled by addition of similar amounts of dFdCTP to each assay.
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PMID:Interference of gemcitabine triphosphate with the measurements of deoxynucleotides using an optimized DNA polymerase elongation assay. 1140 37

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.
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PMID:Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA. 1167 38


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