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:2.7.1.21 (thymidine kinase)
7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Chemical mutagenesis in animal cells is a complex process. Whereas some chemicals are mutagenic in their original form, others such as the nitrosamines and polycyclic hydrocarbon carcinogens are mutagenic only when enzymatically activated. The active form, or ultimate carcinogen, can interact with proteins and nucleic acids, altering amino acids and producing modified bases in DNA. The modified bases do not usually constitute mutations as produced. Instead they are acted on by the DNA enzymes of the cell, which repair most damaged bases but occasionally insert incorrect base sequences at or near the sites of damage. The frequency at which mutant animal cells are recovered depends upon the selection conditions in culture, upon whether the mutation selected is in a gene present in single or multiple active copies, and upon whether expression is dominant or recessive. Many studies depend on selecting for 8-azaguanine- or 6-thioguanine-resistant mutants, which are due to mutations in the HGPRT locus present in a single active copy on the X-chromosome. Other widely used systems depend on selecting for ouabain resistance, which is dominant and results from a change in the sodium/potassium ATPase activity, or on selecting for thymidine kinase mutants in heterozygous Tk+/Tk- mouse cells. Many other types of mutation including nutritional markers are recessive and express only in cells carrying a single active gene copy, as is sometimes the case in established cell lines. The types of base damage causing mutations have been identified in very few cases only, and little is known about the enzymatic mechanisms of mutagenesis. However, chemical mutagenesis in cultured animal cells provide a practical way of testing chemicals and radiations for mutagenicity directly in animal cells, and much has been learned about the mutagenicity of various carcinogenic substances. To date, there is reasonable qualitative agreement between these results and those obtained in the widely used liver microsome-activated bacterial mutagenesis test systems.
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PMID:International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC working paper 2/5: mutagenesis in mammalian cells. 702 63

P388 mouse leukemia lines, one sensitive (P388/S) and the other resistance (P388/R) to vincristine (VCR), cultured in vitro, were hybridized with polyethylene glycol (PEG). A thymidine kinase-deficient mutant (TK-) was isolated from the sensitive line, and a hypoxanthine-guanine phosphoribosyltransferase-deficient mutant (HPRT-) from the resistant line. The hybrid line grows slower than the mutants. The modal chromosome numbers are: TK- = 38, HPRT- = 40, hybrid = 69 (72). The TK- cells contain a large metacentric marker which is missing from the HPRT- cells. Hybrid cells are as resistant to VCR as the P388/R and HPRT- cells.
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PMID:Drug resistance studies on intraspecific hybridomas. 725 34

A human x Chinese hamster (CH) somatic cell hybrid subclone deficient in HPRT and containing only human chromosome 18 was irradiated with 7000 rad and fused to a thymidine kinase deficient CH cell line. Radiation-rescued hybrid cell lines, selected in HAT medium, were analyzed for human DNA with human interspersed-repeat sequence primers. Size and number of human chromosome fragments retained in a subset of hybrids were determined by FISH. A panel of 98 radiation hybrids (RH) was selected and analyzed for 90 chromosome 18-specific STSs by PCR, and for the D18Z1 centromeric marker by Southern blotting. STSs were developed from previously mapped RFLP loci and from published sequences. In addition, 32 novel STSs were generated from an 18-specific lambda library and from 18-specific YACs previously localized to chromosome bands by FISH. Marker retention frequency varied from 8-65% with an average of 24%. In selected RH the STS typing data were correlated with the chromosome 18 regions retained using 'reverse FISH' of IRS-PCR products from the RH to normal metaphase chromosomes. The order and intermarker distances of loci were determined using two-point and multipoint maximum likelihood methods. The resulting RH map covers most of chromosome 18 with four groups of tightly linked markers and three regions of loosely linked markers, one around the centromere and two on the long arm. More than a third of the markers are polymorphic and allow integration with the linkage map. This RH map provides a framework for establishing a clone contig of the entire chromosome 18.
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PMID:A radiation hybrid map of human chromosome 18. 812 19

Human TK6 lymphoblasts were exposed to X radiation or radon, and thymidine kinase negative (TK-/-) mutants were selected, isolated and harvested for analysis of structural changes in the TK gene. A large majority (82%) of the radon-induced mutants, 74% of the X-radiation-induced mutants and 45% of the spontaneous mutants lost the entire active TK allele. To analyze these mutants further we measured the loss of heterozygosity at several loci neighboring the TK locus on chromosome 17q. A greater proportion (61%) of the radon-induced mutants than X-radiation-induced or spontaneous mutants harbored the smaller lesions involving the TK allele alone or extending from the TK locus to one or both of the closest neighboring sequences tested. Further, 21% of the X-radiation-induced mutants but only 5% of the radon-induced mutants lost heterozygosity at the col1A1 locus, 31 Mb from the TK gene. These results are in agreement with a recent analysis of radon- and X-radiation-induced lesions inactivating the HPRT gene of TK6 cells, in which we reported that a lower percentage of radon- than X-radiation-induced mutants showed lesions extending to markers 800 kb or more from the HPRT gene on the X chromosome (Bao et al., Mutat. Res. 326, 1-13, 1995). In the present study, we observed that the percentage of slowly growing and very slowly growing TK-/- mutants was greater after treatment with radon than after treatment with X radiation, regardless of the type of lesion present. It is possible, therefore, that the radon-induced lesions are complex and/or less easily repaired, leading to slow growth in a large proportion of the surviving mutant cells.
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PMID:Characterization of multilocus lesions in human cells exposed to X radiation and radon. 853 34

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.
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PMID:Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor. 1122 43

Stem cells and their progeny constitute a potential resource for replacing damaged tissues or supplying missing functions, but also pose a threat of aberrant behavior, including neoplastic growth or immunopathology. Suicide genes introduced into these cells before transplantation might provide a means of addressing this threat by permitting the ablation of the cells if they subsequently misbehave. Retroviral transduction of the E. coli gpt and herpes thymidine kinase (HSVtk) suicide genes was used to determine the degree to which stem cells could be sensitized to the prodrugs 6-thioxanthine (6TX) and ganciclovir (GCV) respectively, and whether this sensitivity could persist over many cell generations. The ES-E14TG2a murine embryonic stem cell line was rendered sensitive to quantitative ablation at prodrug concentrations well tolerated by untransduced cells (50 microM 6TX, 1 microg/ml GCV). The HSVtk gene also conferred GCV sensitivity on human mesenchymal stem cells and hematopoietic precursors derived from the murine cells, although ablation was not complete. Because ES-E14TG2a cells are deficient in the cellular enzyme HPRT, they are sensitive to hypoxanthine/aminopterin/thymidine (HAT). This property enhanced the persistence of chemosensitivity in gpt-transduced cells by permitting cells that lost 6TX sensitivity to be ablated with HAT.
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PMID:Suicide gene transduction sensitizes murine embryonic and human mesenchymal stem cells to ablation on demand-- a fail-safe protection against cellular misbehavior. 1208 44

Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.
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PMID:Development of an HIV-based cDNA expression cloning system. 1284 40

Induced genomic instability in the human B lymphoblastoid cell line TK6 manifests itself as increases in end-to-end chromosome fusions and non-reciprocal chromosome translocations. It is not associated with elevated frequencies of specific locus mutations or other cytogenetic alterations. Previous studies on a limited number of cells and end-points suggested that induced instability in TK6 mirrors spontaneous instability in terms of the types of alterations observed. In the present study we expanded on our previous analysis to include more cells and more end-points in order to derive a more precise measure of spontaneous instability in TK6 cells. The frequency of normal growth rate thymidine kinase mutants (TK(-/-)), measured in 44 independently isolated clones, was 2.73 +/- 0.78 x 10(-6)/cell, while that for slow growth mutants was 2.39 +/- 0.52 x 10(-6)/cell. These are similar to the frequencies observed for HPRT mutants in primary human cells. There was wide variation in chromatid break frequencies, but the average break frequency, at 0.04+/-0.01 breaks/cell, was only slightly higher than that reported for primary human cells. In contrast, the dicentric frequency of 0.006/cell was more than 10-fold higher for TK6 cells than that reported for normal primary human cells. Furthermore, the dicentrics in TK6 cells are unusual in that they are the result of end-to-end chromosome fusions. TK6 cells also show much higher levels of non-reciprocal chromosome translocations than are usually observed in primary human cells. The results suggest an inherent instability in TK6 cells that differs from what is observed in primary cells in that it affects the frequency of end-to-end chromosome fusions and non-reciprocal chromosome translocations, but not TK gene mutations or other cytogenetic alterations.
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PMID:Baseline levels of chromosome instability in the human lymphoblastoid cell TK6. 1554 60

Gene expression profiles that are anchored to phenotypic endpoints may lead to the identification of signatures that predict mutagenicity or carcinogenicity. The study presented here describes the analysis of DNA adducts in the human TK6 lymphoblastoid cell line after exposure to N-hydroxy-4-aminobiphenyl, a mutagenic metabolite of 4-aminobiphenyl. A validated nano-LC microelectrospray mass spectrometry assay is reported for the detection and quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP), the principal DNA adduct of 4-aminobiphenyl. Limits of quantification, based on a signal-to-noise ratio of 10:1, are determined to correspond to approximately 27 fg of dG-C8-ABP injected on-column. The assay has been used to measure the steady-state levels of the adduct in the human TK6 lymphoblastoid cell line as a function of dose (0.5, 1.0, and 10.0 microM) and time (2, 6, and 27 h) after exposure to N-hydroxy-4-aminobiphenyl. The levels of dG-C8-ABP adducts in the cells, ranging from 18 to 500 adducts in 10(9) nucleotides, were then correlated to cell toxicity, induced mutation at the TK (thymidine kinase) and HPRT loci, and gene expression profiling through microarray analysis. Cell cultures were evaluated for toxicity by growth curve extrapolation, mutation assays were performed on the HPRT and TK loci, and gene expression profiles were generated by analyses using microarray technology. In the mutation assay analysis, as the toxicant concentration increased, there was an increase in mutation fraction, indicating a direct correlation to metabolite dosing level and mutations occurring at these two loci. Statistical analysis of the gene expression data determined that a total of 2250 genes exhibited statistically significant changes in expression after treatment with N-OH-AABP (P < 0.05). Among the genes identified, 2245 were up-regulated, whereas 5 genes that had functions in cell survival and cell growth and, hence, could be indicators of toxicity, were down-regulated relative to controls. The results demonstrate the value of anchoring gene expression patterns to phenotypic markers, such as DNA adduct levels, toxicity, and mutagenicity.
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PMID:Quantification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human lymphoblastoid TK6 cells dosed with N-hydroxy-4-acetylaminobiphenyl and their relationship to mutation, toxicity, and gene expression profiling. 1697 Mar 17

Vinyl acetate monomer (VAM) produced rat nasal tumors at concentrations in the hundreds of parts per million. However, VAM is weakly genotoxic in vitro and shows no genotoxicity in vivo. A European Union Risk Assessment concluded that VAM's hydrolysis to acetaldehyde (AA), via carboxylesterase, is a critical key event in VAM's carcinogenic potential. In the following study, we observed increases in micronuclei (MN) and thymidine kinase (Tk) mutants that were dependent on the ability of TK6 cell culture conditions to rapidly hydrolyze VAM to AA. Heat-inactivated horse serum demonstrated a high capacity to hydrolyze VAM to AA; this activity was highly correlated with a concomitant increase in MN. In contrast, heat-inactivated fetal bovine serum (FBS) did not hydrolyze VAM and no increase in MN was observed. AA's ability to induce MN was not impacted by either serum since it directly forms Schiff bases with DNA and proteins. Increased mutant frequency at the Tk locus was similarly mitigated when AA formation was not sufficiently rapid, such as incubating VAM in the presence of FBS for 4 hr. Interestingly, neither VAM nor AA induced mutations at the HPRT locus. Finally, cytotoxicity paralleled genotoxicity demonstrating that a small degree of cytotoxicity occurred prior to increases in MN. These results established 0.25 mM as a consistent concentration where genotoxicity first occurred for both VAM and AA provided VAM is hydrolyzed to AA. This information further informs significant key events related to the mode of action of VAM-induced nasal mucosal tumors in rats.
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PMID:Nonlinear responses for chromosome and gene level effects induced by vinyl acetate monomer and its metabolite, acetaldehyde in TK6 cells. 2403 27


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