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:2.4.2.8 (hypoxanthine-guanine phosphoribosyltransferase)
2,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potent mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its mutagenic and recombinagenic activity at the heterozygous thymidine kinase (tk +/-) locus and the hemizygous hypoxanthine phosphoribosyltransferase (hprt +/0) locus in the TK6 human lymphoblastoid cell line. TPA at concentrations of 0.01-1.0 micrograms/ml induced a low frequency of tk mutants showing the slow growth phenotype in a dose-dependent manner, but few normal growth tk mutants or hprt mutants. Concentrations of 1.0-10 micrograms/ml TPA induced all three types of mutants. The molecular structure of tk mutants arising spontaneously or induced by 1.0 and 10 micrograms/ml TPA was investigated by Southern hybridization with a human tk cDNA probe: 86% of all mutants arising after incubation with 10 micrograms/ml TPA lost the entire active tk allele, resulting in loss of heterozygosity (LOH), while 71% of spontaneously arising mutants showed LOH. Densitometric analysis indicated that the majority of LOH mutants induced by TPA were homozygous at the tk locus (retained two copies of the mutant allele), consistent with the occurrence of interchromosomal homologous recombination. These results support the hypothesis that tumor promoters such as TPA may increase the rate of chromosomal mitotic recombination and hence facilitate the segregation of recessive mutations. TPA may thus induce a type of genetic instability during the process of tumor promotion that involves enhanced recombinagenic activity.
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PMID:Recombinagenic activity of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in human lymphoblastoid cells. 763 95

We investigated gene-specific damage in adenocarcinoma cells, obtained from pleural effusions of 9 primary lung cancer patients, induced by incubation with cisplatin for 3 h in vitro. The 2.7 kb fragment of the hypoxanthine phosphoribosyltransferase (HPRT) gene was amplified by the polymerase chain reaction (PCR) to quantify the DNA damage. A 7-fold difference in the extent of gene-specific damage among the patients was observed. Mononuclear cells (MNC) were obtained from freshly isolated blood from the same patients before they received chemotherapy. These cells were also incubated with cisplatin in vitro, and PCR amplification of the HPRT gene was carried out. A 4-fold variation of DNA damage among the patients was observed. Moreover, there was a linear correlation between the extents of the DNA damage in the tumor cells and MNCs (R2 = 0.676, P = 0.0016). These results suggest that the PCR-stop assay could be used to detect interindividual variations in the extent of gene-specific damage in both tumor cells and MNC from the same patients induced by cisplatin treatment. In conclusion, MNC could be used to analyze cisplatin-induced gene-specific damage in cancer patients whose tumor cells are inaccessible.
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PMID:Correlation of gene-specific damage with cisplatin between human adenocarcinoma cells and peripheral blood mononuclear cells analyzed by polymerase chain reaction-stop assay. 773 Jan 49

Cysteine conjugate beta-lyase, an enzyme that converts cysteine S-conjugates to free thiols, pyruvate and ammonia, is normally expressed primarily in the liver and kidney. In theory, this selective distribution affords the opportunity to target thiol-containing drugs to these organs and, perhaps, to tumors derived from them. To assess the potential for delivery of such drugs to kidney-derived tissue, we have used a typical beta-lyase substrate, S-(2-benzothiazolyl)-L-cysteine, to measure the beta-lyase activity in normal and tumor tissue of kidneys removed from patients with renal carcinoma. Although considerable heterogeneity in enzyme activity levels was observed in normal and tumor-derived samples, a high proportion of tumor samples had enzyme activity that was at least 50% of that observed in adjacent normal tissue. Frequently, hypoxanthine-guanine phosphoribosyltransferase activity was observed to be greater in the tumor than in normal tissue. These results may aid in the development of therapy for renal carcinomas.
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PMID:Cysteine conjugate beta-lyase activity in human renal carcinomas. 776 99

Loss or inactivation of tumor suppressor genes has been implicated by indirect methods in the etiology of most human cancers. In the functional studies presented here, tumor suppressors on human chromosome 1 were investigated using microcell-mediated chromosome transfer. Translocated chromosomes from normal human cells representing most of 1q, or all of 1p and a small portion of 1q translocated onto the region of the X chromosome encoding HPRT, were transferred into human fibrosarcoma cell line HT1080. Analysis of HT1080 microcell hybrids showed a tumor suppressor activity associated with 1q. All HT1080 cells carrying transferred 1q in a ratio of 1:1 with the HT1080 genome showed a more flattened morphology and a reduced ability to form tumors in nude mice compared to parental HT1080 cells. Diploid HT1080 cells carrying a single extra 1q also had a longer population doubling time and showed a loss of ability to clone in soft agar. Tumors arose from 1q-containing clones with a longer latency period, and a large majority of the cells comprising these tumors had lost the transferred chromosome. These results indicate the presence on chromosome 1q23-qter of a tumor suppressor gene or genes that can act to suppress transformation of a human fibrosarcoma cell line.
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PMID:The distal region of the long arm of human chromosome 1 carries tumor suppressor activity for a human fibrosarcoma line. 817 85

Humans frequently inhale as well as ingest cooked-food mutagens, among which the heterocyclic amines are the quantitatively most important. An extensive systemic distribution of these mutagens implies that most tissues in the body are exposed. Tissues containing cytochrome P450 (CYP) may be particularly susceptible to DNA damage. Accordingly, animal experiments have shown that oral exposure to heterocyclic amines leads to tumor formation at multiple sites. CYP1A2, which has only been demonstrated in the liver, seems to be the isozyme most efficient in metabolically activating the heterocyclic amines. In extrahepatic tissues, however, other CYP forms are likely to be important. Using Salmonella mutagenicity as an endpoint, we have studied the metabolic activation of 2-amino-3-methylimidazo[4,5,f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5,f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by isolated lung microsomes from rats and mice. Our studies show that CYP2A3, an isozyme that has hitherto not been investigated with regard to its capacity to activate heterocyclic amines, catalyses a major part of the IQ activating reactions in the uninduced lung. The formation of mutagens during cooking of meat is highly temperature dependent and meat extracts heated at 200 degrees C show a strong mutagenic activity in the Ames Salmonella assay. These extracts caused mutations at the HPRT locus in normal human fibroblasts as well as a pronounced decrease in survival of the cells. Furthermore, the heated meat extracts caused a decreased proliferative activity in primary cultures of normal mouse colonic epithelial cells as measured by autoradiography.
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PMID:Activation and effects of the food-derived heterocyclic amines in extrahepatic tissues. 884 3

Previous work with two closely related human lymphoblast cell lines demonstrated that WTK1 cells are more resistant than TK6 cells to X-ray-induced cytotoxicity, but more mutable at both the TK and HPRT loci. It was subsequently determined that WTK1 cells overexpress a mutant form of the tumor suppressor gene p53, while TK6 cells correctly express wild-type p53. Thus these two cell lines allowed us to examine the mutational spectra at the HPRT locus in related human lymphoblast cell lines differentially expressing p53. Previously, we isolated sets of X-ray-induced and spontaneous WTK1 mutants and spontaneous TK6 mutants and analyzed the mutational spectra by the combination of multiplex polymerase chain reaction (PCR) and Southern blot analysis. Somewhat unexpectedly, we found that even though there were approximately four times as many mutants induced by X rays in WTK1 cells as in TK6 cells, there was very little difference in the mutational spectra. In the present study, to determine if there was a higher frequency of intragenic deletions among the X-ray-induced WTK1 mutants, we further examined the subsets of mutants that contained HPRT point mutations. cDNA sequence analysis was used to define the mutation precisely in 19 X-ray-induced and 25 spontaneous WTK1 mutants and 25 spontaneous TK6 mutants. While subtle differences exist in the spectra of HPRT mutations between these two cell lines, the data again suggest that p53 is associated with an increase in the frequency of mutations at HPRT without an obvious effect on the types of mutations recovered.
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PMID:Spectra of X-ray-induced and spontaneous intragenic HPRT mutations in closely related human cells differentially expressing the p53 tumor suppressor gene. 900 5

Much recent attention has been paid to the important role of the DNA mismatch repair system in controlling the accumulation of somatic mutations in human tissues and the association of mismatch repair deficiency with carcinogenesis. In the absence of an intact mismatch repair system, cells accumulate mutations at a rate some 1000 times faster than normal cells, and this mutator phenotype is easily measured by the detection of the formation of new variant alleles at microsatellite loci. However, the mismatch repair system is not 100% efficient, even when intact, and the pattern of microsatellite alterations in a wide variety of tumors is consistent with these being due to clonal amplification from tissues that are genetically heterogeneous at microsatellite loci rather than mismatch repair deficiency in the tumor itself. On this basis, it can be estimated that the mutation frequency of microsatellites in normal human tissues is approximately 10(-2) per locus per cell. Similarly, a frequency of mutation at minisatellite loci in normal tissues of around 10(-1) per locus per cell can be estimated. Such elevated levels of mutation are consistent with a recent study of the frequency of HPRT mutation in human kidneys that demonstrated these to be frequent (average 2.5 x 10(-4) in individuals of 70 years or more) and exponentially related to age. Taken as a whole, the data suggest that somatic mutation in human epithelial cells may be some 10-fold higher than in peripheral blood lymphocytes and that the underlying rate of spontaneous mutation is sufficient to account for a large proportion of human carcinogenesis without the need to evoke either stepwise alteration to a mutator phenotype of clonal expansion at all the mutation steps in carcinogenesis. The exponential increase in mutation frequency with age is predictable on the basis that the mutation rate is controlled at the level of repair and that mutation in genes that affect the efficiency of these processes will gradually increase the underlying rate. In addition, the age relatedness of mutation frequency strongly supports the concept that mutation is cell division dependent and that cellular proliferation per se is an important risk factor for cancer. Comparison of somatic mutations with those in the human germline mutation suggests common mechanistic origins and that the high levels of somatic mutation that occur are a direct reflection of the germline mutation rate selected over evolutionary time. Thus, the somatic accumulation of mutations can be seen as a natural process within the human body and cancer a normal part of the human life cycle. This point of view may explain why it has been so difficult to significantly reduce cancer incidence and suggests that, for this to be achieved, the means of altering the natural somatic mutation rate needs to be identified.
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PMID:The natural somatic mutation frequency and human carcinogenesis. 911 67

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSalpha (hMSH2. hMSH6) and hMutSbeta (hMSH2.hMSH3). In HL-60 extracts the hMutSalpha to hMutSbeta ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base-base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSalpha and hMutSbeta display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion-deletion heterology, but only hMutSalpha restores base-base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.
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PMID:DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair. 929 77

The human DNA mismatch repair genes hMSH2 and hMSH6 encode the proteins that, together, bind to mismatches to initiate repair of replication errors. Human tumor cells containing mutations in these genes have strongly elevated mutation rates in selectable genes and at microsatellite loci, although mutations in these genes cause somewhat different mutator phenotypes. These cells are also resistant to killing by certain drugs and are defective in mismatch repair. Because the elevated mutation rates in these cells may lead to mutations in additional genes that are causally related to the other defects, here we attempt to establish a cause-effect relationship between the hMSH2 and hMSH6 gene mutations and the observed phenotypes. The endometrial tumor cell line HEC59 contains mutations in both alleles of hMSH2. The colon tumor cell line HCT15 contains mutations in hMSH6 and also has a sequence change in a conserved region of the coding sequence for DNA polymerase delta, a replicative DNA polymerase. We introduced human chromosome 2 containing the wild-type hMSH2 and hMSH6 genes into HEC59 and HCT15 cells. Introduction of chromosome 2 to HEC59 cells restored microsatellite stability, sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine treatment, and mismatch repair activity. Transfer of chromosome 2 to HCT15 cells also reduced the mutation rate at the HPRT locus and restored sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine treatment and mismatch repair activity. The results demonstrate that the observed defects are causally related to mutations in genes on chromosome 2, probably hMSH2 or hMSH6, but are not related to sequence changes in other genes, including the gene encoding DNA polymerase delta.
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PMID:Correction of hypermutability, N-methyl-N'-nitro-N-nitrosoguanidine resistance, and defective DNA mismatch repair by introducing chromosome 2 into human tumor cells with mutations in MSH2 and MSH6. 930 78

There is increasing evidence that endogenously generated reactive oxygen (ROS) and reactive nitrogen (RNS) species at sites of inflammation and in tumors may be genotoxic. We have developed a murine tumor model (MN-11) in which mutations at the hypoxanthine phosphoribosyltransferase (HPRT) locus, arising both in vitro and in vivo, can be detected. In the present report, we describe an in vitro study of the ability of ROS and RNS to induce mutations in our model system. 137Cs radiation and radiomimetic drugs caused a dose-dependent increase in mutant frequency. At D0, radiation induced about 170 mutants per 10(5) viable cells, compared to 50 and 95 for streptonigrin and bleomycin, respectively. H2O2 induced a lower frequency of mutants, 20-30 per 10(5), for enzymatically generated or bolus, respectively. For the following treatments, mutant frequency at 50% survival is shown. Incubation with human granulocytes induced a low frequency of mutants (about 15 per 10(5)). RNS was tested using a series of NO-donating drugs. Spermine/NO. induced cytotoxicity but no mutants while S-nitroso-N-acetylpenicillamine induced a low level, 10 per 10(5). Both release nitrogen monoxide spontaneously, with a t1/2 < 3 h. Glyceryl trinitrate and sodium nitroprusside are two drugs that were slowly metabolized by MN-11 cells (> 12 h). Glyceryl trinitrate induced about 20 per 10(5) while nitroprusside induced 50 per 10(5). Our results indicate that RNS can readily induce mutations detectable in MN-11 cells. At equicytotoxic doses, the induced mutant frequency varied considerably for different drugs, suggesting that different states of nitrogen monoxide (such as NO+ or NO.) may be generated and these may vary in their mutagenic/cytotoxic potential.
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PMID:Mutagenicity and cytotoxicity of reactive oxygen and nitrogen species in the MN-11 murine tumor cell line. 935 53


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