Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gene amplification contributes to carcinogenesis by enhancing proto-oncogene activity and causing chromosomal instability. The ease of detecting amplified tumor-virus sequences has encouraged use of this system as a surrogate for studying the molecular events involved in endogenous gene amplification. We report here a new system for studying carcinogen-induced amplification of both endogenous and viral sequences in the SV40-transformed human keratinocyte line AG06. Treatment with carcinogens induced a transient dose-dependent amplification of the integrated SV40 sequences. The amplified sequences appeared in the extrachromosomal fraction. Treatment of these cells with carcinogens prior to methotrexate (MTX) selection increased the frequency of MTX-resistant colonies, 67% of which exhibited dihydrofolate reductase (dhfr) amplification. The abilities of five carcinogens with different DNA-damaging activities (the DNA-damaging agents N-methyl-N-nitro-N-nitrosoguanidine, mitomycin C (MMC), ultraviolet light C, and X-rays and the non-DNA-damaging agent arsenite) to induce SV40 and dhfr amplification at concentrations that result in 50% clonal survival were compared. All four DNA-damaging carcinogens (as well as growth arrest) were able to elicit some SV40 amplification, but responses varied markedly, from 1.8-fold for X-rays to sevenfold to eightfold for MMC. There was no correlation between the ability to elicit the two amplification responses. Arsenite, which did not induce SV40 amplification, was the best inducer of MTX resistance. These results point to different controls involved in the induction of viral and dhfr amplification. The signal for amplification of viral genes may be triggered by DNA damage and growth arrest, whereas amplification of dhfr, and perhaps other endogenous sequences, seems to be triggered by other signals as well.
 ...
PMID:Differential susceptibility to carcinogen-induced amplification of SV40 and dhfr sequences in SV40-transformed human keratinocytes. 133 30

A non-enzymatic method that was previously shown to create single-strand DNA breaks at the location of (6-4) photoproducts in the overall genome was adapted to measure (6-4) photoproducts at the level of the gene. This approach employs a photoisomerization step that converts the (6-4) photoproduct into the Dewar valence photoisomer, which is more alkaline labile and allows for the creation of a single-strand DNA break at this site. These strand breaks were quantified via Southern analysis and the DNA repair of (6-4) photoproducts was measured over 2, 4 and 8 h after a UV dose of 40 J/m2. A comparison of repair efficiency in the actively transcribed dihydrofolate reductase (DHFR) gene, a transcriptionally inactive genomic region and the overall genome (as measured by radioimmunoassay) showed preferential repair of the active gene. The active DHFR gene showed 59% repair by 8 h compared to 33% repair in the inactive downstream region. Analysis of (6-4) photoproduct repair in the transcribed and non-transcribed strands of the DHFR gene indicate some strand specificity with 62% repair in the transcribed strand at 8 h compared with 43% repair in the non-transcribed strand. However, this strand bias is much less distinct than has been reported for the major UV photoproduct, the cyclobutane pyrimidine dimer.
Carcinogenesis 1992 Nov
PMID:Preferential and strand-specific DNA repair of (6-4) photoproducts detected by a photochemical method in the hamster DHFR gene. 142 65

Interactions between cisplatin (CDDP) and irradiation are of potential significance for the combined modality treatment of cancer. Previous data have indicated that following in vitro exposure to X-irradiation certain tumour cells expressed resistance to CDDP. To identify parameters associated with this CDDP resistance, the human ovarian carcinoma cell line SK-OV-3/P was pre-exposed to fractionated X-irradiation (total dose: 50 Gy) in vitro. The resultant subline (SK-OV-3/DKR-10) proved 2-fold resistant to CDDP, but not to acute X-irradiation. Consistent with unaltered dihydrofolate reductase and thymidylate synthase activities, SK-OV-3/DXR-10 cells were neither cross-resistant to methotrexate nor to 5-fluorouracil. Verapamil (6.6 microM) significantly (P less than 0.05) enhanced CDDP-induced cytotoxicity in the resistant DXR-10 subline, but not in the parental cells. Total glutathione levels were significantly (P less than 0.01) lower in the resistant subline and BSO pretreatment failed to influence cytotoxicity, whilst related enzyme activities were not consistently modified in the SK-OV-3/DXR-10 cells. Resistance in these cells was associated with significantly decreased cisplatin uptake (P less than 0.002). Immediately following drug exposure the total platination level of the DNA, quantitated immunochemically, was higher (P less than 0.05) in the resistant subline indicative of increased tolerance to DNA damage. After an 18 h post-treatment incubation the parental cell line appeared proficient in the removal of the intrastrand adduct Pt-AG, but deficient in removing the major adduct Pt-GG and the difunctional Pt-(GMP)2 lesion, whilst the DXR-10 resistant subline appeared proficient in removal of all four Pt-DNA adducts. DNA polymerases alpha and beta activities, however, were comparable in both cell lines. These data implicate both enhanced repair and increased tolerance of DNA damage as mechanisms of resistance to CDDP resulting from in vitro exposure of a human ovarian carcinoma cell line to fractionated X-irradiation.
Carcinogenesis 1992 Jul
PMID:Mechanisms associated with the expression of cisplatin resistance in a human ovarian tumor cell line following exposure to fractionated X-irradiation in vitro. 163 88

We previously treated Chinese hamster ovary (CHO) cells with benzo[a]pyrene diol epoxide (BPDE) and mutants at the dihydrofolate reductase (dhfr) locus were isolated. On the basis of Southern blotting and RNA heteroduplex mapping experiments, 14 of the 15 mutants were presumed to carry point mutations. Two restriction fragment length polymorphism mutants were cloned and sequenced; one carried a point mutation, the other a -1 frameshift mutation. Using polymerase chain reaction techniques and direct sequencing of amplified mutant DNA, we have now determined the induced changes in the remaining 12 cell lines. All changes occurred at guanine bases; all target guanines except one were on the non-transcribed coding strand. Most mutants (79%) contained base substitutions; the rest (3/14) carried frameshift mutations. Of the point mutations, all but one (91%) were GC----TA transversions either in the dhfr coding sequence or at splice sites. The single exception was a GC----AT transition. Of the frameshift mutations, two were deletions of a GC pair and the other was an insertion of an AT pair. Four different mutations (29%) were clustered in a 3 bp region in exon 4. Tandem guanine bases adjacent to adenine were favored sites for mutation occurring in 9/14 cases (64%). These results are consistent with data previously obtained by others in the supF shuttle vector system and the CHO aprt gene.
Carcinogenesis 1990 Jan
PMID:DNA base changes in benzo[a]pyrene diol epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells. 168 19

A study of the repair of DNA damage in the dihydrofolate reductase (dhfr) gene of SV40-transformed human fibroblasts after treatment with 8-methoxypsoralen (8MOP) and UVA is described. 8MOP+UVA-induced cross-links in the dhfr gene were completely repaired by 12 h in one normal and one Fanconi's anaemia (FA) group A cell line. In contrast, approximately 35% of cross-links in an episomally maintained Epstein--Barr virus derived plasmid remained unrepaired even after 48 h. Cross-linkable monoadducts in the dhfr gene were repaired more slowly than cross-links, and there was no detectable repair of cross-linkable monoadducts in the plasmid. Thus the ability of a cell to repair 8MOP+UVA-induced cross-links or cross-linkable monoadducts in an episome does not reflect its capacity to repair such lesions in genomic DNA.
Carcinogenesis 1989 Jul
PMID:Repair of 8-methoxypsoralen + UVA-induced damage in specific sequences in chromosomal and episomal DNA in human cells. 254 11

During selection for methotrexate resistance, SV40-transformed human skin fibroblasts from patients with ataxia telangiectasia (A-T) underwent amplification of the dihydrofolate reductase (DHFR) gene, experienced nearly complete loss of the integrated SV40 sequences and showed a 3.6-fold increase in Ki-ras gene copy number. Over a period of months methotrexate-resistant (MTXr) A-T subclones were obtained, which were able to grow in progressively increasing MTX concentrations up to 100 microM. The ED50 values determined as the effective dose of MTX causing 50% growth inhibition in comparison to control cells increased from 3 x 10(-2) microM for MTXs AT5BI-VA cells to 250 microM MTX for the MTXr AX100 subclone. In contrast, human skin fibroblasts of healthy individuals did not show DHFR gene amplification and loss of SV40 sequences under comparable conditions and were unable to grow in MTX concentrations greater than 1 microM. Gene amplification and loss of DNA sequences are features underlying the genomic instability known to be a characteristic property of A-T cells and being probably responsible for the high cancer incidence in these patients.
Carcinogenesis 1987 Dec
PMID:DHFR gene amplification in cultured skin fibroblasts of ataxia telangiectasia patients after methotrexate selection. 282 82

Following carcinogen treatment, elevated expression levels of dihydrofolate reductase (dhfr) were measured by labeling cells with fluorescent methotrexate which binds to this enzyme. Fractionation of carcinogen-treated, Simian virus 40 (SV40)-transformed Chinese hamster embryo cells (C060) into subpopulations differing in their levels of dhfr expression revealed co-expression, at enhanced levels, of dhfr and SV40 T antigen in the same cells. The increased expression of dhfr was amplification independent, while the T antigen coding sequences were amplified. The co-expression of dhfr and the bacterial chloramphenicol acetyltransferase gene linked to the early SV40 regulatory region was measured in CHO cells stably transformed by pSV2CAT-SVgpt (CC24). Both these sequences were expressed at higher levels in treated cells and the elevated expression levels were observed in the same subpopulation of cells, although no increase in their gene copy number was detected. The concomitant activation of enhanced expression of two independent genes in the same cells suggests that cellular factors governing gene expression are activated in the carcinogen-treated cells. The implications of these findings to cellular control mechanisms and to the carcinogenic process are discussed.
Carcinogenesis 1988 Jun
PMID:Carcinogen-mediated co-activation of two independent genes in Chinese hamster cells. 283 8

Novobiocin, an inhibitor of type II topoisomerase, has been reported to inhibit u.v.-induced DNA repair in a number of established mammalian cell lines; we have confirmed this general observation in primary cultures of human epidermal keratinocytes. Using a recently developed technique for measuring pyrimidine dimer frequencies in genomic restriction fragments, we have determined the extent of DNA repair in the active, essential dihydrofolate reductase (DHFR) gene. Novobiocin did not affect repair of the DHFR gene in keratinocytes or in a Chinese hamster ovary (CHO) cell line over a 24-h period following irradiation with 20 J/m2 u.v. These findings suggest that qualitative differences exist in the repair pathways in different genomic regions; topoisomerase II may not have an essential role in repair of active genes but may be required for repair of other regions in the genome.
Carcinogenesis 1986 Nov
PMID:Novobiocin does not inhibit DNA repair in an active gene. 302 54

The pattern of preferential DNA repair of UV-induced pyrimidine dimers was studied in repair-deficient Chinese hamster ovary (CHO) cells transfected with the human excision repair gene, ERCC-1. Repair efficiency was measured in the active dihydrofolate reductase (DHFR) gene and in its flanking, non-transcribed sequences in three cell lines: Wild type CHO cells, a UV-sensitive excision deficient CHO mutant, and the transfected line of the mutant carrying the expressed ERCC-1 gene. The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells. This pattern differs from that previously reported in CHO cells transfected with the denV gene of bacteriophage T4, in which both active and non-transcribed DNA sequences were efficiently repaired (Bohr and Hanawalt, Carcinogenesis 8: 1333-1336, 1987). The ERCC-1 gene product may specifically substitute for the repair enzyme present in normal hamster cells while the denV product, T4 endonuclease V, does not be appear to be constrained in its access to inactive chromatin.
 ...
PMID:Human repair gene restores normal pattern of preferential DNA repair in repair defective CHO cells. 341 90

We have previously demonstrated that the active dihydrofolate reductase (DHFR) gene is efficiently repaired in Chinese hamster ovary (CHO) cells which remove only a small fraction of u.v.-induced pyrimidine dimers from the overall genome. Preferential DNA repair of essential genes may explain why the u.v. resistance of normal CHO cells is as high as that of fully repair-proficient normal human cells. In this report, we have studied the removal of pyrimidine dimers in a CHO cell line expressing the cloned denV gene from bacteriophage T4 which codes for the pyrimidine dimer specific enzyme T4 endonuclease V (T4 endo V). This cell line was derived from a u.v.-sensitive excision deficient mutant of a CHO wild type line by transformation with the denV gene, and partial restoration of u.v. resistance was achieved. We have examined an important aspect of the u.v. excision repair in these denV+ cells by studying the repair efficiencies in the active DHFR gene and in a non-coding sequence located downstream from it. In the u.v.-sensitive CHO mutant cell line from which the denV+ was derived, we detected no pyrimidine dimer removal from the gene or from the downstream sequence after irradiation of the cells with 20 J/m2 u.v. (254 nm) light. In the wild type CHO cells, approximately 50% of the pyrimidine dimers were removed from a sequence in the DHFR gene within 8 h, whereas none were removed from the downstream sequence in that period. This represents the normal pattern of preferential DNA repair of active genes, which we have described in previous communications. In the denV+ cells, approximately 70% of the pyrimidine dimers were removed from both the DHFR gene and from the downstream sequence; these cells thus repair both coding and non-coding regions of the genome and show no pattern of preferential repair. The endogenous activity that initiates excision repair in normal CHO cells is evidently much more restricted in its accessibility to DNA lesions in chromatin than is the activity in cells containing substantial amounts of the small T4 endo V enzyme.
Carcinogenesis 1987 Sep
PMID:Enhanced repair of pyrimidine dimers in coding and non-coding genomic sequences in CHO cells expressing a prokaryotic DNA repair gene. 362 70


1 2 3 4 Next >>