thioguanine-anhydrous and Xeroderma-Pigmentosum

The thiopurines, 6-thioguanine and 6-mercaptopurine, are antileukemic agents that are incorporated into DNA following retrieval by the purine salvage pathway (see [1] for a review). Their toxicity requires active DNA mismatch repair (MMR), and thiopurine resistance is an acknowledged phenotype of MMR-defective cells [2, 3]. In addition to these direct cytotoxic effects, DNA thiobases have distinctive photochemical properties [4], the therapeutic potential of which has not been extensively evaluated. We report here that the thiopyrimidine nucleoside 4-thiothymidine is incorporated into DNA. It does not induce MMR-related toxicity, but it interacts synergistically with UVA light and dramatically sensitizes cultured human cells to very low, nonlethal UVA doses. 4-thiothymidine induced UVA dose enhancements of around 100-fold in DNA repair-proficient cells. Nucleotide excision repair-defective xeroderma pigmentosum cells were sensitized up to 1000-fold, implicating bulky DNA photoproducts in the lethal effect. The synergistic action of thiothymidine plus UVA required thymidine kinase, indicating a selective toxicity toward rapidly proliferating cells. Cooperative UVA cytotoxicity is a general property of DNA thiobases, and 6-thioguanine and 4-thiodeoxyuridine were also UVA sensitizers. Thiobase/UVA treatment may offer a novel therapeutic approach for the clinical management of nonmalignant conditions like psoriasis or for superficial tumors that are accessible to phototherapy.

Topics: Cell Line; DNA; DNA Repair; Humans; Mercaptopurine; Phototherapy; Thioguanine; Thiouridine; Ultraviolet Therapy; Xeroderma Pigmentosum

Xeroderma pigmentosum group A gene (XPA)-deficient mice are defective in nucleotide excision repair (NER) and are therefore highly sensitive to ultraviolet (UV)-induced skin carcinogenesis. We established cell lines from skin cancers of UVB-irradiated XPA-deficient mice to investigate the phenotypic changes occurring during skin carcinogenesis. As anticipated, the skin cancer cell lines were devoid of NER activity but were less sensitive to killing by UV-irradiation than the XPA(-/-) fibroblast cell line. The lines were also more resistant to 6-thioguanine (6-TG) than XPA(-/-) and XPA(+/+) fibroblasts, which was suggestive of a mismatch repair (MMR) defect. Indeed, in vitro mismatch binding and MMR activity were impaired in several of these cell lines. Moreover, these cell lines displayed cell cycle checkpoint derangements following UV-irradiation and 6-TG exposure. The above findings suggest that MMR downregulation may help cells escape killing by UVB, as was seen previously for methylating agents and cisplatin, and thus that MMR deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells.

Topics: Animals; Cell Cycle; Cell Survival; Cell Transformation, Neoplastic; DNA Repair; DNA-Binding Proteins; Drug Resistance; Gene Deletion; Mice; Phenotype; Radiation Tolerance; Skin Neoplasms; Thioguanine; Tumor Cells, Cultured; Ultraviolet Rays; Xeroderma Pigmentosum; Xeroderma Pigmentosum Group A Protein

The mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes from 10 xeroderma pigmentosum patients (including complementation groups D and G and XP variants) has been determined. A highly significantly elevated frequency was observed, compared to age-matched, non-smoking control donors (x 2.1-fold higher than the mutant frequency in normal control donors, adjusted for age and cloning efficiency, p less than 0.001). The mutant frequency of 5 XP heterozygotes was in the normal range, when age, smoking habit and log cloning efficiency were taken into account. A number of possible factors which may account for the elevated mutant frequency seen in the XP donors (including an elevated spontaneous mutation rate, UV mutagenesis of the T-cells as they pass through the skin, an effect of environmental mutagens such as tobacco smoke, or as a consequence of immune deficiency) are discussed.

Topics: Adult; Aged; Child; Drug Resistance; Female; Humans; Hypoxanthine Phosphoribosyltransferase; Male; Middle Aged; Mutation; Smoking; T-Lymphocytes; Thioguanine; Xeroderma Pigmentosum

Topics: Adenosine; Bromodeoxycytidine; Cells, Cultured; Deoxycytidine; DNA, Viral; Herpes Simplex; Humans; Lesch-Nyhan Syndrome; Orotic Acid; Thioguanine; Ultraviolet Rays; Virus Replication; Xeroderma Pigmentosum

Topics: Ataxia Telangiectasia; Cells, Cultured; DNA Repair; Drug Resistance; Gamma Rays; Humans; Immunologic Deficiency Syndromes; Mutagens; Thioguanine; Ultraviolet Rays; Xeroderma Pigmentosum

The cytotoxic and mutagenic effect of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti BPDE) in normally excising diploid human cells treated just prior to onset of S was compared with that of cells allowed approximately 16 h for excision repair before onset of S and with that observed in excision-deficient xeroderma pigmentosum (XP12BE) cells. The cells were synchronized by release from density inhibition of cell replication. DNA synthesis began approximately 22 h after the cells were plated at lower density (i.e., 1.4 x 10(4) cells/cm2). The frequency of thioguanine-resistant mutants induced in normal cells treated just prior to onset of S was approximately 12- to 16-fold higher than that observed in cells treated in early G1 or treated in G0 (confluence) and then plated at lower density. The frequency approximated that expected for XP12BE cells from extrapolation of data obtained at lower doses. The frequency of mutants measured in normal cells treated in exponential growth was also much higher than that in the cells treated in early G1 or in G0. No such difference could be seen in XP12BE cells treated in exponential growth or in G0. In contrast to the mutagenicity data in the normal cells, there was no significant difference in the slope of the survival curve of normal cells treated at various times prior to S phase at low densities. However, normal cells treated even at the onset of S exhibited survival equal to XP12BE cells given a 4- to 5-fold lower dose. The data support the hypothesis that DNA synthesis is the cellular event which converts unexcised DNA lesions into mutations. However, they indicate that S is not the event primarily responsible for translating DNA damage into cell death. Accompanying studies on the rate of excision of anti BPDE adducts from the normal cells during the period prior to S support the conclusions.

Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Benzopyrenes; Cell Cycle; Cell Line; Cell Survival; Cells, Cultured; DNA Repair; Drug Resistance; Humans; Interphase; Male; Mutagens; Mutation; Skin; Skin Physiological Phenomena; Thioguanine; Xeroderma Pigmentosum

If neoplastic transformation of diploid human cells results from carcinogen-induced mutations, cells deficient in excision repair of UV-induced DNA damage should be significantly more sensitive to transformation by UV light than normal cells. We tested this hypothesis by irradiating fibroblasts from a xeroderma pigmentosum patient (XP7BE, complementation group D) with low doses of Uv light (254 nm) and cells from a normal person with much higher doses and comparing the frequency of transformation to anchorage independence. Both sets of cells exhibited a dose-dependent increase in transformation which corresponded to a dose-dependent decrease in survival. At doses that caused equal cell killing, the frequency of anchorage-independent cells was approximately equal. Colonies of XP7BE and normal cells isolated from agar, propagated, and injected into X-irradiated athymic mice produced fibrosarcomas in 100% of the animals. Normal cells irradiated shortly before the onset of DNA synthesis exhibited a high frequency of anchorage-independent cells; cells irradiated in early G1 showed no increase over background. These results agree with those we observed for UV induction of 6-thioguanine-resistant mutants in these cells and support the hypothesis that anchorage independence results from mutations induced by DNA replication on a damaged template.

Topics: Cell Cycle; Cell Line; Cell Survival; Cell Transformation, Neoplastic; DNA Repair; Dose-Response Relationship, Radiation; Humans; Infant, Newborn; Male; Skin; Thioguanine; Ultraviolet Rays; Xeroderma Pigmentosum

Topics: Cell Line; Clone Cells; Drug Resistance; Epithelial Cells; Female; Fibroblasts; Humans; Male; Methylcholanthrene; Mutagens; Ouabain; Thioguanine; Xeroderma Pigmentosum