thioguanine-anhydrous and Carcinoma

In temperature-sensitive (ts) mutants of mouse FM3A cells, the levels of mutagenesis and survival of cells treated with DNA-damaging agents have been difficult to assess because they are killed after their mutant phenotypes are expressed at the nonpermissive temperature. To avoid this difficulty, we incubated the ts mutant cells at the restrictive temperature, 39 degrees C, for only a limited period after inducing DNA damage. We used ts mutants defective in genes for ubiquitin-activating enzyme (E1), DNA polymerase alpha, and p34(cdc2) kinase. Whereas the latter two showed no effect, E1 mutants were sensitized remarkably to UV light if incubated at 39 degrees C for limited periods after UV exposure. Eighty-five percent of the sensitization occurred within the first 12 h of incubation at 39 degrees C, and more than 36 h at 39 degrees C did not produce any further sensitization. Moreover, while the 39 degrees C incubation gave E1 mutants a moderate spontaneous mutator phenotype, the same treatment significantly diminished the level of UV-induced 6-thioguanine resistance mutagenesis and extended the time necessary for expression of the mutation phenotype. These characteristics of E1 mutants are reminiscent of the defective DNA repair phenotypes of Saccharomyces cerevisiae rad6 mutants, which have defects in a ubiquitin-conjugating enzyme (E2), to which E1 is known to transfer ubiquitin. These results demonstrate the involvement of E1 in eukaryotic DNA repair and mutagenesis and provide the first direct evidence that the ubiquitin-conjugation system contributes to DNA repair in mammalian cells.

Topics: Animals; Carcinoma; CDC2 Protein Kinase; DNA Damage; DNA Polymerase II; DNA Repair; Ligases; Mammary Neoplasms, Experimental; Mice; Mutagenesis; Mutagens; Mutation; Temperature; Thioguanine; Tumor Cells, Cultured; Ubiquitin-Activating Enzymes; Ubiquitin-Protein Ligases; Ultraviolet Rays; X-Rays

We have used a primary cloning assay to determine the frequency of 6-thioguanine (TG)-resistant tubular epithelial cells in kidney tissue from 72 human donors ranging in age from 2 to 94 years. The frequency of TG-resistant mutants ranged from approximately 5 x 10(-5) for donors in the first decade of life to approximately 2.5 x 10(-4) for donors in the eighth and later decades of life. Two different statistical analyses indicated that this increase in mutant frequency is exponential with age. We also observed a 2-fold higher TG-resistant mutant frequency in nephrectomy kidneys containing a coincident renal carcinoma. DNA sequence analyses revealed HPRT gene mutations in each of 14 TG-resistant mutants from seven unrelated donors. Thirteen of these 14 mutants resulted from independent mutational events. These results suggest that somatic mutations are common in renal--and perhaps in other human--epithelia, and thus could play an important role in the genesis of age-associated disease.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Base Sequence; Carcinoma; Cells, Cultured; Child; Child, Preschool; DNA Primers; Epithelium; Humans; Hypoxanthine Phosphoribosyltransferase; Kidney; Kidney Cortex; Kidney Neoplasms; Middle Aged; Molecular Sequence Data; Mutation; Nephrectomy; Thioguanine

Topics: Aged; Antineoplastic Agents; Blood Coagulation; Carcinoma; Carcinoma, Bronchogenic; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cyclophosphamide; Drug Therapy, Combination; Fibrinogen; Fluorouracil; Heparin; Humans; Infusions, Parenteral; Lung Neoplasms; Male; Methotrexate; Middle Aged; Neoplasm Metastasis; Radiography; Skin Neoplasms; Thioguanine; Vincristine

Topics: Antimetabolites; Azaguanine; Carcinoma; Cell Division; Cell Line; Cell Survival; Cytarabine; DNA Replication; Dose-Response Relationship, Drug; Floxuridine; Fluorouracil; Humans; Hydroxyurea; Laryngeal Neoplasms; Mercaptopurine; Methotrexate; Mitosis; Ribonucleosides; Thioguanine; Time Factors; Tritium

Topics: Acetamides; Acetates; Animals; Antineoplastic Agents; Arabinose; Carcinoma; Carcinoma 256, Walker; Cell Line; Chlorine; Ethers; Guanosine; Humans; Leukemia L1210; Mice; Mouth Neoplasms; Nucleosides; Purines; Rats; Structure-Activity Relationship; Sulfonamides; Thioguanine; Xylose