Use of a simian virus 40-based shuttle vector to analyze enhanced mutagenesis in mitomycin C-treated monkey cells.

Abstract

When monkey cells were treated with mitomycin C 24 h before transfection with UV-irradiated pZ189 (a simian virus 40-based shuttle vector), there was a twofold increase in the frequency of mutations in the supF gene of the vector. These results suggest the existence of an enhancible mutagenesis pathway in mammalian cells. However, DNA sequence analysis of the SupF-mutants suggested no dramatic changes in the mechanisms of mutagenesis due to mitomycin C treatment of the cells.Exposure of mammalian cells to a wide variety of DNAdamaging agents can lead to mutagenesis and cellular transformation. It is clear that an important component of the mutagenic process is misreplication of damaged DNA (4, 24). The role of indirect effects of DNA-damaging agents in the mutagenic process in mammalian cells is less well understood. In bacterial systems, indirect effects of DNAdamaging agents play a major role in mutagenesis; induction of the bacterial SOS system is required for damage-directed mutagenesis to occur (3,10,31). Evidence for analogous indirect effects in mammalian systems comes primarily from studies on enhanced viral reactivation and mutagenesis (reviewed in references 3, 24, and 25). When mammalian cells are pretreated with a DNA-damaging agent before infection with a UV-irradiated virus, virus survival is enhanced over that observed in untreated cells. Although this enhancement of survival is often accompanied by enhancement of mutagenesis, this correlation has not been observed in all systems (2,20,25,26,30). Nothing is known about the molecular mechanisms involved in enhanced mutagenesis in mammalian cells.To gain a better understanding of enhanced mutagenesis in mammalian cells, we used the simian virus 40-based shuttle vector pZ189 (28). This vector replicates in both primate and bacterial cells and contains the bacterial supF suppressor gene as an easily sequenced mutagenesis target. We analyzed the frequency and sequence characteristics of mutants induced when either UV-damaged or undamaged pZ189 templates replicate in monkey cells pretreated with the DNA-damaging agent mitomycin C. Since the vector DNA and the mammalian cells can be treated independently with DNA-damaging agents, we could assess both direct and indirect effects of the agents on the mutagenic process. UV radiation was chosen as the DNA-damaging agent for the vector DNA because the mutagenic action of this agent has been extensively studied in the pZ189 system (1,6,18 with antibiotics and 5% fetal bovine serum were treated with mitomycin C (1 ug/Iml) in serum-free Dulbecco minimal essential medium for 2.5 h. The mitomycin C-containing medium was then removed, the cells were washed, and fresh medium with 2% serum was added to the dishes. At 24 h later, the medium was removed and saved, the cells were transfected with 100 ng of pZ189 plasmid DNA (either UV irradiated [6] or not irradiated) per 100-mm dish by the DEAE-dextran method (11), and then the conditioned medium was added. At 48-h later, plasmid DNA was extracted from the cells by the...

A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells.

“…Since isolation and cloning of the genes which encode mammalian DNA repair proteins have met with little success, specific information about the formation of DNA repair complexes (in particular those involved in the repair of bulky lesions, such as UV photoproducts) and their regulation in a stress-induced environment is not yet available. Recent studies have indicated, however, that SOS-like inducible phenomena associated with DNA processing occur in mammalian cells exposed to carcinogens or tumor promoters (for a review, see references 5 and 7 and references therein; 26,29,35). Some of the proteins induced in these * Corresponding author.…”