DNA damage induced by 193-nm radiation in mammalian cells.

The contribution of DNA damage to the effects of 193-nm excimer laser radiation on mammalian cells in culture was studied in order to evaluate the mutagenic potential of this UV wavelength in vivo. Two approaches were taken: measurement of pyrimidine dimer-specific endonuclease-sensitive sites/megabase and comparison of the 193-nm radiation-induced cytotoxicity in normal versus DNA repair-deficient cells. The formation of pyrimidine dimer-specific endonuclease-sensitive sites/megabase was inversely related to the thickness of the cytoplasm overlying the nuclei of normal human fibroblasts (NHF) and Chinese hamster ovary cells. The results of these measurements and a calculation of the absorption coefficient of cytoplasm indicate that each 1 micron of cytoplasm attenuates the incident radiation by greater than 90% and, therefore, the nuclear DNA in tissue will be highly protected from 193-nm radiation by overlying cytoplasm. The reduction in colony-forming ability induced by 254-nm, 193-nm, and X-ray radiation was measured in NHF, xeroderma pigmentosum (group A) cells, and ataxia telangiectasia cells. Xeroderma pigmentosum (group A) cells were 16.5 times more sensitive to 254-nm radiation but only 3.5 times more sensitive to 193-nm radiation than NHF cells, indicating that cyclobutylpyrimidine dimers were not the major lethal lesion formed at 193 nm. AT cells were 3.4 times more sensitive to X-rays than NHF cells, but these cell types were almost equally sensitive to 193-nm radiation, indicating that 193 nm did not induce the same type of lethal lesions as X-rays.

Ultraviolet light-induced transformation of human cells to anchorage-independent growth.

We have developed a system for ultraviolet light (UV) transformation of human embryonic cells to anchorage-independent growth. The procedure involves multiple UV irradiations, post irradiation growth, and plating in soft agar. Transformants are obtained at frequencies from 1 to 80 per 10(5) cells at UV exposures to 25 J/sq m. The resulting transformants can be subcultured on solid surfaces. The cells show crisscrossing and piling up; they reach 2- to 5-fold higher saturation densities than the parental cells. Some subcultures show increased plating efficiency in soft agar and increased life span. The susceptibility of the UV transformation process to apparent photoenzymatic reversal implies that purimidine dimers play a role in its induction.

Quantitation of pyrimidine dimer contents of nonradioactive deoxyribonucleic acid by electrophoresis in alkaline agarose gels.

We have developed a method of quantitating the pyrimidine dimer content of nonradioactive DNAs. DNA samples are treated with the UV-endonuclease from Micrococcus luteus and then separated according to molecular weight by electrophoresis on alkaline agarose gels. From their migration relative to known molecular weight standards, their median molecular weights and thus the number of dimers per DNA molecule in each sample can be calculated. Results of action spectra for dimer formation in T7 bacteriophage measured by this method agree well with action spectra for T7 killing. In addition, the method gives dimer yields in good agreement with those obtained by others using alkaline sucrose gradient sedimentation.