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Isolation and Characterization of a 1-ß-D-Arabinofuranosylcytosine-resistant Chinese Hamster Ovary Cell Mutant That Is Also X-Ray Sensitive and Is Noncomplementary with Ataxia Telangiectasia Cells¹

University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences [G. A. P.] and Biology Division, Oak Ridge National Laboratory [H. S. P., R. J. P.], Oak Ridge, Tennessee 37831

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-ß-D-arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray-sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair. Such mutant cells could also be used for the isolation and characterization of human DNA repair genes. In particular, it was predicted that the repair gene defective in individuals with ataxia telangiectasia (AT) might be amenable to study with ara-C-resistant (X-ray-sensitive) mutants, since additional studies, presented here, have shown that AT cells are resistant to ara-C. In the long term, it is hoped that determining the specific defect in AT might lead to an understanding of the possible role of defective repair in tumor induction and/or progression.

The general approach used to isolate ara-C-resistant Chinese hamster ovary cell mutants was to treat cells with ethyl methanesulfonate and select in increasing concentrations of ara-C. Although several mutants were isolated, one in particular, Ara-C^R213, has been studied most extensively. It was selected largely because it shows the greatest sensitivity to X-rays. Ara-C^R213 cells were hypersensitive to the killing effect of X-rays with an LD₁₀ of 2.5 Gy as compared to the wild-type cells that had an LD₁₀ of 6 Gy. The mutant showed an increased frequency of X-ray-induced chromosomal aberrations in the G₁ and G₂ stages of the cell cycle compared to wild-type frequencies. There was no increase in sister chromatid exchange levels. All of these observations in Ara-C^R213 are very similar to those made with AT cells in our and other laboratories. Even more important, complementation analysis of Ara-C^R213 x AT hybrid cells indicated that the gene responsible for X-ray sensitivity of AT is also mutated in Ara-C^R213 cells. Thus, Ara-C^R213 appears to have a mutant phenotype and probably genotype that is very similar to, if not exactly the same as, those of AT. This makes it quite different from other X-ray-sensitive cells that have been isolated in other laboratories.

¹ Research sponsored jointly by the Office of Health and Environmental Research, United States Department of Energy under Contract DE-ACO5-840R21400 with the Martin Marietta Energy System, Inc. (R. J. P. and H. S. P.), and by NIH Training Grant CA09104-13 (G. A. P.).

² Present address and to whom reprint requests should be addressed, at Laboratory of Molecular Carcinogenesis, NIEHS, P.O. Box 1223, Research Triangle Park, NC 27709.

³ Present address: CIIT, 6 Davis Drive, Research Triangle Park, NC 27709.

Received 2/28/91. Accepted 10/25/91.

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