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Induction of Asynchronous Replication of Polyoma DNA in Rat Cells by Ultraviolet Irradiation and the Effects of Various Inhibitors¹

Comprehensive Cancer Center and Institute of Cancer Research, Columbia University, New York, New York 10032 [Z. A. R., M. D. J., E. O., I. B. W.] and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 [M. E. L.]

The ability of various DNA damaging agents to induce asynchronous replication of polyoma DNA (APR) in rat cells carrying integrated copies of these DNA sequences may provide a useful model for understanding mechanisms of gene amplification. The present study has explored in detail the ability of UV irradiation to induce APR in the polyoma transformed rat fibroblast cell line H3. We have found that the optimum condition for induction of APR was obtained by irradiating the H3 cells with UV-C (wavelength, 254 nm) at 1–2 J/m². Irradiation with UV-B (270–360 nm) was much less effective, and no induction of APR was obtained with even high doses of UV-A (345–440 nm). This action spectrum provides evidence that the critical target for induction of APR is DNA. We found that when normal rat fibroblasts were irradiated with UV-C and then fused to H3 cells, this also led to induction of APR. These results provide evidence that the induction of APR by UV-C is mediated by a trans-acting factor. The induction of APR by UV-C was inhibited by high doses of cycloheximide or actinomycin D, suggesting that the production of this trans-acting factor requires de novo protein and RNA synthesis. On the other hand, low doses of cycloheximide or actinomycin D alone were able to induce APR, perhaps by blocking the synthesis of cellular factors that normally inhibit APR. Thus, induction of APR by UV-C provides a useful system for identifying cellular factors that might mediate or prevent the asynchronous replication of various DNA sequences.

¹ This research was supported by National Cancer Institute Grant CA 021111 to I. B. W. and by NIH Grant RR-02188 to M. L.

² To whom requests for reprints should be addressed, at College of Physicians & Surgeons of Columbia University, Comprehensive Cancer Center, 701 West 168th Street, New York, NY 10032.

Received 1/30/87. Revised 5/13/87. Accepted 6/ 5/87.