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Rapid and Slow DNA Rejoining in Nondividing Human Diploid Fibroblasts Treated with Bleomycin and Ionizing Radiation¹

Department of Radiation Biology and Biophysics, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642 [C. W. M.], and Laboratory of Radiobiology, Harvard University School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115 [C. W. M., J. B. L.]

The rejoining of DNA single-strand breaks produced after belomycin treatment or -irradiation of human diploid fibroblasts was studied by the alkaline elution technique. DNA rejoining occurred at slower rates in bleomycin-treated human fibroblasts than in -irradiated fibroblasts. These comparisons were made at similar levels of survival or DNA single-strand breaks (including alkali-labile lesions). Significant numbers of DNA single-strand breaks were detected routinely after 2 µg/ml (1.34 x 10^-6 M) bleomycin treatments (for 30 min, survival greater than 70%). Dose-dependent losses of approximately 3 to 15% of total radioactivity were measured in preelution samples from cells treated with bleomycin (2 to 100 µg/ml), but only 2 to 3.5% of total radioactivity was assayed in lysis samples from cells irradiated with 200 to 1000 rads. This result suggests that DNA was more degraded by or after bleomycin treatment. DNA was rejoined extremely rapidly after bleomycin or radiation treatments, and the rejoining was both agent- and dose-dependent. Over dose ranges yielding surviving fractions of 75 to 0.056%, considerable DNA rejoining occurred after only 2.5 min posttreatment incubation in conditioned medium. Cellular recovery occurred at faster rates after bleomycin treatments than after -irradiation, while DNA rejoining occurred at faster rates after -irradiation, thus uncoupling DNA repair and cellular recovery in relating the cellular action of these 2 agents. No consistent differences were observed among 3 normal fibroblast strains and fibroblasts from a Gardner's syndrome patient (deficient in their capacity for cellular recovery) or a Turcot's syndrome patient in the formation and rejoining of single-strand breaks after bleomycin or radiation treatments.

¹ This paper has been numbered Report UR-3490-2034.

² Supported by USPHS Grant CA25609 awarded by the National Cancer Institute and by grants from the United Cancer Council, Inc. (Rochester) and Rockefeller Foundation to the Harvard Interdisciplinary Programs in Health program. To whom requests for reprints should be addressed.

³ Supported by USPHS Grant CA11751 awarded by the National Cancer Institute.

Received 2/ 4/82. Revised 8/27/84. Revised 12/11/84. Accepted 1/10/85.

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