[CANCER RESEARCH 45,1978-1981, May 1985] Ultrarapid Recovery from Lethal Effects of Bleomycin and 7-Radiation in Stationary-Phase Human Diploid Fibroblasts1 Carol W. Moore,2 Arnold W. Malcolm, Kathleen N. Tomkinson, and John B. Little3 Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642 [C. W. M.J; Harvard University, Laboratory of Radiobiology, School of Public Health, Boston, Massachusetts 02115 [C. W. M., A. W. M., K. N. T., J. B. L]; and Joint Center for Radiation Therapy, Harvard Medical School, Boston, Massachusetts 02115 [A. W. M., K. N. T.] ABSTRACT An ultrarapid phase of cellular recovery, as measured in liquid holding type experiments, was studied in stationary-phase hu man fibroblasts exposed to bleomycin or cobalt-60 ^-irradiation yielding comparable levels of cell killing. This rapid recovery was both faster and considerably greater in magnitude after bleomy cin treatments. Bleomycin survival curves were multiphasic, in dicating the presence of treated cells with varying sensitivities either at the beginning of treatments or as a result of resistance which developed during the treatment period. The amount of both ultrarapid (within 2 to 10 min) and slower recovery was dose dependent after irradiation with 200 to 800 rads or 30-min exposures to bleomycin (5 to 100 ng/ml). Following bleomycin treatments resulting in surviving fractions of 1 to 2%, survival increased up to 8-fold after only 2 min of posttreatment incuba tion. This rapid increase in survival was followed by a slower increase over time periods up to 3 h. In contrast, the rates of cellular recovery after -y-irradiation were more gradual from 0 to 3 h. Recovery at all posttreatment intervals was always greater after bleomycin than after -y-treatments, following doses yielding 1 to 50% survival. The ultrarapid component of cellular recovery after bleomycin treatments may have implications for both clinical cancer management and cellular studies directed toward deter mining mechanisms of action of bleomycin. INTRODUCTION The anticancer antibiotic bleomycin is similar in several re spects to ionizing radiation in its effects upon living cells. Both agents cause the production of DNA strand breaks (2,8-10,21, 22, 26).4 Based upon cell-killing data, bleomycin has been clas sified as an X-ray-like (rather than UV-like) anticancer drug (3). In the eukaryote, Saccharomyces cerevisiae, genes which confer sensitivity to the lethal effects of ionizing radiation also confer sensitivity to killing by the bleomycin-phleomycin group antibiotics (16,17). Similarly, patients with the autosomal-reces- sive disease ataxia telangiectasia exhibit extreme sensitivity to ionizing radiation (23), and their cells exhibit increased sensitivi ties In vitro to the lethal effects of both ionizing radiation (23,27) and bleomycin (11, 28). The apparent similarities of these 2 agents led us to compare the capacity for and kinetics of cellular 1This paper has been numbered University of Rochester Report UR-3490-2036. 2 Supported by USPHS Grant CA25609 awarded by the National Cancer Institute and by grants from the United Cancer Council, Inc. (Rochester), Rockefeller Foundation to the Harvard Interdisciplinary Programs in Health program, and American Cancer Society, Inc. (IN18; New York, NY). To whom requests for reprints should be addressed. 3 Supported by USPHS Grant CA11751 awarded by the National Cancer Institue. 4 C. W. Moore and L. Wall, manuscript submitted for publication. Received 12/14/81; revised 8/27/84,12/11/84; accepted 1/10/85. recovery in human cells after exposure to bleomycin and ionizing radiation. Noncycling cells in murine tumors (in vivo) or in culture (in vitro) recover better than do actively growing cells from the lethal effects of radiation and antibiotics (4, 5, 7, 12, 13). The term "recovery" is used operationally here to indicate a measurable increase in cellular survival which occurs as a result of posttreat ment incubation of noncycling (stationary phase) cells in depleted medium prior to subculturing to low density (a stimulus to DNA synthesis and cell proliferation). Recovery is commonly said to result from "repair of potentially lethal damage" (5, 12, 13, 24, 31,32). At present, however, our knowledge about the molecular bases of cellular damage and recovery remains extremely limited. Human diploid fibroblasts and mouse 10T1/z cells exposed to ionizing radiation during density-inhibition of growth demonstrate 2 phases of recovery during postradiation incubation at 37 °C: one which takes place within 2 to 10 min after exposure, and the second after 30 min (14, 15). The present investigation compares, for the first time, the kinetics of recovery in noncycling cultures of human diploid fibroblasts 2 min to 3 h after exposures to bleomycin and -y-irradiation. Our purpose was to determine if the rapid phase of recovery which occurs after exposures to -y- irradiation also occurs after exposures to bleomycin. We also wished to determine if a slower phase of recovery could be identified and whether or not the magnitude of either the rapid or slow component of recovery after exposures to bleomycin differed from that after 7-irradiation. An ultrarapid component and a slower component of cellular recovery have been identified. An unexpected finding was that, after equivalent killing, the magnitude of cellular recovery after bleomycin treatment always exceeded recovery after -y-irradia tion. MATERIALS AND METHODS Cell Strain and Culturing Conditions. Normal human (ibroblast cell strain AG1518, derived from normal newborn foreskin, was obtained from the Institute for Medical Research, Camden, NJ. Cultures were maintained in Falcon T-75 flasks at 37 °Cin an humidified atmosphere of 95% air and 5% CO2. Cultures were passaged approximately once weekly, suspending and reseeding them at a 1:4 dilution. Cells were used in experiments at passage levels 5 to 10 (1:4 split). The cells were grown in Eagle's MEM5 with Earle's salts (Gioco F-15), supplemented with 10 or 15% fetal calf serum (Microbiological Associates, Walkersville, MO), D-glucose (900 mg/liter), sodium pyruvate (6.6 mg/liter), penicillin (100 /¿g/ml),and streptomycin (100 jig/ml). For each experiment, ap proximately 150,000 cells were seeded into 60-mm Petri dishes. The medium was changed after 3 days. When the cells reached a confluent rnonolayer with an average density of 1 to 2 x 108 cells/plate, they were * The abbreviations used are: MEM, minimal essential medium; EBSS, Earle's balanced salt solution. CANCER RESEARCH VOL. 45 MAY 1985 1978 on March 12, 2021. © 1985 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from