[CANCER RESEARCH 41, 2103-2108, June 1981] 0008-5472/81 /0041-OOOOS02.00 Effects of Protease Inhibitors on Radiation Transformation in Vitro1 Ann R. Kennedy2 and John B. Little Laboratory of Radiobiology, Harvard University School of Public Health. Boston, Massachusetts 02115 ABSTRACT We have investigated the effects of three protease inhibitors, antipain, leupeptin, and soybean trypsin inhibitor, on the induc tion of oncogenic transformation in mouse C3H10T'/2 cells by X-rays. The patterns of inhibition by the three protease inhibi tors were different. Antipain was the most effective, having the ability to suppress completely radiation transformation as well as radiation transformation enhanced by the phorbol ester promoting agent 12-O-tetradecanoylphorbol-13-acetate. The fact that antipain could suppress transformation when present for only 1 day following irradiation suggests that an effect on a DNA repair process might be important in its action. Leupep tin was less effective than antipain in its inhibition of radiation transformation. Soybean trypsin inhibitor suppressed only the promotional effects of 12-O-tetradecanoylphorbol-13-acetate on transformation. Our results suggest that there may be more than one protease involved in carcinogenesis. INTRODUCTION In an attempt to gain more information about the mechanisms of carcinogenesis, we have been studying the effects of various agents on radiation transformation in vitro. It has been shown previously that the development of malignant transformation in mammalian cells irradiated in vitro can be thought of as involv ing at least 2 steps: (a) the induction and fixation of the transformed state as a heritable cellular property; and (b) its subsequent expression in terms of morphologically altered cells (14). We have shown previously that incubation with a nontoxic concentration of TPA3 during the expression period will greatly enhance X-ray transformation in mouse C3H10T1/2 cells (9, 10) as it has been shown to enhance tumor formation in vivo in "2-stage" (initiation-promotion) carcinogenesis ex periments (5). Our interest in the effects of protease inhibitors on radiation transformation was stimulated by the reports that some of these agents could inhibit error-prone DNA repair and mutagenesis in bacteria (16, 30) as well as inhibit TPA-enhanced carcino genesis in vivo (6, 26). Thus, protease inhibitors might influence both the fixation and expression steps in in vitro transformation. It is our hypothesis that much of the transformation observed in rodent cell lines in culture may result from the action of an error-prone DNA repair process such as has been shown to exist in bacterial cells. Supportive evidence for such a mech anism in 1OT'/2 cells was found in previous experiments (14, 24). We have investigated the effects of 3 protease inhibitors, antipain, leupeptin, and SBTI, which specifically inhibit different ' This research was supported by NIH Grants CA-22704 and ES-00002. 2To whom requests for reprints should be addressed. 3 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; SBTI, soybean trypsin inhibitor. Received January 29, 1980; accepted February 25, 1981. proteolytic enzymes. The enzymes known to be inhibited by each agent include: antipain-papain, trypsin, thrombokinase, cathepsin A and B, plasmin, and plasminogen activator (1, 33); leupeptin-papain, trypsin, plasmin, cathepsin B, and plasmin ogen activator (1, 33); and SBTI-trypsin, chymotrypsin, throm- oplastin, plasmin, and elastase (11, 13). SBTI is of particular interest, as it is a naturally occurring protease inhibitor found in the normal diet. It may thus play a role in the prevention of cancer in humans (29). It has been found recently that carci nogenesis in 3 organ systems can be inhibited in animals kept on a soybean diet rich in protease inhibitors (29). Antipain and leupeptin are small-molecular-weight (M.W. approximately 500 to 600) natural products isolated from ac- tinomycetes. They have been characterized by Aoyagi and Umezawa (1 ). Effective concentrations of these 2 inhibitors are nontoxic, either with or without radiation exposure, and have been shown to have no effect on growth, RNA, or protein synthesis in bacteria (16) or mammalian cells (3, 7, 31). SBTI is a high-molecular-weight (M.W. approximately 24,000) mac- romolecular-type protease inhibitor (11 ), which is also nontoxic to C3H10TV2 cells at the concentrations used. We report here the effects of protease inhibitors, added at different times in radiation transformation experiments with and without promo tion induced by TPA. A preliminary report of our results has been published (8). MATERIALS AND METHODS We used a C3H mouse embryo-derived cell line (1 OTVzclone 8) isolated and characterized by Reznikoff ef al. (21, 22) and adapted for studies of radiation transformation (14, 24, 25). Stock cultures were maintained in 60-mm Petri dishes and were passaged by subculturing at a 1:20 dilution every 7 days. The cells used were in passages 9 to 14. They were grown in a humidified 5% CO2 atmosphere at 37° in Eagle's basal medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics. Cells were irradiated 24 hr after seeding and incubated in fresh medium containing the protease inhibi tors. When the cells reached confluence, the serum concentra tion in the medium was changed to 5% for the duration of the transformation assay. Irradiation was carried out at room temperature with a 100- kV constant potential Philips industrial X-ray generator oper ating at 10 ma and yielding a dose rate to the cells of 78 rads/ min. There were 100 to 400 viable cells/dish; plating efficien cies were determined from plates seeded with a cell density one-fifth that of the plates for the transformation assay and terminated 10 days after irradiation. Type II and III foci were scored as transformants. Details of radiation transformation experiments have been described previously (25). TPA treatment at 0.1 jug/ml was begun 48 hr after irradiation and maintained for the entire 6-week expression period as described previously (9, 10), TPA, Lot Nos. 007 and 016, was obtained from Consolidated Midland Co., Brewster, N. Y. Pro- JUNE 1981 2103 Research. on December 15, 2021. © 1981 American Association for Cancer cancerres.aacrjournals.org Downloaded from