[CANCER RESEARCH 48, 1798-1802, April 1, 1988] Potential Intracellular Target Proteins of the Anticarcinogenic Bowman Birk Protease Inhibitor Identified by Affinity Chromatograph}1 Paul C. Billings, William St. Clair, Albert J. Owen,2 and Ann R. Kennedy Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 021 IS ABSTRACT The soybean-derived Bowman Birk inhibitor (BBI) has been shown to inhibit carcinogenesis in both in vitro and in vivo model systems. In the present study, we have utilized a BBI affinity column to determine whether cellular enzymes, present in C3H/10T'/2 cells, specifically inter act with this inhibitor. Using this technique, we have identified three proteins with masses of about 70, 60, and 50 kilodaltons. Cell fractiona- tion experiments demonstrate that the 60- and 50-kilodalton proteins are present in the 10,000 x g pellet (lysosomal/golgi fraction) of C3H/10T'/2 cell homogenates. We have also identified two proteins with masses of 60 and SO kilodaltons which bind to the BBI affinity column in fibroblasts from patients having Bloom syndrome. BBI as well as several other protease inhibitors has been shown previously to reduce the frequency of spontaneous chromosomal aberrations in these cells. Our results indicate that the 50- and 60-kilodalton proteins we have identified by affinity Chromatograph) are present in both mouse and human cells and further suggest that these proteins are potential intracellular targets of the BBI in these cells. INTRODUCTION Protease inhibitors are effective suppressors of carcinogenesis i/i vivo and carcinogen-induced transformation in vitro (1-8). Although much is known about the specific types of protease inhibitors which are potent suppressors of carcinogenesis, rel atively little is known about the precise mechanism(s) by which these compounds exert their anticarcinogenic effects. We hy pothesize that protease inhibitors exert their anticarcinogenic effects by inhibiting cellular enzymes involved in the induction and/or expression of the transformed phenotype. Our labora tory has studied the anticarcinogenic activity of the soybean- derived protease BBI3 both in vivo and in vitro (5, 7, 8). The BBI is an 8000-dalton protein which effectively inhibits both trypsin and chymotrypsin (9). This inhibitor has been shown to suppress dimethylhydrazine-induced colon carcinogenesis in mice when present in the diet (7) and dimethyl- benz(a)anthracene-induced cheek pouch carcinogenesis when topically applied (5). Additionally, BBI has been shown to suppress radiation-induced transformation of C3H/10T'/2 cells at nanomolar concentrations (8). BS is an autosotnal recessive genetic disease characterized by high levels of chromosomal aberrations and sister chromatid exchanges which are thought to predispose affected individuals to a higher than normal incidence of cancer (10). We have demonstrated that the BBI, as well as several other protease inhibitors, will reduce the frequency of chromosomal abnormalities in BS cells when the cells are grown in the presence of the inhibitor (11). Therefore, protease inhibitors have been shown to suppress specific chromosomal abnormal- Received8/24/87;revised12/15/87;accepted12/31/87. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by NIH Grants CA45734, CA40214, CA22704, and ES00002. 1 Present address: Department of Nutrition, Harvard School of Public Health, Boston, M A 02 US. 3 The abbreviations used are: BBI, Bowman Birk inhibitor, BS, Bloom syn drome; DFP, diisopropylfluorophosphate; FITC, fluorescein isothiocyanate; kDa, kilodaltons; MCA, methylcholanthrene. ¡tieswhich are thought to be involved in human cancer devel opment. Previously, defined substrates have been used to identify two enzymatic activities in C3H/10T'/2 cells which are inhibited by several protease inhibitors processing anticarcinogenic activity (12, 13).4 Using the thrombin substrate Boc-Val-Pro-Arg- MCA, our laboratory has identified a 70-kilodalton cytosolic serine protease (12) which is inhibited by several anticarcino genic protease inhibitors. Yavelow et al. (13) and Carew and Kennedy4 have identified a BBI-inhibitable activity which cleaves the chymotrypsin substrate Suc-Ala-Ala-Pro-Phe- MCA. In both of these studies ( 13)4the enzyme was not purified and hence, from the data presented, it is not clear whether this activity represents single or multiple activities. The use of defined substrates is an indirect approach to identifying "target enzymes" of the anticarcinogenic protease inhibitors. This strat egy is inherently limited because it allows one to determine only whether the cleavage of a particular substrate is affected by a specific protease inhibitor and does not directly address the broader and more important question of which cellular proteins actually interact with these inhibitors in the cell. Affinity chromatography represents a more direct approach to identifying those cellular enzymes which interact with the anticarcinogenic protease inhibitors. This technique will poten tially enable us to isolate functionally intact enzymes in large enough quantities for biochemical characterization. In addition, affinity chromatography should allow us to identify all of the cellular proteins which interact with BBI. In the current series of experiments, we have utilized a BBI affinity column as a tool to identify cellular enzymes from C3H/10T'/2 and BS cells which interact with BBI. In this report we describe the results of these experiments. MATERIALS AND METHODS Chemicals. The BBI was obtained as a crude extract from Technion Testing and Research Laboratory, Inc. (Belleville, NJ), and purified by DEAE ion exchange chromatography as previously described (9). S-35 methionine was obtained from New England Nuclear. CNBr-activated sepharose and chymotrypsin (type II) were obtained from Sigma. En lighten was obtained from New England Nuclear. Suc-Ala-Ala-Pro- Phe-MCA was obtained from Peninsula Laboratories, Belmont, CA, and was made up as a 1-mg/ml (1.51 HIM)stock solution in spectrograde dimethylsulfoxide and stored at 4*C. Cells. C3H/10T'/2 cells were grown in basal minimal Eagle's medium containing 10% heat-inactivated fetal calf serum as described (1, 3). BS cells were grown in minimal essential Eagle's medium containing 20% fetal calf serum (11). S-35 Labeling of Cellular Proteins. Log-phase C3H/10T'/2 or BS cells were grown in methionine-free medium containing 10% heat- inactivated fetal calf serum and [3SS]methionine (50 ¿tCi/ml)for 12 h. After labeling, the medium was removed, the cell monolayers were washed twice with ice-cold Earle's balanced salt solution, and the cells were removed from the dishes with a rubber policeman. The cells were pelleted by centrifugation at 800 x g for 5 min. Subcellular Fractionation. Cells were resuspended in 5 ml of ice-cold 4J. A. Carew, and A. R. Kennedy. Identification of a metaloprotease which may be involved in transformation of C3H/10T'/2 cells, submitted for publication. 1798 Research. on November 25, 2021. © 1988 American Association for Cancer cancerres.aacrjournals.org Downloaded from