[CANCER RESEARCH 50. 3915-3920. July I. 1990) 1,2-Dimethylhydrazine-induced Alterations in Protein Kinase C Activity in the Rat Preneoplastic Colon1 Charles L. Baum, Ramesh K. Wali, Michael D. Sitrin, Merry J. G. Bolt, and Thomas A. Brasitus2 Department of Medicine, University of Chicago Hospitals and Clinics, Prit:ker School of Medicine, University of Chicago, Chicago, Illinois 60637 ABSTRACT which indicate that alterations in the activity of this kinase may also be involved in malignant transformation (7-14). Thus, Recently, a number of studies in experimental animals and humans tumor-promoting phorbol esters have been shown to bind to have suggested that alterations in the activity of protein kinase C (PKC) and directly activate PKC (7). Data indicate that PKC may also may be involved in the malignant transformation process. To determine play a role in the in vitro transformation processes produced by whether such alterations in this kinase were present before the develop a number of oncogenes (8-10). Cells that overproduce PKC ment of 1,2-dimethylhydrazine (DMH)-induced colon cancers, rats were given s.c. injections of this procarcinogen (20 mg/kg body weight/week) have, moreover, been shown to be more susceptible to transfor or diluent for 10 or 15 weeks. Animals were sacrificed after these time mation by one such activated oncogene, H-ras (11). Addition periods and colonie epithelium was harvested from each group. The ally, fibroblasts transfected with plasmids containing PKC- activity and distribution of PKC in the cytosolic and membrane fractions complementary DNA, overproduce PKC and demonstrate en of these preparations as well as 1,2-diacylglycerol mass and phosphoi- hanced tumorigenicity (12, 13). Finally, agents which inhibit nositide turnover were then examined and compared in the presence and PKC have recently been shown to possess in vivo antitumor absence of 10 n\i 1,25-dihydroxycholecalciferol, an agent which has activity (14). Taken together, these observations would strongly previously been found to influence these biochemical parameters in the suggest that PKC may be involved in the malignant transfor normal rat colonie epithelium. mation process(es) of various tissues and organs. The results of these studies demonstrate that: (a) the percentage of PKC activity in the membrane fraction was significantly greater in I>M11- Recent studies in experimental animals and cultured cells (15-18) as well as in humans (19) have, in fact, suggested that treated animals compared to their control counterparts at 10 and 15 weeks; (b) the total PKC activity was similar at 10 weeks, but markedly alterations in PKC activity may be involved in colonie carci- reduced in the colonie mucosa of the DMH-treated group at 15 weeks; nogenesis. Both bile acids (15, 17, 18) and free fatty acids (16), (c) 1,2-diacylglycerol mass and phosphoinositide turnover were increased which may act as promoters of colon cancer (20), have been in the colonie mucosa of rats administered this carcinogen at both time shown to affect the activity of this kinase. Furthermore, Guillem points; and (</) in control, but not in DMH-treated animals, in vitro et al. (19) have recently shown a reduction in PKC activity in addition of 1,25-dihydroxycholecalciferol increased PKC activity, 1,2- human colon adenocarcinomas compared to normal adjacent diacylglycerol mass and phosphoinositide turnover at each of the times colonie mucosa. studied. Based on these findings, it would appear that alterations in PKC In this regard, during the past several years, a number of activity may play a role in the malignant transformation process of the colon in animals administered DMH. laboratories, including our own, have utilized the DMH model of experimental colon cancer to study the biological character istics of these tumors (21). DMH-induced neoplasms closely INTRODUCTION parallel human colon cancer with respect to clinical and path PKC3 is a serine/threonine specific protein kinase which is ological features (21). In weekly s.c. doses of 20 mg/kg of body weight, this procarcinogen produces colonie carcinomas in a dependent upon phospholipid and calcium (1). This enzyme is high percentage of susceptible rodent strains (21-23), with a ubiquitous in eukarocytes (2) and appears to play a key role in latency period of approximately 6 months. Moreover, utilizing transmembrane signaling events (1-3). PKC is activated by this model, several laboratories have demonstrated various bio DAG which is formed in response to extracellular signals by chemical changes in rat colonie tissue prior to the development turnover of phosphoinositides (4) as well as other membrane of overt tumors (22-25). While prior investigations have dem phospholipids (5). This activation process involves transloca onstrated early DMH-induced alterations in rat colonie mu- tion of cytosolic PKC to the plasma membrane(s) which, in cosal cyclic AMP-dependent protein kinase activity (25), to turn, leads to phosphorylation of target molecules, thereby date no data are available on the effects of this carcinogen on influencing important cellular processes such as proliferation PKC activity. In the present experiments it was, therefore, of and/or differentiation (6). interest to determine whether alterations in PKC, DAG, or Several lines of evidence have recently been accumulated phosphoinositides exist in colonie cells of rats administered Received 11/17/89; revised 3/13/90. DMH for 10 and 15 weeks, i.e., before the development of The costs of publication of this article were defrayed in part by the payment colon cancer. The data from these experiments demonstrate of page charges. This article must therefore be hereby marked advertisement in that "premalignant" changes in these biochemical parameters accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' These experiments were supported by the Samuel Freedman Laboratories for can, indeed, be detected and serve as the basis for this report. Cancer Research Fund as well as by USPHS Grant CA 36745 awarded by the National Cancer Institute, by USPHS Grant DK 26678 (Clinical Nutrition Research Unit) awarded by the National Institute of Diabetes and Digestive and Kidney Diseases. Department of Health and Human Services, and by USPHS MATERIALS AND METHODS Grant DK 39573 by the National Institute of Diabetes and Digestive and Kidney Diseases. Department of Health and Human Services. 2Recipient of a MERIT award from the National Cancer Institute. NIH. To Animals. Male Sprague Dawley rats weighing 75-100 g were given whom requests for reprints should be addressed, at University of Chicago Hos weekly s.c. injections of diluent or 1,2-dimethylhydrazine dihydrochlo- pitals and Clinics. 5841 S. Maryland Avenue. Box 400. Chicago, IL 60637. ride (Sigma Chemical Co.. St. Louis, MO) at a dose of 20 mg/kg body 3The abbreviations used are: PKC. protein kinase C; DMH. 1.2-dimethylhy- weight for 10 and 15 weeks. The stock solution for injections consisted drazine; DAG. 1,2-diacylglycerol; IP3. inositol-1.4.5-trisphosphate; IP2. inositol- 1,4-bisphosphate; IPt, ¡nositolphosphate; 1.25(OH)2Dj, 1.25-dihydroxyvitamin of 400 mg of DMH dissolved in 100 ml of 1.0 M EDTA adjusted to Dj; HBSS, Hanks' balanced salt solution; EGTA. ethyleneglycol bis(fi-amino- pH 6.5 with sodium hydroxide (23). One week after the last injection, ethylether)-JV,A',A",A''-tetraaceticacid. at each time period studied, rat colonie epithelium was isolated accord- 3915 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. DMH-INDUCED ALTERATIONS IN COLON1C PKC ing to the method of Craven et al. (15) with the following modifications. Same-day aliquots of lipid extract were also assayed for 1,2-diacylglyc- After ether anesthesia the colon was flushed with 50 ml of Ca- and Mg- erol mass by the diacylglycerol kinase procedure of Preiss et al. (30) free HBSS at 37°C.A 16-gauge needle was placed in the left ventricle with the following modifications. S-fW-MorpliolinoÃŒpropanesulfonic and HBSS containing 30 mivi EDTA at 37°Cwas perfused at a flow acid buffer, pH 6.6, was substituted for imidazole buffer, pH 6.6 (31). rate of 20 ml/min for 5-6 min. The colon was then excised, gently For each reaction tube, DAG kinase (20 milliunits in 10 n\) was everted, and applied to a 5-ml glass pipet attached to a rheostat- combined with 50 ß\of reaction buffer (100 mM sodium 3-(/V-mor- controlled electric motor. The everted gut and pipet were then immersed pholino)propanesulfonic acid, pH 6.6, 100 mM NaCl, 25 mM MgCl2, in a 30-ml glass test tube containing cold HBSS. The epithelium was 2 mM EGTA) plus 10 n\ of 20 mM dithiothreitol and 10 n\ of 10 mM removed by rotating the pipet at 1600 rpm in bursts of 5-10 s for 1-2 [7-"P]ATP (5.0 x IO4 cpm/nmol), and the mixture was incubated at min. The epithelium in HBSS was then centrifuged at 500 x g for 5 25°Cfor 30 min as described by Wright et al. (32). Duplicate aliquots min and resuspended in oxygenated Krebs-Ringer bicarbonate buffer at of dioleoylglycerol standards or cellular lipid extracts were transferred pH 7.4. The viability of each preparation was routinely greater than to 75- x 12-mm capped Sarstedt polyethylene tubes and dried under 90% after 2 h, as assessed by Trypan blue exclusion. argon. A 20-^1 aliquot of resuspension buffer (7.5% octyl-/j-D-glucoside, Histológica!Studies. Multiple 1-cm samples (at least 4) were taken 5 mM cardiolipin, 1 mM diethylenetriamine pentaacetic acid) was added, from each colon preparation of each group at 10 and 15 weeks and vortexed, and sonicated as described (30). To this, 80 ^1of the enzyme- were immediately fixed in 4% formaldehyde.