1,2-Dimethylhydrazine-Induced Alterations in Protein Kinase C Activity in the Rat Preneoplastic Colon1

[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 (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, Â¡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 37Â°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 37Â°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 ÃŸ\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 25Â°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, HistolÃ³gica!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. Fixed specimens were ATP mixture were added, followed by vortex mixing and incubation at then embedded for light microscopic examination and stained with 25Â°Cfor 30 min. The reaction was terminated by the addition of 1.67 hematoxylin and eosin as previously described by our laboratory (23). ml of CHCI,/methanol/12 N HCI (66:31:1, v/v), followed by 1.67 ml Assay of Protein Kinase C. Colonie preparations from both groups of methanol/HjO/CHCl, (48:47:3, v/v) as described by MacDonald et of animals at the 10- and 15-week periods were homogenized by 15 al. (31), followed by vortex mixing and centrifugation. The upper phase strokes with a motor-driven Teflon pestle in 5 ml of homogenization was removed and discarded, and the lower phase was reextracted with buffer containing 20 mivi Tris-HCl, pH 7.5, 0.5 mivi EGTA, 2 mivi 1.67 ml of methanol/H2O/CHCl, (48:47:3, v/v). After centrifugation EDTA, 2.0 mivi phenylmethylsulfonyl fluoride, 0.5 mivi benzimidine, and removal of the upper phase, the labeled phosphatidic acid in the 5.0 mM 2-mercaptoethanol, and 10 mg/liter leupeptin (26). Each ho- lower phase was assayed directly by scintillation counting of an aliquot mogenate was centrifuged at 100,000 x g for 60 min and the superna as justified by Muldoon et al. (33). Standard curves were generated tant (cytosolic fraction) was saved. The pellet was gently resuspended from the assay of known amounts of 1,2-diacylglycerol and data were in 5.0 ml of homogenization buffer containing 0.3% Triton X-100 expressed as 1,2-diacylglycerol/lOO nmol of lipid phosphorus (mol%) (w/w) and left on ice for 60 min before recentrifugation at 100,000 x (32). g for 60 min. The supernatant (solubilized membrane fraction) was Determination of Labeled Inositol Phosphates and Phosphoinositides. collected and aliquots of both cytosolic and solubilized membrane Incorporation of pHjmyoinositol into inositol phosphates and mem fractions were assayed for protein (27), using bovine serum albumin as brane phosphoinositides was determined by using colon epithelial prep standard, and applied to DEAE-cellulose columns (Poly-prep columns, arations from both groups at each time period (15, 16). Colonie epithe 0.8 x 4 cm) that had been preequilibrated in the homogenization buffer. lium (10 mg protein) was incubated for 2 h in 2 ml of HBSS containing In preliminary studies, the columns were washed with 16 ml of buffer 180 mg/100 ml of D-glucose and 25 fid of ['Hjmyoinositol (specific and eluted with 32 ml of a linear gradient of 0-0.1 M NaCl in homog activity, 12.8 Ci/mmol). The epithelium was centrifuged at 500 x g for enization buffer. As most of the protein kinase C activity eluted in 10 min at 4Â°Cand washed 3 times with cold HBSS. The preparations 0.035-0.055 M NaCl, for routine purposes the loaded columns were were then resuspended in 2 ml of 20 mM 4-(2-hydroxyethyl)-l-pipera- first washed with 8 ml of homogenizing buffer and then with 4 ml of zineethanesulfonic acid/Tris buffer, pH 7.0, containing 25 mM ÃŸ- the same buffer containing 0.02 M NaCl. Finally, protein kinase C was glycerophosphate, 0.1 M KC1, 5 mM /S-mercaptoethanol, 5 mM MgCl2, eluted in 2 ml of buffer containing 0.08 M NaCl. The eluted cytosolic 0.02% trypsin inhibitor, and 10 mM LiCl. The suspensions were incu or membrane fractions were assayed for protein kinase C activity on bated for 20 min at 37Â°Cand the reactions were terminated by addition the same day, expressed as pmol/mg protein applied to column/min. of 0.67 ml 10% perchloric acid and allowed to stand on ice for 15 min. Protein kinase C activity was determined, using a histone phosphor- Following centrifugation at 500 x g, the supernatant was neutralized ylation assay, as previously described (26). Briefly, the DEAE-cellulose- with 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid/Tris purified fractions were incubated in a reaction mixture (final volume, buffer, pH 8.0, prior to determination of inositol phosphates. The pellet 75 M')containing (final concentrations) 20 mivi Tris-HCl (pH 7.2), 10 was mixed with 0.5 ml chloroform/methanol/12 N HC1 (200:100:0.75, miviMgCl2, 400 Mg/ml histone (type III-S), 1.83 miviCaCl2 (the actual v/v) and the extracted phosphoinositides were saved for later analyses. concentration of unchelated CaCl2 used was only 1.0 mivi, since the Separation of different species of inositol phosphates was achieved by aniÃ³n exchange chromatography on 0.3 ml AG-1-X8 (HCOO~) col contributions of EGTA and EDTA from the column elution buffer were 0.17 and 0.66 mM, respectively), 50 MM(7-"P]ATP (l ÃŸC\;specific umns (200-400 mesh) based on the method of Downes et al. (34). After activity, 3000 Ci/mmol), with and without 80 Mg/m' of phosphatidyl- loading, the columns were eluted serially with 1.5 ml each of (a) water; serine. Reactions were started by adding 25 ^1 of the protein kinase C (b) 0.1 M formic acid-0.2 M ammonium formate; (c) 0. l M formic acid- preparation to 50 /il of the assay mixture and the incubations were 0.4 M ammonium formate; and (d) 0.1 M formic acid-1 M ammonium carried out for 3 min at 30Â°C.Fifty p\ of the assay mixture were blotted formate. IP, IP2, and IP3 eluted in the second, third, and fourth onto a 2.5- x 2.5-cm phosphocellulose papers (Whatman P81) that had fractions, respectively. Radioactivity was assayed by scintillation count been prewashed in 10% trichloroacetic acid-2 mM NaH2PO4 solution. ing. For the separation and quantification of phosphoinositides, 0.2 ml The papers were then washed by agitating in 250 ml of ice-cold 10% of the acidified pellet extract was treated with 1.5 ml of chloroform/ trichloroacetic acid for 12 min and left under running water for 5 min. methanol (2:1, v/v), vortexed, and centrifuged. The upper phase was The filter papers were soaked in 95% ethanol for 5 min, followed by discarded and the lower phase was washed twice with 0.75 ml methanol/ diethyl ether for an additional 5 min, and were then air dried before 0.6 N HCI (1:1, v/v) and then separated on 1% potassium oxalate- taking Cherenkov counts. impregnated Silica Gel G chromatography plates. Unlabeled phos Protein kinase C activity was calculated from the difference in activity phoinositides were added as carriers. The plates were developed in assayed in the presence and absence of phosphatidylserine. Enzyme chloroform:acetone:methanol:glacial acetic acid:H2O (40:15:13:12:7, activity was linear with respect to enzyme concentration in all assays v/v) (22). The labeled phosphoinositides including phosphatidylinosi- and activity was expressed as pmol 32P/mg protein/min. tol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4-5- Measurement of 1,2-Diacylglycerol Mass. Preparations from each bis-phosphate were identified by comparison with phosphoinositide group at 10 and 15 weeks were treated with 10 ml of 2:1 chloro- standards and were quantified by scraping the silica gel from the plates form:methanol (v/v) and total cellular lipids were extracted essentially and counting the radioactivity in a liquid scintillation counter. Rou as described by Folch et al. (28). An aliquot of this extract was assayed tinely, greater than 90% of radioactivity added to the thin layer plates in triplicate for total lipid phosphorus as described by Bartlett (29). was recovered. 3916

Treatment of Colonie Epithelium with 1,25(OH)2D2. Recent studies cytosol to the membrane fraction, whereas DMH tissue did not by our laboratory (35) have demonstrated that in vitro addition of respond to this agent. In contrast to the 10-week data, however, l,25(OH)2Dj (10 nM) to normal colonie epithelial preparations rapidly the total activity of PKC was not significantly reduced (P < increased phosphoinositide turnover and DAG mass and activated PKC 0.001 ) in DMH tissue with and without 1,25(OH)2D, compared in these preparations. In the present studies, it was, therefore, of interest to their control counterparts (Table 1). The decrease was seen to determine whether DMH treatment altered the responsiveness of in cytosolic, not membrane PKC. Background cellular protein the colonie epithelium to this secosteroid. To address this issue, colonie kinase activity, in the absence of phosphatidylserine, was 2-4 epithelium obtained from both groups of animals at 10 and 15 weeks was incubated as described above for the various biochemical analyses, pmol/mg protein/min and did not change with time, DMH, or followed by the addition of a physiological concentration of 1,25(OH)2D, treatment. 1,25(OH)2D3 (10 nM) or ethanol vehicle (final concentration never Colonie DAG Mass. At both 10 and 15 weeks, DMH-treated exceeding 0.004%) for 1 min. This time and concentration were previ colonie mucosa was found to possess a significantly greater ously found to result in a maximum response of PKC, DAG, inositol mass of DAG than control colonie mucosa (Table 2). Moreover, phosphates, and phosphoinositides to this agent (35). The reaction was at each of these times, addition of 10 n\i 1,25(OH)2D, led to a terminated by the addition of ice-cold buffer and the preparation was significant increase in DAG mass in control, but not in DMH- processed as described above for analysis of these biochemical param treated preparations (Table 2). eters. Statistical Methods. All results are expressed as mean values Â±SE. Colonie Phosphoinositides and Inositol Phosphates. As shown in Table 3, at the 10- and 15-week periods, differences were Paired or unpaired r tests were used for all statistical analyses. P < 0.05 noted in the incorporation of [3H]myoinositol into individual was considered significant. Materials. 1,25(OH)2D3 was kindly provided by Dr. M. R. Uskokovic phosphoinositides and inositol phosphates between control and (Hoffmann-La Roche Inc., Nutley, NJ). Leupeptin, phenylmethylsul- DMH-treated preparations. As can be seen in this table, the fonyl fluoride, histone (type III-S), 1,2-dimethylhydrazine, phosphati- turnover of lableled phosphatidylinositol derivatives, repre dylserine, phosphatidylinositol standards, diethylenetriamine pentaa- sented by the ratio of total labeled inositol phosphates/total cetic acid, dithiothreitol, ATP, and DEAE-cellulose were purchased from Sigma Chemical Co. (St. Louis, MO). [7-32P]ATP, ['H]arachidon- labeled phosphoinositides was significantly greater at both time periods in DMH-treated preparations compared to their control ate, and [3H]myoinositol compounds were obtained from New England Nuclear Research Products (Boston, MA). Octyl-0-D-glucoside was counterparts. The majority of the change in inositol phosphate purchased from Boehringer Mannheim (Indianapolis, IN), sn-l-2-di- levels was due to changes in IP with no significant change noted acylglycerol kinase was from Lipidex, Inc. (Westfield, NJ), cardiolipin in the levels of the second messenger, IP,. Finally, control, but and Ã®rt-dioleoylglycerolwere from Avanti Polar Lipids, Inc. (Pelham, not DMH preparations, were found to significantly increase AL). this ratio and IP., levels after in vitro addition of 10 nM 1,25(OH)2D, for 1 min (Table 3). RESULTS DISCUSSION General Observations. In agreement with prior studies by our laboratory (23), at 10 and 15 weeks DMH administration did The present results demonstrate for the first time that alter not significantly affect the weight gain of these animals (data ations in PKC activity could be detected in the preneoplastic not shown). colons of rats after administration of the chemical carcinogen Light Microscopic Studies. In agreement with previous stud DMH for 10 and 15 weeks. At these time points, despite ies from our laboratory (22, 23), despite extensive sampling of extensive histolÃ³gica! sampling of the entire colon in these the colons of each group, no evidence of severe atypia, carci- animals, no evidence of inflammation, severe atypia, carcinoma noma-in-situ, or microscopic carcinomas were seen at 10 and in situ, or microscopic adenocarcinomas were evidenced by light 15 weeks by light microscopy (data not shown). In the present microscopic examination. experiments, minimal inflammation was also noted in the co- At 10 weeks, DMH-treated rats had a different cellular Ionic preparations which did not differ in intensity after 10 or distribution of PKC activity, with a greater percentage in the 15 weeks in each group. These results, therefore, indicate that membrane fraction than in their control counterparts. No dif inflammation per se was not responsible for the biochemical ferences, however, were noted in the total activity of this enzyme alterations noted in the DMH-treated colonie mucosa (see in the colon of these two groups at this time. Previous studies below). have demonstrated that PKC activity is elevated in the panic Colonie Protein Kinase C Activity. After 10 weeks, as shown ulate fraction of cells under a variety of conditions, including in Table 1, approximately 82 and 18% of PKC activity was phorbol ester tumor promotion, viral transformation, and rapid found in the cytosolic and membrane fractions, respectively, of cell growth (reviewed in Ref. 36). Based on these findings, it control colonie preparations. In DMH preparations at this time has been suggested that changes in the distribution of PKC may period, however, only 70% of PKC activity was found in the serve as a regulatory mechanism to alter the endogenous sub cytosolic fraction with 30% of the activity present in the mem strates available for phosphorylation by this kinase, thereby brane fraction. Moreover, upon addition of 10 nM 1,25(OH)2D., leading to tumor promotion and malignant transformation (36). to control tissue, PKC was found to significantly translocate At 15 weeks, the redistribution of PKC seen in the DMH from the cytosolic to membrane fraction at 1 min, whereas colonocytes at 10 weeks was further accentuated and, moreover, DMH-treated tissue failed to respond to this agent (Table 1). the total activity in DMH-treated colonie mucosa was reduced The total activity of PKC was, however, similar in control and to approximately one-half of the activity present in control DMH preparations with and without 1,25(OH)2D., (Table 1). colonie mucosa. It is important to note that changes in crude As can also be seen in this table, after 15 weeks, this pattern PKC activity levels may reflect alterations in the expression of of redistribution of PKC activity was further accentuated in the endogenous PKC inhibitors of phosphatases, both of which DMH preparations compared to their control counterparts. might interfere with the histone phosphorylation assay. Purifi Furthermore, control tissue again responded to in vitro admin cation on DEAE-cellulose, however, likely removes the major istration of 1,25(OH)2D3 with translocation of PKC from the ity of interfering compounds. Of interest, total PKC activity in 3917

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. DMH-INDUCED ALTERATIONS IN COLONIC PKC Table 1 PKC cellular distribution and total activity in colonie epithelium of control and DMH-treated rats after 10 and IS weeks"

PKCPreparations wkMembrane-associatedactivity wkMembrane-associatedactivity

activity activity (pmol/mg (pmol/mg (pmol/mg (pmol/mg nControl protein/min)3.8 protein/min)n20.9 protein/min)6.2 protein/min)27.0 5DMH Â±0.5(18.0 Â±0.8617.7 Â±0.7(23.0 Â±2.515.1 +2.5)5.3 Â±2.6)6.3 Â±2.1*29.6+ 5Control + 0.4 Â±0.5618.9 Â±0.9(42.0 (30.0 Â±2.1)*6.0 +6.2)*12.4

5DMH + 1,25(OH)2D, Â±0.3 Â±1.2617. + 0.7 1.614.4+ (32.0+1.5)*5.0 (42.0 Â±2.4)*7.5

+ 1.25(OH)2Dj 510 Â±0.2 2 Â±2.5 615 Â±0.7 1.0 (29.0+ 1.0)Total (52.0 Â±5.1)Total Â°Values represent means + SE of number of separate animals analyzed at each time point. Values in parentheses, percentage of total activity. * P < 0.05 or less compared to control values of similar time period.

Table 2 DAG mass in colonie epithelium of control and DMH-treated rats after 10 and 15 weeks" not totally clear, based on our study of colonie phosphoinositide turnover in these animals, it would appear that at least a portion wkDAG wkDAG of this DAG may come from the breakdown of these membrane mass(mol mass(mol phospholipids. While prior studies in oncogene-transformed PreparationsControlDMHControl %)0.36 %)0.36 cells have also suggested that phosphoinositides may serve as +0.010.41 Â±0.010.44 Â±0.01*0.53 +0.01*0.62 the source for increased DAG (40), others (5,41) have suggested +1,25(OH)2D,DMH+ +0.01*0.40 +0.01*0.44 that this lipid activator of PKC was derived from the breakdown 1.25(OH)2D3n666810 Â±0.01n888815 Â±0.01 of other membrane phospholipids. It is, therefore, possible that " Values represent means Â±SE of number of separate animals analyzed at the elevations in DAG induced DMH in the present experi each time period. * P < 0.001 compared to control values of similar time period. ments were also derived from these other phospholipids such as phosphatidylcholine and phosphatidylethanolamine as well as conceivably from phosphatidic acid (5). the control rats rose between 10 and 15 weeks, but decreased The exact significance of subsequent elevations in DAG, PKC slightly in DMH-treated rats, thereby illustrating the impor translocation, and down-regulation to colonie malignant trans tance of comparing age-matched animals. The changes in PKC formation in this model remains unclear. The presence of these distribution, followed by a decrease in total kinase activity in changes in the mucosa prior to overt neoplasia, however, sug the preneoplastic colonie mucosa of DMH-treated rats is con gests that they may play a role in the early stage(s) of the sistent with an apparent down-regulation of PKC which has multistage malignant transformation process. Additional fac been noted in several cultured cells treated with tumor-promot tors would then be necessary for the development of carcinoma ing phorbol esters (37-39). For example, MCF-7 cells treated in certain of these cells. with TPA have been shown to initially translocate PKC from Down-regulation in phorbol-treated and ras-transfected cells the cytosolic fraction to the paniculate fraction, followed there appears to be caused by proteolytic cleavage of PKC molecules after by a progressive decline in PKC activity in these cells (39). (42). Furthermore, cleavage of PKC has been associated with Recently, Guillem et ai. (19) also found that human colon the appearance of a protein kinase in the cytosol which was cancer samples that contained an admixture of benign adenom- independent of the activators of PKC, i.e., calcium, phosphati- atous tissue displayed a shift of PKC activity from the cytosolic dylserine and DAG (42). While the physiological significance to membrane fractions, whereas colon cancers lacking this of this protein kinase found by cleavage of PKC remains un benign tissue had reduced levels of total PKC activity. Based clear, Chida et al. (42) have suggested that it may diffuse into on their findings, these investigators (19) suggested that the the cytoplasm and nucleus of the cell and therefore transduce early stages of colonie transformation in humans may involve signals from the cytosolic side of the plasma membrane to the translocation of PKC activity, while later stages might be as nucleus, thereby inducing malignant transformation. Based on sociated with reduced total PKC activity, i.e., down-regulation. studies which have demonstrated synergism between the actions Additionally, initial translocation of PKC followed by reduc of ras oncogenes and phorbol esters on cellular transformation tion of PKC activity has been shown to occur in cells trans (43), Weyman et al. (8) have also recently speculated that down- formed by several oncogenes, particularly Ha-ras and Ki-rai (8, regulation of PKC, or a process closely linked to this phenom 9, 40). Concomitant with these alterations in PKC, DAG levels enon, may be important in the multistage carcinogenesis proc- were increased in these cells, suggesting that at least one mech ess(es). In contrast to these hypotheses, Wolfman et al. (9) have anism responsible for transformation of these cells involves suggested that down-regulation of PKC may actually represent persistent elevation of DAG leading to activation and then an unsuccessful attempt by transformed cells to negatively down-regulation of PKC (40). modulate proliferation signals generated by oncogene products. In the present experiments, DAG mass was found to be The exact relationship between down-regulation of PKC noted increased in the colonie mucosa of rats treated with DMH for in the current experiments to malignant transformation, there 10 and 15 weeks. It would, therefore, appear reasonable to fore, remains unclear. suggest that elevations in DAG might underlie the translocation In agreement with prior studies from our laboratory (35), in of PKC activity to the membrane at these time periods. While the present experiments l,25(OH)2Di was found to stimulate the origin of these DMH-induced elevations in DAG levels is colonie mucosal turnover of phosphoinositides, increase DAG 3918

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. DMH-INDUCED ALTERATIONS IN COLONIC PKC mass, and activate PKC in control animals. In DMH-treated

TO Vi *t O OOOdd rats, however, this secosteroid failed to significantly alter any 6+1 +1 -H+1& of these biochemical parameters in the rat colon. This is of vi mmr*> interest, since it would indicate that DMH-treated preneoplas- 1/1 r--

fNo l/^ â€” agent, well before the development of overt tumors. While the â€”â€”â€”â€¢H -H +1+1â€” exact mechanism(s) responsible for this loss of responsiveness â€¢^oor^r-' to 1,25(OH)2D3 is also unclear at this time, prior studies in od r^oÃ²OS oncogene-transformed cells have demonstrated a similar loss of 3^Ã¬2Â£^C^t1Â£211*%^rCi-3C3|I'â€¢Â§>S.a1'Ãˆ!..;-Â£iia SO ^CO0 response to various growth factors, secondary to a variety of â€” Oâ€”â€¢H alterations involving the inositol phospholipid signal transduc- +1 -H+1tfÃ¬ O00t/Ã¯ f*Ã¬ tion pathways and/or PKC (44-46). It should also be noted Or*1 C> f*Ã¬ that this secosteroid has been shown to possess antiproliferative

o ^soÃ– actions in cell lines derived from human colon cancers (47, 48). IN Ã–â€”+1 -H +1+1â€” It is, therefore, conceivable that this loss of responsiveness to rfÃ¬rt

6 OCâ€”SOÃŒO ogen. In this regard, however, prior studies in this model, using an identical treatment regimen, have demonstrated comparable <->dÃ²- increases in proliferation induced by DMH after 10 and 15 -+1 +1 +1+1rn weekly injections (50). It is, therefore, difficult to reconcile in v,o00 so r*^soTf changes in proliferation per se with the sequential changes noted TO t -t in PKC subcellular distribution and total activity in the present experiments. Moreover, in agreement with previous studies utilizing the phorbol ester 12-0-tetradecanoylphorbol-13-ace- O OOodo â€¢eÂ£'S..2Â§.B"gSf."Ãd+1 â€¢5Sâ€¢cg-5Ci.c\CÃŒ tate as well as deoxycholate (49), in preliminary experiments +1 +1+1â€” m mâ€¢Â»d performed by our laboratory, in vitro addition of 1,25(OH)2D, d ddâ€” was found to activate PKC to a similar extent in proliferative and nonproliferative normal colonie epithelial cells.4 Taken i fi r*linodo d+1 together, these observations, therefore, strongly suggest that +1+1+10 â€”^â„¢mri <â€¢> DMH-induced alterations in cellular proliferation are unlikely 2Â«Â°- to explain all the effects of this agent on PKC activity found in x xr-d o-Ã®Ã¯Iti^ the current studies. d d o +1 +1 +1+1^ In summary, the present results have demonstrated altera 00 00V)â€” 5aÂ« tions in PKC activity, DAG mass, and phosphoinositide turn D.0*- lf>-^ "V TÃ- ^ j3 over in the preneoplastic colons of rats administered DMH for Â« oaâ€¢o J-(â€”a ri riâ€”+1 10 to 15 weeks. Furthermore, in vitro addition of 1,25(OH)2D3 oeÃˆ?_ottQMeCNHCL,_-,a.OuÃ§ueucueueue"scdOÃ•eC&c.CL*Â°Â¿â€¢a. +1 +1+1Vi Ert $O?o.â€¢ooÂ«noÂ£ao,â€¢o"Â«â€” &â€”PÃ’ failed to elicit the normal pattern of response of these biochem O Ô O' CL O..^2 ical parameters in the colons of carcinogen-treated animals. _ r t â€”â€”'S ai Â°'C73 'SiTÃ¬ S p s Based on these observations, it would appear that changes in t/Ã¬oddPO >O PKC activity may play a role in the early stages of the malignant d lilisii!W CLcucu-â€¢CCLCPreparationsr*i+1+1+1+1*â€” transformation process induced by DMH. Further studies con g^55|*â€¢â€¢*POuÂ£.ft_Â¿tr._ti*1fs 00 00 O* f-jcqw od sor--sO Tt Q.rûâ€” cerning this possible relationship are now being conducted in S2Â£Ã•1J1u. '35 aco this experimental model of colonie adenocarcinoma by our 00ÔSodo â€”' Bo 30lili3 â€”*â€¢" laboratory. +1 +1 +1+1fi

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. 1,2-Dimethylhydrazine-induced Alterations in Protein Kinase C Activity in the Rat Preneoplastic Colon

Charles L. Baum, Ramesh K. Wali, Michael D. Sitrin, et al.

Cancer Res 1990;50:3915-3920.

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