[CANCER RESEARCH 50. 3915-3920. July I. 1990)

1,2-Dimethylhydrazine-induced Alterations in Protein Kinase C Activity in the RatPreneoplastic 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

Recently, a number of studies in experimental animals and humanshave suggested that alterations in the activity of protein kinase C (PKC)may be involved in the malignant transformation process. To determinewhether such alterations in this kinase were present before the development of 1,2-dimethylhydrazine (DMH)-induced colon cancers, rats weregiven s.c. injections of this procarcinogen (20 mg/kg body weight/week)or diluent for 10 or 15 weeks. Animals were sacrificed after these timeperiods and colonie epithelium was harvested from each group. Theactivity and distribution of PKC in the cytosolic and membrane fractionsof these preparations as well as 1,2-diacylglycerol mass and phosphoi-nositide turnover were then examined and compared in the presence andabsence of 10 n\i 1,25-dihydroxycholecalciferol, an agent which haspreviously been found to influence these biochemical parameters in thenormal rat colonie epithelium.

The results of these studies demonstrate that: (a) the percentage ofPKC activity in the membrane fraction was significantly greater in I>M11-treated animals compared to their control counterparts at 10 and 15weeks; (b) the total PKC activity was similar at 10 weeks, but markedlyreduced in the colonie mucosa of the DMH-treated group at 15 weeks;(c) 1,2-diacylglycerol mass and phosphoinositide turnover were increasedin the colonie mucosa of rats administered this carcinogen at both timepoints; and (</) in control, but not in DMH-treated animals, in vitroaddition of 1,25-dihydroxycholecalciferol increased PKC activity, 1,2-diacylglycerol mass and phosphoinositide turnover at each of the timesstudied. Based on these findings, it would appear that alterations in PKCactivity may play a role in the malignant transformation process of thecolon in animals administered DMH.

INTRODUCTION

PKC3 is a serine/threonine specific protein kinase which is

dependent upon phospholipid and calcium (1). This enzyme isubiquitous in eukarocytes (2) and appears to play a key role intransmembrane signaling events (1-3). PKC is activated byDAG which is formed in response to extracellular signals byturnover of phosphoinositides (4) as well as other membranephospholipids (5). This activation process involves translocation of cytosolic PKC to the plasma membrane(s) which, inturn, leads to phosphorylation of target molecules, therebyinfluencing important cellular processes such as proliferationand/or differentiation (6).

Several lines of evidence have recently been accumulated

Received 11/17/89; revised 3/13/90.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' These experiments were supported by the Samuel Freedman Laboratories forCancer Research Fund as well as by USPHS Grant CA 36745 awarded by theNational Cancer Institute, by USPHS Grant DK 26678 (Clinical NutritionResearch Unit) awarded by the National Institute of Diabetes and Digestive andKidney Diseases. Department of Health and Human Services, and by USPHSGrant DK 39573 by the National Institute of Diabetes and Digestive and KidneyDiseases. Department of Health and Human Services.

2Recipient of a MERIT award from the National Cancer Institute. NIH. To

whom requests for reprints should be addressed, at University of Chicago Hospitals and Clinics. 5841 S. Maryland Avenue. Box 400. Chicago, IL 60637.

3The abbreviations used are: PKC. protein kinase C; DMH. 1.2-dimethylhy-drazine; DAG. 1,2-diacylglycerol; IP3. inositol-1.4.5-trisphosphate; IP2. inositol-1,4-bisphosphate; IPt, ¡nositolphosphate; 1.25(OH)2Dj, 1.25-dihydroxyvitaminDj; HBSS, Hanks' balanced salt solution; EGTA. ethyleneglycol bis(fi-amino-ethylether)-JV,A',A",A''-tetraaceticacid.

which indicate that alterations in the activity of this kinase mayalso be involved in malignant transformation (7-14). Thus,tumor-promoting phorbol esters have been shown to bind toand directly activate PKC (7). Data indicate that PKC may alsoplay a role in the in vitro transformation processes produced bya number of oncogenes (8-10). Cells that overproduce PKChave, moreover, been shown to be more susceptible to transformation by one such activated oncogene, H-ras (11). Additionally, fibroblasts transfected with plasmids containing PKC-complementary DNA, overproduce PKC and demonstrate enhanced tumorigenicity (12, 13). Finally, agents which inhibitPKC have recently been shown to possess in vivo antitumoractivity (14). Taken together, these observations would stronglysuggest that PKC may be involved in the malignant transformation process(es) of various tissues and organs.

Recent studies in experimental animals and cultured cells(15-18) as well as in humans (19) have, in fact, suggested thatalterations in PKC activity may be involved in colonie carci-nogenesis. Both bile acids (15, 17, 18) and free fatty acids (16),which may act as promoters of colon cancer (20), have beenshown to affect the activity of this kinase. Furthermore, Guillemet al. (19) have recently shown a reduction in PKC activity inhuman colon adenocarcinomas compared to normal adjacentcolonie mucosa.

In this regard, during the past several years, a number oflaboratories, including our own, have utilized the DMH modelof experimental colon cancer to study the biological characteristics of these tumors (21). DMH-induced neoplasms closelyparallel human colon cancer with respect to clinical and pathological features (21). In weekly s.c. doses of 20 mg/kg of bodyweight, this procarcinogen produces colonie carcinomas in ahigh percentage of susceptible rodent strains (21-23), with alatency period of approximately 6 months. Moreover, utilizingthis model, several laboratories have demonstrated various biochemical changes in rat colonie tissue prior to the developmentof overt tumors (22-25). While prior investigations have demonstrated early DMH-induced alterations in rat colonie mu-cosal cyclic AMP-dependent protein kinase activity (25), todate no data are available on the effects of this carcinogen onPKC activity. In the present experiments it was, therefore, ofinterest to determine whether alterations in PKC, DAG, orphosphoinositides exist in colonie cells of rats administeredDMH for 10 and 15 weeks, i.e., before the development ofcolon cancer. The data from these experiments demonstratethat "premalignant" changes in these biochemical parameters

can, indeed, be detected and serve as the basis for this report.

MATERIALS AND METHODS

Animals. Male Sprague Dawley rats weighing 75-100 g were givenweekly s.c. injections of diluent or 1,2-dimethylhydrazine dihydrochlo-ride (Sigma Chemical Co.. St. Louis, MO) at a dose of 20 mg/kg bodyweight for 10 and 15 weeks. The stock solution for injections consistedof 400 mg of DMH dissolved in 100 ml of 1.0 M EDTA adjusted topH 6.5 with sodium hydroxide (23). One week after the last injection,at each time period studied, rat colonie epithelium was isolated accord-

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DMH-INDUCED ALTERATIONS IN COLON1C PKC

ing to the method of Craven et al. (15) with the following modifications.After ether anesthesia the colon was flushed with 50 ml of Ca- and Mg-free HBSS at 37°C.A 16-gauge needle was placed in the left ventricleand HBSS containing 30 mivi EDTA at 37°Cwas perfused at a flow

rate of 20 ml/min for 5-6 min. The colon was then excised, gentlyeverted, and applied to a 5-ml glass pipet attached to a rheostat-controlled electric motor. The everted gut and pipet were then immersedin a 30-ml glass test tube containing cold HBSS. The epithelium wasremoved by rotating the pipet at 1600 rpm in bursts of 5-10 s for 1-2min. The epithelium in HBSS was then centrifuged at 500 x g for 5min and resuspended in oxygenated Krebs-Ringer bicarbonate buffer atpH 7.4. The viability of each preparation was routinely greater than90% after 2 h, as assessed by Trypan blue exclusion.

Histológica!Studies. Multiple 1-cm samples (at least 4) were takenfrom each colon preparation of each group at 10 and 15 weeks andwere immediately fixed in 4% formaldehyde. Fixed specimens werethen embedded for light microscopic examination and stained withhematoxylin and eosin as previously described by our laboratory (23).

Assay of Protein Kinase C. Colonie preparations from both groupsof animals at the 10- and 15-week periods were homogenized by 15strokes with a motor-driven Teflon pestle in 5 ml of homogenizationbuffer containing 20 mivi Tris-HCl, pH 7.5, 0.5 mivi EGTA, 2 miviEDTA, 2.0 mivi phenylmethylsulfonyl fluoride, 0.5 mivi benzimidine,5.0 mM 2-mercaptoethanol, and 10 mg/liter leupeptin (26). Each ho-mogenate was centrifuged at 100,000 x g for 60 min and the supernatant (cytosolic fraction) was saved. The pellet was gently resuspendedin 5.0 ml of homogenization buffer containing 0.3% Triton X-100(w/w) and left on ice for 60 min before recentrifugation at 100,000 xg for 60 min. The supernatant (solubilized membrane fraction) wascollected and aliquots of both cytosolic and solubilized membranefractions were assayed for protein (27), using bovine serum albumin asstandard, and applied to DEAE-cellulose columns (Poly-prep columns,0.8 x 4 cm) that had been preequilibrated in the homogenization buffer.In preliminary studies, the columns were washed with 16 ml of bufferand eluted with 32 ml of a linear gradient of 0-0.1 M NaCl in homogenization buffer. As most of the protein kinase C activity eluted in0.035-0.055 M NaCl, for routine purposes the loaded columns werefirst washed with 8 ml of homogenizing buffer and then with 4 ml ofthe same buffer containing 0.02 M NaCl. Finally, protein kinase C waseluted in 2 ml of buffer containing 0.08 M NaCl. The eluted cytosolicor membrane fractions were assayed for protein kinase C activity onthe same day, expressed as pmol/mg protein applied to column/min.

Protein kinase C activity was determined, using a histone phosphor-ylation assay, as previously described (26). Briefly, the DEAE-cellulose-purified fractions were incubated in a reaction mixture (final volume,75 M')containing (final concentrations) 20 mivi Tris-HCl (pH 7.2), 10miviMgCl2, 400 Mg/ml histone (type III-S), 1.83 miviCaCl2 (the actualconcentration of unchelated CaCl2 used was only 1.0 mivi, since thecontributions of EGTA and EDTA from the column elution bufferwere 0.17 and 0.66 mM, respectively), 50 MM(7-"P]ATP (l ßC\;specificactivity, 3000 Ci/mmol), with and without 80 Mg/m' of phosphatidyl-serine. Reactions were started by adding 25 ^1 of the protein kinase Cpreparation to 50 /il of the assay mixture and the incubations werecarried out for 3 min at 30°C.Fifty p\ of the assay mixture were blotted

onto a 2.5- x 2.5-cm phosphocellulose papers (Whatman P81) that hadbeen prewashed in 10% trichloroacetic acid-2 mM NaH2PO4 solution.The papers were then washed by agitating in 250 ml of ice-cold 10%trichloroacetic acid for 12 min and left under running water for 5 min.The filter papers were soaked in 95% ethanol for 5 min, followed bydiethyl ether for an additional 5 min, and were then air dried beforetaking Cherenkov counts.

Protein kinase C activity was calculated from the difference in activityassayed in the presence and absence of phosphatidylserine. Enzymeactivity was linear with respect to enzyme concentration in all assaysand activity was expressed as pmol 32P/mg protein/min.

Measurement of 1,2-Diacylglycerol Mass. Preparations from eachgroup at 10 and 15 weeks were treated with 10 ml of 2:1 chloro-

form:methanol (v/v) and total cellular lipids were extracted essentiallyas described by Folch et al. (28). An aliquot of this extract was assayedin triplicate for total lipid phosphorus as described by Bartlett (29).

Same-day aliquots of lipid extract were also assayed for 1,2-diacylglyc-erol mass by the diacylglycerol kinase procedure of Preiss et al. (30)with the following modifications. S-fW-MorpliolinoÌpropanesulfonicacid buffer, pH 6.6, was substituted for imidazole buffer, pH 6.6 (31).For each reaction tube, DAG kinase (20 milliunits in 10 n\) wascombined with 50 ß\of reaction buffer (100 mM sodium 3-(/V-mor-pholino)propanesulfonic acid, pH 6.6, 100 mM NaCl, 25 mM MgCl2,2 mM EGTA) plus 10 n\ of 20 mM dithiothreitol and 10 n\ of 10 mM[7-"P]ATP (5.0 x IO4 cpm/nmol), and the mixture was incubated at25°Cfor 30 min as described by Wright et al. (32). Duplicate aliquots

of dioleoylglycerol standards or cellular lipid extracts were transferredto 75- x 12-mm capped Sarstedt polyethylene tubes and dried underargon. A 20-^1 aliquot of resuspension buffer (7.5% octyl-/j-D-glucoside,5 mM cardiolipin, 1 mM diethylenetriamine pentaacetic acid) was added,vortexed, and sonicated as described (30). To this, 80 ^1 of the enzyme-ATP mixture were added, followed by vortex mixing and incubation at25°Cfor 30 min. The reaction was terminated by the addition of 1.67

ml of CHCI,/methanol/12 N HCI (66:31:1, v/v), followed by 1.67 mlof methanol/HjO/CHCl, (48:47:3, v/v) as described by MacDonald etal. (31 ), followed by vortex mixing and centrifugation. The upper phasewas removed and discarded, and the lower phase was reextracted with1.67 ml of methanol/H2O/CHCl, (48:47:3, v/v). After centrifugationand removal of the upper phase, the labeled phosphatidic acid in thelower phase was assayed directly by scintillation counting of an aliquotas justified by Muldoon et al. (33). Standard curves were generatedfrom the assay of known amounts of 1,2-diacylglycerol and data wereexpressed as 1,2-diacylglycerol/lOO nmol of lipid phosphorus (mol%)(32).

Determination of Labeled Inositol Phosphates and Phosphoinositides.Incorporation of pHjmyoinositol into inositol phosphates and membrane phosphoinositides was determined by using colon epithelial preparations from both groups at each time period (15, 16). Colonie epithelium (10 mg protein) was incubated for 2 h in 2 ml of HBSS containing180 mg/100 ml of D-glucose and 25 fid of ['Hjmyoinositol (specific

activity, 12.8 Ci/mmol). The epithelium was centrifuged at 500 x g for10 min at 4°Cand washed 3 times with cold HBSS. The preparations

were then resuspended in 2 ml of 20 mM 4-(2-hydroxyethyl)-l-pipera-zineethanesulfonic acid/Tris buffer, pH 7.0, containing 25 mM ß-glycerophosphate, 0.1 M KC1, 5 mM /S-mercaptoethanol, 5 mM MgCl2,0.02% trypsin inhibitor, and 10 mM LiCl. The suspensions were incubated for 20 min at 37°Cand the reactions were terminated by addition

of 0.67 ml 10% perchloric acid and allowed to stand on ice for 15 min.Following centrifugation at 500 x g, the supernatant was neutralizedwith 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid/Trisbuffer, pH 8.0, prior to determination of inositol phosphates. The pelletwas mixed with 0.5 ml chloroform/methanol/12 N HC1 (200:100:0.75,v/v) and the extracted phosphoinositides were saved for later analyses.Separation of different species of inositol phosphates was achieved byanión exchange chromatography on 0.3 ml AG-1-X8 (HCOO~) col

umns (200-400 mesh) based on the method of Downes et al. (34). Afterloading, the columns were eluted serially with 1.5 ml each of (a) water;(b) 0.1 M formic acid-0.2 M ammonium formate; (c) 0. l M formic acid-0.4 M ammonium formate; and (d) 0.1 M formic acid-1 M ammoniumformate. IP, IP2, and IP3 eluted in the second, third, and fourthfractions, respectively. Radioactivity was assayed by scintillation counting. For the separation and quantification of phosphoinositides, 0.2 mlof the acidified pellet extract was treated with 1.5 ml of chloroform/methanol (2:1, v/v), vortexed, and centrifuged. The upper phase wasdiscarded and the lower phase was washed twice with 0.75 ml methanol/0.6 N HCI (1:1, v/v) and then separated on 1% potassium oxalate-impregnated Silica Gel G chromatography plates. Unlabeled phosphoinositides were added as carriers. The plates were developed inchloroform:acetone:methanol:glacial acetic acid:H2O (40:15:13:12:7,v/v) (22). The labeled phosphoinositides including phosphatidylinosi-tol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4-5-bis-phosphate were identified by comparison with phosphoinositidestandards and were quantified by scraping the silica gel from the platesand counting the radioactivity in a liquid scintillation counter. Routinely, greater than 90% of radioactivity added to the thin layer plateswas recovered.

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DMH-INDUCED ALTERATIONS IN COLON1C PKC

Treatment of Colonie Epithelium with 1,25(OH)2D2. Recent studiesby our laboratory (35) have demonstrated that in vitro addition ofl,25(OH)2Dj (10 nM) to normal colonie epithelial preparations rapidlyincreased phosphoinositide turnover and DAG mass and activated PKCin these preparations. In the present studies, it was, therefore, of interestto determine whether DMH treatment altered the responsiveness ofthe colonie epithelium to this secosteroid. To address this issue, colonieepithelium obtained from both groups of animals at 10 and 15 weekswas incubated as described above for the various biochemical analyses,followed by the addition of a physiological concentration of1,25(OH)2D3 (10 nM) or ethanol vehicle (final concentration neverexceeding 0.004%) for 1 min. This time and concentration were previously found to result in a maximum response of PKC, DAG, inositolphosphates, and phosphoinositides to this agent (35). The reaction wasterminated by the addition of ice-cold buffer and the preparation wasprocessed as described above for analysis of these biochemical parameters.

Statistical Methods. All results are expressed as mean values ±SE.Paired or unpaired r tests were used for all statistical analyses. P < 0.05was considered significant.

Materials. 1,25(OH)2D3 was kindly provided by Dr. M. R. Uskokovic(Hoffmann-La Roche Inc., Nutley, NJ). Leupeptin, phenylmethylsul-fonyl fluoride, histone (type III-S), 1,2-dimethylhydrazine, phosphati-dylserine, phosphatidylinositol standards, diethylenetriamine pentaa-cetic acid, dithiothreitol, ATP, and DEAE-cellulose were purchasedfrom Sigma Chemical Co. (St. Louis, MO). [7-32P]ATP, ['H]arachidon-ate, and [3H]myoinositol compounds were obtained from New EnglandNuclear Research Products (Boston, MA). Octyl-0-D-glucoside waspurchased from Boehringer Mannheim (Indianapolis, IN), sn-l-2-di-acylglycerol kinase was from Lipidex, Inc. (Westfield, NJ), cardiolipinand îrt-dioleoylglycerolwere from Avanti Polar Lipids, Inc. (Pelham,AL).

RESULTS

General Observations. In agreement with prior studies by ourlaboratory (23), at 10 and 15 weeks DMH administration didnot significantly affect the weight gain of these animals (datanot shown).

Light Microscopic Studies. In agreement with previous studies from our laboratory (22, 23), despite extensive sampling ofthe colons of each group, no evidence of severe atypia, carci-noma-in-situ, or microscopic carcinomas were seen at 10 and15 weeks by light microscopy (data not shown). In the presentexperiments, minimal inflammation was also noted in the co-Ionic preparations which did not differ in intensity after 10 or15 weeks in each group. These results, therefore, indicate thatinflammation per se was not responsible for the biochemicalalterations noted in the DMH-treated colonie mucosa (seebelow).

Colonie Protein Kinase C Activity. After 10 weeks, as shownin Table 1, approximately 82 and 18% of PKC activity wasfound in the cytosolic and membrane fractions, respectively, ofcontrol colonie preparations. In DMH preparations at this timeperiod, however, only 70% of PKC activity was found in thecytosolic fraction with 30% of the activity present in the membrane fraction. Moreover, upon addition of 10 nM 1,25(OH)2D.,to control tissue, PKC was found to significantly translocatefrom the cytosolic to membrane fraction at 1 min, whereasDMH-treated tissue failed to respond to this agent (Table 1).The total activity of PKC was, however, similar in control andDMH preparations with and without 1,25(OH)2D., (Table 1).

As can also be seen in this table, after 15 weeks, this patternof redistribution of PKC activity was further accentuated in theDMH preparations compared to their control counterparts.Furthermore, control tissue again responded to in vitro administration of 1,25(OH)2D3 with translocation of PKC from the

cytosol to the membrane fraction, whereas DMH tissue did notrespond to this agent. In contrast to the 10-week data, however,the total activity of PKC was not significantly reduced (P <0.001 ) in DMH tissue with and without 1,25(OH)2D, comparedto their control counterparts (Table 1). The decrease was seenin cytosolic, not membrane PKC. Background cellular proteinkinase activity, in the absence of phosphatidylserine, was 2-4pmol/mg protein/min and did not change with time, DMH, or1,25(OH)2D, treatment.

Colonie DAG Mass. At both 10 and 15 weeks, DMH-treatedcolonie mucosa was found to possess a significantly greatermass of DAG than control colonie mucosa (Table 2). Moreover,at each of these times, addition of 10 n\i 1,25(OH)2D, led to asignificant increase in DAG mass in control, but not in DMH-treated preparations (Table 2).

Colonie Phosphoinositides and Inositol Phosphates. As shownin Table 3, at the 10- and 15-week periods, differences werenoted in the incorporation of [3H]myoinositol into individual

phosphoinositides and inositol phosphates between control andDMH-treated preparations. As can be seen in this table, theturnover of lableled phosphatidylinositol derivatives, represented by the ratio of total labeled inositol phosphates/totallabeled phosphoinositides was significantly greater at both timeperiods in DMH-treated preparations compared to their controlcounterparts. The majority of the change in inositol phosphatelevels was due to changes in IP with no significant change notedin the levels of the second messenger, IP,. Finally, control, butnot DMH preparations, were found to significantly increasethis ratio and IP., levels after in vitro addition of 10 nM1,25(OH)2D, for 1 min (Table 3).

DISCUSSION

The present results demonstrate for the first time that alterations in PKC activity could be detected in the preneoplasticcolons of rats after administration of the chemical carcinogenDMH for 10 and 15 weeks. At these time points, despiteextensive histológica! sampling of the entire colon in theseanimals, no evidence of inflammation, severe atypia, carcinomain situ, or microscopic adenocarcinomas were evidenced by lightmicroscopic examination.

At 10 weeks, DMH-treated rats had a different cellular

distribution of PKC activity, with a greater percentage in themembrane fraction than in their control counterparts. No differences, however, were noted in the total activity of this enzymein the colon of these two groups at this time. Previous studieshave demonstrated that PKC activity is elevated in the paniculate fraction of cells under a variety of conditions, includingphorbol ester tumor promotion, viral transformation, and rapidcell growth (reviewed in Ref. 36). Based on these findings, ithas been suggested that changes in the distribution of PKC mayserve as a regulatory mechanism to alter the endogenous substrates available for phosphorylation by this kinase, therebyleading to tumor promotion and malignant transformation (36).

At 15 weeks, the redistribution of PKC seen in the DMHcolonocytes at 10 weeks was further accentuated and, moreover,the total activity in DMH-treated colonie mucosa was reducedto approximately one-half of the activity present in controlcolonie mucosa. It is important to note that changes in crudePKC activity levels may reflect alterations in the expression ofendogenous PKC inhibitors of phosphatases, both of whichmight interfere with the histone phosphorylation assay. Purification on DEAE-cellulose, however, likely removes the majority of interfering compounds. Of interest, total PKC activity in

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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

nControl

5DMH

5Control

+ 1,25(OH)2D,5DMH

+ 1.25(OH)2Dj 510

wkMembrane-associatedactivity

(pmol/mgprotein/min)3.8

±0.5(18.0+2.5)5.3

+ 0.4(30.0 ±2.1)*6.0

±0.3(32.0+1.5)*5.0

±0.2(29.0+ 1.0)Total

activity(pmol/mg

protein/min)n20.9

±0.8617.7

±0.5618.9

±1.2617.

2 ±2.5 615

wkMembrane-associatedactivity

(pmol/mgprotein/min)6.2

±0.7(23.0±2.6)6.3

±0.9(42.0+6.2)*12.4

+ 0.7(42.0 ±2.4)*7.5

±0.7(52.0 ±5.1)Total

activity(pmol/mg

protein/min)27.0

±2.515.1

±2.1*29.6+

1.614.4+

1.0

°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 after10 and 15 weeks"

PreparationsControlDMHControl

+1,25(OH)2D,DMH+1.25(OH)2D3n666810

wkDAG

mass(mol%)0.36

+0.010.41±0.01*0.53

+0.01*0.40

±0.01n888815

wkDAG

mass(mol%)0.36

±0.010.44+0.01*0.62+0.01*0.44

±0.01" Values represent means ±SE of number of separate animals analyzed at

each time period.* P < 0.001 compared to control values of similar time period.

the control rats rose between 10 and 15 weeks, but decreasedslightly in DMH-treated rats, thereby illustrating the importance of comparing age-matched animals. The changes in PKCdistribution, followed by a decrease in total kinase activity inthe preneoplastic colonie mucosa of DMH-treated rats is consistent with an apparent down-regulation of PKC which hasbeen noted in several cultured cells treated with tumor-promoting phorbol esters (37-39). For example, MCF-7 cells treated

with TPA have been shown to initially translocate PKC fromthe cytosolic fraction to the paniculate fraction, followed thereafter by a progressive decline in PKC activity in these cells (39).

Recently, Guillem et ai. (19) also found that human coloncancer samples that contained an admixture of benign adenom-atous tissue displayed a shift of PKC activity from the cytosolicto membrane fractions, whereas colon cancers lacking thisbenign tissue had reduced levels of total PKC activity. Basedon their findings, these investigators (19) suggested that theearly stages of colonie transformation in humans may involvetranslocation of PKC activity, while later stages might be associated with reduced total PKC activity, i.e., down-regulation.

Additionally, initial translocation of PKC followed by reduction of PKC activity has been shown to occur in cells transformed by several oncogenes, particularly Ha-ras and Ki-rai (8,9, 40). Concomitant with these alterations in PKC, DAG levelswere increased in these cells, suggesting that at least one mechanism responsible for transformation of these cells involvespersistent elevation of DAG leading to activation and thendown-regulation of PKC (40).

In the present experiments, DAG mass was found to beincreased in the colonie mucosa of rats treated with DMH for10 and 15 weeks. It would, therefore, appear reasonable tosuggest that elevations in DAG might underlie the translocationof PKC activity to the membrane at these time periods. Whilethe origin of these DMH-induced elevations in DAG levels is

not totally clear, based on our study of colonie phosphoinositideturnover in these animals, it would appear that at least a portionof this DAG may come from the breakdown of these membranephospholipids. While prior studies in oncogene-transformedcells have also suggested that phosphoinositides may serve asthe source for increased DAG (40), others (5,41) have suggestedthat this lipid activator of PKC was derived from the breakdownof other membrane phospholipids. It is, therefore, possible thatthe elevations in DAG induced DMH in the present experiments were also derived from these other phospholipids suchas phosphatidylcholine and phosphatidylethanolamine as wellas conceivably from phosphatidic acid (5).

The exact significance of subsequent elevations in DAG, PKCtranslocation, and down-regulation to colonie malignant transformation in this model remains unclear. The presence of thesechanges in the mucosa prior to overt neoplasia, however, suggests that they may play a role in the early stage(s) of themultistage malignant transformation process. Additional factors would then be necessary for the development of carcinomain certain of these cells.

Down-regulation in phorbol-treated and ras-transfected cellsappears to be caused by proteolytic cleavage of PKC molecules(42). Furthermore, cleavage of PKC has been associated withthe appearance of a protein kinase in the cytosol which wasindependent of the activators of PKC, i.e., calcium, phosphati-dylserine and DAG (42). While the physiological significanceof this protein kinase found by cleavage of PKC remains unclear, Chida et al. (42) have suggested that it may diffuse intothe cytoplasm and nucleus of the cell and therefore transducesignals from the cytosolic side of the plasma membrane to thenucleus, thereby inducing malignant transformation. Based onstudies which have demonstrated synergism between the actionsof ras oncogenes and phorbol esters on cellular transformation(43), Weyman et al. (8) have also recently speculated that down-regulation of PKC, or a process closely linked to this phenomenon, may be important in the multistage carcinogenesis proc-ess(es). In contrast to these hypotheses, Wolfman et al. (9) havesuggested that down-regulation of PKC may actually representan unsuccessful attempt by transformed cells to negativelymodulate proliferation signals generated by oncogene products.The exact relationship between down-regulation of PKC notedin the current experiments to malignant transformation, therefore, remains unclear.

In agreement with prior studies from our laboratory (35), inthe present experiments l,25(OH)2Di was found to stimulatecolonie mucosal turnover of phosphoinositides, increase DAG

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DMH-INDUCED ALTERATIONS IN COLONIC PKC

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mass, and activate PKC in control animals. In DMH-treated

rats, however, this secosteroid failed to significantly alter anyof these biochemical parameters in the rat colon. This is ofinterest, since it would indicate that DMH-treated preneoplas-tic colonie cells have already lost their responsiveness to thisagent, well before the development of overt tumors. While theexact mechanism(s) responsible for this loss of responsivenessto 1,25(OH)2D3 is also unclear at this time, prior studies inoncogene-transformed cells have demonstrated a similar loss ofresponse to various growth factors, secondary to a variety ofalterations involving the inositol phospholipid signal transduc-tion pathways and/or PKC (44-46). It should also be notedthat this secosteroid has been shown to possess antiproliferativeactions in cell lines derived from human colon cancers (47, 48).It is, therefore, conceivable that this loss of responsiveness tol,25(OH)2Dj may also contribute to the development of DMH-induced colon cancers.

Recently, Craven and DeRubertis (49) have demonstrateddifferences in the subcellular distribution of PKC in rat coloniecells with different proliferative activities. Since DMH is knownto stimulate colonie epithelial cell proliferation (50), it is possible that the PKC alterations noted in the present experimentswere secondary to proliferative changes induced by this carcinogen. In this regard, however, prior studies in this model, usingan identical treatment regimen, have demonstrated comparableincreases in proliferation induced by DMH after 10 and 15weekly injections (50). It is, therefore, difficult to reconcilechanges in proliferation per se with the sequential changes notedin PKC subcellular distribution and total activity in the presentexperiments. Moreover, in agreement with previous studiesutilizing the phorbol ester 12-0-tetradecanoylphorbol-13-ace-tate as well as deoxycholate (49), in preliminary experimentsperformed by our laboratory, in vitro addition of 1,25(OH)2D,was found to activate PKC to a similar extent in proliferativeand nonproliferative normal colonie epithelial cells.4 Taken

together, these observations, therefore, strongly suggest thatDMH-induced alterations in cellular proliferation are unlikelyto explain all the effects of this agent on PKC activity found inthe current studies.

In summary, the present results have demonstrated alterations in PKC activity, DAG mass, and phosphoinositide turnover in the preneoplastic colons of rats administered DMH for10 to 15 weeks. Furthermore, in vitro addition of 1,25(OH)2D3failed to elicit the normal pattern of response of these biochemical parameters in the colons of carcinogen-treated animals.Based on these observations, it would appear that changes inPKC activity may play a role in the early stages of the malignanttransformation process induced by DMH. Further studies concerning this possible relationship are now being conducted inthis experimental model of colonie adenocarcinoma by ourlaboratory.

ACKNOWLEDGMENTS

The authors would like to thank Lynn Nelson for her excellentsecretarial assistance.

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1990;50:3915-3920. Cancer Res   Charles L. Baum, Ramesh K. Wali, Michael D. Sitrin, et al.   Activity in the Rat Preneoplastic Colon1,2-Dimethylhydrazine-induced Alterations in Protein Kinase C

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