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发表于 2009-3-6 00:19 |只看该作者 |倒序浏览 |打印

1 Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, Galveston, TX 775554 r0 w9 j9 I6 t
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2 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555

3 Department of Pharmacology, University of Texas Medical Branch, Galveston, TX 77555+ n; s. j6 E( H  ~, I

4 Molecular and Cell Biology Section, Faculty of Nutrition, Texas A&M University, College Station, TX 77843

Address correspondence to Alan P. Fields, Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1048. Tel.: (409) 747-1935. Fax: (409) 747-1938. E-mail: afields@utmb.edu
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Abstract. Q6 N& j' C+ q0 z3 v( v, v  m
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?ncreasing evidence demonstrates that protein kinase C ?II (PKC?II) promotes colon carcinogenesis. We previously reported that colonic PKC?II is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKC?II in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKC?II represses transforming growth factor ? receptor type II (TGF?RII) expression and reduces sensitivity to TGF-?–mediated growth inhibition in intestinal epithelial cells. Transgenic PKC?II mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGF?RII expression. Chemopreventive dietary -3 fatty acids inhibit colonic PKC?II activity in vivo and block PKC?II-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGF?RII expression in the colonic epithelium of transgenic PKC?II mice. These data indicate that dietary -3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKC?II signaling and restoration of TGF-? responsiveness.
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Key Words: protein kinase C; colon carcinogenesis; -3 fatty acids; transforming growth factor ; hyperproliferation
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Introduction

Epidemiological studies have shown a convincing link between dietary fat intake and colon cancer risk (for review see Bartsch et al., 1999). Consumption of fish oil, which contains a high level of -3 polyunsaturated fatty acids, particularly eicosapentaenoic acid (c20:5, n-3) and docosahexaenoic acid (c22:6, n-3), is associated with reduced colon cancer incidence (Bang et al., 1976; Caygill and Hill, 1995; Caygill et al., 1996). Biochemical studies confirm that -3 fatty acids have potent chemopreventive effects on carcinogen-induced colon cancer in rodents (Deschner et al., 1990; Reddy et al., 1991; Chang et al., 1997). Despite the compelling epidemiological and biochemical evidence demonstrating the efficacy of -3 fatty acids in colon cancer chemoprevention (Rose and Connolly, 1999), little is known about how these dietary lipids mediate their protective effects. -3 fatty acids can inhibit the colonic epithelial hyperproliferation observed during colon carcinogenesis, suggesting that proliferative signaling is an important target of their chemopreventive effects (Anti et al., 1994, 1997; Latham et al., 1999).+ m! o7 Z4 Z# W
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Elevated colonic protein kinase C ?II (PKC?II)* expression and activity correlate with colon carcinogenesis in both rodents and humans (Murray et al., 1999; Gokmen-Polar et al., 2001). Elevated PKC?II expression in the colonic epithelium of transgenic mice induces hyperproliferation and enhances susceptibility to azoxymethane (AOM)-induced colon carcinogenesis (Murray et al., 1999), demonstrating a direct role for PKC?II in colon carcinogenesis. Based on these data, we hypothesized that -3 fatty acids block colon carcinogenesis by interfering with proliferation via inhibition of PKC?II signaling. To test this hypothesis, we evaluated the effect of a diet high in -3 fatty acids on colonic PKC?II activity and signaling. Dietary -3 fatty acids inhibited PKC?II activity and suppressed PKC?II-mediated hyperproliferation and colon carcinogenesis in vivo. In addition, our data demonstrate that PKC?II represses transforming growth factor ? receptor type II (TGF?RII) expression in vitro and in vivo, and that this repression is reversed by dietary -3 fatty acids.- C/ t# ?9 p( i  Z6 l7 u  ~! p

Results and discussion" t! x) j% Y! I2 k2 x8 ?
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PKC?II is a lipid-dependent protein kinase whose activity can be modulated in vitro by -3 fatty acids (Holian and Nelson, 1992; Davidson et al., 2000; Seung Kim et al., 2001). Therefore, we assessed the relationship between colonic PKC?II activity and dietary -3 fatty acids during AOM-induced colon carcinogenesis in vivo. Cytosolic and membrane extracts from colonic epithelia of male Sprague-Dawley rats fed an -6 fatty acid diet (15% maize oil by weight) or -3 fatty acid diet (3.5% maize oil, 11.5% fish oil) were subjected to immunoblot analysis for PKC?II (Fig. 1). A significant decrease in the level of membrane-associated PKC?II was observed in the colonic epithelium of animals fed -3 fatty acids, whereas cytosolic PKC?II levels were similar in both diet groups. The membrane association of conventional PKC isozymes, including PKC?II, is widely accepted as an indirect measure of PKC enzyme activation in vivo, because it is not technically feasible to directly determine the activity of the enzyme in situ. Our results are consistent with the direct inhibition of PKC?II activity by -3 fatty acids in vitro (Holian and Nelson, 1992).
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Figure 1. Dietary -3 fatty acids block AOM-induced PKC?II activity. PKC?II expression was assessed in membrane and cytosolic fractions from colonic epithelial cell extracts from the distal colon of AOM-treated Sprague-Dawley rats fed either an -6 or an -3 fatty acid diet. Values represent means ± SEM, n = 5.3 V% J1 t7 T9 y+ |. Z& n5 j

We previously characterized transgenic mice expressing elevated levels of PKC?II in the colonic epithelium similar to those observed in mouse colon tumors (Murray et al., 1999; Gokmen-Polar et al., 2001). Transgenic PKC?II mice exhibit a hyperproliferative phenotype and enhanced colon carcinogenesis (Murray et al., 1999). Therefore, we assessed the effect of dietary -3 fatty acids on AOM-mediated carcinogenesis in transgenic PKC?II mice. Transgenic PKC?II mice fed a diet rich in -6 fatty acids exhibited enhanced colon carcinogenesis, as evidenced by increased numbers of preneoplastic lesions, aberrant crypt foci (ACF) (Fig. 2 A). In contrast, a diet rich in -3 fatty acids inhibited the enhanced colon carcinogenesis characteristic of transgenic PKC?II mice. Thus, dietary -3 fatty acids block PKC?II activation and attenuate the procarcinogenic effects of PKC?II in the colon in vivo.9 k' U: @5 D/ L  n0 z0 x  q

Figure 2. Dietary -3 fatty acids block PKC?II-enhanced colon carcinogenesis and colonic hyperproliferation. (A) Effect of dietary fat intake on AOM-induced ACF formation in transgenic PKC?II mice. Mice were terminated 12 wk after the final AOM injection and the colons analyzed for ACF formation as described previously (McLellan et al., 1991; Murray et al., 1999). Values represent means ± SEM, n = 6–18 animals/experimental group. (B) Mice were fed an -6 or -3 diet for 18 d. 1 h before sacrifice, mice were injected with 50 mg/kg BrdU and distal colon was isolated, fixed in 4% paraformaldehyde, and analyzed for proliferation as determined by BrdU labeling (Chang et al., 1997). Results respresent proliferative index relative to nontransgenic controls. Values represent means ± SEM, n = 4–5 animals/experimental group.0 \' l  R! ?0 {; q% b4 _9 V
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Transgenic PKC?II mice exhibit hyperproliferation of the colonic epithelium (Murray et al., 1999), whereas dietary -3 fatty acids suppress carcinogen-induced hyperproliferation (Latham et al., 1999). Therefore, we assessed the effect of dietary -3 fatty acids on the hyperproliferative phenotype of transgenic PKC?II mice (Fig. 2 B). Whereas transgenic PKC?II mice exhibited hyperproliferation when fed an -6 fatty acid diet, an -3 fatty acid diet selectively suppressed this hyperproliferative phenotype (Fig. 2 B). These data demonstrate that dietary -3 fatty acids suppress the hyperproliferative phenotype observed in transgenic PKC?II mice and provide a plausible mechanism for the chemopreventive effects of dietary -3 fatty acids on PKC?II-mediated colon carcinogenesis." ]; k# Z" f" d

To gain insight into how PKC?II enhances colon carcinogenesis, we established rat intestinal epithelial (RIE)-1 cells that stably express PKC?II (Fig. 3). Parental RIE-1 cells, which, like most cells of the intestinal epithelium, express little or no PKC?II, were infected with a retrovirus containing the full-length human PKC?II cDNA (Fig. 3 A). Comparative gene microarray analysis of RIE-1 and RIE/PKC?II cells was used to identify potential targets for PKC?II. Among the genes identified through this analysis, TGF?RII expression was found to be markedly repressed in RIE/PKC?II cells, when compared with RIE-1 cells. These results were confirmed by Western blotting using a specific antibody to TGF?RII (Fig. 3 A). Given the importance of TGF-? signaling in colonic epithelial cell proliferation and differentiation, and the frequent loss of TGF-? responsiveness during colon carcinogenesis (Markowitz and Roberts, 1996), we assessed whether this change in TGF?RII expression leads to an alteration in the well-documented response of the RIE-1 cells to TGF-? (Ko et al., 1995). RIE-1 and RIE/PKC?II cells were transfected with a reporter plasmid containing a TGF-?–responsive element from the tissue plasminogen activator promoter linked to luciferase (Wrana et al., 1992). RIE-1 cells respond to TGF-? by activation of this TGF-?–dependent reporter construct (Fig. 3 B). However, RIE/PKC?II cells showed a diminished transcriptional response to TGF-? consistent with repression of TGF?RII expression. Cotransfection of the TGF-? reporter plasmid with a plasmid containing R4TD202, a constitutively active mutant of TGF?RI (Feng and Derynck, 1996), resulted in comparable increases in transcriptional activity in both RIE-1 and RIE/PKC?II cells (Fig. 3 B), indicating that signaling downstream of the TGF-? receptor is intact in both RIE-1 and RIE/PKC?II cells. Consistent with the transcriptional response to TGF-?, RIE-1 cell proliferation was inhibited by TGF-? as determined by a reduction in the number of bromodeoxyuridine (BrdU)-labeled cells after addition of TGF-?1 to mid-log phase cultures (Fig. 3 C). RIE/PKC?II cells, however, were relatively insensitive to TGF-? inhibition of BrdU labeling (Fig. 3 C). Although there was no detectable growth phenotype associated with expression of PKC?II in the absence of added TGF-? (Fig. 3 D), RIE/PKC?II cells were much less sensitive to TGF-?–mediated inhibition of proliferation (Fig. 3 E). Thus, expression of PKC?II leads to repression of TGF?RII and loss of TGF-? responsiveness in RIE-1 cells.

Figure 3. Characterization of TGF-? signaling in RIE/PKC?II cells. (A) Expression of PKC?II, TGF?RII, and actin in RIE-1 and RIE/PKC?II cells. RIE-1 cells were infected with pBABEpuro containing full-length human PKC?II or empty pBABEpuro vector. Puromycin-resistant cell populations were isolated and screened for expression of PKC?II, TGF?RII, and actin by immunoblotting. (B) Effect of PKC?II expression on TGF-?–induced transcription in RIE-1 cells. RIE-1 and RIE/PKC?II cells were transiently transfected with 0.4 μg of 3TP/luc TGF-? reporter plasmid plus 1 μg of TK/renilla as an internal control for transfection efficiency. TGF-?1 (3 ng/ml) was added to cells as indicated. Cellular extracts were prepared after 24 h of TGF-? exposure and assayed for firefly and renilla luciferase activity. In addition, some cells were cotransfected with an expression vector encoding constitutively active TGF-? chimeric receptor R4TD202. Data represent the mean ± SD from three independent experiments measuring firefly luciferase activity normalized to renilla luciferase activity. (C) Effect of PKC?II expression on TGF-?–mediated inhibition of BrdU incorporation in RIE-1 cells. TGF-? responsiveness of RIE-1 and RIE/PKC?II cells was assayed by measuring inhibition of BrdU incorporation. (D) Effect of PKC?II expression on RIE-1 cell proliferation. Growth of RIE-1 and RIE/PKC?II cells in growth medium in the absence of TGF-? was measured by hemocytometer count on a daily basis. Data represent the mean ± SD from three dishes. (E) Effect of PKC?II expression on TGF-?–mediated inhibition of RIE-1 cell proliferation. Cultures of RIE-1 and RIE/PKC?II cells were treated with 3 ng/ml TGF-?1, and cell growth was monitored by measuring OD570 after reduction of MTT. Data represent the mean ± SD from three wells.- T& `0 K/ p0 X! K1 O$ I
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To directly demonstrate that PKC?II activity is responsible for repression of TGF?RII protein expression in RIE/PKC?II cells, we determined the effect of the PKC?-selective inhibitor LY379196 on TGF?RII expression. Treatment of RIE/PKC?II cells with increasing concentrations of LY379196 led to a dose-dependent increase in TGF?RII protein expression (Fig. 4 A). Quantitative analysis of these expression data (Fig. 4 B) indicate an apparent ED50 for re-expression of TGF?RII of 40 nM LY379196, consistent with the reported IC50 of LY379196 for PKC?II of 30 nM (Jirousek et al., 1996). It should also be noted that re-expression of TGF?RII is accompanied by reduced electrophoretic mobility of TGF?RII upon SDS-PAGE. Because TGF?RII is highly glycosylated, it is possible that this shift reflects changes in posttranslational processing of the protein. The significance of this observation to TGF-?–mediated signaling is unclear. Nevertheless, these data provide direct evidence that PKC?II activity is responsible for repression of TGF?RII in these cells.

Figure 4. The selective PKC? inhibitor LY379196 restores TGF?RII expression in RIE/PKC?II cells. (A) RIE/PKC?II cells were treated with the indicated concentration of LY379196 for 48 h, and total cellular protein extracts were subjected to immunoblot analysis for TGF?RII expression. (B) Quantitative analysis of the immunoblot data shown in A.

TGF-? is a potent growth inhibitor in epithelial cells (Lamprecht et al., 1989) and the loss of TGF-? responsiveness is a common feature of many human colon cancers, particularly those exhibiting the microsatellite instability phenotype (Markowitz et al., 1995). Loss of TGF-? responsiveness in hereditary nonpolyposis colon cancer syndrome is due to inactivating mutations within TGF?RII (Markowitz et al., 1995; Kim et al., 2000). Recent studies indicate that transcriptional repression of TGF?RII expression is a common mechanism for loss of TGF-? responsiveness in many tumor types, including colon cancers (Kim et al., 2000). Our data demonstrate that PKC?II represses TGF?RII expression in RIE-1 cells, and indicate that PKC?II-mediated loss of TGF-? responsiveness is involved in the hyperproliferative and procarcinogenic effects of PKC?II in the colonic epithelium. Therefore, we assessed whether TGF?RII is a target for colonic PKC?II in vivo (Fig. 5). Colonic epithelial cell extracts from transgenic PKC?II and nontransgenic mice fed an -6 fatty acid diet were subjected to immunoblot (Fig. 5 A) and immunofluorescence analysis (Fig. 5 B, 1 and 4) using a specific TGF?RII antibody. Extracts from nontransgenic mice contained little PKC?II, but abundant TGF?RII, whereas those from transgenic PKC?II mice contained elevated PKC?II and a reduced level of TGF?RII (Fig. 5 A). Immunofluorescence analysis for TGF?RII confirmed these immunoblotting results (Fig. 5 B). Colonic crypts from nontransgenic mice fed an -6 fatty acid diet (Fig. 5 B, 4) exhibited abundant staining for TGF?RII. Staining was strongest in the upper third and along the luminal surfaces of the colonic crypts, consistent with the reported distribution of TGF?RII in the colonic epithelium (Guda et al., 2001). In contrast, colonic crypts from transgenic PKC?II mice fed an -6 fatty acid diet (Fig. 5 B, 1) expressed much lower levels of TGF?RII, confirming that TGF?RII expression was repressed by PKC?II in the colonic epithelium in vivo.
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Figure 5. -3 fatty acids restore colonic TGF?RII expression in PKC?II mice. (A) Immunoblot analysis of TGF?RII expression. PKC?II transgenic mice and nontransgenic littermates were killed, the colonic epithelia were scraped, and 150 μg of the resulting protein extract was subjected to immunoblot analysis using antibody specific for TGF?RII, PKC?II, or actin (Santa Cruz Biotechnology, Inc.). (B) Immunofluorescence analysis of TGF?RII expression. PKC?II transgenic mice (1, 2, and 3) and nontransgenic littermates (4, 5, and 6) were fed (ad libitum) either an -3– (2, 3, 5, and 6) or -6– (1 and 4) containing diet beginning at 6 wk of age. After 18 d, mice were killed and distal colons were subjected to immunofluorescence analysis using a specific antibody to TGF?RII (1,2, 4, and 5) or secondary antibody only (3 and 6). All panels are at 400x magnification.! Z6 W  ]0 n/ U. q5 l8 `) O' j1 E, Z* z  _
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Because PKC?II mediates repression of TGF?RII expression and dietary -3 fatty acids inhibit PKC?II signaling, we predicted that dietary -3 fatty acids would restore TGF?RII expression in transgenic PKC?II mice. Indeed, transgenic PKC?II mice fed an -3 fatty acid diet showed levels of TGF?RII comparable to those seen in nontransgenic mice (Fig. 5 B, 2). Therefore, TGF?RII is a critical gene target of PKC?II-mediated repression in RIE-1 cells in vitro and in the colonic epithelium in vivo. Our data are consistent with the conclusion that PKC?II down-regulates TGF?RII expression, thereby inducing a TGF-?–insensitive state. Because TGF-? inhibits proliferation and promotes differentiation of intestinal epithelial cells (Lamprecht et al., 1989), PKC?II-mediated repression of TGF?RII imposes a hyperproliferative state that increases sensitivity to carcinogens, such as AOM. Dietary -3 fatty acids inhibit PKC?II activity and restore TGF?RII expression in the colonic epithelium of transgenic PKC?II mice, resulting in reversal of the hyperproliferative phenotype and attenuation of the enhanced colon carcinogenesis characteristic of transgenic PKC?II mice.7 r; y. ?' l$ c  S7 g  |9 N" C$ N

Our results provide a plausible link between chemopreventive dietary -3 fatty acids, colonic PKC?II activity, TGF-? signaling, cellular proliferation, and susceptibility to colon cancer. Therefore colonic PKC?II, which is induced early during colon carcinogenesis, represents a novel and potentially highly effective target for chemopreventive therapy in colon cancer. Given the strong association of dietary -3 fatty acids with the chemoprevention of other epithelial cancers, including breast and prostate cancer (Rose and Connolly, 1999), as well as neurological conditions such as bipolar disorder (Seung Kim et al., 2001), it is possible that selective PKC?II inhibition could prove to be an important therapeutic modality in the treatment and chemoprevention of multiple epithelial cancers and central nervous system disorders. Likewise, because repression of TGF?RII expression has been documented in many cancer cell types, including gastric cancer, colon cancer, small cell lung cancer, esophageal cancer, hepatocellular carcinoma, squamous cell carcinoma, breast cancer, endometrial cancer, bladder cancer, and osteosarcoma (for review see Kim et al., 2000), it is possible that PKC?II inhibition, either through dietary modulation or pharmacological intervention, may be of therapeutic value for a broad range of major cancer types by restoring TGF-? responsiveness in these tumors.
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Materials and methods
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Production and maintenance of transgenic mice0 G1 q. c& K0 G, a5 E; Y! d

Transgenic PKC?II mice were generated and maintained essentially as described previously (Murray et al., 1999), except that the transgene construct used a modified version of the rat liver fatty acid binding protein promoter containing four copies of a heptad repeat enhancer region that directs enhanced and more sustained expression in the entire colonic epithelium (Simon et al., 1997).

Cell fractionation and PKC?II immunoblot analysis
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3-wk-old Sprague-Dawley rats fed either an -6 fatty acid diet (15% maize oil by weight) or an -3 fatty acid diet (3.5% maize oil, 11.5% fish oil, by weight; Chang et al., 1997). After 1 wk on experimental diets, rats were injected with AOM (Chang et al., 1997). Animals were terminated at 16 wk after AOM injection. Colonic epithelial cell extracts from the distal colon were fractionated into membrane and cytosolic fractions and subjected to immunoblot analysis for PKC?II expression (Davidson et al., 2000). Band intensity was quantitated using the Bio-Rad Laboratories Fluor-s imaging system.

ACF analysis% a: P4 O7 S8 J2 c% O

6–8-wk-old transgenic PKC?II mice and nontransgenic littermates were maintained on either an -6 or -3 fatty acid diet (ad libitum) until termination of the experiment. 1 wk after introduction of the diet, mice received intraperitoneal injections of 10 mg/kg AOM 1x/wk for 4 wk. Mice were terminated 12 wk after the final AOM injection and the colons were analyzed for ACF formation (McLellan et al., 1991; Murray et al., 1999)./ S/ c* \! H/ X% [: Y: o8 z: b' ]

Colonic epithelial cell proliferation analysis, D# E7 L' k! f

6–8-wk-old transgenic PKC?II mice and nontransgenic littermates were maintained on an -6 or -3 fatty acid diet for 18 d. 1 h before sacrifice, mice were injected with 50 mg/kg BrdU. Distal colon was isolated, fixed in 4% paraformaldehyde, and analyzed for proliferation as determined by BrdU labeling (Chang et al., 1997).4 L) D2 z% V+ T1 `3 q. L' P( ]9 x) R
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TGF-? transcriptional response assay
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RIE-1 and RIE/PKC?II cells were transiently transfected with 0.4 μg of 3TP/luc TGF-? reporter plasmid (Wrana et al., 1992) plus 1 μg of vector expressing renilla luciferase under control of the thymidine kinase promoter as an internal control for transfection efficiency. TGF-?1 (3 ng/ml) was added to cells. After 24 h of TGF-? exposure, cellular extracts were prepared and assayed for firefly and renilla luciferase activity. Data are presented as firefly luciferase corrected for the level of renilla luciferase. Some cells were cotransfected with 0.1 μg of an expression vector encoding constitutively active TGF-? chimeric receptor R4TD202 (Feng and Derynck, 1996) and assayed for TGF-? reporter activity as described above.
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Effect of PKC?II on RIE-1 cell proliferation and TGF-? responsiveness2 ?% b- v. Y8 E) n- S1 Z
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TGF-? responsiveness of RIE-1 and RIE/PKC?II cells was assayed by measuring inhibition of BrdU incorporation. Midlog phase cells were treated with 3 ng/ml TGF-? for 24 h followed by 1 mM BrdU for 30 min. Cells were then permeabilized and stained with anti-BrdU antibody using the BD PharMingen BrdU FlowKit?, according to the manufacturer's recommendations. Approximately 15,000 cells from each culture were analyzed using a Becton Dickinson flow cytometer. RIE-1 and RIE/PKC?II cells were plated at 105 cells per 6-cm dish. At 24-h intervals, three dishes from each line were harvested by trypsinization and the cells were counted using a hemocytometer. RIE-1 and RIE/PKC?II cells were plated in 96-well plates at 2 x 104 cells in 0.2 ml of medium ± 3 ng/ml TGF-?1. At 24-h intervals, cell growth was assayed in triplicate wells by measuring OD570 after reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

Analysis of TGF-? RII expression/ n0 c3 l1 C/ |: Q

Equal amounts of cell lysates from RIE-1 and RIE/PKC?II cells were subjected to immunoblot analysis using antibody specific for TGF?RII, PKC?II, or actin (Santa Cruz Biotechnology, Inc.). PKC?II transgenic mice and nontransgenic littermates were killed, the colonic epithelia were scraped, and 150 μg of the resulting protein extract was subjected to immunoblot analysis using the same antibodies listed above. Immunoblot images were captured using a Stratagene Eagle Eye imager. To assess the effect of PKC?II inhibition on TGF?RII protein expression, confluent cultures of RIE/PKC?II cells were placed in medium containing 0.2% FBS plus the concentrations of LY379196 indicated in the figure legend. All cultures contained 0.1% DMSO as a diluent control. After 2 d, total cellular protein was extracted and 50 μg of protein per lane was subjected to immunoblot analysis for TGF?RII expression. Chemiluminescent exposure was adjusted to conform to a linear response range, and data were quantified using a Lynx digital image processor.  v% S* |% o3 W+ H, E
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Immunofluorescence analysis of TGF?RII expression1 }' R5 O( L7 K! k

6-wk-old PKC?II transgenic mice and nontransgenic littermates were fed either an -3– or -6–containing diet ad libitum. After 18 d on the defined diets, the mice were killed. Distal colon was isolated, fixed in 4% paraformaldehyde, sectioned, and subjected to immunofluorescence analysis for TGF?RII expression. Images were captured using a CCD color digital camera and digital image capture software.

Footnotes5 c+ E( X7 u9 T/ E1 s* I; F% d0 y
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* Abbreviations used in this paper: ACF, aberrant crypt foci; AOM, azoxymethane; BrdU, bromodeoxyuridine; PKC?II, protein kinase C ?II; RIE, rat intestinal epithelial; TGF?RII, transforming growth factor ? receptor type II./ M; O" r' c/ U" h5 k) P' `
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Acknowledgments1 T8 p9 g- ~, f6 O9 c
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We thank Dr. Jeffrey Gordon (Washington University, St. Louis, MO) for the gift of the modified rat liver fatty acid binding promoter, Dr. Jeffrey Ceci (University of Texas Medical Branch) for generation of the founder transgenic mice, and Brian Bloodworth for excellent technical assistance. The AOM used in these studies was obtained from the National Cancer Institute.2 C9 t& @" h0 ?

This work was supported by National Institutes of Health grants CA81436 (A.P. Fields), CA24347 (E.A. Thompson), and CA59034 (R.S. Chapkin).Revised: 23 April 2002References; C* e1 b+ W3 G% f6 f
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Anti, M., F. Armelao, G. Marra, A. Percesepe, G.M. Bartoli, P. Palozza, P. Parrella, C. Canetta, N. Gentiloni, I. De Vitris, and G. Gasbarrini. 1994. Effects of different doses of fish oil on rectal cell proliferation in patients with sporadic colonic adenomas. Gastroenterology. 107:1709–1718.# n  t4 p2 c/ Z% \3 v: P
! m9 z) H( V! I4 T! L+ V7 m0 g
Anti, M., G. Marra, F. Arlemao, A. Percesepe, and N. Gentiloni. 1997. Modulating effect of omega-3 fatty acids on the proliferative pattern of human colorectal mucosa. Adv. Exp. Med. Biol. 400B:605–610.
5 g7 o: T; H, i  _- h2 x
Bang, H.O., J. Dyerberg, and N. Hjorne. 1976. The composition of food consumed by Greenland Eskimos. Acta. Med. Scand. 200:69–73.

Bartsch, H., J. Nair, and R.W. Owen. 1999. Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis. 20:2209–2218.

Caygill, C.P.J., and M.J. Hill. 1995. Fish, n-3 fatty acids and human colorectal and breast cancer mortality. Eur. J. Cancer Prev. 4:329–332., D' Z  C8 o5 R2 ~
7 S3 G2 N4 g4 t6 A/ |: h, d
Caygill, C.P.J., A.M. Charlett, and J. Hill. 1996. Fat, fish, fish oil and cancer. Br. J. Cancer. 74:159–164.

Chang, W.-C.L., R.S. Chapkin, and J.R. Lupton. 1997. Predictive value of proliferation, differentiation and apoptosis as intermediate markers for colon tumorigenesis. Carcinogenesis. 18:721–730.
# S9 k1 q3 t- g+ O5 L$ d9 h
Davidson, L.A., R.E. Brown, W.C.L. Chang, L.S. Morris, N. Wang, R.J. Carroll, N.D. Turner, J.R. Lupton, and R.S. Chapkin. 2000. Morphodensimetric analysis of protein kinase C ?II expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis. Carcinogenesis. 21:1513–1519.

Deschner, E.E., J.S. Lytle, G. Wong, J.F. Ruperto, and H.L. Newmark. 1990. The effect of dietary omega-3 fatty acids (fish oil) on azoxymethanol-induced focal areas of dysplasia and colon tumor incidence. Cancer. 66:2350–2356.

Feng, X.H., and R. Derynck. 1996. Ligand-independent activation of transforming growth factor (TGF) beta signaling pathways by heteromeric cytoplasmic domains of TGF-beta receptors. J. Biol. Chem. 271:13123–13129.  o( U" S0 l: |3 e

Gokmen-Polar, Y., N.R. Murray, M.A. Velasco, Z. Gatalica, and A.P. Fields. 2001. Elevated protein kinase C ?II is an early promotive event in colon carcinogenesis. Cancer Res. 61:1375–1381.

Guda, K., C. Giardina, P. Nambiar, H. Cui, and D.W. Rosenberg. 2001. Aberrant transforming growth factor-? signaling in azoxymethane-induced mouse colon tumors. Mol. Carcinog. 31:204–213.. B/ |) \6 @" g) O
* ^/ y: h; i. [* W4 }# X
Holian, O., and R. Nelson. 1992. Action of long-chain fatty acids on protein kinase C activity: comparison of omega-6 and omega-3 fatty acids. Anticancer Res. 12:975–980.

Jirousek, M.R., J.R. Gillig, C.M. Gonzalez, W.F. Heath, J.H. McDonald III, D.A. Neel, C.J. Rito, U. Singh, L.E. Stramm, A. Melikian-Badalian, et al. 1996. (S)-13--10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzopyrrolooxadiazacyclohexadecene-1,3(2H)-dione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C?. J. Med. Chem. 39:2664–2671.+ v# O. F0 V/ e- B. M% [
  E( s% K$ D$ ^& @) j
Ko, T.C., H.M. Sheng, D. Reisman, E.A. Thompson, and R.D. Beauchamp. 1995. Transforming growth factor-beta-1 inhibits cyclin D1 expression in intestinal epithelial cells. Oncogene. 10:177–184.  j' O1 \) X  ~( ]; ]" A: ~
/ {! ]0 W8 l6 X. |6 H& o
Lamprecht, S.A., B. Swartz, and A. Glicksman. 1989. Transforming growth factor-? in intestinal epithelial differentiation and neoplasia. Anticancer Res. 9:1877–1882.
& i  X% v4 Y! }) o0 p% f1 v
Kim, S.J., Y.H. Im, S.D. Markowitz, and Y.J. Bang. 2000. Molecular mechanisms of inactivation of TGF-beta receptors during carcinogenesis. Cytokine Growth Factor Rev. 11:159–168.

Latham, P., E.K. Lund, and I.T. Johnson. 1999. Dietary n-3 PUFA increases the apoptotic response to 1,2-dimethylhydrazine, reduces mitosis and suppresses the induction of carcinogenesis in the rat colon. Carcinogenesis. 20:645–650.$ ^8 N! y% b6 r8 u+ x; N- {
3 F0 L/ s% ]4 u# b9 j: R
Markowitz, S., J. Wang, L. Myeroff, R. Parsons, L. Sun, J. Lutterbaugh, R.S. Fan, E. Zborowska, K.W. Kinzler, and B. Vogelstein. 1995. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 268:1336–1338.
& X* E7 l! ?* v$ F, e
Markowitz, S.D., and A.B. Roberts. 1996. Tumor suppressor activity of the TGF-beta pathway in human cancers. Cytokine Growth Factor Rev. 7:93–102.

McLellan, E.A., A. Medline, and R.P. Bird. 1991. Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon. Carcinogenesis. 12:2093–2098.

Murray, N.R., L.A. Davidson, R.S. Chapkin, W.C. Gustafson, D.G. Schattenberg, and A.P. Fields. 1999. Overexpression of protein kinase C ?II induces colonic hyperproliferation and increased sensitivity to colon carcinogenesis. J. Cell Biol. 145:699–711./ N2 Z; m$ Z. J% y! Y1 T

Reddy, B.S., C. Burill, and J. Rigotty. 1991. Effect of diets high in omega-3 and omega-6 fatty acids on initiation and postinitiation stages of colon carcinogenesis. Cancer Res. 51:487–491.( m2 X4 j9 x! ?$ V+ v9 B

Rose, D.P., and J.M. Connolly. 1999. Omega-3 fatty acids as cancer chemopreventive agents. Pharmacol. Ther. 83:217–244.
1 S- }7 }1 M9 I2 e2 G, @. L( _( Y( O( Z
Seung Kim, H.F., E.J. Weeber, J.D. Sweatt, A.L. Stoll, and L.B. Marangell. 2001. Inhibitory effects of omega-3 fatty acids on protein kinase C activity in vitro. Mol. Psychiatry. 6:246–248.& G& L+ _7 D3 w1 _8 v7 F

Simon, T.C., A. Cho, P. Tso, and J.I. Gordon. 1997. Suppressor and activator functions mediated by a repeated heptad sequence in the liver fatty acid-binding protein gene (Fabpl). J. Biol. Chem. 272:10652–10663.) A) ~% z4 {( R1 @
/ \8 L/ n+ C, \+ A& ~. [7 W
Wrana, J.L., L. Attisano, J. Carcamo, A. Zentella, J. Doddy, M. Laiho, X.F. Wang, and J. Massague. 1992. TGF beta signals through a heteromeric protein kinase receptor complex. Cell. 71:1003–1014.(Nicole R. Murray1,2, Capella Weems1, Lu )

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