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© The Rockefeller University Press, 0021-9525/1999//699 $5.00
The Journal of Cell Biology, Volume 145, Number 4, , 1999 699-711

Overexpression of Protein Kinase C β_II Induces Colonic Hyperproliferation and Increased Sensitivity to Colon Carcinogenesis

Nicole R. Murray^*,, Laurie A. Davidson^||, Robert S. Chapkin^||, W. Clay Gustafson^*,, Diane G. Schattenberg^*, and Alan P. Fields^*,,

^* Sealy Center for Oncology and Hematology, Department of Human Biological Chemistry & Genetics, and Department of Pharmacology, University of Texas Medical Branch, Galveston, Texas 77555-1048; and ^|| Faculty of Nutrition, Molecular and Cell Biology Section, Texas A&M University, College Station, Texas 77843-2471

Protein kinase C β_II (PKC β_II) has been implicated in proliferation of the intestinal epithelium. To investigate PKC β_II function in vivo, we generated transgenic mice that overexpress PKC β_II in the intestinal epithelium. Transgenic PKC β_II mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to azoxymethane-induced aberrant crypt foci, preneoplastic lesions in the colon. Furthermore, transgenic PKC β_II mice exhibit elevated colonic β-catenin levels and decreased glycogen synthase kinase 3β activity, indicating that PKC β_II stimulates the Wnt/adenomatous polyposis coli (APC)/β-catenin proliferative signaling pathway in vivo. These data demonstrate a direct role for PKC β_II in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/β-catenin signaling pathway.

Key Words: protein kinase C • colon carcinogenesis • signal transduction • proliferation • transgenic mice

Abbreviations used in this paper: ACF, aberrant crypt foci; AOM, azoxymethane; APC, adenomatous polyposis coli; DAG, diacylglycerol; DBA, dolichos biflorus agglutinin; FABP, fatty acid binding protein; GSK-3β, glycogen synthase kinase 3β; PAS, periodic acid Schiff; PKC, protein kinase C; PKC β_II, protein kinase C β_II isozyme; PNA, peanut agglutinin; RT-PCR, reverse transcriptase PCR; TUNEL, TdT-mediated dUTP-biotin nick end labeling; UEAI, Ulex europaeus-I.

Address correspondence to Alan P. Fields, Sealy Center for Oncology & Hematology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1048. Tel.: 409-747-1940. Fax: 409-747-1938. E-mail: afields{at}utmb.edu

COLON carcinogenesis is a complex multistep process involving progressive disruption of homeostatic mechanisms controlling intestinal epithelial cell proliferation, differentiation, and programmed cell death. This disruption appears to be mediated by dietary and environmental factors that modulate intestinal epithelial cell signaling pathways, as well as genetic mutation of transforming oncogenes and deletion or mutation of DNA repair enzymes and tumor suppressor genes (Bertagnolli et al., 1997). Recent studies have demonstrated the primary importance of the Wnt/APC/β-catenin signaling pathway in colon carcinogenesis (Pennisi, 1998). Mutations in either APC or β-catenin that lead to activation of this pathway are present in the vast majority of colon cancers and colonic carcinoma cell lines (Pennisi, 1998).

Accumulating evidence implicates protein kinase C (PKC)¹ in intestinal epithelial cell proliferation and colon carcinogenesis both in rodents and humans (Weinstein, 1990; Chapkin et al., 1993). PKC activity is higher in actively proliferating colonic epithelial cells than in their quiescent counterparts (Craven and DeRubertis, 1987), suggesting a role for PKC activation in epithelial cell proliferation. A link between PKC and colon carcinogenesis comes from the observation that components of cancer-promoting high fat diets lead to an increase in both colonic epithelial cell PKC activity and cellular proliferation (Craven and DeRubertis, 1988; Reddy et al., 1996). High fat diet-induced hyperproliferation is thought to predispose the colonic epithelium to further genetic and biochemical changes associated with progression along the carcinogenic pathway. PKC has also been shown to play a requisite role in the Wnt/APC/β-catenin proliferative signaling pathway, suggesting a plausible molecular mechanism by which PKC could stimulate colonic epithelial cell proliferation and colon carcinogenesis (Cook et al., 1996).

Several lines of evidence indicate that the PKC β_II isozyme (PKC β_II) is selectively involved in colonic epithelial cell proliferation and colon carcinogenesis. First, PKC β_II is the most responsive of the PKC isozymes expressed in the colonic epithelium to activation by secondary bile acids (Pongracz et al., 1995). Secondary bile acid levels are elevated in rodents fed a cancer-promotive high fat diet and this increase has been implicated in early carcinogenic events (for review see Reddy, 1975). Second, expression of most colonic PKC isozymes (e.g., PKC , , and ) is reduced in the presence of chronically elevated diacylglycerol (DAG), such as is present in preneoplastic colonic epithelial cells (Wali et al., 1991; Jiang et al., 1996; Chapkin et al., 1997; Jiang et al., 1997). However, intestinal PKC β_II is largely resistant to such activator-mediated downregulation (Saxon et al., 1994, Sauma et al., 1996). Third, the levels of PKC β_II are dramatically elevated both during the initial stages of tumorigenesis and in colonic carcinomas when compared with normal colonic tissue (Craven and DeRubertis, 1992; Davidson et al., 1994, 1998). Finally, PKC β_II is directly involved in colon carcinoma cell proliferation in vitro (Lee et al., 1993; Sauma et al., 1996).

These studies provide compelling but indirect evidence that PKC β_II plays an important role in intestinal epithelial cell proliferation and colon carcinogenesis, and are consistent with our studies demonstrating that PKC β_II is required for leukemia cell proliferation (Murray et al., 1993). Therefore, we hypothesized that PKC β_II is directly involved in intestinal epithelial cell proliferation in vivo and that elevated colonic PKC β_II expression and activity would enhance colon carcinogenesis. To directly test this hypothesis, we generated transgenic mice that express elevated levels of PKC β_II in the intestinal epithelium. These animals exhibit both hyperproliferation of the colonic epithelium and an increased susceptibility to colon carcinogenesis. Furthermore, our data indicate that the β-catenin/ APC proliferative signaling pathway is stimulated by PKC β_II in these animals.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Production and Maintenance of Transgenic Mice
A transgene construct consisting of the rat liver fatty acid binding protein (FABP) promoter (–596 to +21; kindly provided by Dr. Jeffrey Gordon, Washington University, St. Louis, MO), the full-length human PKC β_II cDNA, and the SV40 large T antigen polyadenylation signal sequence was produced by conventional cloning methods. The resulting PKC β_II transgene construct was confirmed by direct microsequencing before microinjection. The pFABP/PKC β_II transgene construct was propagated in the mammalian expression vector pREP4 and the transgene insert was excised using NheI (5') and XbaI (3'), purified, and microinjected into C57BL/6J x C3H/HeJ F₂ mouse oocytes as previously described (Hogan et al., 1994). The microinjections and generation of transgenic founder mice were conducted at the University of Texas Medical Branch Transgenic Mouse Facility. Transgenic founder mice were identified by Southern blot analysis. In brief, genomic tail DNA (5 µg) was digested to completion with Taq I (Roche), resolved by agarose gel electrophoresis, transferred to nylon membrane (Amersham), and transgenic DNA detected with a radiolabeled probe corresponding to the SV40 polyadenylation sequence. Three transgenic founder animals were identified from a screen of 120 live births. Transgene copy number was determined for each transgenic line by quantitative Southern blot analysis as previously described (Hogan et al., 1994). Genotype was confirmed by slot blot analysis using a radiolabeled probe corresponding to the polyadenylation sequences within the transgene (Sambrook et al., 1989).

Founder mice were mated with C57BL/6J mice (The Jackson Laboratory) to establish the transgene on a stable genetic background. Transgenic PKC β_II mice and progeny were bred and housed in microisolator cages maintained at constant temperature and humidity on a 12-h on/12-h off light cycle in a pathogen-free barrier facility. Mice were provided a standard autoclavable chow (Purina 7012, 5% fat) and autoclaved water ad libitum.

Detection of Transgenic PKC β_II RNA
Total RNA was extracted from tissue samples using a Totally RNA kit (Ambion). Reverse transcription was carried out using 6 µg RNA, 1 µg oligo(dT) primer, 10 mM dithiothreitol, 0.5 mM dNTPs, and 200 U SuperScript II reverse transcriptase (GIBCO BRL). Amplification of the transgenic RNA was carried out using 20 ng of the following primers, which amplify human PKC β_II but not endogenous mouse PKC β_II: forward, 5' CGTCCTCATTGTCCTC 3'; reverse 5' GACCTTGGTTCCCTGACTG 3'. An optimized amplification program of denaturation (94°C, 15 s), annealing (56°C, 15 s), and extension (74°C, 45 s) for 40 cycles using PCR Supermix (GIBCO BRL) was used. Human brain RNA was used as a positive control; mouse brain RNA and samples incubated without reverse transcriptase served as negative controls.

PKC Immunoblot and Immunohistochemical Analysis
Immunoblot analysis for PKC β_II expression in mouse colonic epithelium was performed essentially as previously described (Davidson et al., 1994). In brief, mice were killed by CO₂ asphyxiation, the colons were isolated and slit open longitudinally and rinsed well with PBS, and the colonic epithelium was scraped using a plastic coverslip. Total cell extracts were prepared in RIPA buffer [50 mM Tris, pH 7.2, 150 mM NaCl, 2 mM EDTA, 0.4 mM EGTA, 20 µM NaF, 0.5% deoxycholate, 1% NP-40, 0.1% SDS, 0.1 mM Na₃VO₄, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 25 µg/ml pepstatin, 1 µg/ml soybean trypsin inhibitor, and 34.5 µg/ml 4-(2-aminoethyl) benzene sulfonyl fluoride]. Equal amounts (30 µg) of protein were subjected to immunoblot analysis using an isotype-specific antibody for PKC β_II (Santa Cruz Biotechnology).

Immunohistochemistry was performed using an enhanced biotinyl tyramide system (New England Nuclear) on sections from the proximal and distal colon fixed in ethanol, embedded in paraffin, and sectioned (5 µm) as previously described (Jiang et al., 1995), with the following modifications. After deparaffinization and rehydration of tissues, sections were treated with 3% hydrogen peroxide in methanol to inhibit endogenous peroxidase, blocked with TNB reagent (Dupont New England Nuclear), and incubated with polyclonal antibody to PKC β_II (Hocevar and Fields, 1991). Specificity was confirmed using antibody preincubated with excess antigen peptide as previously described (Jiang et al., 1995). Sections were incubated with biotinylated secondary antibody followed by addition of streptavidin-conjugated peroxidase. Biotinyl tyramide amplification reagent was then added followed by a second streptavidin-peroxidase incubation. Visualization was with DAB chromagen.

Measurement of Colonic Epithelial Cell Cytokinetics
12-wk-old mice were killed and their colons were dissected and measured for overall length. The distal colon (1 cm from rectal end) was fixed in 4% paraformaldehyde and processed for histology as described previously (Jiang et al., 1995). Tissues were embedded in paraffin, sectioned (5-µm thickness) and stained with hematoxylin and eosin. 25 full-length, longitudinally cut crypts from each animal were analyzed for crypt height (micrometer) and number of cells per crypt height. 25 crypts cut on the cross-section at random height were counted to determine the average crypt circumference (in number of cells). These data were used to calculate cell size (crypt height in micrometer/crypt height in cell number) and estimate the total cells per crypt (mean cells per crypt column x mean crypt circumference).

Proliferation.
Cell proliferation was determined by immunohistochemical detection for proliferating cell nuclear antigen (PCNA) in distal colon sections. Primary antibody against PCNA (PC10 clone; DAKO) was diluted 1:50 in PBS and preincubated with 1:200 biotinylated anti–mouse IgG (Santa Cruz Biotechnology) overnight at 4°C. After deparaffinization, sections were processed for antigen retrieval as described by the manufacturer (DAKO), treated with 1% hydrogen peroxide for 10 min to inactivate endogenous peroxidases, and blocked with normal goat serum. The slides were then incubated with the PCNA/anti–mouse IgG antibody conjugate for 60 min at room temperature. Antigen–antibody complexes were detected with avidin and peroxidase–labeled biotin (ABC staining system; Santa Cruz Biotechnology) and visualized with DAB. Slides were counterstained with hematoxylin to provide contrast. 20 full-length, longitudinally cut crypts were divided into thirds and scored visually for cells staining darkly for PCNA (Lin et al., 1996). The labeling index (percent of labeled cells) and proliferative zone (highest cell from the bottom of the crypt staining for PCNA divided by the total cells per crypt height) were calculated for each set of animals.

Differentiation.
The differentiation status of colonic epithelial cells was measured by detection of the specific binding of three different lectins. After deparaffinization, sections were incubated for 60 min at room temperature in normal goat serum. Three different biotinylated lectins (dolichos biflorus agglutinin [DBA], peanut agglutinin [PNA], and Ulex europaeus-I [UEAI]; Vector Labs.) were diluted to 10 µg/ml in PBS. Sections were incubated with one of the three lectin solutions for 60 min at room temperature. Sections were then washed in three changes of PBS and incubated with 5 µg/ml of rhodamine red-X–conjugated Streptavidin (Jackson Immunoresearch Labs.) in PBS for 30 min at room temperature. After three 5-min washes in PBS, sections were mounted in aqueous media containing 95% glycerol in PBS and analyzed by fluorescence microscopy. Sections were also analyzed histologically by Alcian blue/periodic acid Schiff (PAS) staining for detection of mature, mucin-producing goblet cells.

Apoptosis.
The percentage of cells undergoing apoptosis (apoptotic index) was determined in paraformaldehyde-fixed distal colon tissue by the TdT-mediated dUTP-biotin nick end labeling of fragmented DNA (TUNEL) assay (Gavrieli et al., 1992) using the apoTACS kit from Trevigen. The tissue sections were counterstained with methyl green. 100 longitudinally cut, full-length crypts were scored for apoptotic cells based on a combination of positive staining and morphological criteria as previously described (Kerr et al., 1995).

Carcinogen Treatment and Aberrant Crypt Foci Analysis
40 (20 transgenic PKC β_II mice, 20 nontransgenic littermates) 6–7-wk-old female mice were injected intraperitoneally with azoxymethane (10 mg/kg body wt) or saline weekly for 2 wk as previously described (Chang et al., 1997). At 5 and 20 wk after the second injection, five animals per group were killed by CO₂ asphyxiation and the colons were removed. The colons were flushed with PBS to remove fecal pellets, slit open longitudinally, and fixed flat between two pieces of filter paper under a glass plate in 70% ethanol for 24 h. Fixed colons were stained with 0.2% methylene blue in PBS for 5 min before being mounted on a glass slide for observation at low magnification (x40) on a light microscope. Aberrant crypt foci (ACF) were scored blindly by a single observer (A.P. Fields) for total number and multiplicity (number of crypts/focus) using previously defined criteria (McLellan et al., 1991).

β-Catenin Immunoblot Analysis and Glycogen Synthase Kinase 3β Kinase Assay
Colonic epithelia from transgenic and nontransgenic mice were scraped and equal amounts of protein from total tissue lysates were subjected to immunoblot analysis using a specific β-catenin polyclonal antibody (Santa Cruz Biotechnology Inc.) or a specific GSK-3β monoclonal antibody (Transduction Laboratories). For glycogen synthase kinase (GSK)-3β kinase assay, colonic epithelium scrapings were solubilized in lysis buffer [10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 25 µg/ ml pepstatin, 1 µg/ml soybean trypsin inhibitor, 34.5 µg/ml 4-(2-aminoethyl) benzene sulfonyl fluoride, 20 µM NaF, and 0.1 mM Na₃VO₄]. Lysates containing 300 µg of protein were precleared with 75 µl of protein A agarose and then added to 75 µl of protein A agarose beads that had been preincubated with 5 µg of anti–GSK-3β monoclonal antibody (Transduction Labs.). Samples were incubated for 1 h at 4°C, and beads were pelleted and washed once with lysis buffer and once with kinase assay buffer (8 mM MOPS, pH 7.4, 0.2 mM EDTA, 10 mM Mg acetate, and 0.1 mM ATP). The washed and pelleted beads were then resuspended in 40 µl of kinase assay buffer containing 10 µCi [³²P]ATP and 250 µmol of GSK-3β–specific substrate peptide (Upstate Biotechnology, Inc.). Reactions were incubated for 20 min at 25°C and stopped by pelleting the beads and adding the supernatant to 20 µl of 40% trichloroacetic acid. Reactions were spotted on P-81 filters and washed three times in 0.75% phosphoric acid and once with acetone. Incorporated radioactive phosphate was quantitated by Cerenkov counting. Nonspecific and background counts were calculated by performing parallel assays with a nonphosphorylatable GSK-3β substrate peptide.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Generation and Identification of PKC β_II Transgenic Mice
To investigate the role of PKC β_II in colonic epithelial cell biology, we generated transgenic mice overexpressing PKC β_II in the intestinal epithelium. For this purpose, we used the rat liver FABP promoter, which has been well characterized to target transgene expression to the intestinal epithelium (Cohn et al., 1991; Simon et al., 1993). A schematic diagram of the transgene construct is presented in Fig. 1 A. The FABP promoter (–596 to +21) was fused to the cDNA for human PKC β_II and the SV40 poly A signal sequence by conventional cloning. Southern blot analysis of tail DNA identified four potential transgenic founders (designated Nos. 54, 61, 78, and 92) from 120 live births (Fig. 1 B). Animal 92 gave a reactive band of higher mol wt than the expected 453 bp Taq 1 fragment generated from the intact transgene (Fig. 1, asterisk). Further analysis using overlapping PCR primer sets demonstrated that this animal contained a truncated transgene, whereas animals 54, 61, and 78 contained multiple copies of the intact transgene construct. All three of the founder animals were fertile and subsequent analysis of progeny by quantitative Southern blot analysis demonstrated that they carried 6, 15, and 31 copies of the transgene, respectively. Furthermore, all three transgenic lines exhibit germline transmission of the transgene to subsequent progeny (data not shown).

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 1 (A) Generation and characterization of transgenic PKC βII mice. Schematic diagram of the PKC βII transgene construct. A transgene construct consisting of the rat liver FABP promoter (–596 to +21), full-length human PKC βII cDNA, and the SV40 large T antigen polyadenylation signal sequence was generated. Restriction sites for excision of the construct from the cloning vector (NheI/ XbaI), for generation of radiolabeled probe (BamHI/XbaI), and for digestion of genomic DNA for Southern blot analysis (Taq I) are indicated with arrows. (B) Southern blot analysis to identify founder transgenic mice. Tail DNA was digested with Taq I to generate a 453-bp fragment containing the SV40 polyadenylation sequence from the transgene (see A). 5 µg of digested genomic DNA was resolved by gel electrophoresis and transferred to nitrocellulose, and the membrane was incubated with 32P-labeled probe to the SV40 polyadenylation sequence. M, radiolabeled DNA mol wt markers; 54, 61, 78, and 92 indicate genomic DNA from four founder animals carrying the transgene construct; wt, genomic DNA from a nontransgenic mouse; * indicates an anomalous size band reacting with the transgene probe. (C) Transgenic PKC βII RNA is expressed in the colon of transgenic mice. Total RNA was isolated from scraped colonic epithelium from a litter of mice in the 54 transgenic line. Reverse transcription and amplification was carried out using primers specific to regions of sequence divergence between human and mouse PKC βII. Amplification products were separated on agarose gels and the product was visualized by ethidium bromide staining. Human brain RNA was used as a positive control (+ control); tg, RNA from transgenic animals; wt, RNA from nontransgenic animals. Genotype was confirmed by slot blot analysis.

We next determined the level of PKC β_II protein expression in the colonic epithelium of transgenic mice. Colonic epithelial cell lysates from transgenic and nontransgenic animals from the 54 transgenic line were prepared and subjected to immunoblot analysis using a PKC β_II isozyme-specific antibody (Hocevar and Fields, 1991). Consistent with the presence of transgenic PKC β_II mRNA, PKC β_II protein levels in the colonic epithelium of transgenic mice are elevated relative to their nontransgenic littermates (Fig. 2 A). Transgenic PKC β_II exhibits a relative molecular mass of 85 kD, comigrating with mouse brain PKC β_II used as a positive control. Similar results were obtained in the small intestine of these animals and from animals in the 61 and 78 transgenic lines (data not shown). Quantitation of PKC β_II expression by densitometric analysis of the immunoblots indicated that 54 line transgenic mice express an average of fivefold more PKC β_II protein than do nontransgenic littermates. Immunoprecipitation kinase assays showed an approximately fivefold increase in calcium- and phospholipid-dependent PKC β_II activity in the colonic epithelium of transgenic mice, demonstrating that transgenic PKC β_II is catalytically active and exhibits the same cofactor dependence of endogenous PKC/ β_II (data not shown). Animals in the 54 transgenic line gave a consistently high level of transgene expression and therefore this line was selected for further analysis.

View larger version (102K): [in this window] [in a new window] [Download PPT slide]   Figure 2 PKC βII protein is overexpressed in the colons of transgenic mice. (A) Immunoblot analysis for PKC βII. A litter of mice from the 54 transgenic line was killed, the colons were isolated and scraped, and total cell lysates (30 µg) were subjected to immunoblot analysis using a PKC βII–specific antibody as previously described (Murray et al., 1993). Densitometric analysis indicates that transgenic animals express an average of fivefold higher level of PKC βII than do nontransgenic littermates. tg, lysates from transgenic animals; wt, lysates from nontransgenic animals; +, rat brain extract used as positive control. (B and C) Immunohistochemical localization of PKC βII in proximal colon of transgenic mice. Proximal colon from transgenic mice and nontransgenic littermates in the 54 transgenic line were fixed in 70% ethanol, embedded in paraffin, sectioned, and immunostained for PKC βII as described in Materials and Methods. In nontransgenic colon (B), endogenous PKC βII localizes to the mid-crypt areas and to the luminal surface of the crypts. In transgenic colon (C), PKC βII staining is greater than in the nontransgenic animal and is observed along the entire crypt axis. Original magnification: x400. Bars, 10 µm.

Effect of Transgene Expression on Colonic Crypt Morphometry
To investigate the biological effects of overexpression of PKC β_II in the colonic epithelium, we analyzed the following colonic morphometric parameters: colon length, colonic crypt height (in micrometer and cell number), crypt circumference (in cell number), and cell size (crypt height in micrometer/crypt height in cell number) (Table I). This analysis revealed no statistical difference in the length of the colon, cell size, crypt height in micrometers, or crypt circumference between transgenic and nontransgenic littermates. However, colonic crypts from transgenic mice tended to be longer and have a larger circumference than those from nontransgenic mice. In addition, a highly significant increase in the number of cells per crypt height, and in the total number of cells per crypt, was observed in transgenic mice (Table I). Similar results were obtained in a second transgenic mouse line (line 78; 22.2 cells per crypt height in transgenic versus 20.7 in nontransgenic mice, P = 0.009; and 356.4 total cells per crypt in transgenic versus 321.1 in nontransgenic mice, P = 0.007), indicating that this effect is due to the presence of the PKC β_II transgene rather than an insertional mutagenic event. Both of these cytokinetic parameters are highly regulated and are determined by the balance among cell proliferation, differentiation, and apoptosis. These results demonstrate that increased expression of PKC β_II disrupts one or more of the homeostatic mechanisms regulating cell number in the colonic epithelium.

View larger version (74K): [in this window] [in a new window] [Download PPT slide]   Figure 3 Transgenic PKC βII mice exhibit increased proliferation of the colonic epithelium. 12-wk-old transgenic and nontransgenic mice were killed and their colons were isolated and fixed in paraformaldehyde as previously described (Chang et al., 1997). Sections were stained for PCNA with DAB (brown) using the ABC staining system (Santa Cruz Biotechnology). Sections were counterstained with hematoxylin (blue). A shows the nontransgenic mouse colon, and B shows the transgenic mouse colon. Original magnification: x400. Bars, 10 µm.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Figure 4 Transgenic PKC βII mice show no change in colonic epithelial cell differentiation. (A and B) Alcian blue/PAS staining. Mucin-containing goblet cells in colonic epithelium of nontransgenic (A) and transgenic PKC βII (B) mice were stained with Alcian blue/PAS. (C–H) Lectin staining. Sections from nontransgenic (C, E, and G) and transgenic (D, F, and H) mouse colonic epithelium were incubated with three different biotinylated lectins and detected with avidin-conjugated rhodamine red-X. C and D, DBA; E and F, PNA; G and H, UEAI. Arrowheads indicate Golgi staining in PNA-stained sections. Bars, 10 µm.

View larger version (104K): [in this window] [in a new window] [Download PPT slide]   Figure 5 Transgenic PKC βII mice exhibit no change in apoptosis. (A) Detection of apoptosis in the colonic epithelium by in situ TUNEL. Distal colonic epithelium was isolated and fixed in paraformaldehyde as previously described (Chang et al., 1997) and cells undergoing apoptosis were detected by an in situ TUNEL method. A representative apoptotic cell is indicated by the arrow. (B) Quantitative analysis of in situ TUNEL staining. 100 crypts from transgenic and nontransgenic mice were scored for apoptotic cells and the apoptotic index was calculated (percentage of apoptotic cells). Results are expressed as the mean ± SEM. Bar, 10 µm.

To determine whether transgenic PKC β_II mice differ from nontransgenic mice in their sensitivity to AOM-induced colon carcinogenesis, 6–7-wk-old transgenic PKC β_II mice and nontransgenic littermates (five mice/group) received either AOM (10 mg/kg body wt) or saline by intraperitoneal injection once a week for 2 wk. At 5 and 20 wk after the second AOM injection, mice were killed and their colons were analyzed for the presence of ACF. In agreement with the literature (Bird, 1987; McLellan and Bird, 1988; McLellan et al., 1991), we observed no ACF in saline-injected animals, confirming that ACF arise as a result of AOM exposure. Colons from both transgenic and nontransgenic animals treated with AOM contained ACF exhibiting the distinguishing characteristics described by Bird and colleagues (Bird, 1987; McLellan and Bird, 1988; McLellan et al., 1991). Specifically, ACF appeared as enlarged crypts, often three or four times the size of adjacent crypts, that were raised above the surface of the surrounding mucosa. ACF characteristically stained darker than surrounding crypts, had thicker than normal intercryptal spaces, and exhibited thickening of the crypt wall, suggestive of epithelial stratification. The crypt lumens in ACF were elongated and often serrated, in contrast to the round, smooth lumens of normal crypts. ACF contained either a single aberrant crypt or involved two or more adjacent crypts. Fig. 6 Ashows the morphology of a typical ACF consisting of three crypts from an AOM-treated animal. The total number of ACF/colon and the multiplicity of ACF was determined at 5 and 20 wk after the last AOM injection (Fig. 6, B–D). AOM-treated transgenic mice had a statistically significant increase in the total number of ACF/colon and in the number of ACF of higher multiplicity at both 5 and 20 wk (Fig. 6, B and C). At 20 wk, the total number of ACF did not increase significantly from that measured at 5 wk; however, the number of ACF of higher multiplicity did increase in transgenic PKC β_II mice (Fig. 6 D). Interestingly, at 5 wk, although the total number of ACF and the number of ACF of higher multiplicity were greater in transgenic mice, the average multiplicity of ACF in these two groups did not differ (Fig. 6 D). However, by 20 wk, transgenic mice exhibited an increase not only in the number of ACF but also in the average crypt multiplicity (Fig. 6 D). Since the number of ACF, particularly those of higher multiplicity, are highly predictive of subsequent colon tumor incidence, these data demonstrate that transgenic PKC β_II mice are more susceptible to AOM-induced colon carcinogenesis than nontransgenic littermates. Furthermore, these data suggest that elevated PKC β_II is involved not only in the early promotive phase of ACF development but also in their progression to lesions of higher multiplicity and malignant potential.

View larger version (141K): [in this window] [in a new window] [Download PPT slide]   Figure 6 Transgenic PKC βII mice are more susceptible to AOM-induced ACF formation. (A) An ACF from an AOM-treated mouse. Colons from AOM-treated mice were cut longitudinally from cecum to anus, fixed flat in 70% ethanol, and stained with 0.2% methylene blue for 5 min. Colons were scored for ACF under low magnification (x40) using previously defined criteria for ACF (Bird, 1987; McLellan and Bird, 1988). A representative ACF from an AOM-treated animal is shown. This ACF involves three adjacent crypts. Bar, 100 µm. (B–D) Transgenic PKC βII mice have increased levels of AOM-induced ACF. 6-wk-old female mice from the 54 line (transgenic and nontransgenic littermates) were injected with AOM (10 mg/kg body wt) weekly for 2 wk. At 5 and 20 wk after the second injection the mice were killed and the colons were analyzed for the presence of ACF as described in panel A. wt, nontransgenic; tg, transgenic. (B) ACF analysis at 5 wk after treatment. Total ACF per animal and ACF involving >1 crypt were plotted for both transgenic and nontransgenic animals. For each group of animals, n = 5. (C) ACF analysis at 20 wk after treatment. Total ACF per animal and ACF involving >2 crypt were plotted for both transgenic and nontransgenic animals. For each group of animals, n = 5. (D) Crypt multiplicity of ACF at 5 and 20 wk. The average crypt multiplicity for ACF in nontransgenic and transgenic mice was calculated at 5 and 20 wk. For each group of animals, n = 5. Error bars represent the SEM.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Figure 7 Transgenic PKC βII mice exhibit decreased GSK-3β activity and increased β-catenin levels. (A) Immunoblot analysis for GSK-3β in the colonic epithelium of transgenic (tg) and nontransgenic (wt) mice. (B) Immunoprecipitation kinase assays for GSK-3β were performed on scraped colon extracts from transgenic and nontransgenic mice. Results were normalized to GSK-3β activity in nontransgenic animals. Error bars represent the SEM. (C) Representative immunoblot analysis for β-catenin in the colonic epithelium of transgenic and nontransgenic mice. (D) Densitometric analysis of β-catenin expression. Results were normalized to β-catenin in wild-type mice. n = 6 for each group. Error bars represent SEM. Equal amounts of total protein were analyzed in each immunoblot.

	Discussion

Top Abstract Materials and Methods Results Discussion References

To directly assess the role of PKC β_II in colonic epithelial cell proliferation and colon carcinogenesis, we developed a transgenic mouse model in which PKC β_II is overexpressed in the intestinal epithelium. Transgenic PKC β_II mice exhibit hyperproliferation of the colonic epithelium characterized by an increase in the labeling index and an increase in the number of cells per colonic crypt. Interestingly, no significant changes were observed in colonocyte differentiation status or apoptotic index, indicating a selective effect of PKC β_II on the proliferative program of the colonic epithelium. Although we cannot eliminate the possibility that subtle changes have occurred in the regulation of differentiation or susceptibility to apoptosis, our data clearly demonstrate that the change in proliferation is a major contributing factor to the increased colonic crypt cell number observed in transgenic PKC β_II mice.

Increased proliferation is an important risk factor for induction of colon cancer and is a key biomarker of preneoplastic events (Chang et al., 1997; Einspahr et al., 1997). Our data indicate that PKC β_II acts early in the carcinogenic pathway to increase the proliferation of the colonic epithelium, perhaps making it more susceptible to further genetic mutations and formation of preneoplastic lesions, including ACF. The effect of increased PKC β_II expression on the susceptibility to induction of colon cancer was tested using a well-characterized rodent carcinogenesis model (Deschner and Long, 1977; Deschner et al., 1979). AOM-induced colon tumors are a good model for sporadic human colon cancer because they exhibit many of the same properties as human colon tumors, including increased proliferation, development of tumors predominantly in the distal colon, and the presence of many of the same genetic mutations found in human tumors. In addition, ACF, the earliest preneoplastic lesions observed in this model, are also thought to be preneoplastic lesions in humans (Pretlow et al., 1992; Takayama et al., 1998). ACF exhibit many of the early phenotypic markers of colon cancer including increased proliferation and frequent mutations in the APC and ras genes (Pretlow et al., 1993; Smith et al., 1994; Shivapurkar et al., 1997). We demonstrate that increased PKC β_II expression makes transgenic mice more susceptible to AOM-induced colon carcinogenesis as measured by an increase in the total number of ACF and in the number of ACF of higher multiplicity than nontransgenic mice. ACF are highly predictive of subsequent tumor formation and multiplicity in the rodent carcinogenesis model and of adenoma formation and colon cancer risk in humans (Magnuson et al., 1993; Bird, 1995; Roncucci et al., 1991). Our data indicate that elevated PKC β_II expression not only promotes ACF formation, but also stimulates progression of these lesions. These results suggest that PKC β_II plays a critical role at multiple stages in the colon carcinogenic pathway.

A Model for the Role of PKC β_II in Sporadic Colon Cancer
Accumulating evidence suggests that PKC β_II plays a direct role in intestinal epithelial cell proliferation and colon carcinogenesis in both rodents and humans. PKC β_II levels and activity are elevated in preneoplastic and neoplastic colons, demonstrating that these changes precede colon carcinoma development (Craven and DeRubertis, 1992; Wali et al., 1995; Davidson et al., 1998). Here, we demonstrate that overexpression of PKC β_II in the colonic epithelium leads to hyperproliferation and increased susceptibility to colon carcinogenesis. Furthermore, we demonstrate that elevated PKC β_II leads to inhibition of GSK-3β activity and an increase in β-catenin levels. These observations are consistent with in vitro data demonstrating a requisite role for PKC in the Wnt proliferative signaling pathway (Cook et al., 1995), and suggest that PKC β_II may play such a role in vivo. Further studies will be required to determine whether PKC β_II–mediated activation of this pathway is required for its ability to stimulate proliferation and cancer susceptibility in the transgenic mouse setting.

Taken together, the data lead us to propose a molecular mechanism by which PKC β_II stimulates colonic epithelial cell hyperproliferation and increased colon carcinogenesis in transgenic mice (Fig. 8). In this model, PKC β_II either directly or indirectly leads to GSK-3β inactivation. PKC has been shown to phosphorylate GSK-3β and inactivate the enzyme in vitro (Goode et al., 1992), suggesting that PKC β_II can inhibit GSK-3β by direct phosphorylation and inactivation. Inhibition of GSK-3β leads to an accumulation of β-catenin by decreasing the interaction of β-catenin with APC, which targets β-catenin for degradation. Accumulation of β-catenin causes Tcf-dependent transcriptional activation of growth-related genes to stimulate colonocyte proliferation (Pennisi, 1998). The APC/β-catenin pathway is a major site for mutation during colon carcinogenesis (Pennisi, 1998). Mutations in either APC or β-catenin that disrupt β-catenin degradation are present in the vast majority of colon cancers, providing strong evidence that elevated β-catenin levels are important in colon carcinogenesis (Ilyas et al., 1997; Pennisi, 1998). Furthermore, overexpression of a proteolytically-stable NH₂-terminal truncated β-catenin in the intestinal epithelium of transgenic mice leads to hyperproliferation (Wong et al., 1998). Our data suggest that accumulation of β-catenin through PKC β_II–mediated inhibition of GSK-3β may play an important promotive role in colon carcinogenesis before the acquisition of mutations in members of this critical signaling pathway.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 8 Model of a proposed functional role for PKC βII in colon carcinogenesis. PKC βII is proposed to function in the Wnt signaling pathway in the colonic epithelium. (A) During embryogenesis, Wnt signals through Dsh to PKC βII, which phosphorylates GSK-3β to inhibit its activity. β-catenin levels rise, leading to Tcf-dependent activation of transcription of growth-related genes. (B) In the adult colonocyte, Wnt is not present, GSK-3β activity is constitutively high, and APC binds β-catenin and targets it for degradation. (C) In transgenic PKC βII mice, PKC βII levels and activity are elevated. PKC βII can induce phosphorylation and inactivation of GSK-3β. β-catenin levels rise and Tcf-dependent transcription of growth-related genes is induced, leading to increased proliferation. Similarly, AOM treatment leads to increased expression of PKC βII stimulating this proliferative pathway during colon carcinogenesis. Biochemical evidence suggests that PKC βII can be activated by various lipid components of a cancer-promotive diet, including free fatty acids, secondary bile acids, and bacterially-derived DAG. Direct stimulation of PKC βII by these factors leads to Wnt-independent activation of the APC/β-catenin signaling pathway, hyperproliferation, and enhanced susceptibility to colon carcinogenesis.

Cancer-promotive dietary fats function to increase the level of secondary bile acid and fatty acids in the intestinal lumen. Secondary bile acids can in turn activate colonic PKC by a number of mechanisms. First, secondary bile acids and fatty acids can directly activate PKC β_II activity and stimulate cellular proliferation in the colonic epithelium (DeRubertis et al., 1984; Fitzer et al., 1987; Ward and O'Brian, 1988; Pongracz et al., 1995). Second, bile acids can promote DAG production by intestinal bacteria, which in turn stimulate colonocyte PKC activity (Morotomi et al., 1990; Morotomi et al., 1991). Third, bile acids can stimulate phospholipid breakdown and DAG generation in colonic epithelial cells (DeRubertis and Craven, 1987), leading to PKC activation. Therefore, we hypothesize that these dietary risk factors increase PKC β_II activity in intestinal epithelial cells by multiple mechanisms, resulting in increased epithelial cell proliferation through activation of the APC/β-catenin signaling pathway in a Wnt-independent fashion (Fig. 8 C). This model provides a plausible link between a critical intracellular signaling pathway that is known to be important in colon cancer, and known dietary risk factors for colon carcinogenesis. Our transgenic PKC β_II mice will provide a valuable model to test the hypothesis that PKC β_II is a relevant target for these cancer-promotive dietary risk factors, and to explore the mechanism by which these factors may impinge on the APC/β-catenin signaling pathway.

	Acknowledgments

 
We thank Dr. Jeffrey Gordon for the generous gift of the rat liver FABP promoter; Dr. Jeffrey Ceci for generation of the founder transgenic mice and expert technical advice on genotypic analysis; Jenny Cui, Dr. Wenchi Chang, and Lisa Nash for excellent technical assistance; Dr. Leonard H. Augenlicht for advice on in situ apoptosis measurements; and Dr. Joanne Lupton, and Marco Velasco for helpful discussions.

This work was supported in part by National Institutes of Health grants CA59034 and CA81436. A.P. Fields is a Leukemia Society of America Scholar.

Submitted: 10 March 1999

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