Morphodensitometric analysis of protein kinase C ßII expression in rat colon: modulation by diet and relation to in situ cell proliferation and apoptosis

Laurie A. Davidson, Roxanne E. Brown, Wen-Chi L. Chang, Jeffrey S. Morris1, Naisyin Wang1, Raymond J. Carroll1, Nancy D. Turner, Joanne R. Lupton and Robert S. Chapkin2

Molecular and Cell Biology Section, Faculty of Nutrition and
1 Department of Statistics, Texas A&M University, College Station, TX 77843, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have recently demonstrated that overexpression of PKC ßII renders transgenic mice more susceptible to carcinogen-induced colonic hyperproliferation and aberrant crypt foci formation. In order to further investigate the ability of PKC ßII to modulate colonocyte cytokinetics, we determined the localization of PKC ßII with respect to cell proliferation and apoptosis along the entire colonic crypt axis following carcinogen and diet manipulation. Rats were provided diets containing either corn oil [containing n-6 polyunsaturated fatty acids (PUFA)] or fish oil (containing n-3 PUFA), cellulose (non-fermentable fiber) or pectin (fermentable fiber) and injected with azoxymethane (AOM) or saline. After 16 weeks, an intermediate time point when no macroscopic tumors are detected, colonic sections were utilized for immunohistochemical image analysis and immunoblotting. Cell proliferation was measured by incorporation of bromodeoxyuridine into DNA and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling. In the distal colon, PKC ßII staining was localized to the upper portion of the crypt. In comparison, proximal crypts had more (P < 0.05) staining in the lower tertile. AOM enhanced (P < 0.05) PKC ßII expression in all regions of the distal colonic crypt (upper, middle and lower tertiles). There was also an interaction (P < 0.05) between dietary fat and fiber on PKC ßII expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the distal colonic crypt. With respect to colonic cell kinetics, proliferation paralleled the increase in PKC ßII expression in carcinogen-treated animals. In contrast, apoptosis at the lumenal surface was inversely proportional to PKC ßII expression in the upper tertile. These results suggest that an elevation in PKC ßII expression along the crypt axis in the distal colon is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development. Therefore, select dietary factors may confer protection against colon carcinogenesis in part by blocking carcinogen-induced PKC ßII expression.

Abbreviations: AOM, azoxymethane; BrdU, bromodeoxyuridine; BT, biotinyl tyramide/amplification diluent; DAG, diacylglycerol; PBS, phosphate-buffered saline; PKC, protein kinase C; PUFA, polyunsaturated fatty acids; SA-HRP, streptavidin-conjugated horseradish peroxidase.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Colon cancer is the second leading cause of cancer death in the USA. There were an estimated 129 400 new cases of colon cancer diagnosed in the USA in 1999, resulting in 56 000 deaths (1). Recent human and rodent studies demonstrate that tumorigenesis is a complex multistep process of progressive disruption of homeostatic mechanisms controlling intestinal epithelial cell proliferation, differentiation and programmed cell death (apoptosis) (2). The majority (~80–90%) of colon cancers occur sporadically. They are thought to be caused by non-inherited factors, such as a combination of diet and other environmental factors, which result in somatic mutations of specific genes, i.e. RB1, p16, ras or p53 (3).

Human, rat and mouse colonic mucosa express both calcium-dependent (classical) and calcium-independent protein kinase C (PKC) isoforms with distinct subcellular distributions for each (48). Alterations in PKC isozyme signaling have been linked to the pathogenesis of colon cancer (911). This is consistent with data suggesting that PKC signaling pathways play an important role in colonic cell proliferation, differentiation and apoptosis (1215). With respect to specific isoforms of PKC, accumulating evidence implicates a link between PKC ßII overexpression and colon carcinogenesis (9,11,16,17). For example, in AOM-injected animals, PKC ßII is elevated in colon adenomas and carcinomas compared with uninvolved colonic mucosa (11,18,19). In order to elucidate the exact role of PKC ßII in the colon, we recently generated transgenic mice that overexpress PKC ßII in the colonic epithelium (20). Overexpression of PKC ßII in vivo induced colonic hyperproliferation and increased aberrant crypt foci formation following azoxymethane (AOM) exposure. These data demonstrate a direct role of PKC ßII in colonocyte proliferation and tumorigenesis. However, the strength of this relationship in other model systems, e.g. the rat colonic crypt, has not been determined.

Precisely which environmental and dietary factors are capable of modulating colonic PKC ßII expression in vivo remain speculative. The two dietary components thought to have the most significant effect on colonic cytokinetics and tumor development are dietary fat and fiber (2123). There are considerable data to support the concept that the type of fat or fiber is actually more important to tumor development than is the amount of either of these components in the diet (2224). Specifically, n-3 polyunsaturated fatty acids (PUFA) enhance colonic apoptosis and are protective against colon cancer in experimental studies (2426). Cellulose and wheat bran, non-fermentable fibers, have been shown to be protective against tumor development in carcinogen-induced rat colon cancer models (2730). In contrast, fermentable fibers, such as pectin, oat bran and guar gum, do not protect and may enhance tumorigenesis (29,31,32). However, we have demonstrated that epithelial cell apoptosis in the rat colon can be favorably modulated by feeding a combination of n-3 PUFA (found in fish oil) and pectin, thereby conferring resistance to toxic carcinogenic agents (21). The protective effect of this fat/fiber combination is due to an enhancement of apoptotsis. Therefore, in this study we have probed the linkage between PKC ßII expression and carcinogen and dietary factors that influence cell proliferation and apoptosis along the rat colonic crypt axis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Diet and carcinogen administration
The animal use protocol was approved by the University Animal Care Committee of Texas A&M University and conformed to the National Institutes of Health guidelines. A total of 260 weanling 21-day-old male Sprague–Dawley rats (Harlan Sprague–Dawley, Houston, TX) were used. The experimental format was a 2x2x2 factorial design as previously described (21,24). Cellulose or pectin was used as the fiber source; corn or fish oil was used as the dietary fat source; injection groups were either saline or AOM.

The fat source in the experimental diets was provided at 15% by weight and fiber was provided at 6% by weight (Table IGo). Rats were fed the experimental diets for 1 week and then injected with either saline or AOM at a dose of 15 mg/kg body wt. The animals were injected a second time with either saline or AOM 1 week after the first injection (21,24). Tissues were harvested at 16 weeks after the second injection for the immunohistochemical and immunoblotting analyses described here. Additional experiments were carried out to 34 weeks post-injection, at which time tumor load was determined. These data have been fully described previously (21).


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Table I. Experimental diets
 
The colon was resected proximally from the colon–cecum junction and distally from the most distal portion of the anus. A 1 cm portion of colon was taken from the cecum–colon junction for analysis of the proximal colon and a 1 cm portion from the anus–colon junction was discarded and a second 1 cm portion was removed and designated the distal colon tissue sample. The remaining proximal and distal colon tissue was scraped with a glass slide to remove the mucosal layer, as previously described (33).

Protein extraction and immunoblotting
The scraped proximal and distal mucosae were homogenized separately in 5 vol of homogenizing buffer [50 mM Tris–HCl, pH 7.2, 250 mM sucrose, 2 mM EDTA, 1 mM EGTA, 50 µM sodium fluoride, 25 µg/ml each leupeptin, pepstatin, and aprotinin, 1 µg/ml soybean trypsin inhibitor, 150 µM 4-(2-aminoethyl)benzene sulfonyl fluoride, 10 mM ß-mercaptoethanol] with six strokes of a teflon-in-glass homogenizer (10). The samples were then subjected to ultracentrifugation for 30 min. The 100 000 g supernatant was saved as a cytosolic fraction. The pellet was rehomogenized in buffer containing 1% Triton X-100. The solubilized pellet sample was recentrifuged and the supernatant saved as the membrane fraction. Cellular extracts were stored at –80°C. Protein concentration was determined using the Pierce Coomassie Plus protein assay (Rockford, IL). In order to quantitate the cytosolic and membrane-bound enzyme levels of PKC ßII in the colon following diet and carcinogen manipulation, colonic homogenates were treated with SDS sample buffer and subjected to SDS–PAGE in 4–12% minigels (Novex, San Diego, CA) as per the method of Laemmli (34). Following electrophoresis, the gels were washed in Tris–glycine transfer buffer and electroblotted onto PVDF transfer membranes (Millipore, Bedford, MA). After electroblotting, the PVDF membranes were processed according to the protocol of Davidson (35), including a blocking step with 4% non-fat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and dilution of the primary antibody (1:2000 PKC ßII polyclonal antibody; Gibco BRL, Grand Island, NY) in 4% milk/PBS/Tween. Peroxidase-labeled goat anti-rabbit IgG 2° antibody (Kirkegaard & Perry, Gaithersburg, MD) and the Pierce SuperSignal (Rockford, IL) detection system were subsequently utilized. A range of protein concentrations was loaded onto the gels to ensure a linear response. Specificity was confirmed using antibody preincubated with excess antigen peptide as previously described (12). Rat brain homogenates were used as positive controls on all blots. X-ray films of the blots were scanned using Adobe Photoshop (Salinas, CA) and quantitated using BioImage IQ software (BioImage, Ann Arbor, MI). All results were normalized against a constant amount (3 ng) of recombinant PKC ßII standard (Panvera, Madison, WI).

Immunohistochemistry
In order to assess the expression pattern of PKC ßII within proximal and distal colonic crypts, ethanol-fixed, paraffin-embedded tissue sections were processed as previously described (21,24). Briefly, colonic sections were first deparaffinized in a series of xylene baths and then partially rehydrated in ethanol baths in a gradient of 100–70% ethanol. The tissues were then placed in a 3% hydrogen peroxide/methanol bath for 30 min to inhibit endogenous peroxidase activity and then washed in PBS (36). After washing, the tissues were blocked with TNB blocking buffer [0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking reagent (NENTM Life Science Products, Boston, MA)] for 30 min. Subsequently, a 1:200 dilution of PKC ßII antibody diluted in TNB blocking buffer was applied and allowed to incubate overnight at 4°C (37). Upon completion of an overnight incubation, the primary antibody was rinsed off the tissue using 0.1 M Tris–HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20 (TNT) buffer with gentle agitation. The biotin-conjugated, affinity-purified goat anti-rabbit IgG 2° antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) was applied to the sections at 1:200 dilution in TNB blocking buffer and allowed to incubate at room temperature for 30 min. The tissues were again washed with TNT buffer, followed by incubation with a 1:100 dilution of streptavidin-conjugated horseradish peroxidase (SA-HRP) in TNB blocking buffer at room temperature for 30 min. The tissues were then washed with TNT buffer followed by a 1:50 dilution of biotinyl tyramide/amplification diluent (BT) for 10 min. The BT solution was washed off with TNT buffer. A solution of 1:100 SA-HRP/TNB blocking reagent was again applied for 30 min. PKC ßII was chromagenically detected using 3,3'-diaminobenzadine (Sigma, St Louis, MO), which reacted with the peroxidase to produce a brownish product (38).

Specific PKC ßII detection was assessed with a series of positive and negative controls. Antibodies specific to cytokeratin, which is known to be exclusively expressed in colonic crypts, was used as a positive biological control (12). Negative biological controls consisted of omission of the primary antibody, omission of the secondary antibody, use of an inhibitory peptide for PKC ßII and use of PKC {gamma} antibody, which is not expressed in the colon (8).

Morphodensitometric measurement using computer-assisted image analysis
All colonic crypt images were captured and analyzed using NIH Image v.1.61 (http://rsb.info.nih.gov/nih-image ) as previously described (39). Optimum offset and gain were determined by pre-analysis of multiple darkly and lightly stained tissues to maximize the distribution of stain intensity so that small differences in staining were quantifiable. Once established, the settings remained constant for all images. Background staining intensity was determined on 10 randomly selected images per animal and subtracted from the staining intensity of target cells. Sections were visualized at a 400x magnification using a Microstar IV microscope and Sony DXC-970 MD 3CCD camera. Each tissue section was surveyed from top to bottom to locate crypts that were cut in cross-section, allowing visualization from the lumen to the base of the crypt. A minimum of 20 crypts suitable for analysis was analyzed for each tissue section.

In situ apoptosis measurement
Paraformaldehyde-fixed, paraffin-embedded colon sections were processed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method as previously described (21,40). Positive control slides were treated with DNase I (Ambion, Austin, TX) at 37°C. Negative control slides were incubated without terminal deoxynucleotidyl transferase enzyme. The antibody–antigen complex was visualized by incubation with 3',3-diaminobenzidine. Apoptotic cells were identified based on a combination of positive staining and morphological criteria, as described by Kerr (41). Crypt height in number of cells and the number and location of apoptotic cells were recorded, with 20 crypts analyzed per animal. The apoptotic index was 100 times the mean of the number of apoptotic cells per crypt column divided by the mean of the total number of cells per crypt column.

In situ cell proliferation measurement
Animals were injected i.p. with 5 mg/kg body wt bromodeoxyuridine (BrdU) 1 h prior to killing in order to measure rates of in vivo cell proliferation (21). BrdU incorporation into colonocyte DNA was detected and scored in ethanol-fixed tissues as previously described (21,39).

Statistical analysis
Western blot data were analyzed using a three-way analysis of variance (ANOVA) to determine the effect of dietary fiber, fat and carcinogen on PKC ßII expression. A least squares mean analysis was conducted to examine fatxfiber, fatxinjection and fiberxinjection interactions. The mean immunohistochemical PKC ßII staining intensity was determined for each tissue section by calculating the mean intensity of at least 20 crypts. The mean intensity within the upper (apical), middle and lower tertiles of the crypts in the tissue section was analyzed by least squares mean analysis.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subcellular localization of colonic PKC ßII
Western blot analysis revealed detectable PKC ßII (80 kDa) in the tumor and uninvolved membrane and cytosol of colonic mucosa fractions (Figure 1Go). This is noteworthy because previous studies have shown that PKC ßII translocates to the membrane upon activation (5,6). At 34 weeks post-injection colonic tumors overexpressed PKC ßII in the membrane and cytosol fractions 25- and 29-fold above levels in uninvolved mucosa (n = 3). With regard to uninvolved mucosa, fatxinjection (P = 0.025) and fatxfiber (P = 0.004) interactions in the distal colon were observed only at 16 weeks post-injection. PKC ßII expression data generated from scraped mucosa containing all cell types within the colon are shown in Figures 2 and 3GoGo. Interestingly, fish oil feeding blocked the AOM-induced elevation of membrane PKC ßII expression (Figure 2Go). In addition, the combination of pectin and fish oil feeding resulted in the lowest membrane PKC ßII levels (Figure 3Go).



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Fig. 1. Immunoblot analysis of PKC ßII expression in colonic tumors and uninvolved colonic mucosa 34 weeks after AOM injection. Lanes 1 and 10, recombinant PKC ßII standard (3 ng); lanes 2 and 3, tumor membrane fraction; lanes 4 and 5, uninvolved mucosa membrane fraction from AOM-injected rats; lanes 6 and 7, tumor cytosolic fraction; lanes 8 and 9, uninvolved mucosa cytosolic fraction from AOM-injected rats. All samples contained 5 µg protein.

 


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Fig. 2. PKC ßII expression in colonic mucosa of rats 16 weeks after carcinogen injection. Immunoblot data were generated from distal colon membrane fractions. Values (means ± SEM) with different superscripts are significantly different (P < 0.05, n = 8–16).

 


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Fig. 3. Interactive effects of fat and fiber on PKC ßII expression in distal colon membrane fractions 16 weeks post-injection as assessed by immunoblot analysis. Refer to Figure 2Go for legend details.

 
PKC ßII immunohistochemical staining intensity along the colonic crypt axis
The localization of PKC ßII along the crypt axis is of interest because there is a distinct hierarchical arrangement of epithelial cells in the colon (42). Therefore, treatment effects on PKC ßII immunohistochemical localization exclusively within the epithelial cells which populate colonic crypts were determined at 16 weeks after carcinogen injection. This time point was selected because the balance between cell proliferation and apoptosis is altered without the appearance of macroscopic tumors (21). Representative photomicrographs of PKC ßII localization within proximal and distal colonic crypts are shown in Figure 4Go. Expression patterns were quantitated by tertile, demonstrating contrasting PKC ßII localization between the two tissue locations (Figure 5Go). Specifically, in the distal colon the greatest staining intensity was localized to the upper portion of the crypt. In comparison, proximal crypts had more staining in the lower tertile.



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Fig. 4. Differential localization of PKC ßII in distal (A and B) and proximal (C) rat colon sections by immunohistochemistry. Representative micrograph from sections probed with (A) PKC {gamma} antibody (negative control), (B and C) PKC ßII antibody. Immunoreactivity was detected as a brownish product. 400x magnification.

 


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Fig. 5. Site-specific differences in PKC ßII expression as assessed by quantitative morphodensitometric analysis. Different superscripts indicate significant differences (P < 0.05) between proximal and distal colon (means ± SEM) within each tertile, 16 weeks post-injection. n = 64 animals/colon site (proximal and distal).

 
A statistical evaluation of the overall effects of carcinogen, fat, fiber and their interactions is shown in Table IIGo. With respect to the effect of diet on the localization of PKC ßII in the proximal colonic crypt, there was a significant (P < 0.05) effect of fiber on the colonic crypt (overall), with pectin (18.9 ± 1.1) > cellulose (13.6 ± 1.1), as well as in the upper tertile of the crypt, where pectin (18.4 ± 1.0) > cellulose (10.4 ± 1.0) (n = 8 per group). A carcinogenxfat interaction (P < 0.05) was noted for the proximal crypt overall [AOM/corn oil (19.1 ± 1.6) > AOM/fish oil (13.4 ± 1.6)] and for the lower portion of the crypt [AOM/corn oil (26.8 ± 2.3) > AOM/fish oil (17.6 ± 2.3)]. There was a significant fatxfiber interaction (P < 0.05) with corn oil/cellulose (9.4 ± 1.1) < corn oil/pectin (16.0 ± 1.1), fish oil/cellulose (13.6 ± 1.1) and fish oil/pectin (14.5 ± 1.1) in the lower tertile of the proximal colon. For the distal colon there was a significant (P < 0.05) carcinogen effect and a fatxfiber interaction overall for the upper, middle and lower crypt tertiles (Figures 6 and 7GoGo).


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Table II. P values of type III sum of squares analysis of immunohistochemical PKC ßII expression in the distal and proximal colon
 


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Fig. 6. Effect of carcinogen on PKC ßII expression within different regions of the distal colonic crypt as assessed by morphodensitometric analysis, 16 weeks post-injection. AOM had a uniform effect throughout the crypt. Bars with different superscripts are significantly different within a crypt region (P < 0.05, n = 32 animals per injection type). (Inset) Positive relationship between PKC ßII (all tertiles combined) and the proliferative zone in the distal colon. The proliferative zone was determined by dividing the position of the highest labeled (BrdU) cell by the total cells per crypt column and multiplying by 100. Carcinogen-induced elevation in PKC ßII expression is associated with an expansion of the proliferative zone in the crypt.

 


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Fig. 7. Interactive effects of fat and fiber on PKC ßII expression within different regions of the distal colonic crypt as assessed by morphodensitometric analysis, 16 weeks post-injection. Bars with different superscripts are significantly different within a crypt region (P < 0.05, n = 16 animals per diet group). (Inset) Inverse relationship between PKC ßII expression (all tertiles combined) and apoptosis. Apoptotic index = (no. apoptotic cells ÷ no. cells/column)x100.

 
Association of PKC ßII expression with cell proliferation and apoptosis in the distal colon
In the distal colon the kinetics of colonic cell proliferation (the proliferative zone and the number of proliferative cells in the middle 1/3 of the crypt) paralleled the increase in PKC ßII expression in carcinogen-treated animals (Figure 6Go, inset). Specifically, AOM increased PKC ßII expression compared with saline in all tertiles of the colonic crypt combined (14.3 ± 0.8 versus 10.5 ± 0.8, P < 0.05) and expanded the proliferative zone (32.3 ± 0.7 versus 30.0 ± 0.8, P < 0.05). This relationship was not observed in the proximal colon.

With regard to the interactive effect (P < 0.05) of dietary fat and fiber on PKC ßII expression (corn/pectin > fish/cellulose, fish/pectin > corn/cellulose) in all regions of the crypt, the kinetics of programmed cell death were inversely proportional to the expression of PKC ßII (Figure 7Go, inset). These results suggest that an elevation in PKC ßII expression along the crypt axis is linked to enhancement of cell proliferation and suppression of apoptosis, predictive intermediate biomarkers of tumor development.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have demonstrated that PKC ßII transgenic mice exhibit hyperproliferation of the colonic epithelium and an increased susceptibility to AOM-induced colon carcinogenesis (20). These data indicate that PKC ßII plays a direct role in the development of colon cancer. Recently it has become apparent that oncogenic transformation of the colon is associated with an initial time-dependent accumulation of intracellular diacylglycerol (DAG) (4345), which may be responsible for the observed down-regulation of colonic PKC {alpha}, {delta} and {zeta} expression and activity (8,4648). The exception to this response is PKC ßII, which is not down-regulated efficiently in colonocytes following exposure to PKC activators (13,14). The presence of these changes prior to overt neoplasia suggests that PKC ßII is capable of influencing key molecular events which play a role in the early stages of colonic malignant transformation. Precisely which environmental factors are capable of modulating PKC ßII expression and/or activity in vivo remain speculative. It is well documented that certain types of dietary fat and fiber can modulate the lumenal concentration of several activators of PKC ßII, e.g. free fatty acids, secondary bile acids and DAGs (49,50). Therefore, it is possible that palliative diets may reduce colon cancer incidence in part by antagonizing colonic PKC ßII-related signal transduction.

With respect to the role of PKC ßII in regulating cell proliferation and apoptosis in rat colonic epithelium, our data indicate that the AOM-enhanced expression of PKC ßII along the crypt axis paralleled expansion of the proliferative compartment. Although little is known regarding the mechanisms regulating colonic cell growth, a relationship between PKC ßII expression and cell proliferation has been established in several cell systems (13,20,51,52). Also of interest is the fact that localization of PKC ßII along the crypt axis was inversely proportional to the site of apoptosis (primarily the upper third of the crypt). Therefore, it is possible that activation of PKC ßII antagonizes the induction of apoptosis in the colon. With respect to the regulation of apoptosis by PKC isozymes, several studies indicate that PKC ßII is capable of blocking the induction of programmed cell death (53,54). These data suggest that the homeostatic balance between cell proliferation and apoptosis in the colon may be mediated by PKC ßII. The implication that PKC ßII could be involved in both decreasing apoptosis and increasing proliferation is noteworthy, because these cytokinetic events are predictive biomarkers of the tumorigenic process (17,21,55).

Our results also indicate that there was an interactive effect of fat and fiber on PKC ßII expression, with n-6 PUFA/pectin-treated rats having elevated levels compared with animals fed an n-3 PUFA/pectin diet (Figure 7Go). These data are consistent with previous reports indicating an interactive effect of fat and fiber, whereby n-3 PUFA/pectin versus n-6 PUFA/pectin enhanced apoptosis (24). In contrast, although n-3 PUFA/pectin versus n-6 PUFA/cellulose suppressed tumor development in the colon (21), the n-3 PUFA/cellulose animals consistently had the lowest PKC ßII expression at 16 weeks. Therefore, the use of PKC ßII expression as a predictive biomarker for colon cancer requires further investigation.

Approximately 90–95% of all colon tumors are adenocarcinomas derived from the lumenal epithelium (56,57). With the vast majority of colonic tumors originating from crypt cells, focusing on epithelial cell PKC ßII localization in situ in relation to patterns of cell proliferation, differentiation and apoptosis is of interest. Although immunoblot analysis provides insight into the activation status of PKC ßII, it has limited application in crypt analysis. Colonic scrapings utilized in immunoblot analysis incorporate cells from both the lamina propria and the lumenal epithelium. Because the colon comprises many cell types (smooth muscle, lymphoid, epithelial and nerve cells), differential results between immunoblot and immunohistochemical analyses are not surprising and may explain why identical observations were not noted in the present study.

Differences in the crypt localization of PKC ßII were evident upon immunohistochemical analysis of the proximal and distal colon. This is not entirely atypical, since many biological processes in the proximal and distal colon are distinct (5861). For example, distal colonic epithelial cells are derived from a stem cell population at the base of the crypt and migrate from a region of active cell proliferation in the bottom two thirds of the crypt toward the top of the crypt, obtaining a differentiated or apoptotic phenotype (6264). In contrast, in the proximal colon stem cells are located toward the middle of the crypt and the colonocytes migrate in two directions, up towards the lumenal surface and down towards the crypt base (58). This may explain why a clear pattern linking PKC ßII expression and cytokinetic events emerged only within the distal colonic crypt.

In conclusion, the process of multistage colon carcinogenesis is associated with complex changes in the localization of PKC ßII expression along the crypt axis. In addition, select dietary fats and fibers may confer protection against experimental colon cancer by blocking carcinogen-induced increases in PKC ßII expression.


    Notes
 
2 To whom correspondence should be addressed at: 442 Kleberg Biotechnology Center, Texas A&M University, College Station, TX 77843-2471, USA Email: chapkin{at}acs.tamu.edu. Back


    Acknowledgments
 
We acknowledge Dr Alan Fields for helpful discussions and for providing the polyclonal PKC ßII antibody. We also thank Sid Tracy (Traco Labs) for the generous provision of corn oil. This work was supported in part by NIH grants CA57030, CA59034 and CA61750 and Center for Environmental and Rural Health grant ES09106.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 8, 2000; revised April 27, 2000; accepted May 4, 2000.