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U.S. Department of Agriculture, Agricultural Research Service, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58202-9034
3To whom correspondence should be addressed. E-mail: cdavis{at}gfhnrc.ars.usda.gov.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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protein expression in normal but not in tumor tissue. In contrast, low dietary copper did not affect PKC 
or 
protein expression in either the normal or tumor tissue. PKC and protein and mRNA expression were lower in tumor tissue than in normal tissue. These results along with previous observations suggest that dietary copper-mediated changes in PKC , and protein expression are not as important for colon tumor promotion/progression as they are for tumor initiation.
KEY WORDS: • colon cancer • protein kinase C • copper • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Approximately 130,000 people in the United States were diagnosed with colon cancer in 1999 (1
). Diet is the single greatest contributor to human cancer, including colon cancer, and may be associated with 35–70% of the incidence of the disease (2
). Although various carcinogens are present in foods, their effects are minor compared with dietary components that inhibit the cancer process. One possible dietary factor that may increase the susceptibility to colon cancer is inadequate dietary copper. Recent studies (3
,4
) have shown that ingestion of a diet low in copper significantly increased the formation of 3,2'-dimethyl-4-aminobiphenyl and dimethylhydrazine-induced aberrant crypt foci in rats. Aberrant crypt foci are preneoplastic lesions that have been detected in human colon resections and in experimental rats treated with chemical carcinogens (5
,6
). Two studies have shown that copper deficiency increased the incidence of chemically induced colon cancer in rats (7
,8
). Furthermore, a recent study has shown that copper deficiency significantly increased the small intestine tumor incidence in Min mice, a genetic model for human colon cancer susceptibility (9
). Thus, low dietary copper may be a potential risk factor for colon cancer in humans.
We have recently shown that changes in protein kinase C (PKC)4
isoform protein concentration may be related to increased susceptibility of copper-deficient rats to colon cancer (4
). PKC constitutes a family of serine/threonine protein kinases that play central roles in transmembrane-signaling events and are involved in diverse biological processes, including cellular proliferation and differentiation. We observed that 1 wk after the second dose of dimethylhydrazine (DMH), PKC , and protein content was significantly reduced in rats fed low dietary copper, compared with rats fed adequate dietary copper. There is extensive evidence that signals mediated via PKC serve to regulate colonic tumor development, possibly by influencing the actions of various growth factors and oncogenes (10
–12
). Several laboratories, utilizing DMH or its more proximate metabolite, azoxymethane (AOM), have demonstrated multiple biochemical changes in cellular signaling in both premalignant and malignant colonocytes (13
,14
). Studies in humans as well as in experimental animals have shown that decreased total PKC activity is observed during colonic carcinogenesis (10
,15
–17
). These results suggest that alterations in PKC isoform protein concentration may be related to increased susceptibility of copper-deficient rats to aberrant crypt formation.
Although copper deficiency increases the formation of aberrant crypt foci in rat colon, it is not known whether the increase in preneoplastic aberrant crypts leads to an increase in tumor formation. Accordingly, an objective of the current study was to determine whether dietary copper deficiency would increase AOM-induced intestinal tumor formation. In the previous study (4
), the protein and message expression of various PKC isoforms were investigated in the cytosol of normal colonic tissue after short-term (2 and 8 wk after carcinogen administration) intakes of low dietary copper as a potential indicator of cancer susceptibility rather than actual tumor formation. Thus, a second objective of the current study was to determine the effect of long-term (38 wk after carcinogen administration) intakes of low dietary copper on PKC protein and mRNA expression in normal colonic tissue and colon tumors.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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AOM was purchased from Sigma Chemical (St. Louis, MO.) Xylazine was purchased from Rompan Mobay (Shawnee, KS) and ketamine from Ketaset Aveco (Fort Dodge, IA). Rabbit anti-PKC , and antibodies and Superscript II reverse transcriptase were purchased from Life Technologies (Rockville, MD). Strepavidin conjugated to horseradish peroxidase and vistra green were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Taq DNA polymerase and polynucleotides were purchased from Promega Life Science (Madison, WI).
Eighty weanling, male Fischer-344 rats were purchased from Sasco (Omaha, NE). All rats were housed individually in stainless steel wire-bottomed cages in a room with controlled temperature and light. Rats had free access to demineralized water and purified diet. The rats were randomly assigned to AIN-93G diets that contained either low or adequate concentrations of copper (by analysis, 0.8 and 5.3 µg Cu/g diet) (18
). The protein source was casein and the carbohydrate source was a combination of sucrose and cornstarch as recommended (18
).
After 24 and 31 d of consuming the experimental diets, 30 rats/diet were administered AOM (15 mg/kg body, i.p.) dissolved in sterile saline. Ten additional rats/diet received the comparable vehicle injection of saline. Rats were killed by exsanguination 38 wk after the last AOM or vehicle administration. Four low dietary copper and three high dietary copper rats were killed early and are not included in the analysis.
The study was approved by the Animal Care Committee of the Grand Forks Human Nutrition Research Center, and the rats were maintained in accordance with the National Research Council guidelines for the care and use of laboratory rats.
Food was withheld overnight before rats were anesthetized with xylazine and ketamine and killed by exsanguination. Blood was collected by cardiac puncture into syringes containing 1 g EDTA/L blood. Livers were cleaned of adhering material, weighed and frozen in liquid nitrogen. The entire small and large intestines were removed, opened, spread out with the lumen side up and cleaned and kept on top of ice. Visible tumors along the entire length of the small and large intestines were counted and measured under a stereomicroscope at a magnification of X20. Portions of the tumors were fixed in 10% neutral buffered formalin. If tumors weighed > 0.1 g, a portion was frozen in liquid nitrogen for RNA isolation and if the tumor weighed > 0.3 g, a portion was processed for protein analysis (see below). The mocosa from the remaining colon was scraped off with a microscope slide and was considered normal mucosa. Samples from two to three rats were pooled. A portion of the pooled mucosa was frozen in liquid nitrogen for RNA isolation. The remaining mucosal tissue was weighed and homogenized in nine volumes of cold (4°C) homogenization buffer with six strokes of a Teflon pestle in a Potter-Elvehjem homogenizer. Homogenization buffer consisted of 20 mmol/L HEPES, pH 7.5, 0.25 mol/L sucrose, 10 mmol/L EGTA, 2 mmol/L EDTA, 2 mmol/L dithiothreitol, and 50 TIU/L each of aprotinin, soybean trypsin inhibitor, and leupeptin (19
). A portion of the homogenate was saved for analysis of PKC isoforms. The remaining homogenate was centrifuged at 100,000 x g for 1 h and the supernatant was saved for analysis of PKC isoforms.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.
Homogenate and supernatant fractions from colon mucosa Were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (20
) on 10% polyacrylamide gels. Supernatant samples were loaded (40 µg protein/lane) on 7 x 8 x 0.1-cm gels. Rat brain cytosol (15 µg protein/lane) was used as a control on each immunoblot. Proteins were transferred to polyvinylidene fluoride membrane (Immobilon-P; Millipore Corp., Bedford, MA) by semidry electroblotting (Trans-Blot SD semi-dry transfer cell; Bio-Rad Laboratories, Hercules, CA) and their transfer to the membrane was verified by staining the polyacrylamide gels with Coomassie Blue R250. After the transfer of proteins, the membrane was processed by a method that takes advantage of the hydrophobicity of polyvinylidene fluoride to eliminate the blocking step and to reduce the number of wash steps (Millipore Technical Note RP562; Millipore Corp.). In brief, the dry blots were incubated for 2 h at room temperature with antibodies specific to individual PKC isoforms. The anti-PKC antibodies were diluted 1:1000 in buffer containing 50 g/L nonfat dry milk, 0.5 mL/L Tween-20, 0.081 mol/L Na2HPO4, 0.019 mol/L NaH2PO4 x H2O, and 0.145 mol/L NaCl, pH.7.4. The blots were then incubated for 1 h with horseradish peroxidase-coupled anti-rabbit IgG (Amersham, Arlington Heights, IL) diluted 1:8000 in the buffer described above. Visualization of the PKC isoforms was accomplished by chemiluminescence and exposure of the blots to luminescence detection film (ECL Western Blotting detection reagents and Hyperfilm-ECL; Amersham). Specificity of the antibodies for PKC isozymes was determined by incubating the specific isozyme peptides with antibody and demonstrating the elimination of the specific PKC band on the immunoblot. Immunoreactive bands were analyzed by imaging densitometry (GS-700 Imaging Densitometer; Bio-Rad Laboratories). PKC isoform content was represented as a relative density calculated by dividing the scanned density of the isoform peak in the mucosal samples on a blot by the density of the isoform peak in the brain cytosol standard on the blot. Densities for the peaks representing mucosal PKC were below the film saturation levels and were within the linear range of the densities for the peaks representing PKC in the brain standard.
Total RNA extraction and reverse transcriptase polymerase chain-reaction (RT-PCR) analysis.
Total RNA was isolated by guanidine isothiocyanate lysis and acid phenol extraction (21
). The relative mRNA levels of PKC isozymes were quantified by using rapid competitive PCR as previously described (4
,21
,22
). First strand cDNA was synthesized in 20 µL total volume by using 0.02 µ mol/L 3' oligonucleotide (see below) and SuperScript II reverse transcriptase. Briefly, rat colon total RNA (1 µg) and 0.02 µmol/L 3' oligonucleotide were denatured by heating to 70°C for 10 min, quickly chilled on ice, and subsequently reverse-transcribed by incubation with 0.01 mol/L dithiothrietol, 1 mmol/L dNTP and 200 U SuperScript II reverse transcriptase for 50 min at 42°C, followed by enzyme denaturation at 70°C for 15 min. Incubations containing no reverse transcriptase were used as negative controls.
Primer pairs for construction of internal standards and amplification of PKC isozyme mRNA were: PKC competitor (293bp) forward 5'-TGAACCCTCAGTGGAATGAGT-3', reverse 5'-GGCTGCTTCCTGTCTTCTGAACTTGGCTTTCTCGAAC-3'; PKC mRNA (325 bp) forward 5'–TGAACCCTCAGTGGAATGAGT-3', reverse 5'-GGCTGCTTCCTGTCTTCTGAA-3'; PKC competitor (584 bp) forward 5'-CGATGGGGTGGATGGGATCAAAA-3', reverse 5'-GTATTCATGTCAGGGTTGTCTGGATTTCGGGGGCG-3'; PKC mRNA (680 bp) forward 5'-CGATGGGGTGGATGGGATCAAAA-3', reverse 5'-GTATTCATGTCAGGGTTGTCTG-3'; PKC competitor (299 bp) forward 5'-CACCATCTTCCAGAAAGAACGACATGAGCCCCACC-3', reverse 5'-CTTGCCATAGGTCCCGTTGTTG-3', PKC mRNA (352 bp) forward 5'-CACCATCTTCCAGAAAGAACG-3', reverse 5'-CTTGCCATAGGTCCCGTTGTTG-3' (22
,23
). Internal standards were synthesized using cDNA of the PKC isozyme of interest and the primers listed above. Thus, the internal standard contains the same sequence as the message and utilizes the same primers as the message of interest but is smaller in length.
A 50-µL PCR reaction contained the following: 0.1 mmol/L dNTP, 1.5 mmol/L MgCl2, 1 x Taq DNA polymerase buffer, 20 mL/L DMSO, 0.2 µmol/L of each forward and reverse primer, internal standard generated for the message of interest, 1.25 U of Taq DNA polymerase, and 10 µL of the RT reaction. Quantitative competition experiments were carried out for each message analyzed; a fixed amount of sample RNA (1 µg) was reverse transcribed and coamplified with increasing amounts of synthetic internal standard cDNA, resulting in a product from the endogenous message and a product from the internal standard. Equal amplification efficiency (similar band intensity) of the target sequence and internal standard were obtained when 5, 2.5 and 12.5 pg of PKC , and internal standard cDNA, respectively, were used. PCR was performed on a PTC-100 Programmable Thermal Controller (MJ Research, Incline Village, NV). The reaction was performed for 35 cycles as follows: denaturation, 93°C for 30 s; annealing, 60°C for 45 s; and extension 74°C for 45 s. The final cycle included an additional 10 min at 74°C for complete strand extension. PCR products were incubated 1:10,000 with Vistra Green for 15 min before electrophoresis on 4% agarose gels. The fluorescence intensity of the endogenous target and the internal standards were quantified with a Storm 860 (Molecular Dynamics, Sunnyvale, CA). Results are expressed as the ratio of sample:internal standard for each sample.
Frozen plasma was analyzed for enzymatic ceruloplasmin activity with a Cobas Fara II Centrifugal Analyzer (Roche Diagnostics Systmes, Montclair, NJ) by the method of Sunderman and Nomata (24
), which measures its p-phenylenediamine oxidase activity.
Plasma samples were precipitated with 30 g/L trichloroacetic acid and 6 mol/L HCl for mineral analysis. The supernatant was analyzed by inductively coupled argon atomic emission spectrometer (Liberty Series II; Varian Associates, Sugarland, TX). Control samples containing demineralized water that had been collected through the syringes containing 1 g EDTA/L water and processed similarly to the plasma samples were not found to contain any copper, iron or zinc contamination.
Samples of liver were analyzed for copper, iron and zinc by inductively coupled argon atomic emission spectrometry (Liberty Series II; Varian Associates) as previously described (4
). Liver standard reference material (1577b; National Institute of Standards and Technology, Gaithersburg, MD) was analyzed with each batch of tissue samples for quality control. Liver samples (n = 4) were determined to contain 100%, 103% and 108% of the certified values for copper, iron and zinc, respectively.
The data were analyzed by a two-way ANOVA to determine the significance of the main effects of dietary copper and AOM treatment and the interaction of dietary copper and AOM administration using a SAS general linear model program (SAS Version 6.12; SAS Institute, Cary, NC). Because the data for hematocrit and hemoglobin concentrations did not follow a normal distribution, data were transformed by using the natural log before statistical analysis. Because the variances were proportional to the means, a square root transformation was performed on PKC , and mRNA expression and on PKC protein expression before the analysis. If the interaction was significant, Tukey’s contrasts were used to differentiate among means. Tumor incidence was analyzed by a generalized linear model. Values are reported as means ± SEM except where indicated otherwise.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, beginning at 30 d of age, rats injected with AOM had a significantly (P < 0.05) lower body weight than rats injected with saline.
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). However, this difference reflected a higher incidence of tumors in the small intestine and no difference in colon tumor incidence. There were no differences in tumor mass or total tumor burden between rats fed low and adequate dietary copper and injected with azoxymethane (data not shown). No tumors were observed in the rats injected with saline.
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). There was a significant (P < 0.05) diet by treatment interaction in both ceruloplasmin activity and plasma copper concentrations such that carcinogen treatment increased ceruloplasmin activity and plasma copper concentrations in rats fed adequate dietary copper but decreased ceruloplasmin activity and plasma copper concentrations in rats fed low dietary copper. AOM administration lowered (P < 0.05, ANOVA) hematocrit and hemoglobin concentrations.
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, , and protein expressions were determined by Western blot analysis (Fig. 3
–5
). For the analysis of PKC protein expression, both the cytosolic and homogenate fractions were analyzed. The homogenate includes PKC in both the cytosolic and membrane fractions. Because the data were analyzed per milligram of protein and the different types and amounts of protein (besides PKC) were not the same in the cytosolic and homogenate fractions, it was impossible to statistically compare the percentage of the different PKC isozymes present in the cytosolic verus the homogenate fraction. PKC , , and mRNA expressions were determined by competitive RT-PCR (Fig. 6
).
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protein was predominantly located in the cytosolic compartment (Fig. 3)
. Dietary copper reduced (P < 0.004) PKC protein expression in the homogenate samples in the normal mucosa (Fig. 3B)
. The same trend for decreased PKC protein expression in the cytosolic fraction (Fig. 3A)
and decreased PKC mRNA expression (Table 2
) when rats were fed low dietary copper was observed; however, these effects were not significant (P < 0.05) because of the variability between samples. Tumor tissue had lower PKC protein and mRNA expression than normal mucosa (compare Fig. 3C
with Fig. 3A
and 3B
and Table 3
with Table 2
). Dietary copper did not affect PKC protein and mRNA expression in tumor tissue.
|
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, most of PKC protein was in the homogenate fraction of the normal mucosa samples (Fig. 4
, A and B) but in the cytosolic fraction of the tumor samples (Fig. 4C)
. Dietary copper did not affect PKC protein expression in either the cytosolic or the homogenate fractions of normal or tumor samples (Fig. 4
, A and B). Both dietary copper and carcinogen treatment increased (P < 0.03) PKC mRNA levels (Table 2)
. There was a diet x carcinogen interaction (P < 0.05) such that the effect of dietary copper was more pronounced in rats injected with AOM. Dietary copper did not affect PKC mRNA expression in the tumor samples (Table 3)
.
Although two bands are observed in the representative blots of PKC (Fig. 5)
, only the lower band represents PKC (as demonstrated by its disappearance when specific PKC peptides were incubated with the antibody). Similar to PKC , a large portion of PKC protein was in the homogenate of the normal mucosa samples (Fig. 5B)
but in the cytosolic fraction of the tumor samples (Fig. 5C)
. Dietary copper did not affect PKC protein expression or mRNA levels in the normal mucosa or in the tumor samples (Fig. 5
; Table 2
; Table 3
). However, low dietary copper increased (P < 0.02) the ratio of PKC protein expression in the homogenate to PKC protein expression in the cytosol (3.80 vs 2.53, respectively) compared with rats fed adequate dietary copper. This suggests that low dietary copper caused a translocation of PKC to the membrane fractions.
Carcinogen treatment did not affect PKC , , or protein expression in either the cytosolic or homogenate fractions (Fig. 3
, A and B; Fig. 4
, A and B; Fig. 5
, A and B). Carcinogen treatment increased (P < 0.05) PKC and PKC but not PKC mRNA expression in the normal mucosa (Table 2)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Those results are similar to those of other investigators who have observed that in Min mice, most of the tumors occur in the small intestine rather than in the large intestine (25
). However, because of the genetic and histochemical similarities between small intestinal tumors in the Min mouse and colon cancer in humans, these mice are an accepted model for colon cancer in humans (25
). Although azoxymethane is both a small intestine and a colon carcinogen, the relevance of the increased small intestinal tumor incidence observed in the current study is unknown. In humans, the small intestine contains 75% of the mucosal surface of the gastrointestinal tract, yet it is the site of only 2% as many malignancies as the colon (26
). However, epidemiologic studies suggest that dietary correlates of adenocarcinoma of the small intestine are similar to those of colon cancer and at least of the same magnitude (27
,28
).
In this study, we did not observe an effect of dietary copper on colon tumor incidence. These results are surprising because in two previous studies (3
,4
) we observed increased chemically induced aberrant crypt formation in rats fed a low copper diet. Colon carcinogenesis is regarded as a multistage process. The target cells of colon carcinogens are colonic crypt epithelial cells. Studies in humans have suggested that colonic aberrant crypt foci are precursor lesions from which adenomas and adenocarcinomas will develop. During the last decade, numerous studies including molecular analysis have focused on the importance of aberrant crypt foci as early events in colon carcinogenesis, and aberrant crypt foci are now regarded as putative premalignant lesions for colon cancers (4
,29
). Nevertheless, there is evidence that documents the lack of correlation between tumor development and the expression of aberrant crypt foci (30
,31
). Furthermore, some compounds with the potency to prevent the occurrence of aberrant crypt foci, such as 2-(carboxyphenyl) retinamide or genistein have been found to enhance the development of colon cancers (32
,33
). A recent study (34
) has suggested that ß-catenin-accumulated crypts are independent lesions of aberrant crypt foci and are a more useful marker for premalignant lesions of azoxymethane-induced colon cancer in rats.
Another explanation for the differential effect of dietary copper on aberrant crypt foci formation and tumor development is that dietary copper is having differential effects during different stages of tumorigenesis. Tumorigenesis is believed to be a multistage process. Dietary copper may be protective against aberrant crypt formation because of its effects on antioxidant enzymes. Two copper-containing enzymes, namely copper-zinc superoxide dismutase and ceruloplasmin, that may help protect against oxygen radical-mediated injury are significantly reduced in rats fed low copper diets (3
,4
). Copper-zinc superoxide dismutase functions to eliminate superoxide radicals and ceruloplasmin is hypothesized to inhibit iron-catalyzed radical formation (35
,36
). Substantial evidence suggests that free radicals, particularly oxygen radicals, are involved in both the initiation and promotion stages of carcinogenesis (37
). Thus, low dietary copper may be a risk factor during the early stages of carcinogenesis. However, low dietary copper may be protective during the later stages of tumorigenesis. Both copper and the copper transport protein, ceruloplasmin, have been shown to be involved in angiogenesis and neovascularization (38
–40
). Ceruloplasmin mRNA has been shown to be at least threefold more abundant in tumor cells compared with normal tissue (40
). Furthermore, the copper chelator, tetrathiomolybdate, is being used in the treatment of metastatic solid tumors because it causes mild copper deficiency and impairs neovascularization (40
).
The differential effect of dietary copper on aberrant crypt formation and tumor formation may also be the result of different effects of dietary copper on PKC expression during different stages of tumorigenesis. Alterations in the expression and activation of specific PKC isoforms play a role in the malignant tranformation process of the colon. Decreased levels of PKC activity have been observed in preneoplastic colonic mucosa and in colonic adenocarcinomas, indicating that alterations in PKC isozyme regulation occur early in the multistage process of colon carcinogenesis (11
,19
,41
,42
). In a previous study (4
), we observed that low dietary copper significantly decreased the cytosolic protein concentration of PKC , , and at 2 wk but not at 8 wk after carcinogen administration. In this study, low dietary copper significantly reduced PKC protein expression in the normal mucosa but did not affect PKC , or protein expression. Furthermore, dietary copper did not affect PKC , , and protein or mRNA expression in the tumors. This suggests that dietary copper-mediated changes in PKC , , and protein expression are not important for tumor development but may be important for aberrant crypt formation.
In this study, tumor tissue had a much lower PKC protein expression than normal tissue (Fig. 3)
. Similarly, Gokmen-Polar et al. (43
) observed that PKC protein expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Consistent with these findings, a compelling body of evidence indicates that PKC protein expression is associated with negative growth regulation and cell cycle arrest in various cell lines (44
–48
). In both our study and the study by Gokmen-Polar et al. (43
) quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels. Similarly, in human colonic tumors, down-regulation of PKC protein expression was not associated with decreased mRNA (41
). These results indicate that PKC isozyme expression is likely regulated at the postranscriptional/translational level.
In this study, the majority of PKC and PKC in tumors was found in the cytosolic fraction, whereas these isoforms were found mostly in the homogenate of normal mucosa. The homogenate contains the totality of cellular membranes and organelles. When PKC is activated, there is net translocation of PKC from the cytosol to target membranes and organelles (12
,13
). Thus, the finding that almost all of the PKC and PKC was in the cytosolic fraction of tumors but in the homogenate fraction of the normal mucosa suggests that PKC and PKC activation is greatly reduced in tumor tissue compared with normal mucosal tissue. Consistently, we observed decreased PKC and mRNA in the tumor samples compared with the normal samples (Tables 2
and 3)
. Similarly, previous studies have shown decreased PKC and protein expression in azoxymethane-induced rats and sporadic human colon tumors and decreased PKC mRNA levels in human colorectal tumors than in normal colonic mucosa (10
,49
–51
). PKC is important in cell proliferation and apoptosis (52
) and PKC may play an important role in both inhibiting tumor initiation and promotion by regulating colonic epithelial cell ontogeny along the crypt axis (48
,53
,54
).
Our results suggest that low dietary copper significantly increases small intestinal but not colon tumor development. These results have practical implications because many of the diets consumed in the United States do not contain the recommended amount of copper (55
). Low dietary copper significantly decreased PKC protein expression in normal but not in tumor tissue. However, long-term low dietary copper intake did not affect PKC or protein expression in either the normal or tumor tissue. This result, along with previous observations, suggests that dietary copper-mediated changes in PKC , and protein expression are not as important for colon tumor promotion/progession as they are for tumor initiation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
4 Abbreviations used: AOM, azoxymethane; DMH, dimethylhydrazine; PKC, protein kinase C.
Manuscript received 15 October 2001. Initial review completed 26 November 2001. Revision accepted 14 February 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. American Cancer Society (1999) Cancer Facts and Figures 1999 American Cancer Society Atlanta, GA .
2. Doll, R. & Peto, R. (1981) The causes of cancer: quantitative estimates of avoidable risks in the United States today. J. Natl. Cancer Inst. 66:1192-1200.
3. Davis, C. D. & Feng, Y. (1999) Dietary copper, manganese and iron affect the formation of aberrant crypts in colon of rats administered 3,2'-dimethyl-4-aminobiphenyl. J. Nutr. 12:1060-1067.
4. Davis, C. D. & Johnson, W. T. (2002) Dietary copper and dimethylhydrazine affect protein kinase C isozyme protein and mRNA expression and the formation of aberrant crypts in colon of rats. Biofactors 15:11-26.
5. Bird, R. P. (1995) Role of aberrant crypt foci in understanding the pathogenesis of colon cancer. Cancer Lett. 93:55-71.[Medline]
6.
Pretlow, T. P., O’Rioridan, M. A., Somich, G. A., Amini, S. B. & Pretlow, T. G. (1991) Aberrant crypts: putative preneoplastic foci in human colonic mucosa. Cancer Res. 51:1564-1567.
7. DiSilvestro, R. A., Greenson, J. K. & Liao, Z. (1992) Effects of low copper intake on dimethylhydrazine-induced colon cancer in rats. Proc. Soc. Exp. Biol. Med. 201:94-97.[Abstract]
8. Greene, F. L., Lamb, L. S., Barwick, M. & Pappas, N. J. (1987) Effect of dietary copper on colonic tumor production and aortic integrity in the rat. J. Surg. Res. 42:503-512.[Medline]
9. Davis, C. D. & Newman, S. (2000) Inadequate dietary copper increases tumorigenesis in the min mouse. Cancer Lett. 159:57-62.[Medline]
10.
Wali, R. K., Frawley, B. P., Hartmann, S., Roy, H. K. & Khare, S. (1995) Mechanism of action of chemopreventive ursodeoxycholate in azoxymethane model of rat colonic carcinogenesis: potenital roles of protein kinase C-, ß, and . Cancer Res. 55:5257-5264.
11.
Baum, C. L., Wali, R. K., Sitrin, M. D., Boltm, M.J.G. & Brasitus, T. A. (1990) 1,2'-Dimethylhydrzine-induced alterations in protein kinase C activity in the rat preneoplastic colon. Cancer Res. 50:3915-3920.
12.
Craven, P. A. & DeRubertis, F. R. (1987) Subcellular distribution of protein kinase C in rat colonic epithelial cells with different proliferative activities. Cancer Res. 47:3434-3438.
13. Brasitus, T. A., Dudeja, P. K. & Dahiya, R. (1988) Premalignant alterations in the lipid composition and fluidity of colonic brush border membranes in rats administered 1,2'dimethylhydrazine. J. Clin. Invest. 77:831-840.
14.
DeRubertis, F. R. & Craven, P. A. (1980) Early alterations in rat colonic mucosal nucleotide metabolism and protein kinase activity induced by 1,2'-dimethylhydrazine. Cancer Res. 40:4589-4598.
15. Guillem, J. G., O’Brian, C. A., Fitzer, C. J., Johnson, M. D., Forde, K. A., LoGerto, P. & Weinstein, I. B. (1987) Studies on protein kinase C and colon carcinogenesis. Arch. Surg. 122:1475-1478.[Abstract]
16.
Kopp, R., Noelke, B., Sauter, G., Schildberg, F. W., Paumgartner, G. & Pfeiffer, A. (1991) Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas. Cancer Res. 51:205-210.
17. Kusonoki, M., Sakanoue, Y., Hattada, T., Yanagi, H., Yamamura, T. & Utsonomiya, J. (1992) Protein kinase C activity in human colonic adenoma and colorectal carcinoma. Cancer 69:24-30.[Medline]
18. Reeves, P. G., Nielsen, F. H. & Fahey, G. C. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
19. Johnson, W. T. & Dufault, S. N. (1991) Copper deficiency alters protein kinase C mediation of thrombin-induced dense granule secretion from rat platelets. J. Nutr. Biochem. 2:663-670.
20. Laemmeli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.[Medline]
21. Chirgwin, J. J., Przybyia, A. E., MacDonald, R. J. & Rutter, W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299.[Medline]
22.
Jiang, Y. H., Davidson, L. A., Lupton, J. R. & Chapkin, R. S. (1996) Rapid competitive PCR determination of relative gene expression in limiting tissue samples. Clin. Chem. 42:227-231.
23. Davidson, L. A., Jiang, Y. H., Lupton, J. R. & Chapkin, R. S. (1995) Noninvasive detection of putative biomarkers for colon cancer using fecal messenger RNA. Cancer Epidemiol. Biomarkers Prev. 4:643-647.[Abstract]
24. Sunderman, F. W. & Nomoto, S. (1970) Measurement of human serum ceruloplasmin by its p-phenylenediamine oxidase activity. Clin. Chem. 16:903-910.[Abstract]
25. Shoemaker, A. R., Gould, K. A., Luongo, C., Moser, A. R. & Dove, W. F. (1997) Studies of neoplasia in the Min mouse. Biochem. Biophys. Acta 1332:F25-F48.[Medline]
26. Chen, C. C., Neugut, A. I. & Rotterdam, H. (1994) Risk factors for adenocarcinomas and malignant carcinoids of the small intestine: preliminary findings. Cancer Epidemiol. Biomarkers Prev. 3:205-207.[Abstract]
27. Bosetti, N. E., LaVecchia, C., Fioretti, F., Conti, E. & Franceschi, S. (1999) risk factors for adenocarcinoma of the small intestine. Int. J. Cancer 82:171-174.[Medline]
28. Chow, W. H., Linet, M. S., McLaughlin, J. K., Hsing, A. W., Chien, H. T. & Blow, W. J. (1993) Risk factors for small intestine cancer. Cancer Causes Control 4:163-169.[Medline]
29.
Shivapurkar, N., Tang, Z., Ferreira, A., Nasim, S., Garrett, C. & Alabaster, O. (1994) Sequential analysis of K-ras mutations in aberrant crypt foci and colonic tumors induced by azoxymethane in Fischer-344 rats on high-risk diet. Carcinogenesis 15:775-778.
30. Cameron, I., Garza, J. & Hardman, W. K. (1996) Distribution of lymphoid nodules, aberrant crypt foci and tumours in the colon of carcinogen-treated rats. Br. J. Cancer 73:893-898.[Medline]
31. Takashi, S., Ogawa, K., Oshima, H., Esumi, H., Ito, N. & Sugimura, T. (1991) Induction of aberrant foci in the large intestine of F344 rats by oral administration of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Jpn. J. Cancer Res. 82:135-137.[Medline]
32.
Zheng, Y., Kramer, P., Lubet, R., Steele, V., Kelloff, G. & Pereira, M. (1999) Effect of retinoids on AOM-induced colon cancer in rats: modulation of cell proliferation, apoptosis and aberrant crypt foci. Carcinogenesis 20:255-260.
33.
Rao, C., Wang, C., Simi, B., Lubet, R. & Kelloff, G. (1977) Enhancement of experimental colon cancer by genistein. Cancer Res. 57:3717-3722.
34.
Yamada, Y., Yoshimi, N., Hirose, Y., Matsunaga, K., Katayama, M., Sakata, K., Shimizu, M., Kuno, T. & Mari, H. (2001) Sequential analysis of morphological and biological properties of ß-catenin-accumulated crypts, provable premalignant lesions independent of aberrant crypt foci in rat colon carcinogenesis. Cancer Res. 61:1874-1878.
35. Oberley, T. D. & Oberley, L. W. (1997) Antioxidant enzyme levels in cancer. Histol. Histopathol. 12:525-535.[Medline]
36. Sun, Y., Li, Y. & Oberley, L. W. (1993) Superoxide dismutase activity during dimethylhydrazine colon carcinogenesis and the effect of cholic acid and indole. Free Radic. Res. Commun. 4:299-310.
37. Guillem, J. G., O’Brian, C. A., Fitzer, C. J., Johnson, M. D., Forde, K. A., LoGerto, P. & Weinstein, I. B. (1987) Studies on protein kinase C and colon carcinogenesis. Arch. Surg. 122:1475-1478.
38. Raju, K. S., Alessandri, G., Ziche, M. & Guillino, P. M. (1982) Ceruloplasmin, copper ions and angiogenesis. J. Natl. Cancer Inst. 69:1183-1188.
39. Kunapuli, S. P., Singh, H., Singh, P. & Kumar, A. (1987) Ceruloplasmin gene expression in human cancer cells. Life Sci. 40:2225-2228.[Medline]
40.
Brewer, G. J., Dick, R. D., Grover, D. K., LeClaire, V., Tseng, M., eWicha, M., Pienta, K., Redman, B. G., Jahan, T., Sondak, V. K., Strawderman, M., LeCarpentier, G. & Merajver, S. D. (2000) Treatment of metastatic cancer with tetrathiomolybdate, an anticopper, antiangiogenic agent: phase I study. Clin. Cancer Res. 6:1-10.
41. Kopp, R., Noelke, B., Sauter, G., Schildberg, F. W., Paumgartner, G. & Pfeiffer, A. (1991) Altered protein kinase C activity in biopsies of human colonic adenomas and carcinomas. Cancer Res. 51:205-210.
42.
Kahl-Rainer, P., Karner-Hanusch, J., Weiss, W. & Marian, B. (1994) Five of six protein kinase C isoenzymes present in normal mucosa show reduced protein levels during tumor development in the human colon. Carcinogenesis 15:779-782.
43.
Gokmen-Polar, Y., Murray, N. R., Velasco, M. A., Gatalica, Z. & Fields, A. P. (2001) Elevated protein kinase C âII is an early promotive event in colon carcinogenesis. Cancer Res. 61:1375-1381.
44.
Mischak, H., Pierce, J. H., Goodnight, J., Kazanietz, M. G., Blumberg, P. M. & Mushinski, J. F. (1993) Phorbol ester-induced myeloid differentiation is mediated by protein kinase C- and - but not by protein kinase C-ßII, -
, , and 
. J. Biol. Chem. 268:20110-20115.
45. Sasaguri, T., Kosaka, C., Hirata, M., Masuda, J., Shimokado, K., Fujishima, M. & Ogata, J. (1993) Protein kinase C-mediated inhibition of vascular smooth muscle cell proliferation: the isoforms that may induce G1/S inhibition. Exp. Cell Res. 208:311-320.[Medline]
46.
Borner, C., Ueffing, M., Jaken, S., Parker, P. J. & Weinstein, I. B. (1995) Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts. J. Biol. Chem. 270:78-86.
47.
Abraham, C., Scalgione-Sewell, B., Skarosi, S. F., Qin, W., Bissonette, M. & Brasitus, T. A. (1998) Protein kinase C modulates growth and differentiation in Caco-2 cells. Gastroenterology 114:503-509.[Medline]
48.
Scaglione-Sewell, B., Abraham, C., Bissonette, M., Skarosi, S. F. & Hart, J. (1995) Decreased PKC- expression increases cellular proliferation, decreases differentiation, and enhances the transformed phenotype of CaCo-2 cell. Cancer Res. 58:1074-1081.
49. Kuranami, M., Powell, C. T., Hug, H., Zeng, Z., Cohen, A. M. & Guillen, J. G. (1995) Differential expression of protein kinase C isoforms in human colorectal cancers. J. Surg. Res. 58:233-239.[Medline]
50.
Jiang, Y. H., Lupton, J. R. & Chapkin, R. S. (1997) Dietary fish oil blocks carcinogen-induced down-regulation of colonic protein kinase C isozymes. Carcinogenesis 18:351-357.
51. Wali, R. K., Baum, C. L., Bolt, M.J.G., Dudeja, P. K., Sitrin, M. D. & Brasitus, T. A. (1991) Down-regulation of protein kinase C activity in 1,2-dimethylhydrazine-induced rat colonic tumors. Biochim. Biophys. Acta 1092:119-123.[Medline]
52. Cerda, S. R., Bissonnette, M., Scaglione-Sewell, B., Lyons, M. R., Khare, S., Mustafi, R. & Brasitus, T. A. (2001) PKC-delta inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in Caco-2 cells. Gastroenterology 120:1700-1712.[Medline]
53.
Craven, P. A. & DeRubertis, F. R. (1992) Alterations in protein kinase C in 1,2-dimethylhydrazine induced colonic carcinogenesis. Cancer Res. 52:2216-2221.
54. Jiang, Y. H., Aukema, H. M., Davidson, L. M., Lupton, J. R. & Chapkin, R. S. (1995) Localization of protein kinase C isozymes in rat colon. Cell Growth Differ 6:1381-1386.[Abstract]
55. Klevay, L. M. (1998) Lack of a recommended dietary allowance for copper may be hazardous to your health. J. Am. Coll. Nutr. 4:322-326.
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0.05) in the cytosolic fraction or in the tumor samples. Neither carcinogen administration nor the interaction of carcinogen and copper affected PKC 
