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Correlation between protein kinase C activity and histopathological criteria in human colorectal adenoma.

We examined protein kinase C (PKC) activity in the cytosolic and particulate fractions of homogenates obtained from 25 colorectal adenomas and adjacent normal mucosa in patients with colorectal carcinoma. The total PKC activity of colorectal adenomas was significantly reduced compared with that of normal mucosa in all cases (122 +/- 45.8 vs 174 +/- 50.5 pmol min-1 mg-1) (means +/- s.d.) (P less than 0.001). The particulate fraction PKC activity of adenomas was also significantly lower than in normal mucosa (71.4 +/- 31.3 vs 115 +/- 39.6 pmol min-1 mg-1) (P less than 0.001). Adenomas were classified by size, histological type and degree of dysplasia. The average particulate PKC activity ratio (adenoma/normal mucosa) of tubulovillous adenomas or those with severe dysplasia was significantly reduced compared with that of tubular adenomas or tumours with mild and moderate dysplasia (both P less than 0.001), while there were no significant differences in the cytosolic PKC activity ratio. The particulate PKC activity ratio decreased significantly with increasing adenoma size (P less than 0.001), while the cytosolic ratio again showed no difference. These findings suggested that the particulate PKC activity ratio had a possible correlation with the malignant potential of colorectal adenomas and that this ratio may be a useful biological indicator of colorectal carcinogenesis.

kinase, the activity of which is dependent upon Ca2" and phospholipid (Takai et al., 1979;Nishizuka, 1984). This enzyme is ubiquitous in eukaryotes (Kuo et al., 1980) and appears to play a key role in transmembrane signaling (Nishizuka, 1986). PKC is activated by 1,2-diacylglycerol, which is formed in response to extracellular signals by the turnover of phosphoinositides (Berridge & Irvine, 1984) and other membrane phospholipids (Lacal et al., 1987). This activation process involves translocation of cytosolic PKC to the plasma membrane, which in turn leads to the phosphorylation of target molecules, thereby influencing important cellular processes such as proliferation and/or differentiation (Weinstein, 1987). It has also been suggested that alterations in PKC activity may play a role in the early stages of malignant transformation (Ashendel, 1985). Kopp et al. showed that in vitro membrane-bound PKC activity was reduced in colonic adenomas and carcinomas when compared to the adjacent normal colonic mucosa, suggesting that alterations within the PKC pathway occurred as an early event in the adenoma-carcinoma sequence (Kopp et al., 1991).
The purpose of this study was to investigate the PKC activity of colorectal adenomas in comparison with their clinicopathological findings to determine whether or not activation of PKC was a useful biological marker of colonic tumorigenesis. Materials

Sample collection
All of the patients gave informed consent for this study and it was also approved by the College ethics committee. Operations were performed on 25 patients with adenocarcinoma and co-existing adenomas (polyps). All operations were performed at the Second Department of Surgery of Hyogo College of Medicine, between August 1990 and March 1991. Samples of adenoma tissue and samples of normal mucosa located about 10 cm from the tumours were quickly removed and immediately rinsed with cold phosphate-buffered saline. To ensure that only intact tumour tissue and normal mucosa were used for analysis, all ulcerated and necrotic tissue was removed from the tumour specimens and the submucosa and muscularis were removed from the normal tissue samples. The specimens had a wet weight ranging from 250-800 mg.
All specimens were sufficiently large to allow the histological evaluation of tumour tissue adjacent to the tissue sample subjected to enzyme assay. A histological diagnosis was made according to the criteria of Morson (Morson et al., 1979). To assess the degree of tissue heterogeneity, the amount of each of the following tissue components present was estimated: stromal elements, nontumorous epithelium, and tumorous epithelium. For this purpose a grid on the microscopic field was used (Hennipman et al., 1989) and the proportion of each tissue element was expressed as a percentage of the total. If stromal tissue exceeded 20%, the tumour was excluded from the study. Tissue samples were frozen in acetone-dry ice within 30 min of resection and then stored at -80'C until analysis. The size of adenomas was determined by measuring the longest diameter.
Preparation of subcellular fractions from tissue specimens All procedures were done at 4'C. The tissues were cut into small pieces and homogenised in 5 ml of Buffer A (25 mM Tris-HCI at pH 7.5, 5 mM EDTA, 5 mM ethyleneglycol bis (B-aminoethylether)-N, N, N', N', -tetra-acetic acid, 0.25 mM phenylmethylsulfonyl fluoride, 10 ml-' leupeptin, 15 mM 2mercaptoethanol, and 0.25 M sucrose) for 1 min at low speed and then for 5 min at full speed using a Polytron homogeniser. Homogenates were centrifuged at 1,000 g for 10 min to remove the nuclear fraction and any unhomogenised tissue. The resultant supernatant was filtered through glass wool and then centrifuged at 100,000 g for 1 h at 4°C.
The supernatant fraction was then stored at 4°C for use as the 'cytosolic' fraction, while the pellet was solubilised in buffer A containing 1% Triton X-100 (5 ml of Buffer A per gram of tissue) by continuous stirring for 1 h at 4C.
The solubilised pellet was then centrifuged at 100,000 g for 1 h at 4°C, and the resulting supernatant was used as the solubilised 'particulate' fraction (Sakanoue et al., 1991b).

Mini DEAE-Sephacel column purification
The cytosolic and particulate fractions were further purified by mini DEAE-Sephacel column chromatography in order to remove inhibitors of PKC or phosphoprotein phosphatase (Sakanoue et al.,199 la). Fractions containing 2 or 5 mg of protein were applied to a 0.5 ml mini DEAE-Sephacel column which was equilibrated in Buffer B (25 mM Tris-HCI at pH 7.5, 0.5 mM EGTA, 0.5 mM EDTA, and 10 mM 2-mercaptoethanol).
The column was then washed with 7.5 ml of Buffer B, and PKC was eluted in 2 ml of Buffer B containing 0.15 M NaCl. The purified fractions thus obtained were used to determine the cytosolic and particulate PKC activity, as described below (Sakanoue et al.,199 la).
Peptide phosphorylation assay PKC activity was assayed as described previously (Sakanoue et al., 1987). The reaction mixture (20 pl) containing 50 mM Tris-HCI (pH 7.5) with 15 mM 2-mercaptoethanol, 10 mM MgC92, 1 mM CaCI2, 8 yg ml-' phosphatidyl serine, 0.8pgmlm' diolein, 5011M [F -_32p] ATP (400-800c.p.m. pmol-'), and 100mM histone III-s. For control reactions, phosphatidyl serine and diolein were omitted, and EGTA was added at a final concentration of 0.5 mm along with the same volume of phospholipid suspension buffer (20 mM Tris-HCI at pH 7.5). The protein concentration was 10-40 tg tube-' for cytosolic enzymes and 5-25 g tube-' for particulate enzymes. Each reaction was conducted at 30°C for 10 min and was terminated onto 2 x 2 cm squares of P-81 paper. These paper squares were then washed four times for 2 min each in 75 mM phosphoric acid (10 ml filter-'), and the radioactivity was determined with a liquid scintillation counter by counting Cerenkov radiation. PKC activity was calculated by subtracting the phosphotransferase activity observed in the presence of EGTA from the activity observed in the presence of Ca2", phosphatidyl serine, and diolein.
PKC activity was a linear function of time and protein with this assay, up to 10 min and 100 pg of protein in both the cytosolic and particulate fractions of adenoma tissue.
Other techniques The protein concentration was determined using a Bio Rad Protein assay kit and bovine serum albumin as the standard (Bradford, 1976). All experiments were carried out with at least two tissue preparations and the assays were carried out in duplicate. Specimens were stained with hematoxylin eosine according to standard methods for histological examination. Data are given as the mean ± s.d. and were evaluated by analysis of variance using the Mann-Whitney U-test. The likelihood ratio test (chi-square statistic) was used to provide a statistical assessment of whether adenoma size, histological type, or degree of dysplasia was an independent risk factor for the PKC activity ratio, and P values <0.05 were considered significant.

Results
We examined PKC activity in the cytosolic and particulate fractions extracted from colonic adenomas and the adjacent normal mucosa. The total PKC activity in the adenomas was significantly lower than that in the normal mucosa (122 ± 45.8 vs 174 ± 50.5 pmol min' mg-1) (P<0.001) (Figure 1). Figure 2 shows that the particulate PKC activity was also The total and particulate PKC activity of both fractions did not differ with the age and sex of the patients or the location of the adenoma. When the specific activity ratio (adenoma/normal mucosa) was evaluated with respect to adenoma size using linear regression, the particulate fraction PKC activity ratio was found to decrease significantly as the adenoma size increased (Figure 3a) (test for association: P<0.001, test for linear trend: P<0.001), while the cytosolic fraction ratio was not significantly altered (Figure 3b).
The relationship of the PKC activity and it's ratio (adenoma/normal mucosa) to the histological appearance of the adenomas is shown in Table I. The particulate specific PKC activity of tubulovillous adenomas was significantly lower than that of tubular adenomas (55.2 ± 28.7 vs 80.4 ± 29.7) (pmol min' mg-') (P<0.05), while there was no significant difference in the cytosolic PKC activity (55.7 ± 25.4 vs 48.0 ± 26.4) (pmol min-' mg-'). The particulate PKC activity ratio (adenoma/normal mucosa) of tubulovillous adenomas was significantly lower than that of tubular adenomas (0.42 ± 0.11 vs 0.77 ± 0.21) (P<0.05), while there was no significant difference in the cytosolic PKC activity ratio (0.78 ± 0.21 vs 0.89 ± 0.34). Table II shows the relationship of the PKC activity and its ratio to the grade of dysplasia. The particulate specific PKC activity with severe dysplasia was lower than that of those with mild dysplasia (55.0 ± 28.1 vs 89.5 ± 33.5) (pmol min-' mg-') (P<0.05), while the cytosolic PKC activity showed no  (pmol min-' mg-'). The particulate PKC activity ratio of adenomas with severe dysplasia was lower than that of those with mild dysplasia (0.45 ± 0.13 vs 0.81 ± 0.21) (P<0.001), while the cytosolic ratio of showed no difference between the various degrees of dysplasia (mild: 1.02 ± 0.36, moderate: 0.75 ± 0.26, and severe: 0.74 ± 0.17).

* *0
This study showed that the total PKC activity and the par-* * ticulate fraction PKC activity of colorectal adenomas was * * 4_ lower than that of adjacent normal mucosa. The percentage of PKC activity in the particulate fraction in adenomas was also lower than in the normal mucosa. These findings indicate that a greater proportion of the membrane-bound Down-regulation of PKC has been demonstrated in several lines of cultured cells treated with tumour promoting phorbol esters (Jaken et al., 1981;Rodriguez & Rozengurt, 1984). In addition, down-regulation of PKC has been demonstrated in several cell lines transformed by the ras oncogene (Hsiao et al., 1989). Moreover, cells with PKC down-regulation have * been shown to be more susceptible to transformation. Kopp et al. have also reported that the total PKC activity was significantly reduced in human colorectal carcinomas and a adenomas as compared to the adjacent normal mucosa, and that the particulate PKC activity was equally decreased in these two types of tumours when compared to the adjacent * 3 .00 * | | normal mucosa. They suggested that alterations within the 0 | protein kinase C pathway occur as an early event in the adenoma-carcinoma sequence in the intestinal mucosa, and * that PKC had an important role in epithelial differentiation * and growth (Kopp et al., 1991)  suggested that the early stages of colonic transformation in humans may involve the translocation of PKC activity, while the later stages might be associated with a reduction in total PKC activity, i.e., down-regulation (Guillem et al., 1987). However, we did not confirm such a translocation of PKC activity in adenoma tissue and the reason for this is currently unclear. In this content, we showed previously that the cellular distribution (% particulate fraction) of PKC activity in the normal-looking colonic mucosa of patients with colon cancer was significantly higher than in patients without cancer, suggesting that the translocation of PKC activity had already occurred in apparently normal mucosa in the cancer patients (Sakanoue et al., 1991a). Moreover, Baum et al. have reported that the initial translocation of PKC activity in rat colonic tissue occurred prior to the development of overt tumours (Baum et al., 1990). Several reports have shown that normal appearing mucosa of carcinoma patients might alternations promoting malignant transformation (Terpstra et al., 1987). Extensive pathological research has accumulated evidence that small adenomas (1-2 cm in diameter) have a low malignant potential, whereas adenomas over 2 cm in diameter have a much higher rate of malignant transformation, and has shown that the malignant potential of adenomas with severe dysplasia is significantly greater than that of those with mild or moderate dysplasia (Muto et al., 1975;O'Brien et al., 1990).
In conclusion, we utilised the specific PKC activity ratio (adenoma/normal mucosa) in the particulate and cytosolic fractions to assess the relationship between PKC activity and malignant potential without bias due to individual variations in activity. Our data showed that a close correlation existed between a decrease in the particulate PKC activity ratio and an increase in the risk of malignant transformation, as predicted by adenoma size, histological type, and degree of dysplasia. In contrast, the cytosolic ratio did not differ significantly with any of these three risk factors. These results suggested a role for down-regulation of particulate PKC in the progression of colonic tumours which appears to be an early event in colonic carcinogenesis. Thus, the PKC activity of colorectal adenomas may potentially be a useful biological indicator of tumour progression.
We are grateful to Ms. Takegawa for excellent technical assistance.