Shuho Semba

Reduced expression of cyclin-dependent kinase inhibitor p27Kip1 is associated with advanced stage and invasiveness of gastric carcinomas

Reduced expression of a cyclin–dependent kinase inhibitor p27Kipl has recently been shown to predict poor survival of patients with breast and colorectal cancers. We studied the expression of p27Kipl in gastric carcinomas by northern blotting, western blotting and immunohistochemistry to determine whether lack of p27 has implications for aggressiveness of gastric cancer. Reduced expression of p27 was detected in 40% of the gastric carcinomas at the mRNA level, while it was detected in 57% at the protein level. No gross alterations of the p27 gene were observed in any of the cases examined by Southern blot analysis. Inimunohistochemical studies revealed that the expression of p27 was well preserved in most of the gastric adenomas, whereas it was so in only 26% of the gastric carcinomas. Fifty–six percent of the carcinomas showed almost no p27–positive cells. Decrease of p27–positive cells significantly correlated with advanced stage, depth of tumor invasion and lymph node metastasis. The expression of p27 showed an inverse correlation with the expression of cyclin E. These findings suggest that reduction of p27Klpl protein may reflect the progression of gastric carcinomas and may be an indicator of high–grade malignancy.

Expression of cyclin-dependent kinase inhibitor p21WAF1/CIP1 in non-neoplastic mucosa and neoplasia of the stomach: relationship with p53 status and proliferative activity.

The expression of the p53‐inducible cyclin‐dependent kinase inhibitor p21WAF1/CIP1 in non‐neoplastic mucosa, adenoma, and adenocarcinoma of the stomach was examined immunohistochemically and its relationship with p53 expression and proliferative activity was analysed. In normal gastric mucosa as well as in intestinal metaplasia the epithelial cells at the surface which showed no proliferative activity expressed p21whereas the cells in the deep area of the glands expressing Ki‐67 did not. In the neoplastic lesions, the expression of p21WAF1/CIP1 was detected in 78 per cent (112/144) of the adenomas and 76 per cent (262/343) of the adenocarcinomas. The incidence of p21WAF1/CIP1 expression did not differ among histological types of gastric carcinoma. The strong expression of p21WAF1/CIP1 was more frequently observed in carcinomas invading into submucosa or in cases of stages 2, 3, and 4 than in carcinomas limited to the mucosa or in stage 1 cases. The incidence of strongly positive cases was higher in carcinomas with lymph node metastasis than in those without metastasis. There was no apparent correlation between the expression of p21WAF1/CIP1 and the abnormal accumulation of p53 or with proliferative activity measured by Ki‐67 expression. These findings overall suggest that p21WAF1/CIP1 might be associated with the senescence of non‐neoplastic gastric epithelial cells; that a p53‐independent pathway might be substantially involved in the induction of p21WAF1/CIP1 in gastric neoplasia; and that the proliferative activity of gastric cancer might not be solely dependent on control of the cell cycle by p21WAF1/CIP1.

Altered microsatellites in incomplete-type intestinal metaplasia adjacent to primary gastric cancers.

AIM: To investigate the presence of genetic instability in precancerous lesions of the stomach. METHODS: Fifteen cases of sporadic gastric cancers with a background of intestinal metaplasia were studied by microsatellite assay at nine loci. Altered metaplastic mucosa was microdissected, reconstructed topographically, and examined immunohistochemically with an anti-p53 antibody, comparing its positive area with foci of microsatellite instability in each individual. RESULTS: Alterations at one or more loci were observed in seven of 15 cancers (46.7%) and four of 15 intestinal metaplasias (26.7%). Two cases of replication error positive phenotype had no microsatellite alterations in their metaplastic mucosa. All the microsatellite alterations in the metaplastic mucosa were restricted to incomplete-type intestinal metaplasia around the respective cancers. Moreover, in one case, an identical pattern of microsatellite alteration was detected in the cancer tissue and in the adjacent metaplastic mucosa, suggesting the sequential development of gastric cancer from intestinal metaplasia. Frequent alteration was found at the locus D1S191 (1q), indicating that this locus might be altered early in the development of intestinal-type gastric cancer. No significant association between microsatellite instability and p53 immunoreactivity was observed in the cases examined. CONCLUSION: These results indicate that microsatellite instability may be an early event in stomach carcinogenesis, especially in intestinal-type cancers.

Expression of cyclin E in colorectal adenomas and adenocarcinomas: correlation with expression of Ki-67 antigen and p53 protein

The expression of cyclin E in human colorectal adenomas and adenocarcinomas was examined immunohistochemically to elucidate the role of cyclin E in the colorectal carcinogenesis. The expression of cyclin E was detected in 25% (91/358) of the adenomas and 56% (149/267) of the adenocarcinomas. The incidence of strongly positive cases was significantly higher in the adenocarcinomas (20%) than in the adenomas (5%) (P<0.01). Among adenomas, a significant correlation was noticed between the expression of cyclin E and the grade of atypia. The incidence of cyclin E expression was significantly higher in the adenocarcinomas without an adenoma component (62%; 104/169) than in those with this component (46%; 45/98) (P<0.05). Furthermore, the incidence of the cyclin E expression was higher in stages 1 and 2 carcinoma than in stage 0 and stages 3 and 4 carcinoma. The expression of cyclin E was the most prominent in tumors invading the submucosa and muscularis propria. The expression of cyclin E was significantly correlated with the proliferative activity of the tumor cells measured by Ki-67 antigen expression (P<0.01). It was also correlated with the expression of p53 protein in the tumor cells (P<0.01). Overexpression of cyclin E and subsequent deregulation of cell cycle may contribute to the development and early progression of the colorectal carcinomas.

EXPRESSION OF P21WAF1/CIP1 IN COLORECTAL ADENOMAS AND ADENOCARCINOMAS AND ITS CORRELATION WITH P53 PROTEIN EXPRESSION

The expression of p53‐Inducible cylln‐dependent kinase Inhibitor, p21WAF1/CIP1 in non‐neopiastic mucosa, adenoma and adenocarclnoma of the colorectum was examined by immunohistochemistry and western bootting and Its relation with the expression of p53 protein was analyzed. Non‐neoplastic epithelial cells at the surface area showing no proitferative activity expressed p21WAF1/CIP1.The expression of p21WAF1/CIP1 was lmmunohistochemlcally detected in 55% (206/377 of the adenomas and 66% (190/289) of the adenocarcinomas, respectively. The lncldence of strongly positive cases was significantly higher In the adenocarcinomas (27%) than In the adenomas (18%) (P<.05). The incidence of cases wtth strong p21WAF1/CIP1 expression was higher In stages 0,1 and 2 carcinomas than in stages 3 and 4 carcinomas (P<0.05). A decrease in the incidence of cases with strong expression was detected in carclnomas Invading deeper than muscularis propria. The influence of strongly positive cases was signiflcantly lower in carcinomas with lymph node metastasis than those without metastasls (P<0.05). The expression of p21 as well as p53 detected by western blotting was compatlble with the results of lmmunohistochemlstry in most cases examined. However, there was no significant correlatlon between the expression of p21WAF1/CIP1 and the abnormal accumulation of p53. These findlngs overall suggest that: (i) the physiological expression of p21WAF1/CIP1 may be associated with cellular senescence of colorectal mucosa; (ii) reduced expression of p21WAF1/CIP1 participate in the progression of colorectal carcinoma; and (iii) p53‐Independent paulway may be considerably Involved In the inductions of p21WAF1/CIP1.

Molecular Bases of Human Stomach Carcinogenesis

Multiple genetic alterations, including inactivation of tumor-suppressor genes, activation of oncogenes, and reactivation of telomerase, are implicated in human stomach carcinogenesis. Among them, replication errors (RERs) at microsatellite loci, reactivation of telomerase, activation of c-met, inactivation of p53, and deranged CD44 transcription are common events of both welldifferentiated and poorly differentiated gastric carcinomas. In addition to these common events, K-ras mutation, APC inactivation, loss of DCC, and amplification of c-erbB2 are preferentially found in well-differentiated gastric carcinomas, whereas gene mutations, loss of the cadherin/ catenin system, and amplification of K-sam are frequently observed in poorly differentiated cancers. In addition, a paracrine loop formed between cancer cells and stromal cells through the hepatocyte growth factor/c-met system plays an important role in morphogenesis and invasion of gastric carcinoma with different status of adhesion molecules and signal transduction systems in vivo. Reduction or loss of p27 protein, associated with overexpression of cyclin E, may confer the progression and metastasis. In regard to precancerous lesions of the stomach, some of the intestinal metaplasias and adenomas exhibited the same genetic alterations (e.g. mutations of APC and p53, RERs, and reactivation of telomerase) and telomere shortening as those found in well-differentiated carcinomas. Moreover, human telomerase RNA overexpression, which correlates well with the number of Helicobacter pylori present, may precede telomerase reactivation in human stomach carcinogenesis. These observations suggest overall that there are two distinct genetic pathways in human stomach carcinogenesis, and some well-differentiated cancers share the same multistep genetic alterations as those established for colorectal cancers.

20 – Role of Immunohistochemical Expression of PRL-3 Phosphatase in Gastric Carcinoma

This chapter discusses the role of immunohistochemical detection of phosphatase of regenerating liver (PRL)-3 in human gastric carcinomas. Protein tyrosine phosphatases play a fundamental role in regulating diverse proteins that participate in every aspect of cellular physiologic and pathogenic processes. The proteins PRL-1, -2, and -3 represent a novel class of protein tyrosine phosphatise superfamily members, in that they possess a unique COOH-terminal prenylation motif, with a protein tyrosine phosphatase-active site, signature sequence CX5R. The PRLs are found to be associated with the early endosome and plasma membrane in their prenylated state, whereas nuclear localization of these phosphatases may occur in the absence of prenylation. PRLs share 75% amino-acid sequence similarity, the ScanProsite analysis reveals that the potential sites of phosphorylation, by several kinases, are quite different. Moreover, Northern blot analysis demonstrates that the preferential messenger ribonucleic acid (mRNA) expression pattern of these PRLs also differed among the organs, indicating that PRLs are quite divergent in their functions. The PRL family tyrosine phosphatases, PRL-3 enhance the growth of human embryonic kidney fibroblasts.