Takenori Ishikawa

Co‐expression of osteopontin and CD44v9 in gastric cancer

We have examined the expression of osteopontin (OPN) in 40 human primary gastric carcinoma tissues, 5 metastatic foci (lymph nodes) and corresponding normal mucosas. Twenty‐nine of 40 primary tumors (72.5%) and 3 of 5 lymph node metastases (60%) overexpressed OPN mRNA in comparison with those of the corresponding normal mucosa. The incidence as well as relative expression level of OPN mRNA was higher in well differentiated gastric cancers than poorly differentiated ones. Moreover, increased OPN mRNA expression in primary tumor specimens was observed along with the advancement of the clinico‐pathological stage. Using in situ hybridization (ISH) analysis, not only inflammatory cells in tumor stroma but also tumor cells showed positive signals for OPN mRNA. By immunohistochemistry, co‐immunoreaction of OPN and CD44v9 in tumor cells obviously correlated with the degree of lymphatic vessel invasion or long distant lymph node metastases in poorly differentiated gastric cancer. Interestingly, strong co‐immunoreaction of OPN and CD44v9 of tumor cells was concommitant with cluster formation in the lymphatic vessels. Our results suggest that overexpression of OPN correlated with the progression of human gastric carcinoma. Especially in CD44‐bearing poorly differentiated gastric cancer, interaction between OPN and CD44 may parallel lymphogenous metastasis. Int. J. Cancer (Pred. Oncol.) 79:127–132, 1998.© 1998 Wiley‐Liss, Inc.

Frequent amplification of the cyclin E gene in human gastric carcinomas

We searched for genetic alterations of the cyclin D1 and cyclin E genes in 45 human gastric carcinoma tissues. Expression of cyclin E mRNA and protein was also analyzed in eight of them by Northern and Western blots and immunohistochemical staining. The cyclin E gene was amplified 3–10 fold in seven gastric cancer tissues (15.6%), of which six were advanced gastric cancers. All of the cases with the cyclin E gene amplification displayed lymph node metastasis. Moreover, the case with the gene amplification overexpressed the cyclin E mRNA and protein. One of eight gastric cancer cell lines, MKN‐7, shared the cyclin E gene amplification, and all of the gastric cancer cell lines expressed high levels of the cyclin E mRNA and protein even without gene amplification. Amplification of the cyclin D1 gene was not observed in any of the gastric carcinoma tissues or gastric carcinoma cell lines. These results suggest that the gene amplification and overexpression of cyclin E play an important role in the abnormal growth and progression of gastric carcinoma.

Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9‐cis‐retinoic acid resistant (MKN‐7 and Ho‐1‐N‐1) and IFN‐β resistant cell lines (MKN‐7, ‐28 and ‐45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP‐ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21WAF1, CREB‐binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F‐1, E2F‐4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell‐to‐cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis‐regulating proteins. Int. J. Cancer 88:992–997, 2000.

Expression of the E2F family in human gastrointestinal carcinomas.

The E2F family of transcription factors plays a key role in the control of cell‐cycle progression. Some family members may act as oncogenes, others as tumor‐suppressor genes. The genetic changes and the expression of E2F‐1 and ‐3 were examined in human gastric and colorectal carcinomas by Southern, Northern and Western blots. Gene amplification of E2F‐1 was detected in 4% (1/23) of gastric carcinomas and 25% (3/12) of the colorectal carcinomas. Increased expression of E2F‐1 mRNA was observed in 40% (12/30) of the gastric carcinomas and in 60% (3/5) of the colorectal carcinomas in comparison with the corresponding non‐neoplastic mucosa. Over‐expression of E2F‐1 protein was confirmed in many of the gastric carcinomas. In contrast, expression levels of E2F‐3 mRNA were lower in 70% (21/30) of the gastric carcinomas and in 20% (1/5) of the colorectal carcinomas than in their corresponding normal conterparts, while gross alteration of the E2F‐3 gene was not detected. These results suggest that gene amplification and anomalous expression of the E2F gene may permit development of gastrointestinal carcinomas. Int. J. Cancer 81:535–538, 1999.

Evaluation of CD44 transcription variants in human digestive tract carcinomas and normal tissues

We generated DNA probes for CD44 variable region exons II (v6) to 14 (v9) and also for intronic sequences and examined the expression of aberrant CD44 transcripts in digestive tract carcinomas, colorectal adenomas and intestinal metaplasia of the stomach. The study used the reverse transcription‐polymerase chain reaction/Southern blot technique. Among the probes generated, the CD44 intron 9 probe was the best for distinguishing cancer tissue from normal tissue in adenocarcinomas of the colorectum or stomach. All the colorectal adenocarcinomas revealed overexpression of CD44 variants containing the intron 9 sequence compared with the corresponding normal colorectal mucosa. The overexpression of such abnormal CD44 variants was observed from an early stage in colorectal cancer and did not correlate with nodal or distant metastatic status. In intestinal metaplasia of the stomach, aberrant CD44 transcripts with characteristic 2 or 3 peaks containing intron 9 were observed. On the other hand, in oral or esophageal squamous cell carcinomas and corresponding normal squamous epithelia, overexpression of CD44 variants with variable exons and retained intron 9 sequence was frequently observed in both cancer tissue and corresponding normal mucosa. Examination of the expression of CD44 variants in a screening panel of normal tissues from the whole body revealed that overexpression of transcripts containing exon 11 to 14 as well as of the intron 9 sequence was constantly observed in tissues with squamous epithelia like skin, oral mucosa, esophagus and uterine cervix. Expression of these variants was also found in urinary bladder, respiratory tract, pancreas and salivary glands. Our results overall indicate that detecting the overexpression of abnormal CD44 transcripts, especially ones containing the intron 9 sequence, could be a powerful indicator for the presence of adenocarcinomas in the digestive tract. However, it is not applicable for the diagnosis of malignancies originating from squamous epithelia.

Genetic Status and Expression of the Cyclin‐dependent Kinase Inhibitors in Human Gastric Carcinoma Cell Lines

Deregulation of cyclin, cyclin‐dependent kinases (CDKs) and their inhibitors could have a pivotal role in the development of diverse human cancers. We examined the genetic status and the expression of CDK inhibitors (p21, p27, pl6 and p15), CDK2 and cyclins (A, D1 and E) in eight gastric carcinoma cell lines, in comparison with the status of p53 gene alterations. All the cell lines (except MKN‐28) that contained a p53 gene abnormality expressed very low or undetectable levels of p21 mRNA, while the cell lines (MKN‐45 and ‐74) with wild‐type p53 gene expressed high levels of p21 mRNA. An inverse correlation was found between the level of p21 mRNA and the expression of mRNAs for CDK2 and G1 cyclins. MKN‐28 was an exception; it contained mutated p53, and expressed mRNAs for p21, CDK2 and G1 cyclins at high levels. Only MKN‐45 and ‐74, with wild‐type p53, expressed considerable levels of p21 protein. Homozygous deletion of the p16 and p15 genes was detected in two (MKN‐45 and HSC‐39) of the eight gastric carcinoma cell lines. p16 protein was not expressed in three cell lines (MKN‐28, MKN‐74 and KATO‐III), as well as MKN‐45 and HSC‐39. Rearrangement of the p15 gene was found in TMK‐1. Rearrangement of the p27 gene was detected in MKN‐45, although the expression of p27 protein was well preserved in all the gastric carcinoma cell lines. The expression of pRb was also preserved in all the cell lines except KATO‐III. No obvious correlation was observed between the p53 gene status and the expression of p27 and p16. These findings suggest that abnormal regulation of CDK2/cyclins and CDK inhibitors might be involved in deregulated growth of gastric carcinomas.

Possible identity of diffuse sclerosing osteomyelitis and chronic recurrent multifocal osteomyelitis. One entity or two.

On the basis of the findings of nine of our patients and our review of previously reported cases of diffuse sclerosing osteomyelitis and chronic recurrent multifocal osteomyelitis, we discuss the similarity of these two entities. Our nine patients had initially been given diagnoses of diffuse sclerosing osteomyelitis on the basis of their clinicopathologic findings. However, technetium 99m-MDP bone scans performed on four of them revealed multiple bone lesions leading to the diagnosis of chronic recurrent multifocal osteomyelitis. Furthermore, no clear difference between clinical features in the patients with multiple bone lesions and those in the patients with diffuse sclerosing osteomyelitis was found. We conclude that diffuse sclerosing osteomyelitis is an expression of chronic recurrent multifocal osteomyelitis.

Expression of Bub1 Gene Correlates with Tumor Proliferating Activity in Human Gastric Carcinomas

Bub1 plays an important role at the spindle assembly checkpoint to prevent cell cycle progression following spindle damage. We examined the expression of human Bub1 mRNA in 20 gastric carcinoma tissues and corresponding nonneoplastic mucosas by reverse transcriptase-polymerase chain reaction and analyzed the relation with proliferative activity monitored by the expression of proliferating cell nuclear antigen (PCNA) on Western blotting as well as Ki-67 labeling index by immunohistochemistry. Increased expression of Bub1 mRNA was detected in 8 (40%) of the gastric carcinomas in comparison with their nonneoplastic counterparts, while 4 (20%) expressed Bub1 at lower levels. The expression of Bub1 mRNA was confirmed by in situ hybridization. The expression levels of Bub1 mRNA were well correlated with the levels of PCNA protein in 16 (80%) gastric carcinoma cases. The examination of Ki-67 labeling indices proved the close correlation between the expression levels of Bub1 and proliferating activity. These findings suggest that mRNA expression of human Bub1 gene is closely associated with the tumor-proliferating activity. Since genetic alterations of human Bub1 rarely occur in gastrointestinal cancers, the functional machinery of Bub1 to prevent cell cycle progression into anaphase might be well preserved in gastric carcinomas even with high proliferative activity.

Chronic recurrent multifocal osteomyelitis involving the mandible

A patient with chronic recurrent multifocal osteomyelitis is presented for the first time in the dental literature. On the basis of the initial diagnosis of diffuse sclerosing osteomyelitis of the mandible, the clinical course was protracted and refractory to surgical and antibiotic therapies. The literature is reviewed to evaluate the relationship between chronic recurrent multifocal osteomyelitis and diffuse sclerosing osteomyelitis. Distinguishing features between the two entities could not be found. It is recommended that a skeletal survey be performed when the diagnosis of diffuse sclerosing osteomyelitis is established.

Increased expression of CHK2 in human gastric carcinomas harboring p53 mutations

Human Chk1 and Chk2 are DNA damage–activated protein kinases that function as downstream mediators of ataxia‐telangiectasia mutated (ATM), which is involved in G2/M cell cycle arrest. To clarify the relation between the expression of Chk1/Chk2 and p53 gene status in human gastric carcinomas, we examined expression of Chk1, Chk2 and p53 proteins in 87 gastric carcinomas by Western blotting and immunohistochemistry. We found a significant correlation between the expression levels of Chk1 and p53 proteins in gastric carcinomas (p = 0.016). Significant statistical association was also observed between levels of Chk2 and p53 proteins (p = 0.00024). To clarify the genetic alterations of p53 in gastric carcinomas, we performed PCR‐SSCP analysis on 47 gastric carcinomas. Although we found that 5 of 7 (71%) gastric cancers expressed elevated levels of Chk1 had p53 mutation, there was not a statistically significant correlation between expression of Chk1 and genetic status of p53. We also found that 7 of 11 (78%) gastric carcinomas expressed elevated levels of Chk2 had p53 mutation, and this correlation was significant (p = 0.0157). We used a highly quantitative 5′ nuclease fluorogenic RT‐PCR method (TaqMan) to analyze the expression of Chk2 mRNA in 22 gastric carcinomas. Chk2 mRNA expression was higher in gastric carcinomas with p53 mutations compared to those harboring wild‐type p53. A significant association was recognized between the expression of Chk2 mRNA and p53 mutational status (p = 0.031). Our findings support the hypothesis that expression of Chk2 protein is increased in gastric carcinomas with mutant p53. Chk1 and Chk2 may play important roles in the checkpoint function in human gastric carcinomas harboring p53 mutation when their functions are preserved to prevent cell cycle progression.