Hideki Kakiuchi

DISTINCT METHYLATION PATTERN AND MICROSATELLITE INSTABILITY IN SPORADIC GASTRIC CANCER

Aberrant 5′ CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor‐suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5′ CpG island hypermethylation of hMLH1, E‐cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation‐specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E‐cadherin and p16 methylation respectively, and 8 (13.1%) presented high‐frequency MSI (MSI‐H). Of the 8 MSI‐H patients, 5 presented hMLH1 methylation, whereas no low‐frequency MSI (MSI‐L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E‐cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers. Int. J. Cancer 83:309–313, 1999.

Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5′ CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in 1 out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of 18 (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P<0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (−300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2′-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.

p53 Gene Mutations in Human Prostate Cancers in Japan: Different Mutation Spectra between Japan and Western Countries

The involvement of p53 mutations in prostate cancers in Japan was investigated. To evaluate any possible clinicopathological significance, p53 mutations in 40 samples from 36 Japanese prostate cancers of different stages (five cases of latent tumors, three of stage A cancers, 10 of stage B, five of stage C and 13 of stage D), including four lymph node metastases of stage D cases, were examined by polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) analysis and sequencing. Mutations were detected in five of 40 samples (12.5%); four were in primary cancers and the other in a lymph node metastasis from one of them. All mutation‐positive cases were in stage D, and the mutation frequency in stage D cases was 31%. This result indicates that p53 mutations may play a role in the progression of a subgroup of prostate cancers in Japanese, as observed for Americans and Europeans. However, a difference was noted between Japanese and Americans in the p53 mutational spectrum (at CpG site), presumably arising from variation in the underlying etiotogic factors.

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer.

A novel DNA methylation assay technique, termed bisulfite single‐strand conformation polymorphism (bisulfite‐SSCP), is a combination of sodium‐bisulfite modification and fluorescence‐based polymerase chain reaction (PCR)‐SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite‐SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite‐SSCP.

Absence of Microsatellite Instability and Germline Mutations of E-Cadherin, APC and p53 Genes in Japanese Familial Gastric Cancer

To evaluate the genetic factors of familial predisposition to gastric cancer, genetic alterations in the surgically resected stomach samples from gastric-cancer-prone families were investigated. Familial gastric cancer (FGC) was defined as gastric cancer occurring in a family with 3 or more gastric cancer patients over at least two successive generations. We examined replication error (RER) of six microsatellite markers and screened mutations of the 10-(A) repeat sequence in the transforming growth factor-β receptor type II (TGF-βRII) gene in individuals from seven unrelated FGC families. Three cases showed RER at one of the six (CA)n microsatellite markers but the other 4 cases showed no RER at any of these loci. No mutation was found in the 10-(A) repeat of the TGF-βRII gene. Additionally, no germline mutation was found by polymerase chain reaction-single strand conformation polymorphism in exons 1–16 of E-cadherin, exons 5–8 of p53 and in the mutation cluster region of APC. These results indicate that disorders in the DNA mismatch repair system, E-cadherin, p53 and APC may be infrequently involved in the carcinogenesis of Japanese FGC.

Expression and identification of aberrant c-kit transcripts in human cancer cells

We have previously cloned and sequenced a novel 3.5 kb c-kit mRNA expressed in a colon carcinoma cell line Colo201. Here we examined the expression of this truncated form of c-kit in 14 gastrointestinal cancer cells and 16 hematopoieic cancer cells by RT-PCR. Expression of the aberrant c-kit transcript was observed in various cancer cell lines. Furthermore, a new transcript which is 78 bp shorter than the transcript previously described was identified and characterized. These results indicate that two kinds of aberrant c-kit transcript produced by alternative promoter in intron 15 are expressed in human cancer cells.

Infrequent alterations of the p16 (MTS-1) gene in human gastric cancer.

p16 (MTS-1, multiple tumor suppressor gene 1), a putative tumor suppressor gene, is one of the cyclin-dependent kinase inhibitors (CDI) and it regulates the G1/S transition of the cell cycle. To clarify the role of p16 in primary gastric cancer, we have investigated somatic mutations of this gene by using the polymerase chain reaction/single strand conformation polymorphism (PCR-SSCP) method. In 23 surgical specimens of primary gastric cancer, none were detected in exon1 and exon 2. Among the 6 human gastric cancer cell lines examined, PCR products were not found in 2, MKN28 and MKN45, suggesting the presence of homozygous deletions. No mutation was found in the other 4 cell lines. Furthermore, decreased expression levels were not observed in 13 gastric cancer tissues by reverse transcription PCR (RT-PCR). Considering the above results of PCR-SSCP and RT-PCR, genetic alterations of the p16 gene are rarely implicated in human gastric cancer tumorigenesis.

Identification of genes highly expressed in G2-arrested Chinese hamster ovary cells by differential display analysis

Abnormal cell cycle regulation is believed to be an important step in tumorigenesis. In mammalian cells, DNA damage commonly leads to cell cycle arrest in G2; however, little is known about the detailed biochemical mechanisms underlying the DNA damage‐induced G2 arrest. In order to identify genes differentially expressed in association with G2 arrest, differential display analysis was performed between exponentially growing Chinese hamster ovary (CHO) cells and G2‐arrested CHO cells induced by etoposide, SN‐38, or X‐radiation. We identified five cDNA clones whose expression was up‐regulated in G2‐arrested CHO cells. Sequence analysis revealed that three clones were homologous to known genes: isogene I of translation initiation factor eIF‐4A, ribosomal protein L13, and translation repressor NAT1. The remaining two clones showed no homology to known genes. These results indicate that DNA damage can alter the expression of multiple genes, including translational regulators. J. Clin. Lab. Anal. 14: 314–319, 2000.

Circulating tumor-associated antigens detected by monoclonal antibodies against the polypeptide core of mucin-comparison of antigen MUSE11 with CA15-3

SummaryThe antigen MUSE11 detected by a monoclonal antibody (MAb) is an adenocarcinoma-associated antigen, while CA15-3 is a representative breast cancer-associated antigen detected by MAbs 115D8 and DF3. MAb MUSE11 showed higher binding activity to a synthetic peptide corresponding to the tandem repeat motif of the mucin core protein than that of MAb DF3, although MAb DF3 also had a significant binding activity indicating that MAbs MUSE11 and DF3 could recognize an identical polypeptide core. The reactivity of MAb DF3 to a breast cancer cell line MRK-neu-1 was completely abolished by neuraminidase treatment whereas that of MAb MUSE11 was partly conserved. The simultaneous measurement of the antigens MUSE11 and CA15-3 in sera from 35 cancer patients demonstrated that the incidence of abnormal serum level of CA15-3 was lower than that of antigen MUSE11. These data suggest that at least a part of the structural basis for the difference between the serum levels of antigen MUSE11 and CA15-3 could be carbohydrate side chains including sialic acids.

Familial gastric cancer in the Japanese population is frequently located at the cardiac region.

The clinical features of familial gastric cancer are still unknown. To approach this question, we investigated the clinicopathological characteristics of 16 cases of familial gastric cancer. In this study the criteria used to define familial gastric cancer was the existence of three or more family members with gastric cancer in at least two successive generations. The clinicopathological characteristics of cases who fulfilled this criteria were studied. This study contained 16 familial gastric cancer probands. Seven cases (44%) of gastric cancer had developed at the cardiac region of the stomach. This frequency was significantly higher than for gastric cancer in the general population in Japan (15.4%, p < 0.01). Undifferentiated types were dominant in familial gastric cancer (69%, p < 0.05). Furthermore, the frequency of disseminated peritoneal (40%) and liver metastases (20%) in familial gastric cancer was also significantly higher than for gastric cancer in the general population in Japan (10.9%, p < 0.01, and 4.4%, p < 0.05, respectively). Familial gastric cancers were frequently located at the cardiac region and appeared to be more aggressive than sporadic gastric cancers. The unique characteristics of familial gastric cancer suggest a genetic background in their etiology.