PubMed Health⌕ Search

Biomedical subjects

M Miyaki

Publications and source records attributed to M Miyaki.

At least 55 records · Page 3Linked to original sources
PubMed1995

Malignant transformation of rat embryo fibroblasts by cotransfection with eleven human mutant p53 cDNAs and activated H-ras gene.

Eleven different missense and one nonsense mutant-type p53 cDNAs, which have been frequently detected in human colorectal carcinomas, were constructed and examined for their ability to cooperate with activated human H-ras genes, pSK2 and pHs49, in transfection of rat embryo fibroblasts (REF). Each missense mutant-type p53 cDNA with either of the two activated H-ras genes transformed REF with a different frequency of transformation depending on the different kind of mutation, whereas wild-type p53 (with ras), nonsense mutant-type p53 (with ras), as well as mutant-type p53 (alone) and ras (alone), did not transform REF. Six transformed REF cell lines were established from cotransfection with missense mutant-type p53 cDNA and ras gene; all of them exhibiting exogenous human p53 DNA, RNA, protein, and H-ras DNA and RNA. All six transformed cell lines showed both tumorigenicity and lung metastatic potential in nude mice. They also exhibited 92 kilodalton gelatinase activity, which was not detected in parental REF. These results suggest that missense mutations in p53 gene have a role in malignant transformation as well as metastatic potential.

Animals↗
PubMed1995

Somatic mutations of the adenomatous polyposis coli gene in gastroduodenal tumors from patients with familial adenomatous polyposis.

We analyzed somatic mutations of the adenomatous polyposis coli (APC), p53, and K-ras genes in gastroduodenal polyps and normal gastroduodenal mucosa from 21 familial adenomatous polyposis patients, using PCR-single-strand conformation polymorphism and direct sequencing methods. Seventy-five polyps were obtained from these patients endoscopically or surgically, and they were histopathologically diagnosed as mild adenoma, moderate adenoma, severe adenoma, adenocarcinoma, and fundic gland polyp. Examining the APC-coding region where somatic mutations in colorectal tumors are known to be clustered, we detected 47 somatic mutations. The frequency of mutation detected was 6 of 9 (67%) in ampullary adenomas, 1 of 2 (50%) in ampullary adenocarcinoma, 11 of 24 (46%) in non-ampullary adenomas, 26 of 29 (90%) in gastric adenomas, and 3 of 11 (27%) in gastric fundic gland polyps. These mutations frequently occurred at codons 1450, 1462-1465, and 1554-1556, the third being a newly found hot spot. All mutations formed stop codons that resulted in truncated APC proteins. K-ras mutation was detected only in an ampullary adenocarcinoma, and p53 mutation was not detected in any of the tumors analyzed. There was no somatic mutation detected in samples of flat mucosa that were diagnosed as normal mucosa both endoscopically and histopathologically. Frequent APC mutations in mild and small adenomas, similar to the findings in severe and large adenomas, suggested that the genetic change in the APC gene occurs in an early stage of forming gastroduodenal adenomas. Moreover, the presence of somatic APC mutations in fundic gland polyps suggests that inactivation of the APC gene plays a role not only in forming adenomas but also in forming hyperplastic polyps in fundic gland mucosa, and there may be some additional steps to the adenoma-carcinoma sequence.

Adenomatous Polyposis Coli↗
PubMed1995

Germ line mutations of hMSH2 and hMLH1 genes in Japanese families with hereditary nonpolyposis colorectal cancer (HNPCC): usefulness of DNA analysis for screening and diagnosis of HNPCC patients.

Mutations in hMSH2 and hMLH1 genes were analyzed in patients from 11 Japanese families that had been diagnosed as carrying hereditary nonpolyposis colorectal cancer (HNPCC) by clinical examination. Germ line mutations of hMSH2 gene were identified in 5 independent families in which colorectal (87% of patients), endometrial (30%), ovarian (17%), gastric (14%), and other cancers existed. Five mutations detected between codons 136 and 811 included single-base substitutions (C-->T and T-->G), a T deletion, and an A insertion, all of which produced stop codons resulting in truncated proteins, and an A-->T substitution at splice donor site of exon 5 which resulted in deletion of this exon. Moreover, one HNPCC family was presumed to have germ line mutation of hMSH2 gene because a somatic mutation of hMSH2 gene was detected in a cancer from a patient in this family. In addition to these 11 families already diagnosed with HNPCC, 3 new families with germ line mutations of hMSH2 gene and hMLH1 gene were found through analysis of DNA from patients who had multiple cancers with alteration in microsatellite DNA. These mutations included an AG deletion at codons 877-878 of hMSH2 gene, an AAG deletion at codons 616-618 of hMLH1 gene, and a C-->T single-base substitution at codon 217 of hMLH1 gene. Seven of eight germ line mutations found in this study are new mutations that have not been reported previously. In families in which germ line mutations were identified presymptomatic examination was then carried out using polymerase chain reaction single-strand conformation polymorphism analysis of DNA from peripheral blood, and the result was the detection of family members predisposed to HNPCC who did not yet show signs of cancer. These results indicate the value of DNA analysis in the screening and diagnosis of HNPCC patients and families.

Adult↗
PubMed1995

[Increased cell adhesiveness and decreased tumorigenicity induced in human colon carcinoma cells by transfection with E-cadherin cDNA].

Metastasis of colon carcinomas is assumed to be caused by multiple steps, which include a loss of cell adhesion that results in the release of carcinoma cells from the original tumor tissue. A human colon carcinoma cell line was established from a poorly differentiated metastatic adenocarcinoma without cell-cell adhesion and without expression of E-cadherin mRNA. To this cell line, mouse E-cadherin cDNA in a expression vector was co-transfected with a neomycin-resistant gene. The transfected cells, which expressed exogenous E-cadherin gene, showed a compact shape with strong cell-cell adhesion and with increased cell-substratum adhesion. These cells showed a significantly low anchorage independency and decreased invasiveness compared to the parental carcinoma cells. Their growth rate was decreased both in vitro and in the subcutis of nude mice.

Adenocarcinoma↗
PubMed1994

Microsatellite instability in the progression of gastric carcinoma.

Seventy-six gastric carcinomas were analyzed with regard to whether or how microsatellite instability was associated with the development of the carcinoma. Microsatellite instability occurred as a late genetic alteration, with an incidence significantly higher in the advanced stage (17 of 51) than in the early stage (3 of 25; P < 0.05). Chromosomal losses on 5q and 17p, detected by polymerase chain reaction-restriction fragment length polymorphism, more frequently accompanied microsatellite instability (9 of 15 and 8 of 11, respectively), compared with carcinomas which lacked instability (5 of 28 and 9 of 30, respectively; P < 0.01 and P < 0.05, respectively). Epstein-Barr virus was observed in only 8 of 76 carcinomas, none of which was associated with microsatellite instability. No significant correlation was found between instability and the familial tendency to develop gastric carcinomas. Our results suggest that microsatellite instability might play a role in the progression of gastric carcinomas but not in Epstein-Barr virus-associated gastric carcinomas.

Adult↗
PubMed1994

Characteristics of somatic mutation of the adenomatous polyposis coli gene in colorectal tumors.

Mutation of the adenomatous polyposis coli (APC) gene was analyzed in 500 colorectal tumors from 70 familial adenomatous polyposis (FAP) and 102 non-FAP patients and in normal tissues from 119 FAP patients, using polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods. These tumors were histopathologically diagnosed. Sixty-eight germ line mutations (62% deletion, 9% insertion, and 29% single-base substitution) and 241 somatic mutations (56% deletion, 12% insertion, and 32% single-base substitution) were detected. All mutations formed stop codons resulting in truncated APC proteins, except for one germ line mutation. Differences were found between somatic and germ line mutations, including 3 new hot spots of mutation at codons 1378, 1450, and 1487-1490, which frequently occurred in somatic mutations but not in germ lines. The frequency of mutation in each histopathological type of FAP tumor was 53% in moderate adenoma, 64% in severe adenoma, 52% in intramucosal carcinoma, and 33% in invasive carcinoma, whereas the loss of heterozygosity including the APC gene increased with development to each histopathological type. A similar tendency was observed in non-FAP tumors. Additionally, we found 10 FAP tumors that had both somatic mutation and loss of heterozygosity. These tumors were assumed to have developed from moderate adenomas with germ line and somatic mutations, followed by deletion of the allele with germ line mutation. These results suggest that inactivation of the APC gene by two mutations is involved in the development of moderate adenoma, and loss of heterozygosity of the APC gene is associated with further development to carcinoma. It was also observed that the distribution of 75 somatic mutations from one FAP patient on the APC sequence was similar to the distribution of 159 somatic mutations from 83 patients with FAP and non-FAP, which suggests that the position of somatic mutation is mostly due to the APC sequence itself.

Adenoma↗
PubMed1994

Molecular genetics for clinical management of colorectal carcinoma. 17p, 18q, and 22q loss of heterozygosity and decreased DCC expression are correlated with the metastatic potential.

BACKGROUND: The molecular genetic changes associated with colorectal carcinoma are among the best understood of any common human cancer. The genetic changes during the late stages of colorectal carcinomas may be useful in clinical management for determining the metastatic potential of the carcinoma. METHODS: Tumor tissues were evaluated by restriction fragment length polymorphism (RFLP) analysis of chromosomes 5q, 17p, 18q, and 22q (n = 98), by reverse transcription-polymerase chain reaction (RT-PCR) analysis of messenger RNA expression of the DCC gene (deleted in colorectal carcinoma) (n = 27) and by immunohistochemical analysis of p53 protein expression (n = 44). RESULTS: Loss of heterozygosity (LOH) on chromosomes 17p, 18q, and 22q, but not on 5q, was much more frequently detected in advanced carcinomas than in intramucosal carcinomas (P < 0.01). 17p LOH was significantly correlated with vascular invasion (P < 0.001), whereas 18q LOH was correlated with lymphatic invasion and hepatic metastasis (P < 0.01), and 22q LOH was correlated with lymph node metastasis (P < 0.05). LOH on 5q did not show a significant correlation with any factors of tumor invasion or metastasis. DCC expression was not observed in any of five hepatic metastasis or in five of seven advanced carcinomas that were accompanied by hepatic metastasis (10 of 12). However, a similar lack of expression was observed in only 5 of 15 carcinomas without hepatic metastasis (P < 0.05). p53 Expression was found to vary in both primary and metastatic carcinomas by immunohistochemistry. CONCLUSIONS: The clinical application of molecular genetics (i.e., RFLP analysis of chromosome 17p, 18q, and 22q and RT-PCR analysis of DCC expression into messenger RNA) can be used to determine the metastatic potential of colorectal carcinomas.

Adult↗
PubMed1994

Epstein-Barr virus-associated gastric carcinoma and Epstein-Barr virus infection of the stomach.

BACKGROUND: Epstein-Barr virus (EBV) has been found to be associated with a type of gastric carcinoma (EBVaGC). However, many questions remain unanswered, such as epidemiology, and pathologic features of EBVaGC and the significance of EBV in the genesis of EBVaGC. EXPERIMENTAL DESIGN: Gastric carcinoma and non-neoplastic mucosa were evaluated to reveal the following issues: the incidence of EBVaGC in Japanese population, pathologic features and EBV genotype, clonality, and gene-expression in EBVaGC, localization of EBV in non-neoplastic stomach, and serum titer of anti-EBV antibodies in EBVaGC-carrying patients. RESULTS: Using PCR and EBER1 in situ hybridization, EBVaGC (definitely amplifiable EBV-DNA and positive EBER1-signal in the nuclei of carcinoma cells) was found in 8 of 72 gastric carcinomas (11%). The dominant genotype of EBV was type A (7/8), with type C (6/8), and F (8/8) restriction enzyme polymorphism, which are the predominant type of EBV found in throat washing of the general population in Japan. EBVaGC was found in the cardia (4/8) or body (4/8) of the stomach, and consisted of 7 advanced and 1 intramucosal carcinoma. By Southern blot analysis of EBVaGC hybridized with right- and left-side probe adjacent to the terminal repeats, EBV was present in a monoclonal episomal form in all of the EBVaGC. EBVaGC lacked expression of EBNA2 (0/8) and LMP1 (0/8) by immunocytochemistry. In non-neoplastic mucosa, EBER1 signal was identified in the infiltrating lymphocytes and shedding epithelial cells predominantly in fundic gland mucosa of patients with EBVaGC (8/8). Patients with EBVaGC showed high titers of anti-VCA IgG (8/8), anti-VCA IgA (2/8) and anti-EA IgG (7/8) antibodies just before surgery. CONCLUSIONS: EBV may infect the surface epithelium of the stomach through the reactivated EBV-carrying lymphocytes. EBV may be a factor initiating EBVaGC. Anti-EBV antibodies or EBER1 in situ hybridization may help to identify patients at high risk for EBVaGC.

Antibodies, Viral↗
PubMed1993

Coexistence of somatic and germ-line mutations of APC gene in desmoid tumors from patients with familial adenomatous polyposis.

Desmoid tumors, which are locally invasive with recurrence but without metastasis, are frequently observed in patients with familial adenomatous polyposis after abdominal surgery or during pregnancy. This study analyzed mutation of the adenomatous polyposis coli gene in 8 desmoid tumors from 7 familial adenomatous polyposis patients using polymerase chain reaction-single-strand conformation polymorphism and the direct sequencing method. Seven somatic mutations, 1 somatic allele loss, and 6 germ-line mutations were detected. The majority of adenomatous polyposis coli gene mutations were deletions of 1 to 19 base pairs in exon 15, and all mutations led to the formation of stop codons. A somatic mutation with repetition of 82 base pairs from codon 1399 to 1426 was also observed in a desmoid, which was most likely caused by an error during replication or repair replication. No mutation was detected in exons 1 to 2 of H-ras, K-ras, and N-ras genes and in exons 5 to 8 of p53 gene, in these tumors. The simultaneous existence of somatic and germ-line alterations of adenomatous polyposis coli gene observed in all 8 tumors strongly suggests that inactivation of both alleles of adenomatous polyposis coli gene is involved in the development of desmoid tumors.

Adenomatous Polyposis Coli↗
PubMed1993

Allelic loss at chromosome band 8p21.3-p22 is associated with progression of hepatocellular carcinoma.

Human hepatocellular carcinomas (HCC) frequently show loss of heterozygosity at loci on the short arm of chromosome 8. To define a region on 8p commonly deleted in HCC, we used 20 restriction fragment length polymorphism markers to carry out detailed deletion mapping in 142 HCC. Of the 124 informative cases, 56 (45%) showed allelic losses in tumors. While more than half of them had lost alleles at every locus on the short arm, others lost them partially or interstitially. Common deletion of an 8-cM region flanked by cMSR-32 and cC18-245, at 8p21.3-p22, suggested the presence of a tumor suppressor gene(s) in this interval. Correlation between allelic losses on 8p and clinicopathological parameters were examined in the 51 cases for which detailed clinical data were available; allelic losses on 8p were observed in none of five tumors at stage T1 (0%), nine of 20 tumors at stage T2 (45%), and 17 of 26 tumors at stage T3/T4 (65%). A higher frequency of allelic losses on 8p was observed in poorly differentiated tumors (10/14, 71%) than in well differentiated tumors (3/13, 23%). The association of allelic losses on 8p with advanced clinical stage and poor differentiation implies that loss or inactivation of a tumor suppressor gene(s) on 8p may play a role in the progression of HCC.

Carcinoma, Hepatocellular↗
PubMed1993

No allelic loss at the p53 locus in 1,2-dimethylhydrazine-induced mouse colon tumours: PCR-SSCP analysis with sequence-tagged microsatellite site primers.

We examined allelic loss in colon tumors induced by 1,2-dimethylhydrazine (DMH) in F1 hybrid mice, using sequence-tagged microsatellite site (STMS) primers derived from the chromosomal region closely linked to the p53 locus. Polymerase chain reaction--single-strand conformation polymorphism (PCR-SSCP) analysis of 155 colonic tumors with two STMS markers revealed that no genetic alterations had occurred in these tumors, except for one case where one of the markers detected an increase of one CA repeat unit in one allele. No allelic loss at the loci closely linked to the p53 locus strongly suggests that allelic loss at the p53 locus is not involved in DMH-induced colon carcinogenesis in mice.

1,2-Dimethylhydrazine↗
PubMed1993

Suppression of tumourigenicity in human colon carcinoma cells by introduction of normal chromosome 1p36 region.

Development of colon carcinomas appears to be associated with inactivation of multiple tumour suppressor genes. Cytogenetic and DNA analyses of colon carcinomas have detected a high frequency of chromosome 1p deletion, which suggests the presence of a tumour suppressor gene. We therefore introduced normal human chromosome 1 into colon carcinoma COKFu cells, through microcell hybridization. Six clones of hybrid cells containing normal chromosome 1 were obtained, four of which had a small fragment of the introduced chromosome 1, including 1p36-34. The morphology of hybrid cells with chromosome 1 markedly altered to a flat shape. The cloning efficiency of all six hybrid cells in soft agar was significantly reduced, and the tumourigenicity in athymic nude mice was completely suppressed. Hybrid cells containing only the region of 1p36-34, as well as those containing intact chromosome 1, showed suppressed transformed phenotype. Furthermore, several tumourigenic revertant cells were obtained from the hybrid cells. These revertant cells had a morphology similar to that of COKFu cells, and were found to have lost the 1p36 region from the introduced chromosome 1. These results indicate that a normal chromosome 1p36 carries a tumour suppressor gene for colon carcinogenesis.

Adenocarcinoma↗
PubMed1992

Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorectal cancer, and lung cancer.

Frequent loss of heterozygosity at chromosomal loci in a specific tumor type may indicate the presence of a tumor suppressor gene. We have examined loss of heterozygosity on chromosome 8p in paired tumor and constitutional DNA from 346 patients representing seven different types of human cancer. Frequent allelic losses were observed in hepatocellular carcinoma (22 of 46 cases, 47.8%), in colorectal cancer (12 of 26, 46.2%), and in non-small cell lung cancer (14 of 35, 40.0%), in contrast to low frequencies detected in breast cancer (5 of 56, 8.9%) and renal cell carcinoma (2 of 27, 7.4%). Ovarian cancer and gastric cancer showed intermediate frequencies of 33.3% and 22.2%. Subsequent analysis of 120 hepatocellular carcinomas and 94 colorectal cancers with five polymorphic markers along the short arm of chromosome 8 defined commonly deleted regions within the same chromosomal interval, 8p23. 1-8p21.3, suggesting that one or more tumor suppressor genes for both cancers may be present in that region.

Alleles↗
PubMed1992

Loss of expression of the DCC gene during progression of colorectal carcinomas in familial adenomatous polyposis and non-familial adenomatous polyposis patients.

We have previously observed that the frequency of loss of heterozygosity (LOH) on chromosome 18q was low in adenomas and intramucosal carcinomas, whereas invasive carcinomas exhibited a high frequency in familial adenomatous polyposis patients (M. Miyaki et al., Cancer Res., 50: 7166-7173, 1990). In the present study, LOH at the DCC locus on chromosome 18q and the expression of DCC gene into mRNA were analyzed in colorectal tumors with distinct histopathological types. The carcinomas that showed 18q LOH also lost the DCC locus. The expression of DCC gene into mRNA was examined at the level of 233-base pair fragments of nucleotide 986-1218 in DCC complementary DNA. In a moderate-to-severe adenoma, 5 carcinoma-in-adenomas, and 4 intramucosal carcinomas, the level of expression was as high as in normal colorectal mucosa, whereas it was greatly reduced or not detectable in most (13 of 16) invasive carcinomas. Among these invasive carcinomas, 7 of 11 showed 18q LOH, but 4 showed no LOH. These results suggest that the DCC gene is included in the allelic deletion on chromosome 18q, and that the progression of colorectal carcinoma from early stage to advanced stage accompanies the inactivation of the DCC gene through LOH and other mechanisms.

Adenoma↗
PubMed1992

Loss of normal allele of the APC gene in an adrenocortical carcinoma from a patient with familial adenomatous polyposis.

Endocrine neoplasms have been reported occasionally in patients with familial adenomatous polyposis (FAP). An adrenocorotical carcinoma was studied in a patient with a family history of FAP. Loss of heterozygosity (LOH) in the region close to the adenomatous polyposis coli (APC) gene was detected in this carcinoma, and evidence was obtained that there was a loss of the normal allele of the APC gene. This is the first demonstration of LOH at the APC locus in adrenocortical tumors. The present results and our previous data on LOH in a recurring desmoid tumor suggest that the heterozygous mutant/wild-type condition of the APC gene may give rise to benign tumors, and that functional loss of this gene leads to development of tumors not only in the colon but also in other various parts of the body in FAP patients.

Adenomatous Polyposis Coli↗
PubMed1991

Suppression of tumorigenicity in human colon carcinoma cells by introduction of normal chromosome 5 or 18.

Development of colon carcinomas can be associated with allelic deletions on several chromosomes, including 5q and 18q. The APC gene on 5q and the DCC gene on 18q have been identified as potential tumour suppressor genes, whose suppression contributes to colon carcinogenesis. To investigate the role of genes in these deleted regions, we have now introduced a single normal human chromosome into a human colon carcinoma cell line, COKFu, through microcell hybridization. Several clones of hybrid cells containing normal chromosome 5, and others containing normal chromosome 18, were obtained. The morphology of the hybrid cells was markedly altered: the hybrids with chromosome 5 exhibited a closely packed polygonal morphology, and the hybrid cells with chromosome 18 were flattened. The cloning efficiency of the hybrid cells in soft agar was reduced from 0.46 to 0% of that of the parental carcinoma cells, and the tumorigenicity of these hybrid cells in athymic nude mice was completely suppressed. The growth properties of the hybrid cells with chromosome 11 were not substantially changed. These results strongly suggest that the genes on normal chromosome 5 and 18 function as tumour suppressors in colon carcinogenesis.

Animals↗