Aikou Okamoto

Heat shock protein 27 was up-regulated in cisplatin resistant human ovarian tumor cell line and associated with the cisplatin resistance

To understand the molecular basis for failure of cisplatin (CDDP) based chemotherapy, we compared gene expressions between CDDP sensitive and resistant ovarian tumor cell line, 2008 and 2008/C13*5.25, by mRNA differential display. We detected both up-regulated and down-regulated bands in the resistant cell and found some of them to be positive on Northern blotting. DNA sequencing revealed one to be mitochondrial heat shock protein 75. We found that HSP27 and HSP70 were also up-regulated in the resistant cell by Western blotting. Further, transient transfection with the HSP27 sense gene made the sensitive cell more resistant, while transient transfection with the antisense gene made it more sensitive.

Allelic imbalance and mutations of the PTEN gene in ovarian cancer

The PTEN/MMAC1/TEP1 tumor‐suppressor gene, which maps to chromosome 10q23.3, is mutated and homozygously deleted in a variety of human tumors, including endometrioid‐type ovarian tumors. We examined 33 primary ovarian cancers and 3 ovarian borderline tumors for allelic imbalance (AI) of the 10q23.3 region using 5 polymorphic markers, including an insertion/deletion‐type polymorphic marker identified in intron 4 of the PTEN gene. AI at one or more loci was detected in 12 of 31 (39%) informative ovarian cancers and none of 3 ovarian borderline tumors. The commonly deleted region was mapped between the D10S215 and D10S541 loci, including the PTEN locus. Moreover, the incidence of AI at the PTEN locus (38%) was the highest among the 5 loci examined. Therefore, we searched for mutations in the entire coding region of the PTEN gene by PCR‐SSCP and sequencing analyses in these tumors and 7 ovarian cancer cell lines. Mutations were detected in 3 of the 33 (9%) ovarian cancers: 2 cases with double mutations and 1 case with a mutation on 1 allele accompanied by deletions on both alleles in the poly T tract preceding the splice acceptor site in intron 7. An intragenic deletion was detected in 1 of the 7 (14%) ovarian cancer cell lines. PTEN mutations were detected not only in the endometrioid type but also in the serous and mucinous types of ovarian cancer. However, PTEN was not mutated in the 12 tumors that showed AI of the PTEN locus. Our results suggest that the PTEN gene plays an important role in the development of a subset but diverse histological types of ovarian tumors. However, it is possible that another tumor‐suppressor gene in the close vicinity of the PTEN gene is also inactivated by AI of the 10q23.3 region. Int. J. Cancer 85:160–165, 2000. ©2000 Wiley‐Liss, Inc.

Molecular analysis of the cyclin-dependent kinase inhibitor genesp15INK4b/MTS21,p16INK4/MTS1,p18 andpl9 in human cancer cell lines

Cyclin‐dependent kinase‐4 inhibitor genes (INK4) regulate the cell cycle and are candidate tumor‐suppressor genes. To determine if alterations in the coding regions of the pl8 and pl9 genes, which are novel members of the INK4 family and if they correlate with the development of human cancer, 100 human cancer cell lines were analyzed. Two other INK4 gene family members, p15INK4b/MTS2 and pl6INK4/MTS1 genes were also analyzed. Homozygous deletions of the p15INK4b/MTS2 gene were detected in 29 cancer cell lines. Thirty‐five homozygous deletions and 7 intragenic mutations of the pl6INK4/MTS1 gene were also detected in these cell lines. Neither homozygous deletions nor intragenic mutations of the p18 and p19 genes were found except in an ovarian cancer cell line, SKOV3, harboring a single base pair deletion in exon 1 of p19. In p16INK4/MTS1 expression analysis, 5 cell lines with both authentic and alternative spliced pl6INK4/MTS1 mRNA had no detectable pl6INK4/MTS1 protein. These results suggest the hypotheses that either post‐translational modification or enhanced degradation may be responsible for the lack of detection of the pl6INK4/MTS1 protein. Using Western blot analysis, subsets of 26 human cancer cell lines were examined for pl8 expression and 39 cell lines for p19 expression. All of these cell lines expressed the pl8 or pl9 protein, with the exception of SKOV3, which did not express pl9. Therefore, the INK4 gene family may be divided into 2 groups. One group includes p15INK4b/MTS2 and pl6INK4/MTS1 in which genetic and epigenetic alterations might contribute to the development of human cancers. The other group includes p18 and p19, in which somatic mutations are uncommon in many types of human cancer, and their role in human carcinogenesis and cancer progression is uncertain. (This article is a US Government work and, as such, is in the public domain in the United States of America.)

Somatic mutations and genetic polymorphisms of the PPP1R3 gene in patients with several types of cancers

Recently, we found nonsense and missense mutations of the PPP1R3 (protein phosphatase 1, regulatory subunit 3) gene in diverse human cancer cell lines and primary lung carcinomas, indicating that PPP1R3 functions as a tumor suppressor in human carcinogenesis. In this study, to assess the prevalence of PPP1R3 mutations in human primary cancers and the genetic diversity of the PPP1R3 gene in the human population, somatic mutations and genetic polymorphisms in the PPP1R3 gene were examined in 137 pairs of cancerous and non-cancerous tissues of patients with cancers of colon, ovary, and liver. Five somatic mutations including two missense mutations were detected in three cancerous tissues consisting of two colorectal carcinomas and one ovarian carcinoma. Five novel single nucleotide polymorphisms (SNPs) associated with the substitution of amino acids were also identified in cancer patients, in addition to five known nonsynonymous SNPs, including three previously reported ones as having an impact on the susceptibility to insulin resistant disorders. Differences in the activities and properties of multiple PPP1R3 proteins, which are produced in human cells due to variable somatic mutations and genetic polymorphisms in the PPP1R3 gene, can be involved in human carcinogenesis and susceptibility to diseases.

Low specificity of cytokeratin 19 mRNA expression in the peripheral blood cells from patients with ovarian tumors

We examined CK19 mRNA expression in the peripheral blood mononuclear cells (PBMC) from patients with ovarian tumor by reverse transcriptase-polymerase chain reaction (RT-PCR) using primers which did not cross-amplify pseudogenes of CK19 gene. The incidence of positive CK19 mRNA expression in healthy individuals, benign ovarian tumor patients and ovarian cancer patients were 60% (12/20), 71% (10/14) and 84% (21/25), respectively. Although the frequency of positive CK19 mRNA expression in PBMC from ovarian tumor patients was higher than that from healthy individuals, there was no statistically significant difference between the frequencies. Moreover, one healthy control showed CK19 mRNA expression only in her menstrual period, not in her proliferative phase or secretory phase. These results suggested that CK19 is not a suitable target to detect the presence of tumor cells in the PBMC from patients with ovarian tumors.

Upregulation of endothelial ICAM-1 expression by plasma from somatic mutations and genetic polymorphisms of the PPPR1R3 gene in patients with several types of cancers

P1.08.23 UPREGULATION OF ENDOTHELIAL ICAbEXPRESSION BY PLASMA FROM SOMATIC MUTATIONS AND GENETIC POLYMORPHISMS OF THE PPPRlR3 GENE IN PATIENTS WITH SEVERAL TYPES OF CANCERS S. Takakura (11, A. Okamoto (2), .I. Yokota (l), T. Tanaka (2), T. Kohno (l), T. Yamada (l), K. Shimizu (l), S. Ohwada (l), (1)National Cancer Center Research Institute, l-l, Tsukiji 5-chome, Chuo-ku, Tokyo, Japan, 1040045, (2) Jikei University School of Medicine, Tokyo, Japan.

Somatic mutations and genetic polymorphisms of the PPPRIR3 gene in patients with several types of cancers

Objective: Recent studies on mEN and PPP2RlB (a regulatory subunit of PPZA) mutations in human cancer indicate that aberrations of intracellular signaling pathways via protein phosphatases (PP) are involved in human carcinogenesis. We examined genetic alterations of the PPPlR3 gene located at chromosome 7q31, which encodes the regulatory subunit 3 of PPl, in various types of human cancers to asses the role of this gene in human carcinogenesis. Study Methods: All of coding exons of PPPlR3 were examined for mutations and polymorphisms in 104 cancer cell lines and 192 primary tumors by PCR-SSCP and direct sequencing. Results: Sixteen mutations of the PPPlR3 gene were detected in 9 of 104 cancer cell lines and 5 of 192 primary tumors, and they occurred in a subset of ovarian cancer, lung cancer, colorectal cancer, and gastric cancer. Sixteen mutations detected consisted of 3 nonsense mutations, 10 missense mutations, 2 silent mutations and a mutation in intron sequence. Seven novel single nucleotide polymorphisms (SNPs) associated with the substitution of amino acids were also identified in cancer patients, in addition to three known nonsynonymous SNPs, including three previously reported ones as having an impact on the susceptibility to insulin resistant disorders. Conclusions: Differences in the activities and properties of multiple PPPlR3 proteins, which are produced in human cells due to variable somatic mutations and genetic polymorphisms in the PPPlR3 gene, can be involved in human carcinogenes