Showing papers by "Seiji Isonishi published in 2000"

Allelic imbalance and mutations of the PTEN gene in ovarian cancer

Abstract: 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.

Mithramycin represses MDR1 gene expression in vitro, modulating multidrug resistance.

Abstract: The effect of an aureolic acid, mithramycin (MTM) on multidrug resistance (MDR) was investigated. At a concentration of 0.02--0.1 mg/ml (about 20--90 microM), MTM repressed MDR1 gene transcription of SBC-3/ADM, a MDR-phenotype subline derived from human small cell lung tumor. Under the same conditions, another aureolic acid, chromomycin A3, showed potent cytotoxicity. FACS analysis revealed that 5 microm MTM depleted the P-glycoprotein (Pgp) and lowered the efflux activity of SBC-3/ADM cells. Furthermore, MTM sensitized the cells against adriamycin. These results suggest that MTM would be a useful modulator of MDR induced by Pgp.

Depletion of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances platinum drug sensitivity in human ovarian carcinoma cells.

Abstract: Down-regulation of protein kinase C (PKC) by 12- O -tetradecanoylphorbol-13-acetate (TPA) enhances the sensitivity of human ovarian carcinoma 2008 cells to various types of platinum compounds such as cisplatin (DDP), carboplatin and (–)-(R)-2-aminomethylpyrrolidine (1,1-cyclobutanedicarboxylato)-platinum(II) monohydrate (DWA) by a factor of two- to threefold. TPA enhanced the sensitivity of the DDP-resistant 2008/C13*5.25 subline to each of these three drugs to the same extent as for the 2008 cells. The extent of PKC down-regulation and drug sensitization depended on the duration of TPA exposure; maximum effect was achieved with a 48 h pretreatment. Sensitization was TPA concentration-dependent and was maximal at 0.05 μM TPA. 2008 cells expressed only the PKCα and PKCζ isoforms. Western blot analysis revealed that whereas the expression of PKCα was reduced by TPA the level of PKCζ was not affected. These results suggest that PKCα is the isotype responsive to TPA in these cells and that platinum drug sensitivity can be modulated by this isoform alone. In parallel to its effect on PKCα, TPA decreased cellular glutathione content by 30 ± 3 (standard deviation (s.d.) % in 2008 cells and by 41 ± 3 (s.d.) % in 2008/C13*5.25 cells. TPA also increased accumulation of DDP and DWA by 70%, although this effect was limited to the 2008/C13*5.25 cells. TPA rendered 2008 and 2008/C13*5.25 cells resistant to cadmium chloride by a factor of 3.7 and 3.6-fold respectively, suggesting a significant increase in cellular metallothionein content. Although the mechanism of TPA induced sensitization is not yet fully understood, this study points to a central role for PKCα in modulating platinum drug sensitivity. © 2000 Cancer Research Campaign

Two novel C-glycosides of aureolic acid repress transcription of the MDR1 gene.

Abstract: In the search for compounds which repress MDR1 gene expression, two novel aryl C-glycosides were isolated from a broth of Streptomyces sp. They had the characteristic structure of a dideoxy-carbohydrate (oliose or olivose) linked directly to chromomycinone, an aglycone of aureolic acids. Further investigation revealed that they were artifacts yielded from an aureolic acid, mithramycin. Acid and methanol were necessary to yield the C-glycosides. This reaction would contribute to the design of useful aryl C-glycosides.

New ovarian cancer cell strain

Abstract: PROBLEM TO BE SOLVED: To obtain a new cell strain clarifying natural resistant mechanism of ovarian clear cell adenocarcinoma to anticancer agent and developing an effective treating method. SOLUTION: Cell strain derived from ovarian clear cell adenocarcinoma or its variant is established preferably from primary tumor by using serum-free culture medium from primary culture and preferably repeating passage. The cell strain preferably produces at least one of tumor marker selected from a group ErbB-2, CA-125, epithelial membrane antigen, cytokeratin and EGF receptor and preferably exhibits resistance to cisplatin and/or doxorubicin and preferably has accession numbers of FERN P-17237 and FERM P-17238. A medicine resistant mechanism of cisplatin and/or doxorubicin is analyzed by using the above cell strain.

Glutathione biosynthesis inhibitor

Abstract: PROBLEM TO BE SOLVED: To obtain the subject inhibitor having the activity to scavenge the SH groups of SH group-contg compounds in vivo and therefore useful for overcoming drug resistance in therapies using anticancer agent by including an L-hydroxyoxonorvaline as an active substance SOLUTION: This inhibitor is obtained by including L-5-hydroxy-4- oxonorvaline of the formula which is available by isolation from an Actinomycetes KS6701 or KS8846 strain or Streptomyces akiyoshiensis novsp as a well-known Actinomycetes strain The above compound of the formula can induce overcoming drug resistance through reducing cancer cell glutathione level by its administration to cancer tissues which have acquired drug resistance, thereby improving therapeutic effect as a result of using carcinostatics Also, drug resistance overcoming agents and SH group scavengers can be obtained by including the above compound of the formula It is preferable that the above inhibitor is administered orally or percutaneously, and its daily dose being 1-2,000 mg or 01-900 mg for oral administration or intravenous administration, respectively

Base sequence of chromosomal domain 9p11 participating in cancer and abortion

Abstract: PROBLEM TO BE SOLVED: To obtain a novel base sequence which consists of a human DNA sequence on ninth chromosome monobrachius containing a specific base sequence, participates in cancer and abortion and is useful in the detection or the like of ninth chromosome aberration or KGF-like gene expression aberration and used for the gene diagnosis or the like for cancer and abortion SOLUTION: This base sequence is a novel human DNA sequence on ninth chromosome monobrachius represented by the formula, participates in cancer and abortion and is used in the production or the like of a PCR primer or the like capable of detecting ninth chromosome aberration or keratinocyte growth factor(KGF)-like gene expression aberration and useful in the gene diagnosis or the like for cancer and abortion This human DNA sequence is obtained by selecting YAC clone peculiar to human chromosome from a YAC (yeast artificial chromosome) library originating from the yeast that holds human genomic DNA, preparing a cosmid library by the use of this clone followed by restricting the target domain of the resultant chromosomal breakage point- containing clone by means of FISH analysis and finally specifying the sequence participating in cancer and abortion

Method of testing susceptibility to carcinostatic

Abstract: PROBLEM TO BE SOLVED: To test the susceptibility of cancer cells to carcinostatics in order to judge the resistance ability of the cancer cells to the carcinostatics by detecting the expression amount of a gene in heat shock protein 75 (mt HSP 75) localized in mitochondria. SOLUTION: The susceptibility of cancer cells to carcinostatics is tested by detecting the expression amount of a gene in heat shock protein 75 (mt HSP 75) localized in mitochondria by the use of northern plotting for the transcriptional product of the gene in mt HSP 75 or western plotting for the translation product of the gene in mt HSP 75, because the expression amount of the gene in mt HSP 75 increases in the cancer cells which show resistance to carcinostatics, e.g. cisplatin and the like, and is on a low level in the cancer cells which show susceptibility to carcinostatics. Further, by this susceptibility test for carcinostatics, it is possible to previously judge whether the cancer cells have resistance ability to carcinostatics or not and more effectively carry out treatment.

Phosphatidylinositol 4-kinase assay in ovarian carcinoma cells.

Abstract: Membrane-constituting phospholipids include glycerol phospholipids such as phos- phatidylcholine and phosphatidylinositol (PI) and sphingolipids. Recently, signal transduction starting from hydrolysis of these phospholipids have attracted attention as regulatory mechanisms for cell growth, differentiation, and apoptosis. During this process, phosphatidylcholine is hydrolyzed by phospholipase-D into phosphatidic acid and choline, and the former is dephosphosphorylated into diacylglycerol (DAG). DAG stimulates protein kinase C and often promotes cell growth or differentiation. In case of PI, it is first phosphorylated by PI 3-kinase or PI 4-kinase. PI 3-kinase is often activated by phosphotyrosine of the activated growth factor receptors. Metabolic pathways including PI 4-kinase are now known as classical PI turnover pathways. PI-4-P formed with PI 4-kinase is then phosphorylated by PI-4-P kinase into PI-4,5-P(2). Phosphatidylinositol-specific phospholipase C(PI-PLC) is the rate-limiting enzyme of PI turnover (1), and catalyzes the hydrolysis of PI-4,5-P(2) to produce two second messengers, inositol 1,4,5-trisphosphate (IP(3)) and DAG. The former mobilizes Ca(2+)from internal stores by binding to the receptor on endoplasmic reticulum. PI-PLC is activated in response to a wide variety of physiological stimuli such as growth factors and hormones and often shows enhanced activity in transformed cells.