2004 Fiscal Year Final Research Report Summary
Cloning of tissue-specific expressing genes in huma ovarian cancer cells and basic study of the adenovirus-mediaetd p16 gene therapy.
| Project/Area Number |
15591730
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Gunma University |
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Principal Investigator |
KANUMA Tatsuya Gunma University Graduate School of Medicine, Department of Gynecology and Reproductive Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (90241885)
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| Co-Investigator(Kenkyū-buntansha) |
MINEGISHI Takashi Gunma University Graduate School of Medicine, Department of Gynecology and Reproductive Medicine, Professor, 大学院・医学系研究科, 教授 (00209842)
AOKI Hiroshi Gunma University School of Medicine, Department of Obstetrics and Gynecology, Assistant, 医学部, 助手 (30344948)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Ovarian cancer / Clear cell carcinoma / Chemo-resistance / SP17 / Gene therapy / Adenovirus / Estrogen receptor / p16INK4A gene |
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| Research Abstract |
Clear cell adenocarcinoma of the ovary is the poor prognostic tissue subtypes due to the chemo-resistance and/or the early lymph node metastases. It is sometimes affected from the endometriosis and frequent subtypes in Japanese. On the other hand, serous adenocarcinoma of the ovary is common epithelial cancer tissue-subtypes, and it is highly sensitive for the chemotherapy though the peritoneal dissemination is frequent. We tried to identify the specifically expressing genes in clear cell adenocarcinoma by using Differential Display method and SP17 gene was identified as one candidate gene. The function of SP17 gene was unknown, so the full length of SP17 gene was inserted into the Adenovirus vector, and it was infected to the serous adenocarcinoma cells. As further confirmative experiment, RNAi interference of SP17 gene was introduced in the clear cell adenocarcinoma, and the function of SP17 gene was analyzed. MTS assay and Cell-death detection assay were performed in the ovarian can
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cer cells, with or without treatment of Paclitaxel, most useful anticancer drug for the ovarian cancers. As results, it was strongly suggested that SP17 plays an important role of chemotherapy-resistance of clear cell adenocarcinoma of the ovary.
P16INK4A gene is a cyclin-dependent kinase inhibitor, one of the tumor suppressor genes, and a key molecule of cell cycle G1-S entry. P16 gene function was loss in about 30% of the epithelial ovarian cancers, due to the loss of heterozygosity, methylation of the promoter region and some missense mutations of coding regions. It was inserted into Adenovirus vector as a fundamental experiment of the gene supplementary therapy. In order to control the function of p16 gene, we made the construct of the ER gene which was lack of DNA binding domain combined with full length of p16 gene. The ER-p16 fusion protein which function would be expressed in only estrogen-treatment condition, was considered to suitable for in vivo application. Theoretically, p16 function was expressed only under the estrogen existence condition. The G1 cell cycle arrest due to p16 gene was observed only in estrogen positive condition because of the estrogen receptor internalization carrying p16 protein. The growth arrest of ovarian cancer cells lack of wild type p16 gene was observed by MTT assay only in estrogen existence condition due to infected ER-p16 fusion proteins. Intracellular switching system by the estrogen stimulus was confirmed by Western Blot analyses. The possibility to the gene therapy application was reported. Less
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