{"created":"2023-06-20T13:21:04.043369+00:00","id":1163,"links":{},"metadata":{"_buckets":{"deposit":"325936b1-c4f3-4237-93f7-1ec79b93fa64"},"_deposit":{"created_by":1,"id":"1163","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"1163"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00001163","sets":["2:430:22"]},"author_link":["0","0","0"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"LEE, Elbert Lan"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"リー, エルベルト ラン"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2005-09-30"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"Resistance of cancer cells to drug treatment is a major problem in cancer therapy. The platinum-based drug cisplatin (cis-diamminedichloroplatinum(II)) is a widely used anticancer drug introduced more than 20 years ago which acts mainly by forming adducts with DNA that cause the induction of apoptosis. However, some types of cancer have high intrinsic resistance to cisplatin; in addition, resistance to the drug frequently appears in initially responsive tumors. The mechanisms of resistance to cisplatin are the subject of intense study because their elucidation will lead to the development of better, more effective treatments for cancer. <br />　A model of cisplatin resistance is provided by the cisplatin-resistant KB (KB/CP4) cell line, which is derived from human KB epidermoid cancer cells. It was found previously that activity of the volume-sensitive, outwardly rectifying (VSOR) chloride channel is virtually absent in these cells. Because VSOR chloride current activation is known to be essential to the progression of apoptosis, it was hypothesized that the absence of the current contributes to the cisplatin resistance of these cells. <br />　An attempt was first made to determine the molecular identity of the VSOR chloride channel or a regulator of it. A strategy for identifying the molecule(s)was suggested by the fact that the KB/CP4 cell line, unlike the KB cell line from which it is derived, does not express volume-sensitive chloride current. Using a subtractive hybridization method, candidates for differentially expressed genes were identified. With semi-quantitative RT-PCR, seven genes were confirmed as being expressed at a higher level in KB cells than in KB/CP4 cells; five of these were genes which had been identified by subtractive hybridization. The genes confirmed as having higher expression in KB cells were lactate dehydrogenase A (LDHA), S100 calcium binding protein A4 (S100A4), thymosin ,,4 (TMSB4X), argininosuccinate synthetase(ASS), placental alkaline phosphatase (ALPP), folate receptor 1 (FOLR1) and GPI-specific phospholipase D1 (GPLD1). All of these genes, except for ALPP, were then cloned into expression vectors and overexpressed in KB/CP4 cells to determine if they could restore VSOR chloride current expression. The overexpression of these genes did not result in restoration of the current in KB/CP4 cells, however. <br />　Because VSOR chloride current could not be restored in KB/CP4 cells by gene overexpression, treatment with drugs that remove gene silencing was tested in an attempt to restore the current. Epigenetic changes which alter chromatin structure--gene promoter methylation and histone deacetylation, in particular--are known to cause inhibition of gene expression, and could potentially contribute to the silencing of genes in KB/CP4 cells. 5-aza-2'-deoxycytidine(Aza-dC) is a drug that reverses DNA methylation by inhibiting DNA methyltransferases, and trichostatin A (TSA) is a drug that reverses histone deacetylation by inhibiting histone deacetylases(HDACs). In KB/CP4 cells treated with Aza-dC, a volume-sensitive chloride current was not induced; however, it was found that the current could be induced in cells treated with TSA. This current was confirmed as VSOR chloride current based on its volume sensitivity, intracellular ATP dependency and sensitivity to two blockers, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. It was found that in addition to this phenotypic change in the expression of VSOR chloride current, alterations in the expression of some genes occurred due to TSA treatment. As assessed by semi-quantitative RT-PCR, two genes previously identified as differentially expressed in KB and KB/CP4 cells, FOLR1 and GPLD1, were upregulated in TSA-treated KB/CP4 cells. <br />　The effects of restoration of VSOR chloride current on cisplatin resistance in these cells were checked by a cell viability assay and caspase-3 activity assay. The viability of cells treated for 48 h with both TSA and cisplatin decreased to a greater extent than that of cells treated with TSA alone, and this decrease could be blocked by treatment with DIDS. Moreover, simultaneous treatment with both TSA and cisplatin for 24 h was found to induce apoptosis in this cisplatin-resistant cell line: caspase-3 activity in cells treated for this length of time with both drugs was elevated compared to that in cells treated with TSA alone. This increase in caspase-3 activity could also be blocked by DIDS. These results demonstrate that cisplatin resistance in KB/CP4 cells treated with the HDAC inhibitor TSA decreases as a result of an increase in apoptosis, at least in part; in other words, TSA caused a sensitization of the cisplatin-resistant tumor cells to cisplatin-induced apoptosis. It is thus suggested that the activity of the VSOR chloride channel is essential for the TSA-induced increase in apoptosis.<br />　In conclusion, this study provides evidence that the absence of VSOR chloride channel activity contributes to cisplatin resistance in a cisplatin-resistant KB cell line, and that restoration of VSOR chloride current in these cells is involved in sensitization to cisplatin-induced apoptosis in this tumor cell line. The identities of genes involved in the functioning of the channel remain to be clarified. ","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第910号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"20 生理科学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2005"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"LEE, Elbert Lan","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲910_要旨.pdf","filesize":[{"value":"265.3 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨","url":"https://ir.soken.ac.jp/record/1163/files/甲910_要旨.pdf"},"version_id":"3b3dd809-89a9-40ea-be9e-696abd05de61"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲910_本文.pdf","filesize":[{"value":"2.1 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/1163/files/甲910_本文.pdf"},"version_id":"31c6cefb-6b77-4ac2-bc6d-03e1a5ce6ddd"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"eng"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Alteration of gene expression in cisplatin-resistant KB cells and functional consequences","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Alteration of gene expression in cisplatin-resistant KB cells and functional consequences"},{"subitem_title":"Alteration of gene expression in cisplatin-resistant KB cells and functional consequences","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["22"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"1163","relation_version_is_last":true,"title":["Alteration of gene expression in cisplatin-resistant KB cells and functional consequences"],"weko_creator_id":"1","weko_shared_id":-1},"updated":"2023-06-20T16:07:44.078717+00:00"}