The mechanism of regulation of P-glycoprotein (Analysis of multidrug-resistant mutants that do not express P-glycoprotein)

P-糖蛋白的调控机制（不表达P-糖蛋白的多重耐药突变体分析）

• 批准号: 06044188
• 负责人: HARAGUCHI Misako
• 金额: $ 0.96万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06044188/
• 关键词: P-glycoprotein; Multidrug resistance; Adriamycin; MRP; Glutathione-S-transferase; Topoisomerase; Progesterone; アドリアマイシン

1) We have isolated human epidermoid KB cell lines resistant to high levels of adriamycin, C-A90, C-A120, C-A500 and C-A1000. They were isolated in selection medium containing increasing concentrations of adriamycin, 1mug/ml of cepharanthine, a multidrug-resistance (MDR) reversing agent, and 100nM mezerein, a protein kinas C activating agent. One of the adriamycin-resistant KB cell lines, C-A500, was cross-resistant to drugs that typify the classical multidrug resistance phenotype, such as vincristine, actinomycin D,VP-16 and colchicine.2) The accumulation of adriamycine and vincristine was decreased in C-A500 cells and the efflux of adriamycine from C-A 500 was enhanced compared with parental KB-3-1 cells.These adriamycin-resistant KB cells did not contain detectable levels of P-glycoprotein or overexpress MDR1. Multidrug-resistance-associated protein (MRP) and MRP mRNA were expressed in the adriamycin-resistant KB-cells, C-A120, C-A500 and C-A1000, but not in parental KB-3-1 and reve … More rtant C-AR cells. The MRP gene was amplified in these cells that overexpressed MRP mRNA.4) DNA topoisomerase II levels were markedly decreased in C-A500 and C-A1000 cells but only slightly decreased in C-A120 cells. These results indicate that MRP overexpressed in the resistant cells may be responsible for the reduced accumulation of adriamycin and vincrisrine and that both the increased expression of MRP and decreasd levels of topoisomerase II underlie te drug resistance in C-A120, C-A500 and C-A1000 cell lines.5) We investigated the expression of MRP mRNA in multidrug-resistant KB cell line (KB-8-5, KB-C2, C-A40 and C-A120), human non-small-cell lung carcinomas (NSCLC), gastric and colorectal carcinomas and compared it with that in drug-sensitive human KB cells. MRP gene expression was elavated in 8 of 9 (89%) squamous-cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous-cell carcinomas (L13,18,19 and 20) was more than 3.6 times higher than in KB-3-1 cells, and the average MRP mRNA expression level of all squamous-cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. Less

1) 我们分离了对高水平阿霉素、C-A90、C-A120、C-A500 和 C-A1000 具有抗性的人表皮样 KB 细胞系。它们在含有浓度递增的阿霉素、1mug/ml千金藤素（一种多药耐药（MDR）逆转剂）和100nM mezerein（一种蛋白激酶C激活剂）的选择培养基中分离。其中一种阿霉素耐药 KB 细胞系 C-A500 对经典多药耐药表型的药物具有交叉耐药性，如长春新碱、放线菌素 D、VP-16 和秋水仙碱。 2) C-A500 细胞中阿霉素和长春新碱的积累减少，C-A 500 中阿霉素的外流减少 与亲本 KB-3-1 细胞相比增强。这些阿霉素耐药 KB 细胞不包含可检测水平的 P-糖蛋白或过度表达 MDR1。多药耐药相关蛋白 (MRP) 和 MRP mRNA 在阿霉素耐药 KB 细胞 C-A120、C-A500 和 C-A1000 中表达，但在亲本 KB-3-1 和 reve … More rtant C-AR 细胞中不表达。 MRP 基因在这些过表达MRP mRNA 的细胞中扩增。4) DNA 拓扑异构酶II 水平在C-A500 和C-A1000 细胞中显着降低，但在C-A120 细胞中仅略有降低。这些结果表明，MRP在耐药细胞中过表达可能是导致阿霉素和长春新碱积累减少的原因，并且MRP表达增加和拓扑异构酶II水平降低是C-A120、C-A500和C-A1000细胞系耐药的基础。 5)我们研究了多重耐药KB细胞系中MRP mRNA的表达 (KB-8-5、KB-C2、C-A40 和 C-A120)、人非小细胞肺癌 (NSCLC)、胃癌和结直肠癌，并将其与药物敏感的人 KB 细胞进行比较。 MRP 基因表达在 9 例肺鳞状细胞癌中的 8 例 (89%) 中升高。此外，4种鳞状细胞癌（L13、18、19和20）中的MRP表达量比KB-3-1细胞高3.6倍以上，并且所有鳞状细胞癌的平均MRP mRNA表达水平显着高于肺腺癌、结直肠癌和胃癌。较少的

项目成果

Miyadera,K.,Sumizawa,T.,Haraguchi,M.,Yoshida,H.,Konstanty,W.,Yamada,Y.and Akiyama,S.: "Role of thymidine phosphorylase activity in the angiogenic effect of platelet-derived endothelial cell growth factor/thymidine phosphorylase." Cancer Res.55. 1687-1690

Miyadera，K.，Sumizawa，T.，Haraguchi，M.，Yoshida，H.，Konstanty，W.，Yamada，Y.和 Akiyama，S.：“胸苷磷酸化酶活性在血小板源性内皮细胞的血管生成作用中的作用

Miyadera, K., Dohmae, N., Takio, K., Sumizawa, T., Haraguchi, M., Furukawa, T., Yamada, Y.and Akiyama, S.: "Structural characterization of thymidine phosphorylase purified from human placenta." Biochem.Biophys.Res.Commun.212. 1040-1045 (1995)

Miyadera, K.、Dohmae, N.、Takio, K.、Sumizawa, T.、Haraguchi, M.、Furukawa, T.、Yamada, Y. 和 Akiyama, S.：“从人胎盘中纯化的胸苷磷酸化酶的结构表征

Haraguchi M.,Miyadera K.,Uemura K.,Sumizawa T.,Furuakawa T.,Yamada Y.,Ymada K.,and Akiyama S.: "Angiogenic activity of enzymes" Nature. 368. 198-198 (1994)

原口 M.、Miyadera K.、Uemura K.、Sumizawa T.、Furuakawa T.、Yamada Y.、Ymada K. 和 Akiyama S.：“酶的血管生成活性”自然。