Cloning of tissue-specific expressing genes in huma ovarian cancer cells and basic study of the adenovirus-mediaetd p16 gene therapy.

卵巢透明细胞腺癌是由于化疗耐药和/或早期淋巴结转移而预后较差的组织亚型。它有时会受到子宫内膜异位症和日本常见亚型的影响。另一方面，卵巢浆液性腺癌是常见的上皮性癌组织亚型，虽然腹膜播散频繁，但对化疗高度敏感。我们尝试用差异显示法鉴定透明细胞腺癌中特异性表达的基因，SP17基因被确定为候选基因之一。SP17基因的功能尚不清楚，因此将SP17基因全长插入腺病毒载体，感染浆液腺癌细胞。作为进一步的确证性实验，我们在透明细胞腺癌中引入RNAi干扰SP17基因，并分析SP17基因的功能。应用紫杉醇治疗或不应用紫杉醇治疗卵巢癌，对卵巢癌细胞进行MTS试验和细胞死亡检测。提示SP17在卵巢透明细胞腺癌化疗耐药过程中发挥重要作用。P16INK4A基因是周期蛋白依赖性激酶抑制剂，是肿瘤抑制基因之一，是细胞周期G1-S进入的关键分子。在大约30%的上皮性卵巢癌中，由于杂合性缺失、启动子区域甲基化以及编码区域的一些错义突变，P16基因功能丧失。将其插入腺病毒载体，作为基因补充治疗的基础实验。为了控制p16基因的功能，我们将缺乏DNA结合域的ER基因与p16基因全长结合构建。ER-p16融合蛋白仅在雌激素处理条件下才有表达功能，适合在体内应用。理论上，p16功能仅在雌激素存在的条件下表达。p16基因引起的G1细胞周期阻滞仅在雌激素阳性的情况下观察到，这是因为雌激素受体内化携带p16蛋白。MTT法仅在雌激素存在条件下观察到缺乏野生型p16基因的卵巢癌细胞因感染ER-p16融合蛋白而生长停滞。Western Blot分析证实了雌激素刺激下的细胞内转换系统。报道了基因治疗应用的可能性。少

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 … More 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