Identification of A Group of Genes Associated with Oncogenesis of Ovarian Cancers

在这项研究中，我们专注于端粒酶基因在一组与卵巢癌的肿瘤发生有关。通过各种可用的分子和细胞遗传学技术，细胞系和卵巢癌手术标本进行了分析，以临床研究以下问题。1)端粒长度的缩短和端粒酶活性的加速是否与癌变相关，2)如果相关，肿瘤进展到哪个阶段，3)它们如何与其他癌基因和抑癌基因沟通。我们设计了不同于TRAP检测中使用的端粒酶活性的PCR引物，并利用这些荧光标记引物在自动测序仪上成功地进行了电泳和计算机分析。片段分析程序对端粒酶产生的荧光阶梯带进行了准确、方便的定量测定。这个新开发的检测系统应用于细胞系、原发性或转移性肿瘤、化疗后复发肿瘤和福尔马林固定组织切片的端粒酶活性的定量分析。在6个卵巢癌细胞系中，我们检测到高水平的端粒酶活性。然而，对于卵巢癌的手术和病理标本，我们没有得到稳定的结果。这种失败被认为是由于一些抑制物质的端粒酶或PCR反应在组织提取物。因此，研究了几种提取组织蛋白的技术，但这一问题在研究期间尚未得到解决。我们还开展了卵巢癌和宫颈癌涂片细胞端粒酶活性原位检测，并取得了具有临床应用前景的初步结果。计划继续改进，以建立端粒酶活性的定量测量，并将其活性程度与临床背景进行比较，包括分期，组织类型和分级，以及一组癌基因和细胞周期相关基因的表达。我们的最终目的是阐明端粒酶在肿瘤发生机制中的作用，并探讨其临床应用的可能性。少

In this study we focused on the telomerase gene among a group of those associated with oncogenesis of ovarian cancers. By a variety of available molecular and cytogenetic techniques, cell lines and surgical specimens of ovarian cancers have been analyzed to clinically investigate following questions. 1) whether the shortening of telomere length and the acceleration of telomerase activity are correlated to carcinogenesis or not, 2) if so, to which stage in tumor progression, 3) how they communicate with other oncogenes and tumor suppresser genes.We designed new PCR primers for detecting telomerase activity, which are different from ordinary ones used in TRAP assay and succeeded in performing electrophoresis and computer analysis on an autosequencer using theses fluorescence-labeled primers. Fragment analysis program led to accurate and convenient quantitative measurement of fluorescent ladder bands produced by telomerase. This newly developed assay system was applied to quantitative ana … More lysis of telomerase activity in cell lines, primary or metastatic tumors, recurrence tumors after chemotherapy, and formalin-fixed tissue sections.In 6 ovarian cancer cell lines we examined high degrees of telomerase activity were detected as expected. However, we failed to obtain stable results for surgical and pathological specimen of ovarian cancers. This failure was thought to result from some inhibitory substance for telomerase or PCR reaction in tissue extracts. Several extraction techniques for tissue proteins were thus examined, but this problem has not been overcome in the research period. We also developed in situ detection of telomerase activity in smear cells of ovarian and cervical cancers, and obtain preliminary results promising clinical application.It is scheduled to make continuous improvements to establish quantitative measurement of telomerase activity in povarian cancers and, compare degrees of their activity with clinical background including stage, tissue type and grade, and expression of a panel of oncogenes and cell cycle-related genes. Our final goal is to clarify the roles of telomerase in oncogenesis mechanism and examine the ossibility of its clinical application. Less