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

在这项研究中，我们重点研究了一组与卵巢癌发生有关的端粒酶基因。通过各种可用的分子和细胞遗传学技术，对卵巢癌的细胞系和手术标本进行了分析，以期在临床上探讨以下问题。1)端粒长度缩短和端粒酶活性加速是否与肿瘤的发生有关；2)如果是，与肿瘤进展的哪个阶段有关；3)它们与其他癌基因和抑癌基因的通讯方式。我们设计了不同于常规TRAP方法的检测端粒酶活性的新的PCR引物，并成功地在自动测序仪上进行了电泳和计算机分析。片段分析程序实现了对端粒酶产生的荧光梯带的准确和方便的定量测量。将该检测系统应用于定量分析…端粒酶活性在细胞系、原发或转移性肿瘤、化疗后复发肿瘤和福尔马林固定的组织切片中有更多的裂解。在我们检测的6个卵巢癌细胞系中，如预期的那样检测到高度的端粒酶活性。然而，对于卵巢癌的手术和病理标本，我们未能获得稳定的结果。这一失败被认为是由于组织提取物中某些端粒酶或聚合酶链式反应的抑制物质所致。因此，研究了几种提取组织蛋白质的技术，但这一问题在研究期间没有被克服。我们还建立了卵巢癌和宫颈癌涂片细胞端粒酶活性的原位检测，并取得了具有临床应用前景的初步结果。我们计划不断改进，建立卵巢癌端粒酶活性的定量检测方法，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。我们的最终目标是阐明端粒酶在肿瘤发生机制中的作用，并检查其临床应用的可行性。较少

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