Biomarkers of ovarian cancer & molecular genetics of HOSE cell transformation

卵巢癌的生物标志物

• 批准号: 6667422
• 负责人: ANDREW K. GODWIN
• 金额: $ 16.54万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-27 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6667422
• 关键词: biomarker; brca gene; gene expression; genetic mapping; human tissue; immunocytochemistry; in situ hybridization; molecular cloning; molecular genetics; neoplastic transformation; northern blottings; ovary neoplasms

DESCRIPTION: (Applicant's Description) The goal of this project is to elucidate the genetic and biological determinants of ovarian cancer, focusing on an in vitro model for ovarian cancer that we have developed (1-4). First, we propose to identify genes that are differentially expressed upon malignant transformation of HOSE cells using a modified suppression subtractive hybridization (SSH) approach. We will use consolidative SSH (CSSH) to identify genes that are either over-expressed or under-represented upon immortalization and malignant transformation of HOSE cells. Second, we propose to identify genes that are differentially expressed in HOSE cells heterozygous for BRCA1 (+/-) using CSSH. We have previously reported that the surface epithelial cells from ovaries of BRCA1 mutant allele carriers have a different phenotype in vitro compared to control surface epithelial cells and these features are present without the inactivation of the remaining wild-type allele of BRCA1. These results suggest that other genetic changes might precede loss of both copies of BRCA1 in the genesis of familial ovarian cancer. We will compare populations of HOSE cells for differentially expressed genes related to their BRCA1 carrier status. Third, we propose to determine the expression pattern of candidate ovarian cancer-causing genes identified by CSSH in benign and borderline ovarian tumors and ovarian carcinomas. To find the subset of genes discovered in our tissue culture model, which are relevant to clinical ovarian tumors, cDNA arrays representing the differentially expressed clones will be created. Reverse transcribed RNAs derived from benign, borderline, early and late stage ovarian tumors will be hybridized to individual arrays and the patterns of expression determined. Clones showing expression patterns consistent with the findings in the model will be evaluated for tissue specific expressions by multiple tissue northern blot analysis. Fourth, we propose to establish the pattern of expression of candidate ovarian cancer-causing genes in preneoplastic lesions by performing in situ hybridization on cancer prone ovaries. We have previously identified a preneoplastic phenotype in the ovaries from women at increased risk for developing ovarian cancer. Once we have confirmed the expression of several genes, in situ hybridization and immunohistochemical approaches will be used to evaluate their RNA and protein expression patterns at potentially the earliest stages of cancer development. Overall, the studies proposed should enable us to (i) determine if the histological phenotype we have previously described is likely to be a true precursor of ovarian cancer, (ii) establish whether other genetic changes precede BRCA1 inactivation in ovarian carcinogenesis, (iii) identify genes that are specifically expressed in the ovarian surface epithelium as opposed to the entire ovary, and (iv) identify early surrogate intermediate endpoint bio- markers of impending ovarian cancer.

描述：(申请人描述)这个项目的目标是阐明卵巢癌的遗传和生物学决定因素，重点是我们开发的卵巢癌体外模型(1-4)。首先，我们建议使用一种改进的抑制消减杂交(SSH)方法来识别软管细胞恶性转化过程中差异表达的基因。我们将使用整合SSH(CSSH)来识别在软管细胞永生化和恶性转化过程中过度表达或表达不足的基因。其次，我们建议使用CSSH来鉴定BRCA1(+/-)杂合子在软管细胞中差异表达的基因。我们以前报道过BRCA1突变等位基因携带者的卵巢表面上皮细胞在体外具有与对照表面上皮细胞不同的表型，这些特征在没有BRCA1剩余野生型等位基因失活的情况下存在。这些结果表明，在家族性卵巢癌的发生中，BRCA1的两个拷贝丢失之前可能存在其他基因变化。我们将比较软管细胞群体中与其BRCA1携带者状态相关的差异表达基因。第三，我们建议确定CSSH确定的候选卵巢癌致癌基因在良性和交界性卵巢肿瘤和卵巢癌中的表达模式。为了找到在我们的组织培养模型中发现的与临床卵巢肿瘤相关的基因子集，将创建代表差异表达克隆的cDNA阵列。来自良性、交界性、早期和晚期卵巢肿瘤的逆转录RNA将与单独的阵列杂交，并确定表达模式。表现出与模型中发现一致的表达模式的克隆将通过多组织Northern印迹分析来评估组织特异性表达。第四，我们建议通过对有癌倾向的卵巢进行原位杂交来建立候选卵巢癌致癌基因在癌前病变中的表达模式。我们之前已经在卵巢中发现了一种癌前表型，这些表型来自卵巢癌风险较高的女性。一旦我们确认了几个基因的表达，将使用原位杂交和免疫组织化学方法来评估它们在潜在的癌症发展的最早阶段的RNA和蛋白质表达模式。总体而言，建议的研究应该使我们能够(I)确定我们先前描述的组织表型是否可能是卵巢癌的真正先兆，(Ii)确定在卵巢癌发生中是否有其他基因变化先于BRCA1失活，(Iii)识别在卵巢表面上皮而不是整个卵巢中特异表达的基因，以及(Iv)确定即将发生的卵巢癌的早期替代中间终点生物标记物。