Biomarkers of ovarian cancer & molecular genetics of HOSE cell transformation

卵巢癌的生物标志物

• 批准号: 6230162
• 负责人: ANDREW K. GODWIN
• 金额: $ 5.07万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2004-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6230162
• 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）该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，我们建议使用改进的抑制减法杂交（SSH）方法鉴定在HOSE细胞恶性转化中差异表达的基因。我们将使用整合SSH （CSSH）来鉴定在HOSE细胞的永生化和恶性转化中过度表达或代表性不足的基因。其次，我们建议使用CSSH鉴定BRCA1杂合的HOSE细胞中差异表达的基因（+/-）。我们之前报道过，与对照表面上皮细胞相比，BRCA1突变等位基因携带者的卵巢表面上皮细胞在体外具有不同的表型，这些特征在BRCA1野生型等位基因失活的情况下仍然存在。这些结果表明，在家族性卵巢癌的发生过程中，BRCA1的两个拷贝丢失之前可能存在其他遗传变化。我们将比较HOSE细胞群体中与BRCA1载体状态相关的差异表达基因。第三，我们建议确定CSSH鉴定的卵巢癌候选致癌基因在卵巢良性、交界性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将被杂交到单个阵列并确定表达模式。表达模式与模型结果一致的克隆将通过多次组织northern blot分析来评估组织特异性表达。第四，我们建议通过对易患癌症的卵巢进行原位杂交，建立候选卵巢癌致癌基因在肿瘤前病变中的表达模式。我们之前已经在患卵巢癌风险增加的女性卵巢中发现了一种肿瘤前表型。一旦我们确定了几个基因的表达，原位杂交和免疫组织化学方法将用于评估它们的RNA和蛋白质表达模式，可能在癌症发展的早期阶段。总的来说，提出的研究应该使我们能够(i)确定我们之前描述的组织学表型是否可能是卵巢癌的真正前兆，（ii）确定在卵巢癌发生过程中BRCA1失活之前是否有其他遗传变化，（iii）确定在卵巢表面上皮中特异性表达的基因，而不是整个卵巢，以及（iv）确定早期替代中间终点的卵巢癌生物标志物。