MOLECULAR MARKERS FOR PROSTATE CANCER

前列腺癌是男性中最常见的癌症，但目前可用于前列腺癌早期检测的方法不足且准确性有限。该项目的总体目标是识别前列腺癌中的分子异常，以开发新的工具来早期诊断和评估前列腺癌患者的预后。这将通过监测不同类型的前列腺组织中基因表达的差异来实现。差异表达的候选基因可能与肿瘤的发生没有直接关联，但可能有助于开发诊断标记。我们将使用差异显示、逆转录聚合酶链式反应和基因发现阵列筛选来比较5个人前列腺肿瘤细胞系的mRNA丰度。可识别的cdna片段的表达模式将通过细胞系的RNA印迹分析来确认。阳性候选基因将被克隆、测序并通过BLAST分析进行鉴定。下一步，将评估识别出的cDNA作为前列腺组织中肿瘤标志物的潜力。前瞻性标记物将用于筛选前列腺癌患者的组织样本。石蜡包埋的档案或新鲜样本将通过原位杂交进行筛选。新鲜组织将通过RT-PCR进行筛选。筛选未受累、结节状增生、不同级别PIN和前列腺癌5组组织，并对结果进行统计分析。一个或两个最突出的候选cdna片段将被用于开发优化的诊断测试。已知同源性的潜在标记基因的全长或部分cDNA将通过RT-PCR被克隆。通过快速扩增或筛选文库的方法，可以克隆出身份未知的基因。克隆的cDNA将在大肠杆菌中表达，更多常见的蛋白将用于制备多克隆抗体。如果真的可以使用合成肽来提高抗体，根据表达模式，将开发一种基于多克隆抗体的ELISA法或更灵敏的基于RTPCR的分析法。

DESCRIPTION: (adapted verbatim from the investigator's abstract) Prostatic adenocarcinoma is the most common cancer diagnosed in males, yet currently available methods for early detection of prostate cancer are inadequate and limited in accuracy. The overall goal of this project is to identify molecular abnormalities in prostate tumors to develop new tools for early diagnosis and evaluating prognosis of patients with prostate cancer. This will be accomplished by monitoring differences in gene expression in various types of prostatic tissues. Differentially expressed candidate genes may not be directly associated with tumorigenesis but may be useful for developing diagnostic markers. We will use differential display, reverse transcriptase PCR and gene discovery array filter screening to compare mRNA abundance in 5 human prostate tumor cell lines. Expression patterns of identifiable cDNA fragments will be confirmed by RNA blot analysis of cell lines. Positive candidates will be cloned, sequenced and identified by BLAST analysis. Next, identified cDNAs will be evaluated for their potential as tumor markers in prostate tissues. Prospective markers will be used to screen tissue samples from patients with prostate cancer. Paraffi- embedded archival or fresh samples will be screened by in situ hybridization. Fresh tissues will be screened by RTPCR. Five groups of tissue including uninvolved, nodular hyperplasia, different grades of PIN and prostatic adenocarcinoma will be screened, and results evaluated by statistical analysis. One or two of the most prominent candidate cDNA fragments will be pursued to develop an optimized diagnostic test. Either a full length or partial cDNA of a potential marker gene with known identity will be cloned by RTPCR. cDNA of genes with unknown identity will be cloned by rapid amplification of cDNA or by screening a cDNA library. Cloned cDNAs will be expressed in E. coli and more common proteins will be used to raise polyclonal antibodies. If indeed synthetic peptides may be used to raise antibody, depending upon the expression pattern, either an ELISA based assay using a polyclonal antibody or a more sensitive RTPCR based assay will be developed.