| The LNCaP cell line is a versatile and useful model that is suitable for the study of human prostate cancer in vitro. It has been determined that the elevation of LNCaP intracellular cAMP levels through the addition of membrane permeable cAMP analogues, phosphodiesterase inhibitors, adenylate cyclase activators, or components of the cAMP signal transduction pathway can induce reversible neuroendocrine differentiation. Elucidation of those genes that are differentially expressed between undifferentiated prostate cancer cells and prostate cancer cells that have been induced to differentiate may present new insights for the molecular mechanisms governing neuroendocrine differentiation, early detection of prostate cancer, and/or potential targets for gene therapy. In this study, differential display PCR was used to identify 226 differentially expressed PCR products. Twelve of the differential display PCR products were confirmed by Northern blot analysis and cloned. DNA sequencing and database comparisons were performed. Among the differentially expressed genes, I identified the human ribosomal S3a gene as being down regulated in response to LNCaP differentiation. In order to better ascertain the mechanism by which HRS3a gene expression is decreased during differentiation, the promoter region for this gene was analyzed. Electrophoretic mobility shift assay, antibody supershift assays, site directed mutagenesis, and luciferase reporter gene analysis were employed to authenticate the roles of several transcription factors in the regulation of the S3a gene. I found that two cyclic AMP response elements, a Sp1 element, and a GA-binding protein element were involved in the regulation of HRS3a gene expression. In order to ascertain the effect of HRS3a down regulation in LNCaP cells, antisense phosphorothioate oligonucleotides were designed to inhibit HRS3a gene expression. Treatment of LNCaP cells with antisense HRS3a oligonucleotides did not influence cAMP induced neuroendocrine differentiation but antisense treatment did result in a decrease in LNCaP cell growth. In addition, I demonstrate that morphological changes that are associated with cAMP induced differentiation of LNCaP cells from the epithelial to the neuroendocrine state do not require alterations in gene expression nor the expression of novel proteins. |
