The main goal of this proposal is to identify and characterize the epigenetic alterations that are involved in aging and race-related prostate cancer. Specific hypotheses: 1) The DNA methylation status of most frequently deleted genes [ERs, E-cadherin, CD44 and GSTPI (glutathione S-transferase pi)] will be different in aging and race-related prostate cancer. 2) Identification of novel DNA methylated genes will provide us the potential biomarkers of aging and race-related prostate cancer. 3) DNA methyltransferase, DNA demethylase and histone deacetylase activities will correspond to DNA methylation status in aging and race-related prostate cancer.
Specific Aim number 1. To test the hypothesis that inactivation of ER genes by DNA methylation can be used as biomarkers for aging and race-related prostate cancer. We hypothesize that ERalpha and ERbeta are differentially expressed in different aging and race related prostate cancer. To test this hypothesis we will investigate the mRNA and protein expression of ER types (alpha and beta) in different stages and grades of prostate cancer. Protein expression of ERs will be analyzed by immunohistochemistry (for localization) and western blotting (for quantitation). RT-PCR (for screening) and northern blotting (for quantitation) will analyze Gene expression. DNA methylation will be analyzed by sodium bisulfite methylation techniques and confirm by direct DNA sequencing.
Specific Aim number 2. To test the hypothesis that inactivation of E-cadherin, CD44 and GSTPI genes by DNA methylation can be used as biomarkers for aging and race-related prostate cancer. Based on our preliminary data and prior publications, E-cadherin, CD44 and GSTPI genes are most frequently inactivated by DNA methylation in prostate cancer. We hypothesize that the expression or inactivation of these genes will be different in aging and race- related prostate cancer. To test this hypothesis, we will first determine the protein expression of these genes in prostate cancer using immunohistochemistry (for localization), and western blotting (for quantitation). RNA expression will be analyzed by RT-PCR (for screening) and northern blotting (for quantitation). CpG methylation will be analyzed by sodium bisulfite methylation techniques and confirm by direct DNA sequencing.
Specific Aim number 3. To test the hypothesis that identification of new DNA methylated genes by restriction landmark genomic scanning (RLGS) can be used as novel biomarkers for aging and race-related prostate cancer. We hypothesize that identification of novel genes that are inactivated by DNA methylation will be important in understanding the mechanisms of aging and race-related prostate cancer. To test this hypothesis, we will analyze new genes that are methylated in aging and race-related prostate cancer. We will use Restriction landmark genomic scanning for methylation (RLGS) to survey genome wide methylation alterations in aging and race-related prostate cancer and subsequently verify potentially hypermethylated gene loci using sodium bisulfite sequencing technique.
Specific Aim number 4. To investigate the mechanisms of DNA methylation in aging and race-related prostate cancer. We hypothesize that DNA methyltransferase, DNA demethylase, histone deacetylase are responsible for regulation of DNA hypermethylation. To test this hypothesis, we will analyze DNA methyltransferase, DNA demethylase and histone deacetylase enzyme activity, mRNA and protein expression in different age and race related prostate cancer. Accomplishment of these experiments will demonstrate whether DNA methylated genes are involved in aging and race-related prostate cancer. This information can be used for better management of prostate cancer using DNA methylated genes as diagnostic or prognostic biomarkers.
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