Young, Charles YF

AbstractBackground: Human glandular kallikrein (hK2) is a serine protease that has 79% amino acid identity with prostate-specific antigen (PSA). Both free hK2 and hK2 complexed to α1-antichymotrypsin (ACT) are present in the blood in low concentrations. We wished to measure hK2 in serum with limited contribution from hK2-ACT for the results.Methods: We developed an automated assay for hK2 with use of a select pair of monoclonal antibodies. The prototype assay was implemented on a Beckman Coulter ACCESS® analyzer.Results: The detection limit of the assay was 1.5 ng/L, the “functional sensitivity” (day-to-day CV <15%) was <4 ng/L, cross-reactivity with PSA and PSA-ACT was negligible, and cross-reactivity with hK2-ACT was 2%. After surgical removal of prostate glands, serum hK2 was <7 ng/L and was <15 ng/L in most healthy women. The median serum concentration of hK2 in healthy men without prostate cancer was 26 ng/L. The median concentration of hK2 was 72 ng/L for men having prostate cancer with lower Gleason scores compared with 116 ng/L for men with more advanced cancer. The concentration of hK2 correlated weakly with PSA, with the mean hK2 concentrations generally 30- to 80-fold lower than PSA concentrations.Conclusion: The availability of a robust, high sensitivity automated assay for hK2 should facilitate further investigations of the role of hK2 measurements in the management of patients with prostate disease.

ScopeAndrogen receptor (AR) signaling is critical for all aspects of prostate growth and tumorigenesis. The glucosinolate‐derived phenethyl isothiocyanate (PEITC) has recently been demonstrated to reduce the risk of prostate cancer (PCa) and inhibit PCa cell growth. We previously reported that p300/CBP‐associated factor (PCAF), a co‐regulator for AR, is upregulated in PCa cells through suppression of the mir‐17 gene. Here, we assessed the effects of PEITC on PCAF expression and AR‐regulated transcriptional activity in PCa cells.Methods and resultsUsing AR‐responsive LNCaP cells, we observed the inhibitory effects of PEITC on the dihydrotestosterone‐stimulated AR transcriptional activity and cell growth of PCa cells. Interestingly, overexpression of PCAF attenuated the inhibitory effects of PEITC on dihydrotestosterone‐stimulated AR transcriptional activity. Expression of PCAF was upregulated in PCa cells through suppression of miR‐17. PEITC treatment significantly decreased PCAF expression and promoted transcription of miR‐17 in LNCaP cells. Functional inhibition of miR‐17 attenuated the suppression of PCAF in cells treated by PEITC.ConclusionOur results indicate that PEITC inhibits AR‐regulated transcriptional activity and cell growth of PCa cells through miR‐17‐mediated suppression of PCAF, suggesting a new mechanism by which PEITC modulates PCa cell growth.

Abstract DNA methylation abnormalities, either gain or loss of methylation in promoters or other regulatory regions, contribute to tumorigenesis by altering the activity of tumor suppressor or oncogenes. To identify the molecular alterations in prostate cancer, we have profiled gene methylation using Infinium Human Methylation27 bead chips in benign prostate tissue, primary prostate tumor tissue from patients who developed metastasis within 5 years of prostatectomy, and serum from patients who developed metastasis. Tissue DNA isolation and bisulfite conversion was done using ZR Genomic DNA kit and EZ methylation kit. Serum DNA isolation and bisulfite conversion was done using ZR serum DNA kit and EZ methylation kit. Methylation data was processed using the Beadstudio average normalization algorithm and differentially methylated genes identified using a two-group T-test p-value < 0.05. To evaluate the impact of methylation on mRNA expression levels, we isolated RNA from LCM-derived epithelial cells of 124 patients using Picopure RNA isolation kit and measured expression using HG-U133+2 microarrays. The gene expression data was preprocessed using GCRMA background correction, FASTLO normalization, affinities-only PM correction, and median polish summarization, and differential gene expression was defined by a two-group T-test p-value < 0.05, a log2 fold change > 1, and a maximum average expression value > log2 7. Comparison of the methylation and gene expression data identified 321 differentially expressed Affymetrix probesets which mapped to genes differentially methylated genes; 272 with differential methylation in the prostate cancer tissue and 97 with differential methylation in the prostate cancer serum. In general, we expected a complementary effect between gene methylation and expression, with hyper-methylation driving decreased expression, and hypo-methylation driving increased expression. This was seen in the prostate cancer tissue methylation data, where 228/272 (84%) probesets possessed this methylation-expression complementarity; 212/238 hyper-methylated genes and 16/34 hypo-methylated genes. The effect was not as strong in the prostate cancer serum methylation data, where 30/97 (31%) probesets displayed the methylation-expression complementarity; 19/79 hyper-methylated genes and 11/18 hypo-methylated genes. Overall, our results revealed a significant correlation of the DNA methylation with the down regulation in the mRNA expression levels of the genes, which could play potential roles in prostate cancer progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4950.