• Gene expression analysis in prostate cancer: the importance of the endogenous control.

      Vajda, Alice; Marignol, Laure; Barrett, Ciara; Madden, Stephen F; Lynch, Thomas H; Hollywood, Donal; Perry, Antoinette S; Prostate Molecular Oncology, Academic Unit of Clinical and Molecular Oncology, Institute of Molecular Medicine, Trinity College Dublin, Ireland. vajdaa@tcd.ie (2013-03)
      Aberrant gene expression is a hallmark of cancer. Quantitative reverse-transcription PCR (qRT-PCR) is the gold-standard for quantifying gene expression, and commonly employs a house-keeping gene (HKG) as an endogenous control to normalize results; the choice of which is critical for accurate data interpretation. Many factors, including sample type, pathological state, and oxygen levels influence gene expression including putative HKGs. The aim of this study was to determine the suitability of commonly used HKGs for qRT-PCR in prostate cancer.
    • Gene expression and epigenetic discovery screen reveal methylation of SFRP2 in prostate cancer.

      Perry, Antoinette S; O'Hurley, Gillian; Raheem, Omer A; Brennan, Kevin; Wong, Simon; O'Grady, Anthony; Kennedy, Anne-Marie; Marignol, Laure; Murphy, Therese M; Sullivan, Linda; et al. (2013-04-15)
      Aberrant activation of Wnts is common in human cancers, including prostate. Hypermethylation associated transcriptional silencing of Wnt antagonist genes SFRPs (Secreted Frizzled-Related Proteins) is a frequent oncogenic event. The significance of this is not known in prostate cancer. The objectives of our study were to (i) profile Wnt signaling related gene expression and (ii) investigate methylation of Wnt antagonist genes in prostate cancer. Using TaqMan Low Density Arrays, we identified 15 Wnt signaling related genes with significantly altered expression in prostate cancer; the majority of which were upregulated in tumors. Notably, histologically benign tissue from men with prostate cancer appeared more similar to tumor (r = 0.76) than to benign prostatic hyperplasia (BPH; r = 0.57, p < 0.001). Overall, the expression profile was highly similar between tumors of high (≥ 7) and low (≤ 6) Gleason scores. Pharmacological demethylation of PC-3 cells with 5-Aza-CdR reactivated 39 genes (≥ 2-fold); 40% of which inhibit Wnt signaling. Methylation frequencies in prostate cancer were 10% (2/20) (SFRP1), 64.86% (48/74) (SFRP2), 0% (0/20) (SFRP4) and 60% (12/20) (SFRP5). SFRP2 methylation was detected at significantly lower frequencies in high-grade prostatic intraepithelial neoplasia (HGPIN; 30%, (6/20), p = 0.0096), tumor adjacent benign areas (8.82%, (7/69), p < 0.0001) and BPH (11.43% (4/35), p < 0.0001). The quantitative level of SFRP2 methylation (normalized index of methylation) was also significantly higher in tumors (116) than in the other samples (HGPIN = 7.45, HB = 0.47, and BPH = 0.12). We show that SFRP2 hypermethylation is a common event in prostate cancer. SFRP2 methylation in combination with other epigenetic markers may be a useful biomarker of prostate cancer.
    • In silico analysis and DHPLC screening strategy identifies novel apoptotic gene targets of aberrant promoter hypermethylation in prostate cancer.

      Murphy, Therese M; Sullivan, Linda; Lane, Caroline; O'Connor, Lisa; Barrett, Ciara; Hollywood, Donal; Lynch, Thomas; Lawler, Mark; Perry, Antoinette S; Prostate Molecular Oncology, Institute of Molecular Medicine, Trinity College, Dublin, Ireland. murphyth@tcd.ie (Wiley, 2011-01-01)
      Aberrant DNA methylation has been implicated as a key survival mechanism in cancer, whereby promoter hypermethylation silences genes essential for many cellular processes including apoptosis. Limited data is available on the methylation profile of apoptotic genes in prostate cancer (CaP). The aim of this study was to profile methylation of apoptotic-related genes in CaP using denaturing high performance liquid chromatography (DHPLC).