Semiquantitative RT-PCR was performed on diluted templates of 16 cell lines and one blood sample using primers specific to CSR1 and -actin. 60C for 12 hours and deparaffinized in xylene and ethanol. Antigen retrieval was performed using 25 mmol/L sodium citrate buffer (pH 9.0) at 90C for 15 minutes, followed by treatment of 3% H2O2 to block endogenous peroxidase. The slides were incubated at room heat for 2 hours with anti-CSR1 antibodies at a 1:400 dilution. The sections were NBD-556 then incubated with horseradish peroxidase-conjugated anti-rabbit IgG for 30 minutes at room heat. For visualization, horseradish peroxidase was reacted with 3,3-diaminobenzidine answer (DAKO, Carpinteria, CA). Cells were counterstained with hematoxylin. Immunohistochemical specificity was verified by incubating comparable slides with preimmune sera. Results CSR1 Expression Is usually Down-Regulated in Prostate Malignancy The CSR1 NBD-556 Rabbit Polyclonal to BMX gene was originally identified as a cellular stress response protein. It is widely expressed in most tissues, and it functions to protect cells from your damaging effects of reactive oxygen intermediates.7 Analysis of three data sets of Affymetrix oligo array indicates that CSR1 mRNA expression was down-regulated in the prostate cancer tissues (Table 1). CSR1 was down-regulated in 9 of 15 prostate malignancy samples overall in the Luo et al8 data set and in 5 of 7 relapsed prostate cancers. An average 1.93-fold decrease was observed for nonrelapsing prostate cancer, and an average 4.3-fold decrease was observed for relapsing prostate cancer. In the data of Yu et al,3 CSR1 was down-regulated 3.26-fold in prostate cancer that did not metastasize within 5 years of prostatectomy. CSR1 was down-regulated 4.49-fold in prostate cancer samples with PSA relapse. The higher differential data of the most recent analysis is a result of using completely normal prostate tissues as the controls. When using 23 prostate malignancy samples from LaTulippe et al9 to compare with donors samples, comparable results were obtained. To validate the microarray data, semiquantitative reverse transcriptase-PCR (RT-PCR) on eight selected prostate samples and three prostate malignancy cell lines were performed. The results showed consistent and substantial down-regulation of CSR1 mRNA in cancerous prostate tissues (Figures 1A and 2C). Open in a separate window Physique 1 Down-regulation of CSR1 expression NBD-556 in prostate malignancy. A: Semiquantitative RT-PCR. Total RNA (1 g) of normal prostate tissues (PD14 and PD16) and prostate malignancy samples (47T, 101T, 83T, 85T, 91T, and 68T) were reverse-transcribed. PCR was performed on diluted themes using primers specific to CSR1 and -actin. B: Immunostaining of CSR1 protein on prostate samples. Representative images of CSR1 expression scored as 3+ (normal), 1+ (malignancy), and 0 (malignancy) are shown. C: Expression of CSR1 protein was down-regulated in prostate malignancy samples (reddish) relative to normal prostate (blue). Open in a separate window NBD-556 Physique 2 Methylation of CpG island in the promoter region of CSR1. A: Diagram of CpG island in promoter/exon1 region of CSR1. Orange spikes show CpG. Straight arrows indicate the position of the PCR primers. Putative mRNA start site is usually indicated by right angle arrow. B: Primer specificity of CSR1 methylation and nonmethylation primers. Unmethylated DNA themes were treated with (lanes 1 and 2) or without (lanes 3C6) SSSI methylase. The samples were subsequently treated with (lanes 1C4) or without (lanes 5 and 6) NBD-556 sodium bisulfite. PCR was performed with methylation primers (lanes 1, 3, and 5) or unmethylation primers (lanes 2, 4, and 6). C: Correlation of methylation status and suppression of CSR1 expression. Semiquantitative RT-PCR was performed on diluted.