Molecular Features of Hormone-Refractory Prostate Cancer Cells by Genome-wide Gene-expression Profiles

To characterize the molecular features of clinical (Hormone-Refractory Prostate Cancers) HRPCs, we generated the precise gene-expression profiles of 25 clinical HRPCs and 10 hormone-sensitive prostate cancers (HSPCs) by genome-wide cDNA microarrays combining with laser microbeam microdisection. Keywords: disease status analysis 25 clinical HRPC tissues were procured from 18 HRPC patients through prostate biopsy, TUR-P, bone biopsy, or warm autopsy. We microdissected cancer cells from these frozen slices by means of LMM (laser microbeam microdissection) system (EZ cut system with a pulsed ultraviolet narrow beam-focus laser, SL Microtest GmbH, Germany). Simultaneously, 10 hormone-sensitive or -naïve prostate cancers (HSPCs) were also microdissected from 10 untreated operable cases undergoing radical prostatectomy. Normal prostatic epithelial cells were also microdissected from one benign prostatic hyperplasia (BPH) patient and four bladder cancer patients, where we confirmed no apparent prostate cancers or PINs histopathologically, and we used the RNA mixtures of these microdissected normal prostate epithelial cells as a reference of cDNA microarray analysis. Extraction protocol 100,00-200,000 tumor cells or normal cells were isolated selectively using the EZ cut system with a pulsed ultraviolet narrow beam-focus laser (SL Microtest GmbH, Germany) in accordance with the manufacturer’s protocols. Total RNAs were isolated from captured cells with QIAGNE RNAeasy Kit (QIAGEN). After DNAse I treatment, total RNAs were subjected to two rounds of T7-based amplification using MEGAscript High Yield Transcription Kit (Ambion), which yielded 50-100 ug of amplified RNA (aRNA) from each sample. Label protocol 2.5 ug aliquots of aRNA from tumor cells or normal cells were annealed with random primer for 10 min at 70oC. They were labeled in a 50ul volume by reverse transcription reaction for 2 hours at 37oC using Superscript II reverse transcriptase (Invitrogen) with 1mM Cy5-dCTP for tumor cells or Cy3-dCTP for normal cells (Amersham) and 25mM each dATP, dGTP, and dTTP. The labeling reaction was stopped by adding 6ul 2.5 N NaOH and incubation for 30 min at 65oC and neutralized by 20ul 1M Tris-HCl (pH7.4) and 7ul 2.5N HCl. The labeled cDNAs were purified by QIAquick PCR Purification Kit (QIAGEN). Hybridization protocol Labeled cDNAs were mixed with microarray hybridization solution version 2 (Amersham) and formamide (Sigma) to a final concentration of 50%. After hybridization for 14-16 hours at 42 oC in Automated Slide Processor, the slides were washed in 2xSSC and 1% SDS for 10min at 55 oC, washed in 0.2xSSC and 0.1% SDS for 10min at 55 oC, and washed in 0.1xSSC for 1 min at room temperature in Automated Slide Processor. Scan protocol Microarray slides were scanned using the Array Scanner Generation III (Amersham) after hybridization. Data processing Images were gridded and the fluorescent intensity of each spot (Cy5/Cy3) was evaluated photometrically by the ArrayVision computer program (Imaging Research, Inc., St. Catharines, Ontario, Canada) . The fluorescent intensities of Cy5 (tumor) and Cy3 (control) for each target spot were adjusted so that the mean Cy3/Cy5 ratio of the 52 housekeeping genes was equal to one. Because data derived from low signal intensities are less reliable, we determined a cut-off value on each slide and excluded genes from further analysis when both Cy3 and Cy5 dyes yielded signal intensities lower than the cut-off value. For other genes, we calculated the Cy5/Cy3 ratio using the raw data of each sample.