Amplification of Ribosomal RNA Gene Loci in Human Prostate Cancer
Q Zheng, S Yegnasubramanian, AM De Marzo. Johns Hopkins, Baltimore, MD
Background: Nucleolar enlargement is a consistent structural change in many neoplastic cells, and this is especially common in prostatic adenocarcinoma (CaP) and prostatic intraepithelial neoplasia (PIN). Since the nucleolus is organzied by the rDNA, this raises the question of whether part of the nucleolar size expansion is cancer is related to amplification of the rDNA locus. The aim of this study was to quantify ribosomal gene copy number in clinical samples of prostate cancer.
Design: Copy number of rDNA repeats was quantified by real-time PCR using separate primer sets designed to amplify regions coding for all 3 rDNA gene products (28S, 18S, and 5.8S). To control for overall ploidy, 3 different single copy reference genes were used for normalization (HBB, PTGS2, and STK19). Copy numbers were determined using the using the relative standard curve method. Genomic DNA was isolated from frozen tissue sections of radical prostatectomy specimens from n = 21 patients (Gleason scores 6-9). Relative normalized target gene quantity in tumor DNA was determined from the standard curves and divided by the relative normalized target gene quantity of the calibrator, which consisted of DNA from each patient's matched normal prostate tissue.
Results: The median ratio of rDNA copies in tumor vs. normal was 1.45 for 5.8S, 1.43 for 18S, and 1.56 for 28S. The difference between these values and the expected tumor/normal ratio of 1.0 (null hypothesis) was significant for each target (5.8S p=0.0055, 18S p=0.0072, 28S p=0.0129; Wilcoxon Sign Rank test). Overall tumor/normal ratios were increased in 16 of the 21 cases. There was a high level of concordance in median relative copy number (tumor/normal) using different reference genes (e.g. HBB, PTGS2, RP1) with the same target (e.g. 18S rDNA), and using the different target genes in the locus (e.g. for a given patient if 5.8S rDNA was amplified then the 18S and 28S genes were also amplified).
Conclusions: In the majority of cases of prostate cancer there was an increase in copy number of the entire rDNA locus (relative increase ∼1.5 fold). Since there are approximately 400 copies of this repeat unit in normal cells, these increases are on the order of hundreds of extra copies. These results raise the possibility that rDNA gene copy number may: i) influence nucleolar size expansion in cancer cells, and/or ii) play an important role in the development and/or progression prostate cancer.
Category: Genitourinary (including renal tumors)
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 120, Tuesday Afternoon