Nguyen T T, Nguyen C T, Gonzales F A, Nichols P W, Yu M C, Jones P A
Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, CA 90089-9181, USA.
Prostate. 2000 May 15;43(3):233-42. doi: 10.1002/(sici)1097-0045(20000515)43:3<233::aid-pros10>3.0.co;2-s.
Downregulation of genes which negatively control cell cycle progression represents a possible mechanism for prostate tumorigenesis. We examined the expression levels of the p16, p15, p14, and retinoblastoma-susceptibility (RB) genes in primary prostate cancers and human prostate cancer cell lines, and correlated this with the DNA methylation levels of two loci in p16.
The mRNA levels of p16, p15, and p14 were examined by reverse transcriptase-PCR (RT-PCR). DNA methylation of the p16 5' CpG island was determined by bisulfite genomic sequencing, while methylation of exon 2 shared by the p16 and p14 genes was measured by a quantitative bisulfite-based technique, methylation-sensitive single-nucleotide primer extension (Ms-SNuPE). RB protein levels were assessed by immunohistochemical staining of histologic sections of normal and tumor prostate tissues, using a monoclonal antibody (mAB).
Overexpression of p16 mRNA was found in 6/9 (67) of prostate tumors compared to the adjacent normal prostate, whereas elevated p14 and p15 levels were only observed in 2/9 (22) and 1/6 (17) of prostate cases, respectively. There was no statistically significant association of grade (P = 0.18) and stage (P = 1.00) of prostate cancer to the elevated p16 levels in the tumors. The p16 5' CpG island was completely unmethylated in these tissues. In contrast, exon 2 of p16/p14 was methylated in both the tumor and normal adjacent prostates, and was increased in 8/11 (73) of tumors relative to normal tissues. There was no association between p16 overexpression and increased p16/p14 exon 2 methylation in these tumors (P = 1.00). Diminished RB levels in prostate tumors that had upregulated p16 mRNA were found, although absent RB was also detected in tumors without elevated p16 levels. The expression levels of the two genes, RB and p16, were not correlated statistically (P = 0.16).
Our studies show that although the levels of the cell cycle regulators p16, p15, p14, and Rb are altered in prostate cancers, there is no apparent correlation to grade, stage, or any pattern of regulation between the related genes. Exon 2 of p16/p14 is methylated in a majority of prostate tumors compared to the unmethylated upstream 5' region, and may be a potential tumor marker for human prostate cancer.
负向调控细胞周期进程的基因下调是前列腺癌发生的一种可能机制。我们检测了原发性前列腺癌和人前列腺癌细胞系中p16、p15、p14和视网膜母细胞瘤易感（RB）基因的表达水平，并将其与p16基因两个位点的DNA甲基化水平相关联。
采用逆转录聚合酶链反应（RT-PCR）检测p16、p15和p14的mRNA水平。通过亚硫酸氢盐基因组测序确定p16 5'CpG岛的DNA甲基化，而通过基于亚硫酸氢盐的定量技术——甲基化敏感单核苷酸引物延伸（Ms-SNuPE）检测p16和p14基因共有的外显子2的甲基化。使用单克隆抗体（mAB）通过对正常和肿瘤前列腺组织的组织学切片进行免疫组织化学染色来评估RB蛋白水平。
与相邻正常前列腺相比，在6/9（67%）的前列腺肿瘤中发现p16 mRNA过表达，而p14和p15水平升高分别仅在2/9（22%）和1/6（17%）的前列腺病例中观察到。前列腺癌的分级（P = 0.18）和分期（P = 1.00）与肿瘤中p16水平升高无统计学显著关联。这些组织中p16 5'CpG岛完全未甲基化。相反，p16/p14的外显子2在肿瘤和相邻正常前列腺中均甲基化，并且相对于正常组织，8/11（73%）的肿瘤中其甲基化增加。这些肿瘤中p16过表达与p16/p14外显子2甲基化增加之间无关联（P = 1.00）。在p16 mRNA上调的前列腺肿瘤中发现RB水平降低，尽管在p16水平未升高的肿瘤中也检测到RB缺失。RB和p16这两个基因的表达水平无统计学相关性（P = 0.16）。
我们的研究表明，尽管前列腺癌中细胞周期调节因子p16、p15、p14和Rb的水平发生了改变，但与分级、分期或相关基因之间的任何调控模式均无明显相关性。与未甲基化的上游5'区域相比，p16/p14的外显子2在大多数前列腺肿瘤中甲基化，可能是人类前列腺癌的潜在肿瘤标志物。
