Youssef Emile M, Estecio Marcos R H, Issa Jean-Pierre J
Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030, USA.
Cancer Biol Ther. 2004 Jan;3(1):82-6. doi: 10.4161/cbt.3.1.591. Epub 2004 Jan 9.
Tumor suppressor genes can become inactivated in cancer via hypermethylation of their promoter. The retinoic acid receptor beta (RARbeta) gene is expressed from two distinct promoters, both of which have CpG islands. RARbeta1 is expressed primarily during embryogenesis, whereas RARbeta2 is expressed in adult tissues and hypermethylated in a number of cancer cells. We used combined bisulfite restriction analysis to evaluate their methylation in colorectal mucosa and tumors. Methylation of RARbeta1 was detected, with a mean of 2% in normal colon tissues in young subjects (< 32 years), and 16% in older subjects (> 75 years) (P < 0.001). Using paired normal/tumor tissue samples, we found higher mean methylation rate in tumors than in adjacent normal tissue (mean, 46% versus 16%; P < 0.001) and hypermethylation of RARbeta1 in all eight cell lines examined. By RT-PCR, RARbeta1 was not expressed in normal adult colon tissues and its expression could not be efficiently activated in most cell lines by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR). RARbeta2 methylation was also observed in normal colon tissues and was lower in young individuals than in older ones (mean, 11% versus 23%; P < 0.05). Among paired samples, RARbeta2 methylation was higher in tumor tissue than in normal tissue in 14 cases, vice versa in 7 cases, and equal in 6 cases. All eight cell lines were hypermethylated and did not express RARbeta2, but RARbeta2 expression could be reactivated easily by 5-Aza-CdR. We suggest that the embryonic RARbeta1 isoform is readily hypermethylated in aging colon mucosa and all colorectal cancers because of its lack of expression in normal tissues. The adult RARbeta2 isoform also shows age-related methylation in normal tissues but more variable methylation in colorectal cancer, perhaps because its expression offers continued protection against methylation or its silencing does not provide a selective advantage in the early stages of the disease.
肿瘤抑制基因可通过其启动子的高甲基化在癌症中失活。维甲酸受体β（RARβ）基因由两个不同的启动子表达，这两个启动子都有CpG岛。RARβ1主要在胚胎发育过程中表达，而RARβ2在成人组织中表达，并在许多癌细胞中发生高甲基化。我们使用联合亚硫酸氢盐限制性分析来评估它们在结直肠黏膜和肿瘤中的甲基化情况。检测到RARβ1的甲基化，年轻受试者（<32岁）正常结肠组织中的平均甲基化率为2%，老年受试者（>75岁）为16%（P<0.001）。使用配对的正常/肿瘤组织样本，我们发现肿瘤中的平均甲基化率高于相邻正常组织（平均分别为46%和16%；P<0.001），并且在所检测的所有8种细胞系中RARβ1均发生高甲基化。通过逆转录聚合酶链反应（RT-PCR），RARβ1在正常成人结肠组织中不表达，并且在大多数细胞系中其表达不能被DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-CdR）有效激活。在正常结肠组织中也观察到RARβ2的甲基化，年轻个体中的甲基化率低于老年个体（平均分别为11%和23%；P<0.05）。在配对样本中，14例肿瘤组织中的RARβ2甲基化高于正常组织，7例相反，6例相等。所有8种细胞系均发生高甲基化且不表达RARβ2，但RARβ2的表达可被5-Aza-CdR轻易重新激活。我们认为，胚胎型RARβ1同工型在衰老结肠黏膜和所有结直肠癌中容易发生高甲基化，因为它在正常组织中不表达。成人型RARβ2同工型在正常组织中也显示出与年龄相关的甲基化，但在结直肠癌中甲基化变化更大，这可能是因为其表达持续提供抗甲基化保护，或者其沉默在疾病早期不提供选择优势。
