Kim Young Il, Shin Min-Kyung, Lee Jin-Woo, Chung Joo-Ho, Lee Mu-Hyoung
East-West Medical Research Institute, Kyunghee University, College of Medicine, Seoul 130-702, Korea.
Oncol Rep. 2009 Jan;21(1):159-64.
KAI1/CD82, a metastasis suppressor gene of prostate cancer, is located on the human chromosome 11p11.2. Down-regulation of KAI1/CD82 during tumor progression and metastasis has been reported in several cancers, but the mechanism of this down-regulation remains unknown. The relationship between down-regulation of KAI1/CD82 mRNA expression and KAI1/CD82 gene alterations in human melanoma cell lines were investigated. The promoter methylation status was examined after a 331-bp GC-rich fragment of the promoter region was amplified in G361, SK-MEL-24 and SK-MEL-28 cell lines treated with bisulfite. In order to detect methylated CpGs in all three cell lines, 331-bp fragments were sequenced. To examine the restoration of KAI1/CD82 mRNA and protein expression, the cells were exposed to methylase inhibitor, 5-aza-2'-deoxycytidine (5-AzaC). Bisulfite-sequencing data showed no methylation in G361 and SK-MEL-24 cells, and slight methylation in SK-MEL-28 cells at CpG sites 23-26 in the promoter. Real-time PCR and flow cytometry analysis showed that 5-AzaC-treated cells restored KAI1/CD82 mRNA and protein expression in SK-MEL-24 and SK-MEL-28 cells, compared to the controls. The restoration of KAI1/CD82 mRNA and protein expression detected no significant difference between SK-MEL-24 and SK-MEL-28 cells. This means that 5-AzaC did not affect the methylated cells only. Loss of heterozygosity (LOH) at polymorphic microsatellite loci on the human chromosome 11 in the human melanoma cells was also examined. Microsatellite analysis showed LOH at D11S1344 in SK-MEL-24 and SK-MEL-28 cells, and G361 showed allelic imbalance. In conclusion, this study suggests that down-regulation of KAI1/CD82 mRNA expression in human melanoma cell lines is related to LOH or allelic imbalance, but not to methylation of the KAI1/CD82 gene region.
KAI1/CD82是一种前列腺癌转移抑制基因，位于人类染色体11p11.2上。已有报道称，在几种癌症的肿瘤进展和转移过程中，KAI1/CD82会下调，但其下调机制尚不清楚。本研究调查了人类黑色素瘤细胞系中KAI1/CD82 mRNA表达下调与KAI1/CD82基因改变之间的关系。在用亚硫酸氢盐处理的G361、SK-MEL-24和SK-MEL-28细胞系中，扩增启动子区域富含GC的331bp片段后，检测启动子甲基化状态。为了检测所有三个细胞系中的甲基化CpG，对331bp片段进行了测序。为了检测KAI1/CD82 mRNA和蛋白表达的恢复情况，将细胞暴露于甲基化酶抑制剂5-氮杂-2'-脱氧胞苷（5-AzaC）中。亚硫酸氢盐测序数据显示，G361和SK-MEL-24细胞中无甲基化，而SK-MEL-28细胞在启动子的CpG位点23-26处有轻微甲基化。实时PCR和流式细胞术分析表明，与对照组相比，5-AzaC处理的细胞在SK-MEL-24和SK-MEL-28细胞中恢复了KAI1/CD82 mRNA和蛋白表达。在SK-MEL-24和SK-MEL-28细胞中检测到的KAI1/CD82 mRNA和蛋白表达的恢复没有显著差异。这意味着5-AzaC不仅影响甲基化细胞。还检测了人类黑色素瘤细胞中人类染色体11上多态微卫星位点的杂合性缺失（LOH）。微卫星分析显示，SK-MEL-24和SK-MEL-28细胞在D11S1344处存在LOH，而G361显示出等位基因失衡。总之，本研究表明，人类黑色素瘤细胞系中KAI1/CD82 mRNA表达下调与LOH或等位基因失衡有关，但与KAI1/CD82基因区域的甲基化无关。
