Guan Xin, Sagara Junji, Yokoyama Taro, Koganehira Yoko, Oguchi Misae, Saida Toshiaki, Taniguchi Shun'ichiro
Department of Molecular Oncology, Institute on Aging and Adaptation, Shinshu University Graduate School of Medicine, Matsumoto, Nagano, Japan.
Int J Cancer. 2003 Nov 1;107(2):202-8. doi: 10.1002/ijc.11376.
ASC/TMS1 is an adaptor protein activating caspase-1 that stimulates processing of proIL-1beta and proIL-18. ASC was reported to be aberrantly methylated and silenced in human breast cancers. In our present study, ASC expression was examined in 12 melanoma cell lines by Western blot analysis and in 18 benign melanocytic nevi and 32 melanoma tissues by immunohistochemical staining. ASC expression was absent or reduced in 7 of 12 (58.3%) cell lines and in 20 of 32 (62.5%) melanoma tissues, whereas all 18 benign melanocytic nevi showed intensive ASC expression. To investigate whether ASC silencing in melanoma is involved in aberrant methylation, methylation specific PCR was carried out. Five of ten (50%) melanoma tissues exhibited methylation in CpG island of ASC companied with reduced ASC expression. Six of twelve (50%) melanoma cell lines showed aberrant methylation in the ASC gene, and 4 of the 6 (66.7%) methylation positive cell lines exhibited reduced ASC expression. We characterized methylation patterns in melanoma cell lines by using bisulfite genomic sequencing, and found that the degree of aberrant methylation correlated with the level of reductive ASC expression. Treatment with demethylating agent 5-aza-2'-deoxycytidine resulted in both demethylation of the ASC gene and the upregulation of ASC expression in the methylation positive melanoma cell lines. Our study shows that ASC is downregulated in melanoma, and that its suppression is partially mediated by aberrant methylation.
ASC/TMS1是一种激活半胱天冬酶-1的衔接蛋白，可刺激白细胞介素-1β前体（proIL-1β）和白细胞介素-18前体（proIL-18）的加工。据报道，ASC在人类乳腺癌中存在异常甲基化并沉默。在我们目前的研究中，通过蛋白质免疫印迹分析检测了12种黑色素瘤细胞系中的ASC表达，并通过免疫组织化学染色检测了18例良性黑素细胞痣和32例黑色素瘤组织中的ASC表达。12种细胞系中有7种（58.3%）以及32例黑色素瘤组织中有20例（62.5%）ASC表达缺失或降低，而所有18例良性黑素细胞痣均显示ASC表达强烈。为了研究黑色素瘤中ASC沉默是否与异常甲基化有关，进行了甲基化特异性PCR。10例黑色素瘤组织中有5例（50%）在ASC的CpG岛出现甲基化，同时ASC表达降低。12种黑色素瘤细胞系中有6种（50%）在ASC基因中出现异常甲基化，6例甲基化阳性细胞系中有4例（66.7%）ASC表达降低。我们通过亚硫酸氢盐基因组测序对黑色素瘤细胞系中的甲基化模式进行了表征，发现异常甲基化程度与ASC表达降低水平相关。用去甲基化剂5-氮杂-2'-脱氧胞苷处理导致甲基化阳性黑色素瘤细胞系中ASC基因去甲基化以及ASC表达上调。我们的研究表明，ASC在黑色素瘤中表达下调，其抑制部分由异常甲基化介导。
