Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL
Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL
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DOI：
10.1038/sj.onc.1210655
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发表时间：
2008-01-17
期刊：
影响因子：
8
通讯作者：
Borden, E. C.
中科院分区：
文献类型：
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作者：
Bae, S. I.;Cheriyath, V.;Borden, E. C.
人黑色素瘤细胞株SK-Mel-3和SK-Mel-28，尽管有促凋亡细胞因子Apo2L/TRAIL的诱导，但在干扰素(干扰素-α2b或干扰素-β)的作用下不发生凋亡。假设IFNS诱导细胞凋亡的重要基因可能被甲基化沉默，DNA去甲基化试剂5-氮杂-2‘-脱氧胞苷(5-AZAdC)被评估。DR4(TRAIL-R1)是被5-AZAdC重新激活的基因之一，在10个黑色素瘤细胞系中，有8个细胞株的TRAIL-R1基因表达增加了3倍。5-AZAdC使SK-Mel-3和SK-Mel-28细胞对干扰素-a2b和干扰素-b诱导的细胞凋亡敏感，甲基化特异的聚合酶链式反应和亚硫酸氢盐测序证实DR4的5‘CpG岛去甲基化，流式细胞仪显示细胞表面DR4蛋白表达增加。在DR4重新激活的细胞中，中和TRAIL的单抗可减少干扰素-b或载脂蛋白2L/TRAIL诱导的细胞凋亡。为了进一步证实DR4的作用，用逆转录病毒载体在对干扰素-b和载脂蛋白2L/TRAIL耐药逆转的SK-MEL-3和SK-MEL-28细胞中表达DR4。因此，在启动子甲基化抑制了DR4表达的黑色素瘤细胞中，通过5-AZAdC重新表达DR4或逆转录病毒转染法，可增强干扰素-α2b、干扰素-β和载脂蛋白2L/TRAIL诱导的细胞凋亡。用DNA甲基化抑制剂重新激活沉默的促凋亡基因可能会增强对IFNS或Apo2L/TRAIL的临床反应。
Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons ( IFN-alpha 2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine ( 5-AZAdC) was assessed. DR4 ( TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a > 3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-a2b and IFN-b; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-b or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-b and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha 2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.