A novel mechanism for acquired cisplatin-resistance: suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells.
A novel mechanism for acquired cisplatin-resistance: suppressed translation of death-associated protein kinase mRNA is insensitive to 5-aza-2'-deoxycitidine and trichostatin in cisplatin-resistant cervical squamous cancer cells.
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DOI：
10.3892/ijo.28.2.497
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发表时间：
2006-02
影响因子：
5.2
通讯作者：
T. Bai;Tetsuji Tanaka;K. Yukawa;N. Umesaki
中科院分区：
文献类型：
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作者：
T. Bai;Tetsuji Tanaka;K. Yukawa;N. Umesaki
尽管进行了广泛的研究，但癌细胞对顺铂（CDDP）耐药的分子机制尚未阐明。在这里，我们通过检查 ME180 人宫颈鳞状癌细胞系和 6 个单克隆 ME180 衍生的 CDDP 抗性亚克隆，研究了死亡相关蛋白 (DAP) 激酶（一种凋亡调节剂）是否参与 CDDP 抗性。 CDDP 和去甲基化剂 5-aza-2'-脱氧胞苷 (5-aza-CdR) 共同处理可显着增强亲本细胞和 CDDP 抗性亚克隆的 CDDP 敏感性。随后去除 5-aza-CdR 迅速将 CDDP 抗性亚克隆的 CDDP 敏感性逆转至原始水平，而亲本细胞保留增强的 CDDP 敏感性至少 24 小时。定量 RT-PCR 显示，CDDP 抗性亚克隆比亲本细胞表达更高的 DNA 甲基转移酶 (DNMT) mRNA 水平，表明 DNMT 表达增加很容易恢复 CDDP 抗性亚克隆在 5-aza-CdR 去除后的 CDDP 抗性。尽管亲本细胞在 DAP 激酶启动子区域显示出高甲基化，但通过甲基化特异性 PCR 在 6 个 CDDP 抗性亚克隆中的 2 个中未检测到相应的甲基化条带。通过定量 RT-PCR 评估，所有 6 个 CDDP 抗性亚克隆均表达比亲代细胞更高的 DAP 激酶 mRNA 水平。尽管 DAP 激酶蛋白表达在亲本细胞和 CDDP 抗性亚克隆中受到强烈抑制，但亲本细胞的 5-aza-CdR 处理剂量依赖性地刺激了 DAP 激酶蛋白表达，并且通过除 5-aza-CdR 之外的曲古抑素处理抑制组蛋白脱乙酰化，可协同增强这种作用。然而，CDDP 抗性亚克隆中的 DAP 激酶蛋白表达并未受到 5-aza-CdR 和/或曲古抑菌素处理的刺激。这些结果表明，在由 ME180 人宫颈鳞状癌细胞建立的 CDDP 抗性亚克隆中，DAP 激酶 mRNA 的转录后翻译受到强烈抑制，并且对 5-aza-CdR 和曲古抑菌处理不敏感。这种 CDDP 抗性伴随着干扰 DAP 激酶 mRNA 转录后翻译的分子变化，并且这些分子变化可通过去甲基化暂时恢复。
The molecular mechanism for cisplatin (CDDP)-resistance of cancer cells has not yet been clarified, despite extensive studies. Here, we investigated whether death-associated protein (DAP) kinase, an apoptosis modulator, was involved in CDDP-resistance by examining the ME180 human cervical squamous cancer cell line and 6 monoclonal ME180-derived CDDP-resistant subclones. Co-treatment with CDDP and 5-aza-2'-deoxycytidine (5-aza-CdR), a demethylating agent, significantly enhanced the CDDP-sensitivities of the parent cells and CDDP-resistant subclones. Subsequent removal of 5-aza-CdR rapidly reversed the CDDP-sensitivity of the CDDP-resistant subclones to their original levels, whereas the parent cells retained the enhanced CDDP-sensitivity for at least 24 h. Quantitative RT-PCR revealed that the CDDP-resistant subclones expressed higher DNA methyltransferase (DNMT) mRNA levels than the parent cells, suggesting that increased DNMT expressions easily restored the CDDP-resistance of the CDDP-resistant subclones following 5-aza-CdR removal. Although the parent cells showed hypermethylation in the DAP kinase promoter region, corresponding methylated bands were not detected in 2 of the 6 CDDP-resistant subclones by methylation-specific PCR. All 6 CDDP-resistant subclones expressed higher DAP kinase mRNA levels than the parent cells, as evaluated by quantitative RT-PCR. Although DAP kinase protein expression was strongly suppressed in the parent cells and CDDP-resistant subclones, 5-aza-CdR treatment of the parent cells dose-dependently stimulated the DAP kinase protein expression, and this was synergistically enhanced by inhibiting histone deacetylation via trichostatin treatment in addition to 5-aza-CdR. However, DAP kinase protein expression in the CDDP-resistant subclones was not stimulated by treatment with 5-aza-CdR and/or trichostatin. These results indicate that post-transcriptional translation of DAP kinase mRNA is strongly suppressed and insensitive to treatment with 5-aza-CdR and trichostatin in the CDDP-resistant subclones established from ME180 human cervical squamous cancer cells. This CDDP-resistance is accompanied by molecular changes that disturb the post-transcriptional translation of the DAP kinase mRNA, and these molecular changes are transiently restored by demethylation.