Institute of Pathology, University of Magdeburg, 39120 Magdeburg, Germany.
J Neurooncol. 2012 May;107(3):503-16. doi: 10.1007/s11060-011-0791-2. Epub 2012 Jan 20.
Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.
胶质母细胞瘤已知具有高度的化疗耐药性，但组蛋白去乙酰化酶抑制剂（HDACi）已被证明对这种侵袭性肿瘤类型具有治疗相关性。我们用曲古抑菌素 A（TSA）处理 U87 胶质母细胞瘤细胞，以确定 HDACi 介导的抗肿瘤作用的潜在表观遗传靶标。使用涵盖 96 个细胞周期基因的 cDNA 阵列分析，鉴定出细胞周期蛋白依赖性激酶抑制剂 p21（WAF1）是 TSA 诱导的细胞周期阻滞中的主要参与者。TSA 轻度抑制 U87 细胞的增殖和活力，导致 G1/S 细胞周期阻滞。这种作用伴随着 p53 及其转录靶标 p21（WAF1）的显著上调，以及关键 G1/S 调节剂如 CDK4、CDK6 和 cyclin D1 的下调。然而，TSA 并未诱导 U87 细胞凋亡。正如预期的那样，TSA 促进了总乙酰化组蛋白 H3 和 H4 的积累，并降低了内源性 HDAC 活性。使用染色质免疫沉淀法在 TSA 处理后对 p21（WAF1）启动子周围的染色质调节进行表征，我们发现：（1）HDAC1 的释放；（2）乙酰化 H4 结合的增加；（3）p53 的募集增强。p53 耗尽的 U87 细胞显示出 G1/S 阻滞的消除并重新进入细胞周期。免疫荧光染色显示 TSA 诱导 p21（WAF1）的核易位，验证了细胞周期阻滞。另一方面，相当一部分 p21（WAF1）存在于细胞质部分，导致抗凋亡。此外，TSA 处理的 p53 突变细胞系 U138 未能诱导 p21（WAF1），显示出有缺陷的 G2/M 检查点，并发生有丝分裂灾难。我们认为，HDAC 抑制与其他临床使用的药物联合使用可能被认为是克服胶质母细胞瘤细胞化疗耐药性的有效策略。
