Blocking Hedgehog survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells.
Blocking Hedgehog survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells.
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DOI：
10.1158/0008-5472.can-10-4173
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发表时间：
2011-09-01
期刊：
影响因子：
11.2
通讯作者：
Houghton JA
中科院分区：
文献类型：
--
作者：
Mazumdar T;Devecchio J;Agyeman A;Shi T;Houghton JA
典型刺猬（HH）信号的特征是调节HH靶基因的转录因子Gli 1和Gli 2的Smoothened（Smo）依赖性激活。在人结肠癌细胞中，与Smo抑制剂环巴胺相反，用Gli小分子抑制剂GANT 61处理诱导广泛的细胞死亡。在这里，我们阐明了由GANT 61引起的细胞死亡上游的细胞事件，这揭示了其在结肠癌细胞中独特的细胞毒活性的基础。与环巴胺不同，GANT 61在G1/S（24小时）和早期S期（32小时）诱导短暂的细胞积累，在HT 29细胞中升高p21 Cip 1、细胞周期蛋白E和细胞周期蛋白A。GANT 61在24小时内诱导DNA损伤，出现p-ATM和p-Chk 2。GANT 61对Gli 1和Gli 2的药理学抑制或瞬时转染Gli 3阻遏物（Gli 3R）的遗传学抑制可下调Gli 1和Gli 2的表达，诱导γ H2 AX、PARP裂解、caspase-3激活和细胞死亡。GANT 61诱导γ H2 AX核灶，而瞬时转染Gli 3R证实Gli 3R和γ H2 AX灶在HT 29、SW 480和HCT 116的相同核内表达。GANT 61特异性靶向Gli 1和Gli 2，通过特异性抑制1）Gli 1和Gli 2与靶基因HIP 1和BCL-2的启动子的直接结合，2）Gli-fluorescence活性，和3）BCL-2的转录激活来证实。综上所述，这些发现确定了在Smo下游GLI基因水平上抑制HH信号传导在早期S期诱导DNA损伤中是至关重要的，从而导致人结肠癌细胞的细胞死亡。
Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small molecule inhibitor GANT61 induces extensive cell death, in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G1/S (24 hr) and in early S-phase (32 hr), with elevated p21Cip1, cyclin E and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hr, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R), downregulated Gli1 and Gli2 expression, induced γH2AX, PARP cleavage, caspase-3 activation and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R demonstrated expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480 and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of 1) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, 2) Gli-luciferase activity, and 3) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.