Department of Biology, American University of Beirut, Beirut, Lebanon.
Radiat Oncol. 2010 Nov 15;5:107. doi: 10.1186/1748-717X-5-107.
We have shown that the radio sensitizer DCQ enhances sensitivity of HCT116 human colon cancer cells to hypoxia. However, it is not known whether the p53 or p21 genes influence cellular response to DCQ. In this study, we used HCT116 that are either wildtype for p53 and p21, null for p53 or null for p21 to understand the role of these genes in DCQ toxicity.
HCT116 cells were exposed to DCQ and incubated under normoxia or hypoxia and the viability, colony forming ability, DNA damage and apoptotic responses of these cells was determined, in addition to the modulation of HIF-1α and of p53, p21, caspase-2, and of the ataxia telangiectasia mutated (ATM) target PIDD-C.
DCQ decreased colony forming ability and viability of all HCT116 cells to a greater extent under hypoxia than normoxia and the p21-/-cell line was most sensitive. Cells had different HIF-1α responses to hypoxia and/or drug treatment. In p53+/+, DCQ significantly inhibited the hypoxia-induced increases in HIF-1α protein, in contrast to the absence of a significant HIF-1α increase or modulation by DCQ in p21-/- cells. In p53-/- cells, 10 μM DCQ significantly reduced HIF-1α expression, especially under hypoxia, despite the constitutive expression of this protein in control cells. Higher DCQ doses induced PreG1-phase increase and apoptosis, however, lower doses caused mitotic catastrophe. In p53+/+ cells, apoptosis correlated with the increased expression of the pro-apoptotic caspase-2 and inhibition of the pro-survival protein PIDD-C. Exposure of p53+/+ cells to DCQ induced single strand breaks and triggered the activation of the nuclear kinase ATM by phosphorylation at Ser-1981 in all cell cycle phases. On the other hand, no drug toxicity to normal FHs74 Int human intestinal cell line was observed.
Collectively, our findings indicate that DCQ reduces the colony survival of HCT116 and induces apoptosis even in cells that are null for p53 or p21, which makes it a molecule of clinical significance, since many resistant colon tumors harbor mutations in p53.
我们已经证明，放射增敏剂 DCQ 增强了 HCT116 人结肠癌细胞对缺氧的敏感性。然而，p53 或 p21 基因是否影响细胞对 DCQ 的反应尚不清楚。在这项研究中，我们使用了野生型 p53 和 p21、p53 缺失或 p21 缺失的 HCT116 细胞，以了解这些基因在 DCQ 毒性中的作用。
将 HCT116 细胞暴露于 DCQ 中，并在常氧或缺氧条件下孵育，然后测定这些细胞的活力、集落形成能力、DNA 损伤和凋亡反应，以及 HIF-1α 和 p53、p21、半胱天冬酶-2 和共济失调毛细血管扩张突变 (ATM) 靶标 PIDD-C 的调节。
与常氧相比，DCQ 更显著地降低了所有 HCT116 细胞在缺氧条件下的集落形成能力和活力，p21-/-细胞系最为敏感。细胞对缺氧和/或药物处理有不同的 HIF-1α 反应。在 p53+/+ 细胞中，DCQ 显著抑制了缺氧诱导的 HIF-1α 蛋白增加，而在 p21-/-细胞中，HIF-1α 增加或 DCQ 调节不显著。在 p53-/-细胞中，尽管在对照细胞中 HIF-1α 蛋白组成性表达，但 10 μM DCQ 显著降低了 HIF-1α 表达，尤其是在缺氧条件下。较高的 DCQ 剂量诱导 PreG1 期增加和凋亡，但较低的剂量导致有丝分裂灾难。在 p53+/+ 细胞中，凋亡与促凋亡半胱天冬酶-2 的表达增加和抗凋亡蛋白 PIDD-C 的抑制相关。p53+/+ 细胞暴露于 DCQ 后，诱导单链断裂，并在所有细胞周期阶段通过 Ser-1981 磷酸化激活核激酶 ATM。另一方面，对正常 FHs74 Int 人肠细胞系没有观察到药物毒性。
总之，我们的研究结果表明，DCQ 降低了 HCT116 的集落存活率，并诱导了凋亡，即使在缺失 p53 或 p21 的细胞中也是如此，这使得它成为一种具有临床意义的分子，因为许多耐药结肠肿瘤都存在 p53 突变。
