Abstract

Purpose

Caspases are common mediators of cell death. Evasion of cell death including apoptosis are considered to be hallmarks of cancer. A deeper understanding of the apoptotic cascade may aid improving cancer therapies. Our aim was to characterize the progression of cell death following UV-induced genotoxic injury in a defined cell culture model.

Materials and methods

Hela cells were UV-irradiated with doses ranging from 0.1 to 60 mJ/cm². Cells were counted and colony forming assays were performed with caspase inhibitors.

Results

In our model of HeLa cells, cells remain >90% viable until 6 hrs after UV radiation (UVR), but more than half of the cells are dead after 12 − 72 hrs after UVR. Within a dose range between 0.1 and 50 mJ/cm², viability ranges roughly between 20 and 30%. The difference between the lowest dose applied (0.1 mJ/cm²) and the other doses applied is significant, with the exception of the next higher dose of 1 mJ/cm². The activation of caspases precedes the cell death induction by several hrs. Caspase-9 starts to be activated at 1 hr after UVR followed by caspases 3, 6 and 7 which are fully active at 2 hrs after UVR while caspase-8 is fully active only 3 hrs after UVR. Most caspases are only weakly or not active at 0.1 mJ/cm² after 3 hrs, but fully active at the same time point with increased radiation doses. PARP-1, a caspase substrate, is cleaved immediately after activation of the caspases. Colony formation activity of the tumor cells decreases exponentially after UVR dropping down to < 0.01% plating efficiency at a dose of 60 mJ/cm². Interestingly, this drop in plating efficiency cannot be rescued by any of the two caspase inhibitors tested.

Conclusions

UV-induced cell death in this model involves the activation of apoptosis-related caspases, but this activation seems to be dispensable for the execution of cell death. Further experiments should clarify which mechanisms of cell death are really necessary for the execution of this type of cell death.

中文翻译：

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紫外线诱导的基因毒性损伤后半胱天冬酶活化的动力学

摘要

目的

半胱天冬酶是细胞死亡的常见介质。包括细胞凋亡在内的细胞死亡的逃避被认为是癌症的标志。对凋亡级联反应的更深入了解可能有助于改进癌症治疗。我们的目标是在确定的细胞培养模型中表征紫外线诱导的基因毒性损伤后细胞死亡的进展。

材料和方法

Hela 细胞用 0.1 至 60 mJ/cm ^{2 的}剂量进行紫外线照射。计数细胞并用半胱天冬酶抑制剂进行集落形成试验。

结果

在我们的 HeLa 细胞模型中，细胞在紫外线辐射 (UVR) 后 6 小时前仍保持 >90% 的活力，但在紫外线辐射 (UVR) 后 12 - 72 小时后，超过一半的细胞死亡。在 0.1 和 50 mJ/cm ²之间的剂量范围内，存活率范围大约在 20% 和 30% 之间。施加的最低剂量 (0.1 mJ/cm ² ) 和施加的其他剂量之间的差异是显着的，除了下一个更高的剂量 1 mJ/cm ²。半胱天冬酶的激活先于细胞死亡诱导数小时。Caspase-9 在 UVR 后 1 小时开始激活，然后是 caspase 3、6 和 7，它们在 UVR 后 2 小时完全活跃，而 caspase-8 在 UVR 后仅 3 小时完全活跃。大多数半胱天冬酶仅在 0.1 mJ/cm ^{2 时}微弱或无活性3 小时后，但在辐射剂量增加的同一时间点完全活跃。PARP-1，一种半胱天冬酶底物，在半胱天冬酶激活后立即被切割。UVR 在 60 mJ/cm ²的剂量下降至 < 0.01% 电镀效率后，肿瘤细胞的集落形成活性呈指数下降。有趣的是，测试的两种半胱天冬酶抑制剂中的任何一种都无法挽救这种电镀效率的下降。

结论

该模型中紫外线诱导的细胞死亡涉及细胞凋亡相关半胱天冬酶的激活，但这种激活似乎对于细胞死亡的执行是可有可无的。进一步的实验应该阐明哪些细胞死亡机制对于执行这种类型的细胞死亡是真正必要的。