Temporal Dynamic Regulation of Autophagy and Senescence Induction in Response to Radiation Exposure.

Autophagy and senescence are closely related cellular responses to genotoxic stress, and play significant roles in the execution of cellular responses to radiation exposure. However, little is known about their interplay in the fate-decision of cells receiving lethal doses of radiation. Here, we report that autophagy precedes the establishment of premature senescence in normal human fibroblasts exposed to lethal doses of radiation. Activation of the p53-dependent DNA damage response caused sustained dephosphorylation of RB proteins and consequent cell cycle arrest, concurrently with Ulk1 dephosphorylation at Ser638 by PPM1D, which promoted autophagy induction 1-2 days after irradiation. In addition, mitochondrial fragmentation became obvious 1-2 days after irradiation, and autophagy was further enhanced. However, Ulk1 levels decreased significantly after 2 days, resulting in lower LC3-II levels. An autophagic flux assay using chloroquine (CQ) also revealed that the flux in irradiated cells gradually decreased over 30 days. In contrast, lysosomal augmentation started at 1 day, became significantly upregulated after 5 days, and continued for over 30 days. After a rapid decrease in autophagy, p16 expression increased and senescence was established, but autophagic activity remained reduced. These results demonstrated that X-ray irradiation triggered two processes, autophagy and senescence, with the former being temporary and regulated by DNA damage response and mitophagy, and the latter being sustained and regulated by persistent cell cycle arrest. The interplay between autophagy and senescence seems to be essential for the proper implementation of the cellular response to radiation exposure.

Mitochondrial Metabolism in X-Irradiated Cells Undergoing Irreversible Cell-Cycle Arrest.

Irreversible cell-cycle-arrested cells not undergoing cell divisions have been thought to be metabolically less active because of the unnecessary consumption of energy for cell division. On the other hand, they might be actively involved in the tissue microenvironment through an inflammatory response. In this study, we examined the mitochondria-dependent metabolism in human cells irreversibly arrested in response to ionizing radiation to confirm this possibility. Human primary WI-38 fibroblast cells and the BJ-5ta fibroblast-like cell line were exposed to 20 Gy X-rays and cultured for up to 9 days after irradiation. The mitochondrial morphology and membrane potential were evaluated in the cells using the mitochondrial-specific fluorescent reagents MitoTracker Green (MTG) and 5,5',6,6'-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1), respectively. The ratio of the mean MTG-stained total mitochondrial area per unit cell area decreased for up to 9 days after X-irradiation. The fraction of the high mitochondrial membrane potential area visualized by JC-1 staining reached its minimum 2 days after irradiation and then increased (particularly, WI-38 cells increased 1.8-fold the value of the control). Their chronological changes indicate that the mitochondrial volume in the irreversible cell-cycle-arrested cells showed significant increase concurrently with cellular volume expansion, indicating that the mitochondria-dependent energy metabolism was still active. These results indicate that the energy metabolism in X-ray-induced senescent-like cells is active compared to nonirradiated normal cells, even though they do not undergo cell divisions.