title: Activation of ER stress and apoptosis by hydrogen peroxide in HeLa cells: protective role of mild heat preconditioning at 40°C.
abstract: The accumulation of misfolded proteins in the endoplasmic reticulum (ER) during stress conditions causes activation of the unfolded protein response (UPR). If this adaptive response cannot restore ER homeostasis, cells undergo ER-mediated apoptosis. This study determines whether thermotolerance developed at a mild temperature (40°C) can alter induction of ER-mediated stress and apoptosis by H(2)O(2) in HeLa cells. Protein expression of PERK, p-PERK, eIF2α and p-eIF2α was increased in thermotolerant compared to non-thermotolerant cells. Thus, mild thermotolerance enhanced pro-survival effects of the PERK/eIF2α branch of the UPR. A short exposure (15 min) of cells to H(2)O(2) (15-50 μM) activated the UPR: expression of p-PERK, p-eIF2α and p-IRE1α increased, and ATF6 cleavage occurred. Longer exposure (1-3h) to H(2)O(2) induced ER-mediated apoptosis, whereby CHOP expression increased, and enzymatic activity of calpain, caspase-7, -4, -12 and -9 also increased. These pro-apoptotic events and clonogenic cell killing were all diminished in thermotolerant cells. Activation of caspases-4/-12 was decreased by the calcium chelator BAPTA-AM, and by inhibitors of calpain and caspase-7, confirming the roles of calcium, calpain and caspase-7 in activation of ER-mediated apoptosis by H(2)O(2). In thermotolerant cells with decreased levels of PERK by siRNA, there was partial reversal of resistance to H(2)O(2)-induced apoptosis. Hence, a causal connection exists between the ER stress response and resistance to H(2)O(2)-induced apoptosis. Mild thermotolerance plays a protective, anti-apoptotic role by increasing the threshold for induction of ER-mediated apoptosis by H(2)O(2). Moreover, the adaptive response (UPR) dominates during milder H(2)O(2) stress, whereas ER-mediated apoptosis occurs during more severe stress.
authors.affiliation: Départment des sciences biologiques and TOXEN, Université du Québec à Montréal, Montréal, Québec, Canada.
authors.forename: Pragathi
authors.initials: P
authors.lastname: Pallepati
authors.forename: Diana A
authors.initials: DA
authors.lastname: Averill-Bates
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007124:Immunoenzyme Techniques; D016877:Oxidants / Q000494:pharmacology*; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D056811:Unfolded Protein Response / Q000187:drug effects*; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D006861:Hydrogen Peroxide; D016877:Oxidants; D034741:RNA, Small Interfering; D019892:eIF-2 Kinase