Abstract
Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.
Highlights
Cell death processes have been classified as apoptosis or necrosis
DOX induced DNA damage through the topoisomerase II (TOP II) complex, which is related to DNA damage response (DDR) signaling pathways involving a DNA-damage sensor, mediator, and effector proteins, including Poly-(ADP-ribose) polymerase 1 (PARP1), H2A histone family member X (H2AX), ataxia telangiectasia mutated (ATM), checkpoint kinase 2 (CHK2), p53, p53-binding protein 1 (53BP1), and breast cancer 1 early onset (BRCA1)[28,29,30]
PARP1 is a nuclear protein, which by utilizing NAD+ and adenosine triphosphate (ATP) is involved in single- and double-strand DNA break recognition, DNA damage repair, chromatin modification, and transcriptional regulation by poly-(ADP-ribosyl)ation[17,48]
Summary
Cell death processes have been classified as apoptosis or necrosis. Apoptosis is the process of regulated cell death, while necrosis refers to unregulated cell death triggered by chemotherapeutic drugs or other insults[1]. The activation of TLR3 by polyinosinic:polycytidylic acid [poly(I:C)] and of TLR4 by LPS was reported to induce necrosis through RIP3-mediated ROS generation in caspase-inhibited macrophage cells[13]. Taken together, these findings showed that different pathways are associated with necrosis, resulting in the onset of various diseases, such as cardiovascular disease, Alzheimer’s disease, and cancer[14,15]. The zinc fingers of the DNA-binding domain recognize DNA breaks, and result in sequential poly-(ADP-ribosyl)ation using nicotinamide adenine dinucleotide (NAD+) and adenosine triphosphate (ATP) via the catalytic domain This process is involved in DDR signaling pathways, such as DNA damage repair and cell death[16]. We examined the morphological changes that occur during necrotic cell death by using carbon nanotube (CNT) atomic-force microscopy (AFM) probes
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