ROS Production Is Essential for the Apoptotic Function of E2F1 in Pheochromocytoma and Neuroblastoma Cell Lines

Abstract

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

Reactive Oxygen Species– and DNA Damage Response–Dependent NK Cell Activating Ligand Upregulation Occurs at Transcriptional Levels and Requires the Transcriptional Factor E2F1

“…This is consistent with numerous observations indicating that ROS generation is a critical event regulating E2F1-dependent apoptosis (52)(53)(54), and mitochondrial stress response pathway activates E2F1 in an ROS-dependent manner (55).…”

Hypoxia potentiates the cytotoxic effect of piperlongumine in pheochromocytoma models

“…Currently, there is no SDHB -mutated PHEO/PGL cell line available; therefore, we used N2a cells, which are derived from mouse neuroblastoma. Neuroblastoma-derived cells are frequently used in studies of PHEO/PGL as neuroblastoma is a tumor of neural crest origin that shares numerous similarities with PHEO/PGL [31, 32]. A viability assay was performed on N2a cells with silenced Sdhb (si Sdhb -N2a) to model tumors lacking functional SDHB, similar to SDHB -related PHEO/PGL (Supplementary Figure S1F, S1G).…”

Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration

“…We considered the E2F1 transcription factor as a potential candidate to establish a link between pVHL activity and OMA because pVHL is a direct downstream target of E2F1 [63]. In addition, recently it was reported that the phosphorylated and active form of E2F1 is regulated in ROS-dependent manner [64], [65]. ROS-dependent phosphorylation of E2F1 has previously been investigated in some detail.…”

“…ROS-dependent phosphorylation of E2F1 has previously been investigated in some detail. ATM protein kinase is phosphorylated by cellular ROS to act as a redox sensor in cells, and ROS-dependent phosphorylation of ATM activates the effector kinase Chk2, which in turn phosphorylates its downstream target, E2F1 [32], [64], [66], [67].…”