Suppression of cell cycle progression by poly(ADP-ribose) polymerase inhibitor PJ34 in neural stem/progenitor cells

Highlights

• Inhibition of poly(ADP-ribosyl)ation in NSCPs decreases cyclin B1 promoter activity.

• Down-regulation of FoxM1 and B-MYB by PJ34 in NSPCs reduces cyclin B gene expression.

• Up-regulation of GADD45 by PJ34 in NSPCs suppresses cell cycle progression at G2/M.

Abstract

Neural stem/progenitor cells (NSPCs) express higher levels of poly(ADP-ribose) polymerase 1 (PARP1) than mouse embryonic fibroblasts (MEFs). Inhibition of PARP induces the expression of several genes in the p53 signaling pathway, including p21, which is critical for cell cycle control at the G1/S phase, triggers apoptosis, and suppresses cell cycle progression in NSPCs. However, upon the up-regulation of p21, the cell cycle does not arrest at any specific phase. In the present study, the expression of genes specific to the G1/S and G2/M phases of the cell cycle were analyzed following treatment with PJ34 (N-[6-oxo-5,6-dihydro-phenanthridin-2-yl]-N,N-dimethylacetamide), an inhibitor of PARP. PJ34 treatment dramatically down-regulated cyclin B1 expression in NSPCs, but not in MEFs, which was confirmed by a promoter assay. Down-regulation of FoxM1 and B-MYB revealed that the down-regulation of cyclin B occurs at the transcriptional level. GADD45 was also specifically up-regulated in NSPCs. Taken together, the activation of p53 by PJ34 treatment in NSPCs induced changes in the expression of genes involved in the cell cycle. Fluorescence-activated cell sorting analysis revealed that PJ34 treatment suppressed G2/M to G1 progression in NSPCs, but not in MEFs. These data indicate that PJ34 treatment inhibits cyclin expression at the mRNA level and suppresses cell cycle progression in NSPCs.

Introduction

Poly(ADP-ribose) polymerase 1 (PARP1), which catalyzes the poly(ADP-ribosyl)ation of proteins by using NAD⁺ as a substrate and transfers them to the glutamate, aspartate, or lysine residues of acceptor proteins [1], plays a key role in several nuclear events, including DNA repair, replication, transcription, and chromatin modifications. DNA double-strand breaks increase PARP1 activation by 10–500-fold [[2], [3], [4]]. Neuronal cells injured by ischemia and reperfusion are, to a certain extent, committed to die via necrosis following the excessive activation of PARP1; the depletion of NAD⁺ by PARP1 after severe DNA damage results in the consumption of ATP in an attempt to replenish NAD⁺, leading to an energy crisis and necrotic cell death. Previously, we reported that the activation of PARP1 after oxygen and glucose deprivation impairs mitochondrial function in cortical neurons, leading to death via apoptosis [5]. This cell death pathway, which is dependent on the activity of PARP and the nuclear translocation of the mitochondrial-associated apoptosis-inducing factor, is called parthanatos [6]. PARP inhibitors (1,5-isoquinolinediol, benzamide, and short hairpin RNAs) have been shown to inhibit neuronal cell death following cerebral ischemia. Therefore, PARP1 inhibitors could be a potential therapeutic intervention for cerebral infarction.

PARP inhibitors are also being developed and approved for cancer therapy. Breast cancer 1, early onset (BRCA1), breast cancer 2, early onset (BRCA2), and partner and localizer of BRCA2 have roles in the repair of double-strand DNA breaks by homologous recombination [7]. PARP1 is required for the repair of single-strand breaks, and unrepaired DNA causes double-strand breaks during replication, which ultimately lead to cell death. Normal cells that do not replicate their DNA as often as cancer cells and that lack mutated BRCA1 or BRCA2 still undergo repair via homologous recombination. Thus, PARP inhibitors are becoming an attractive option in cancer therapy.

Previously, we reported that neural stem/progenitor cells (NSPCs) in the mouse brain express more PARP1 protein than mouse embryonic fibroblasts (MEFs), and abundant levels of poly(ADP-ribosyl)ated proteins are found in a steady state in NSPCs [8]. The PARP inhibitor PJ34 (N-[6-oxo-5,6-dihydro-phenanthridin-2-yl]-N,N-dimethylacetamide) induces apoptosis in NSPCs, and also suppresses cell cycle progression at the G1/S and/or G2/M phase in these cells. PJ34 treatment and PARP1 knockdown induces the gene expression of factors in the p53 signaling pathway for apoptosis, including tumor necrosis factor (TNF) receptor superfamily member 6 (Fas), p53-induced death domain protein 1 (PIDD), TNF receptor superfamily member 10b, phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), p53-upregulated modulator of apoptosis (PUMA), and p53 apoptosis effector (PERP), and the expression of genes involved in G1/S cell cycle arrest including cyclin-dependent kinase inhibitor 1A (p21). On the basis of the finding that PARP inhibitors activate the p53 signaling pathway, we propose that poly(ADP-ribosyl)ation contributes to the proliferation and self-renewal of NSPCs through the suppression of p53 activation. p53 phosphorylation is considered to be inactivated by PARP1 through the poly(ADP-ribosyl)ation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related (ATM/ATR), which are p53 kinases.

Although the suppression of cell cycle progression at the G1/S stage can be mediated by p21, the molecular mechanism underlying its role at the G2/M stage is not clear. In the present study, we investigated the effects of the PARP inhibitor PJ34 on the gene expression of cell cycle-related proteins in NSPCs from the mouse embryonic brain, and focused on the factors responsible for the G2/M phase.