ATM/ATR-related checkpoint signals mediate arsenite-induced G2/M arrest in primary aortic endothelial cells

Abstract

Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular disease. Our recent in vitro studies have linked this vascular damage to vascular endothelial dysfunction induced by arsenic exposure. However, cell-cycle arrest induced by arsenic and its involvement in vascular dysfunction remain to be clarified. In this study, we employed primary porcine aortic endothelial cells to investigate regulatory mechanisms of G2/M phase arrest induced by arsenite. Our study revealed that lower concentrations of arsenite (1 and 3 microM) increased cell proliferation, whereas higher concentrations of arsenite (10, 20, and 30 microM) inhibited cell proliferation together with correlated increases in G2/M phase arrest. We found that this arsenite-induced G2/M phase arrest was accompanied by accumulation and/or phosphorylation of checkpoint-related molecules, including p53, Cdc25B, Cdc25C, and securin. Inhibition of activations of these checkpoint-related molecules by caffeine significantly attenuated the 30-microM arsenite-induced G2/M phase arrest by 93%. Our data suggest that the DNA damage responsive kinases ATM (ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) play critical roles in arsenite-induced G2/M phase arrest in aortic endothelial cells possibly via regulation of checkpoint-related signaling molecules including p53, Cdc25B, Cdc25C, and securin.

ATM directs DNA damage responses and proteostasis via genetically separable pathways

“…We have previously shown that treatment of cells with low levels of hydrogen peroxide induces ATM activation via the oxidation pathway independent of DNA damage (3). Here we compared peroxide to an alternative method of inducing ROS with sodium arsenite (26) and found that both treatments induce ATM-dependent phosphorylation of CHK2 on thr68 but not KAP1 phosphorylation (Fig. 2E, F).…”

“…The cells were then treated with DNA damaging agents including ionizing radiation (IR) and camptothecin (CPT) and oxidizing agents, including hydrogen peroxide (H 2 O 2 ) and sodium arsenite, to examine the effects of the ATM mutations on substrate phosphorylation in cells. We used arsenite here because work from other groups has indicated that arsenite stimulates checkpoint activation responses that are dependent on ATM (26) and that ATM is required for survival of human cells to arsenite even though arsenite does not detectably generate DNA double-strand breaks (Fig. S2)(27).…”

“…Arsenite is thought to induce ROS through activation of NADPH oxidase (Nox) isoforms as well as through the mitochondria (28, 29) and can produce both superoxide as well as hydrogen peroxide in the cytoplasm. Arsenite is stable in serum-containing media over long periods unlike exogenously added hydrogen peroxide (30, 31) and has been reported to induce checkpoint responses mediated by caffeine-sensitive protein kinases (26). As expected, ATM depletion resulted in a complete defect in the phosphorylation of known ATM substrates including KAP1 and CHK2 after each treatment we used (Fig.…”

p53 Regulates Hsp90β during arsenite-induced cytotoxicity in glutathione-deficient cells

“…Activation of p53 is involved in a variety of cellular processes including cell differentiation, cell cycle regulation, DNA repair, apoptosis, and different stress response programs triggered by endogenous and exogenous stressors [20, 21]. Studies on arsenic cytotoxicity have suggested that inorganic arsenic and its dimethylated derivatives could induce DNA damage and the subsequent activation of ATM followed by stabilization of p53 [13, 14, 22–24]. Recent findings indicate the involvement of ATR, PML, and Chk2 in arsenic trioxide-induced apoptosis [25].…”

Pituitary tumor-transforming gene 1: Perspective on an apoptosis pathway

“…Tsou et al demonstrated that the protein kinases ataxiatelangiectasia mutated (ATM) and ATM and Rad3-related (ATR) play critical roles in arsenite-induced G 2 /M arrest in aortic endothelial cells, possibly via regulation of checkpoint-related signaling molecules including p53, cdc25B/C and securin. 18 This study brings us closer to the hypothesis that PTTG1 disorder causes apoptosis via the DNA damage pathway. In tumor tissues with high PTTG1 expression, regulating PTTG1 may interrupt the cell cycle and induce apoptosis.…”