Monochloramine inhibits ultraviolet B-induced p53 activation and DNA repair response in human fibroblasts

Abstract

Monochloramine (NH 2Cl) is one of the inflammation-derived oxidants, and has various effects on cell cycle, apoptosis and signal transduction. We studied the effects of NH 2Cl on DNA repair response induced by ultraviolet B (UVB) irradiation in normal human diploid fibroblasts, TIG-1. TIG-1 irradiated with 20 mJ/cm 2 UVB showed marked increase in thymine dimer, which decreased by about 50% after 24 h. This decrease in thymine dimer was significantly attenuated ( P < 0.05) by the pretreatment of NH 2Cl (200 μM), which indicated DNA repair inhibition. UVB induced p53 phosphorylation at Ser15, Ser20 and Ser37, and p53 accumulation, and NH 2Cl also inhibited these changes. Consequently, UVB-induced increase in the downstream effectors of p53, namely p21 Cip1 and Gadd45a, were almost completely inhibited by NH 2Cl. Immunoprecipitation study indicated that the association of p53 and MDM2, an E3 ubiquitin ligase for p53, did not change substantially by NH 2Cl and/or UVB. The phosphorylation of p53 (Ser15 and Ser37) by UVB is catalyzed by ATR (ataxia telangiectasia mutated and Rad3 related kinase), which works as DNA damage sensor, and ATR also phosphorylates checkpoint kinase 1(Chk1) at Ser345. NH 2Cl also inhibited the phosphorylation of Chk1 (Ser345). As UVB-induced DNA damage is repaired by nucleotide excision repair (NER) in human cells, these findings indicated that NH 2Cl inhibited NER through the inhibition of p53 phosphorylation and accumulation, and NH 2Cl probably impaired DNA damage recognition and/or ATR activation. NH 2Cl may facilitate carcinogenesis through the inhibition of NER that repairs DNA damages from various carcinogens.