Calmodulin, poly(ADP-ribose)polymerase and p53 are targets for modulating the effects of sulfur mustard

We describe two pathways by which the vesicating agent sulfur mustard (HD) may cause basal cell death and detachment: induction of terminal differenti ation and apoptosis, Following treatment of normal human epidermal keratino cytes (NHEK) with 10 or 100 muM HD, the differentiation-specific keratin pa ir K1/K10 was induced and the cornified envelope precursor protein, involuc rin, was crosslinked by epidermal transglutaminase, Fibronectin levels were reduced in a time- and dose-dependent manner. The rapid increase in p53 an d decrease in Bcl-2 levels was consistent not only with epidermal different iation but with apoptosis as well. Further examination of biochemical marke rs of apoptosis following treatment of either NHEK or human papillomavirus (HPV)-immortalized keratinocytes revealed a burst of poly(ADP-ribose) synth esis, specific cleavage of poly(ADP-ribose)polymerase (PARP) in vivo and in vitro into characteristic 89 and 24 kDa fragments, processing of caspase-3 into its active form and the formation of DNA ladders. The intracellular c alcium chelator BAPTA suppressed the differentiation markers, whereas antis ense oligonucleotides and chemical inhibitors specific for calmodulin block ed both markers of differentiation and apoptosis, Modulation of p53 levels utilizing retroviral constructs expressing the E6, E7 or E6 + E7 genes of H PV-16 revealed that HD-induced apoptosis was partially p53-dependent. Final ly, immortalized fibroblasts derived from PARP -/- 'knockout mice' were exq uisitely sensitive to HD-induced apoptosis, These cells became HD resistant when wild-type PARP was stably expressed in these cells. These results ind icate that HD exerts its effects via calmodulin, p53 and PARP-sensitive pat hways. Copyright (C) 2000 John Wiley & Sons, Ltd.