Sulfur Mustard Induces Markers of Terminal Differentiation and Apoptosis in Keratinocytes Via a Ca2+-Calmodulin and Caspase-Dependent Pathway

Abstract

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.

A Convenient Fluorometric Method to Study Sulfur Mustard-Induced Apoptosis in Human Epidermal Keratinocytes Monolayer Microplate Culture

“…Following 0.3 mM SM exposure, which is considered to be the in vitro equivalent of a vesicating dose in vivo, a very small but statistically significant increase in fluorescence was observed at 6 to 8 h, which was markedly higher at 12 h. At 24 h, all SM concentrations increased fluorescence. These results are in agreement with those reported by Rosenthal et al (1998) on the concentration and time dependence of SMinduced caspase-3 processing (conversion of the inactive 32 kDa procaspase-3 to the active 17 kDa caspase-3) and activation as well as other apoptosis indicators in HEK studied by the Western blotting method. The time-course and concentration dependence of SM-induced caspase-3 activation in HEK observed by our fluorometric method, therefore, appear to be valid indicators of apoptosis.…”

“…Apoptosis contributes to the mechanisms of removal of unwanted cells in normal physiological processes (e.g., during organ development, sloughing of the endometrium in early menstruation period, immune response, etc.). Apoptosis also is the mechanism of cell death responsible for pathological conditions such as cancer, Alzheimer disease, ischemic cardiac damage, oxidative stress, autoimmune syndromes (Hollinger, 2002), or chemical toxicity, for example, due to the highly reactive alkylating compound sulfur mustard (SM) (Rosenthal et al, 1998). SM reacts with multiple targets in the cell and, therefore, causes toxicity via different mechanisms such as DNA damage, disturbance in intracellular calcium homeostasis, abnormal cellular energy metabolism to include oxidative stress, and upregulation of death receptors (Fas) and Fasligand (Ray et al, 2002).…”

“…Any of these mechanisms may independently contribute to apoptosis by initially activating a pathway that is characteristic of the mechanism involved, but later converging to a common pathway culminating into the final apoptotic manifestations. There are reports showing that in cultured human epidermal keratinocytes (HEK), SM induces apoptosis via at least two separate mechanisms: 1) an intrinsic mechanism involving mitochondria, Ca 2 ÷-calmodulin (Ca2+-CaM), and caspase-3/poly (ADP-ribose) polymerase (PARP), and 2) an extrinsic mechanism involving Fas (death) receptors and Fas ligand (FasL) (Rosenthal et al, 1998(Rosenthal et al, , 2003. These reports also show that inhibiting the Ca 2 +-CaM or the Fas response attenuates the biochemical markers of apoptosis and pathology due to SM.…”

The E7 protein of human papillomavirus type 16 sensitizes primary human keratinocytes to apoptosis

“…Caspase-3 appears to be a converging point for distinct apoptotic pathways (Nicholson et al, 1995) and, in an number of analysed systems, caspase-3 cleaves key proteins involved in the structure and integrity of the cell, including PARP. We treated transduced keratinocytes with sulfur mustard in order to induce a higher rate of apoptosis in these cells (Dabrowska et al, 1996;Rosenthal et al, 1998) and thereby allow us to determine whether the caspase pathway was activated during E7-augmented apoptosis. Preliminary studies showed that the treatment of the cells with 50 mM sulfur mustard was sucient to induce apoptosis (by bisbenzimide staining) in 40% of the E7-transduced cells (data not shown).…”

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions

“…Several previous studies reported PAR formation after SM treatment, however at present those remain incidental and lack any systematic time and dose dependent analysis (Debiak et al, 2009;Hinshaw et al, 1999;Kehe et al, 2008;Rosenthal et al, 1998). In terms of CEES induced PAR formation, Paromov analyzed PARP activity in HaCaT cell extracts using biotinylated NAD + , leading to a maximum of a $10 fold in signal intensities at 5 mM CEES treatment (Paromov et al, 2011a).…”