Biological and molecular mechanisms of sulfur mustard analogue-induced toxicity in JB6 and HaCaT cells: possible role of ataxia telangiectasia-mutated/ataxia telangiectasia-Rad3-related cell cycle checkpoint pathway

Abstract

Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity.

Figure 1. CEES treatment induced changes in cell morphology, dose-dependent decreases in cell viability, growth inhibition, and decrease in DNA synthesis in JB6 and HaCaT cells

JB6 and HaCaT cells were treated with DMSO (control) or with 0.1–1 mM concentrations of CEES and then examined under a light microscope for morphological analysis (A and B). After similar treatments to JB6 and HaCaT cells in 96 well plates, MTT assay (C and D) was carried out at 2, 4 and 24 h as described under ‘Experimental Procedures’. At the end of each treatment time, both floaters and attached cells were collected and counted on hemocytometer after Trypan blue staining for total number of cells (E and F) and percent of dead cells (G and H) as described in the ‘Experimental Procedures’. JB6 (I) and HaCaT (J) cells were treated with DMSO (control) or 0.1, 0.25, 0.5 and 1 mM concentrations of CEES for 2, 4 and 24 h in 96 well plates. At desired time points, cells were incubated with BrdU for 2 h, fixed and DNA denatured and labeled with anti-BrdU mouse monoclonal Ab-Fab. Further product quantification was done by measuring the absorbance at 370 nm (reference wavelength: 492 nm) as described under ‘Experimental Procedures’. Black arrow, cell death as floating cells; white arrow, elongation of cells; black arrowhead, cell swelling; 4 h (magnification ×100), 24 h (magnification ×200). Data shown are mean ± SEM of eight independent samples for each treatment, (C and D), mean ± SEM of three independent samples for each treatment (E–H), and mean ± SEM of six independent samples for each treatment (I and J).*, P<0.001; ψ, P<0.005; $, P<0.01 as compared to control. UC, untreated control; VC, DMSO treated vehicle control. Similar results were obtained in two independent experiments.

Figure 2. CEES treatment caused S and G2-M phase arrests during cell cycle progression of JB6 and HaCaT cells

JB6 (A) and HaCaT (B) cells were cultured and treated with DMSO (Control) or 0.25, 0.5 and 1 mM concentrations of CEES. Following 2, 4 and 24 h treatments, cells were collected and incubated with saponin/PI at 4°C for 24 h in the dark and subjected to FACS analysis as detailed in ‘Experimental Procedures’. Data shown are mean ± SEM of three independent samples.*, P<0.001; ψ, P<0.005; $, P<0.01; #, P<0.05 as compared cells in G1, S and G2-M phases in control samples.

Figure 3. CEES treatment induced ATM ser1981 and/ATR ser428 phosphorylation in JB6 and HaCaT cells

JB6 and HaCaT cells were treated with DMSO (control) or 0.25, 0.5 and 1 mM concentrations of CEES, and harvested 30 min thereafter. Total cell lysates were prepared, subjected to SDS-PAGE followed by western immunoblotting with 120 μg of protein for phosphorylated ATM and ATR. Total protein levels were determined after stripping and reprobing with total ATM and ATR antibodies. C, DMSO treated control.

Figure 4. CEES treatment modulated the expressions of S and G2-M cell cycle regulatory proteins in JB6 and HaCaT cells

JB6 (A and C) and HaCaT (B and D) cells were treated with DMSO (control) or 0.25–1 mM concentrations of CEES and harvested at indicated time-points. Total cell lysates were prepared, subjected to SDS-PAGE followed by Western immunoblotting with 60–80 μg of protein, and membranes were probed for phosphorylated and total Chk1 and Chk2, Cdc25C and Cdc25A, Cdc2 and Cdk2, and cyclin A and B1 levels as described under ‘Experimental Procedures’. Protein loading was checked by stripping and re-probing the membranes with β-actin antibody. C, DMSO treated control.

Figure 5. CEES treatment caused both apoptotic and necrotic cell death in JB6 and HaCaT cells

Cells were treated with DMSO or 0.25–1 mM concentrations of CEES for 2, 4 and 24 h. At the end of each treatment time, both floaters and attached cells were collected and stained with Hoechst+PI dye as detailed in ‘Experimental Procedures’, counted manually and percent live, necrotic and apoptotic cells were calculated (A and D). Cells were also observed under a fluorescence microscope and pictures were taken as detailed in ‘Experimental Procedures’ at magnification ×100 (B and E) and ×400 (C and F). Data shown are mean ± SEM of three independent samples. *, P<0.001; ψ, P<0.005; $, P<0.01; #, P<0.05 as compared to control. C, DMSO treated control; 1, 0.25 mM CEES; 2, 0.5 mM CEES; 3, 1 mM CEES; PI, only PI staining indicating the necrotic cells; HO+PI, Hoechst and PI staining recording both apoptotic and necrotic cells; red arrows, apoptotic cells; white arrows, necrotic cells.

Figure 6. CEES treatment caused an increase in Caspase-3 activity and induction of Caspase-3 and PARP cleavage in JB6 and HaCaT cells

For measuring caspase-3 activity, JB6 and HaCaT cells were collected at desired time points after DMSO (control) and CEES treatments, and cell lysates prepared in the cell lysis buffer. About 150 μg of protein lysate per sample was taken and assayed (A and B) as described under ‘Experimental Procedures’. For caspase-3 and PARP cleavage, JB6 and HaCaT cells were treated with DMSO (control) or 0.25, 0.5 and 1 mM concentrations of CEES and harvested at indicated time-points. Total cell lysates were prepared, subjected to SDS-PAGE followed by western immunoblotting with 80 μg of protein lysate, and membranes were probed for cleaved caspase-3 and PARP (C and D) as described under ‘Experimental Procedures’. Protein loading was checked by stripping and reprobing the membranes with β-actin antibody. Data shown in A and B are mean ± SEM of three independent samples. *, P<0.001; ψ, P<0.005; #, P<0.05 as compared to control; C, DMSO treated control.

Figure 7. Proposed mechanism of CEES-caused molecular alterations leading to the observed biological events in JB6 and HaCaT cells

Our results indicate that CEES-induced decrease in cell viability and DNA synthesis, and S and G2-M phase cell cycle arrest were associated with a DNA damage followed by the activation of DNA-damage sensor kinases ATM and ATR, and modulation of S and G2-M phase regulatory molecules in both JB6 and HaCaT cells. In addition, CEES-induced apoptosis/necrosis in both cell lines was associated with cleavage of caspase-3 and PARP.