DNA damage signaling in the cellular responses to mustard vesicants

Abstract

Mustard vesicants, including sulfur mustard (2,2'-dichlorodiethyl sulfide, SM) and nitrogen mustard (bis(2-chloroethyl)methylamine, HN2) are cytotoxic blistering agents synthesized for chemical warfare. Because they contain highly reactive electrophilic chloroethyl side chains, they readily react with cellular macromolecules like DNA forming monofunctional and bifunctional adducts. By targeting DNA, mustards can compromise genomic integrity, disrupt the cell cycle, and cause mutations and cytotoxicity. To protect against genotoxicity following exposure to mustards, cells initiate a DNA damage response (DDR). This involves activation of signaling cascades including ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related) and DNA-PKcs (DNA-dependent protein kinase, catalytic unit). Signaling induced by the DDR leads to the recruitment and activation of repair related proteins such as phospho H2AX and phospho p53 to sites of DNA lesions. Excessive DNA modifications by mustards can overwhelm DNA repair leading to single and double strand DNA breaks, cytotoxicity and tissue damage, sometimes leading to cancer. Herein we summarize DDR signaling pathways induced by SM, HN2 and the half mustard, 2-chloroethyl ethyl sulfide (CEES). At the present time, little is known about how mustard-induced DNA damage leads to the activation of DDR signaling. A better understanding of mechanisms by which mustard vesicants induce the DDR may lead to the development of countermeasures effective in mitigating tissue injury.

Keywords: Cell cycle; DNA damage response; H2AX; Nitrogen mustard; Sulfur mustard; Vesicants; p53.

Fig. 1.. Summary of DNA damage signaling following exposure of cells to vesicants.

Mustard vesicants cause DNA damage forming both signal-strand breaks (SSBs) and double-strand breaks (DSBs). In response, cells activate DNA damage response (DDR) signaling pathways. This is initiated by DNA damage sensors such as ATRIP (ATR interacting protein), MRN (MRE11–RAD50–NBS1) complex and the Ku70/Ku80 complex. These proteins are recruited to sites of DNA damage where they trigger DNA damage signaling transducers, including apical phosphatidylinositol 3-kinase-related kinases (PIKKs) such as ATM, ATR, and DNA-PKcs. ATM and DNA-PKcs respond to DNA double strand breaks, while ATR responds to replication protein A (RPA)-coated single strand DNA (ssDNA). These are activated by phosphorylation. ATR and ATM activate downstream cell cycle regulators CHK1 and CHK2, respectively, which in turn signals downstream regulatory proteins such as p53 to regulate cell cycle transit and to activate DNA repair, apoptosis and/or senescence. Activated PIKKs also phosphorylate H2AX, a histone H2A variant, at S139 to promote DNA repair.

Fig. 2.. Cell cycle-dependent induction of DNA damage response proteins by HN2 in A549 cells.

A549 cells were treated with increasing concentrations of HN2 or vehicle control in serum-free medium. After 2 h and 4 h, cells were harvested and stained with Alexa Fluor 488-conjugated phospho H2AX (S139) antibody or Alexa Fluor 647-conjugated phospho p53 (S15) antibody. DNA was then stained with propidium iodide and the cells analyzed by flow cytometry. (A) Representative bivariate distributions of DNA and expression of phospho proteins. Cells shown in blue express phospho H2AX (S139), while cells in red express phospho p53 (S15). Cells that do not express or express very low levels of phospho H2AX or phospho p53 are shown in green for cells in G0/G1 phase, cyan for cells in S phase, and magenta for cells in G2/M phase. The percentages of immunopositive cells are shown at the top of each panel. (B) Relative phospho H2AX and phospho p53 protein expression. Data are presented as means ± SE, n = 4. Data adapted from Jan et al., 2019.