Sulfur Mustard Analog Mechlorethamine (Bis(2-chloroethyl)methylamine) Modulates Cell Cycle Progression via the DNA Damage Response in Human Lung Epithelial A549 Cells

Chem Res Toxicol. 2019 Jun 17;32(6):1123-1133. doi: 10.1021/acs.chemrestox.8b00417. Epub 2019 Apr 22.

Abstract

Nitrogen mustard, mechlorethamine (bis(2-chloroethyl)methylamine; HN2), and sulfur mustard are potent vesicants that modify and disrupt cellular macromolecules including DNA leading to cytotoxicity and tissue injury. In many cell types, HN2 upregulates DNA damage signaling pathways including ataxia telangiectasia mutated (ATM), ataxia telangiectasia mutated- and Rad3-related (ATR) as well as DNA-dependent protein kinase (DNA-PK). In the present studies, we investigated crosstalk between the HN2-induced DNA damage response and cell cycle progression using human A549 lung epithelial cells. HN2 (1-20 μM; 24 h) caused a concentration-dependent arrest of cells in the S and G2/M phases of the cell cycle. This was associated with inhibition of DNA synthesis, as measured by incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into S phase cells. Cell cycle arrest was correlated with activation of DNA damage and cell cycle checkpoint signaling. Thus, HN2 treatment resulted in time- and concentration-dependent increases in expression of phosphorylated ATM (Ser1981), Chk2 (Thr68), H2AX (Ser139), and p53 (Ser15). Activation of DNA damage signaling was most pronounced in S-phase cells followed by G2/M-phase cells. HN2-induced cell cycle arrest was suppressed by the ATM and DNA-PK inhibitors, KU55933 and NU7441, respectively, and to a lesser extent by VE821, an ATR inhibitor. This was correlated with abrogation of DNA damage checkpoints signaling. These data indicate that activation of ATM, ATR, and DNA-PK signaling pathways by HN2 are important in the mechanism of vesicant-induced cell cycle arrest and cytotoxicity. Drugs that inhibit activation of DNA damage signaling may be effective countermeasures for vesicant-induced tissue injury.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • A549 Cells
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / pharmacology*
  • Cell Cycle Checkpoints / drug effects*
  • Cell Proliferation / drug effects
  • Chemical Warfare Agents / chemistry
  • Chemical Warfare Agents / pharmacology*
  • DNA Damage*
  • Dose-Response Relationship, Drug
  • Drug Screening Assays, Antitumor
  • Humans
  • Mechlorethamine / chemistry
  • Mechlorethamine / pharmacology*
  • Signal Transduction / drug effects
  • Structure-Activity Relationship
  • Time Factors
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • Chemical Warfare Agents
  • Mechlorethamine