Inturi Swetha, Tewari-Singh Neera, Agarwal Chapla, White Carl W, Agarwal Rajesh
Department of Pharmaceutical Sciences, University of Colorado Anchutz Medical Campus, Skaggs School of Pharmacy and Pharmaceutical Sciences, Aurora, CO 80045, USA.
Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
Mutat Res. 2014 May-Jun;763-764:53-63. doi: 10.1016/j.mrfmmm.2014.04.002. Epub 2014 Apr 13.
Nitrogen mustard (NM), a structural analog of chemical warfare agent sulfur mustard (SM), forms adducts and crosslinks with DNA, RNA and proteins. Here we studied the mechanism of NM-induced skin toxicity in response to double strand breaks (DSBs) resulting in cell cycle arrest to facilitate DNA repair, as a model for developing countermeasures against vesicant-induced skin injuries. NM exposure of mouse epidermal JB6 cells decreased cell growth and caused S-phase arrest. Consistent with these biological outcomes, NM exposure also increased comet tail extent moment and the levels of DNA DSB repair molecules phospho H2A.X Ser139 and p53 Ser15 indicating NM-induced DNA DSBs. Since DNA DSB repair occurs via non homologous end joining pathway (NHEJ) or homologous recombination repair (HRR) pathways, next we studied these two pathways and noted their activation as defined by an increase in phospho- and total DNA-PK levels, and the formation of Rad51 foci, respectively. To further analyze the role of these pathways in the cellular response to NM-induced cytotoxicity, NHEJ and HRR were inhibited by DNA-PK inhibitor NU7026 and Rad51 inhibitor BO2, respectively. Inhibition of NHEJ did not sensitize cells to NM-induced decrease in cell growth and cell cycle arrest. However, inhibition of the HRR pathway caused a significant increase in cell death, and prolonged G2M arrest following NM exposure. Together, our findings, indicating that HRR is the key pathway involved in the repair of NM-induced DNA DSBs, could be useful in developing new therapeutic strategies against vesicant-induced skin injury.
氮芥（NM）是化学战剂硫芥（SM）的结构类似物，可与DNA、RNA和蛋白质形成加合物并交联。在此，我们研究了NM诱导皮肤毒性的机制，该毒性是对双链断裂（DSB）的反应，导致细胞周期停滞以促进DNA修复，以此作为开发针对糜烂性毒剂所致皮肤损伤对策的模型。用NM处理小鼠表皮JB6细胞会降低细胞生长并导致S期停滞。与这些生物学结果一致，NM处理还增加了彗星尾矩以及DNA DSB修复分子磷酸化H2A.X Ser139和p53 Ser15的水平，表明NM诱导了DNA DSB。由于DNA DSB修复通过非同源末端连接途径（NHEJ）或同源重组修复（HRR）途径发生，接下来我们研究了这两条途径，并分别通过磷酸化和总DNA-PK水平的增加以及Rad51灶的形成来确定它们的激活。为了进一步分析这些途径在细胞对NM诱导的细胞毒性反应中的作用，分别用DNA-PK抑制剂NU7026和Rad51抑制剂BO2抑制NHEJ和HRR。抑制NHEJ并未使细胞对NM诱导的细胞生长降低和细胞周期停滞敏感。然而，抑制HRR途径导致细胞死亡显著增加，并且在NM处理后G2M停滞延长。总之，我们的研究结果表明HRR是参与修复NM诱导的DNA DSB的关键途径，这可能有助于开发针对糜烂性毒剂所致皮肤损伤的新治疗策略。
