In vitro evidence for homologous recombinational repair in resistance to melphalan

Background: The generation of DNA interstrand cross-links is thought to be important in the cytotoxicity of nitrogen mustard alkylating agents, such a s melphalan, which have antitumor activity. Cell lines with mutations in re combinational repair pathways are hypersensitive to nitrogen mustards. Thus , resistance to melphalan may require accelerated DNA repair by either reco mbinational repair mechanisms involving Rad51-related proteins (including x -ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by non homologous endjoining involving DNA-dependent protein kinase (DNA-PK) and K u proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines. Methods: Melphalan cytotoxicity was determi ned in 14 epithelial tumor cell lines by use of the sulforho-damine assay. Homologous recombinational repair involving Rad51-related proteins was inve stigated by determining the levels of Rad51, Rad52, and Xrcc3 proteins and the density of nuclear melphalan-induced Rad51 foci, which represent sites of homologous recombinational repair. Nonhomologous endjoining was investig ated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activit y. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melpha lan cytotoxicity. All statistical tests were two-sided. Results: Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the dens ity of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correla tion between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity. Conclusion: Correlations of melphalan resistance in epith elial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad5 1 foci density suggest that homologous recombinational repair is involved i n resistance to this nitrogen mustard.