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 as melphalan, which have antitumor activity. Cell lines with mutations in recombinational repair pathways are hypersensitive to nitrogen mustards. Thus, resistance to melphalan may require accelerated DNA repair by either recombinational repair mechanisms involving Rad51-related proteins (including x-ray repair cross-complementing proteins Xrcc2, Xrcc3, and Rad52) or by nonhomologous endjoining involving DNA-dependent protein kinase (DNA-PK) and Ku proteins. We investigated the role of DNA repair in melphalan resistance in epithelial tumor cell lines.


METHODS
Melphalan cytotoxicity was determined in 14 epithelial tumor cell lines by use of the sulforhodamine assay. Homologous recombinational repair involving Rad51-related proteins was investigated 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 investigated by determining the levels of Ku70 and Ku86 proteins and DNA-PK activity. Linear regression analysis was used to analyze correlations between the various protein levels, DNA-PK activity, or Rad51 foci formation and melphalan cytotoxicity. All statistical tests were two-sided.


RESULTS
Melphalan resistance was correlated with Xrcc3 levels (r =.587; P =.027) and the density of melphalan-induced Rad51 foci (r =.848; P =.008). We found no correlation between melphalan resistance and Rad51, Rad52, or Ku protein levels or DNA-PK activity.


CONCLUSIONS
Correlations of melphalan resistance in epithelial tumor cell lines with Xrcc3 protein levels and melphalan-induced Rad51 foci density suggest that homologous recombinational repair is involved in resistance to this nitrogen mustard.
Wang
Z M
ZM
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, PQ, Canada.
Chen
Z P
ZP
Xu
Z Y
ZY
Christodoulopoulos
G
G
Bello
V
V
Mohr
G
G
Aloyz
R
R
Panasci
L C
LC
D034961:Antigens, Nuclear; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D015153:Blotting, Western; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004265:DNA Helicases; D004260:DNA Repair; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D000072200:Ku Autoantigen; D008558:Melphalan / Q000494:pharmacology*; D018613:Microscopy, Confocal; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000032:analysis; D051135:Rad51 Recombinase; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid; D014407:Tumor Cells, Cultured / Q000187:drug effects
D034961:Antigens, Nuclear; D018906:Antineoplastic Agents, Alkylating; D003432:Cross-Linking Reagents; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C110901:X-ray repair cross complementing protein 3; C113187:XRCC2 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D008558:Melphalan