Mutant Nbs1 enhances cisplatin-induced DNA damage and cytotoxicity in head and neck cancer.
OBJECTIVE
Enhanced DNA double-strand break (DSB) repair could be a primary cause for development of resistance in tumor cells to cisplatin, which induces crosslinks and DNA DSBs. A protein complex consisting of hMre11, hRad50, and Nbs1 (MRN) has been identified as a critical component in repair of DNA DSBs. The present study investigates whether the expression of a truncated form of Nbs1 interrupts the function of the MRN complex and therefore enhances cisplatin-induced DNA damage and cytotoxicity in human head and neck squamous cell carcinoma (HNSCC).


METHODS
Two human HNSCC cell lines, JHU006 and JHU029, were used. A dominant negative recombinant adenovirus expressing domains of Nbs1 was constructed. Adenovirus-mediated mutant Nbs1 (Ad-Nbs1) gene transfer was performed with replication-defective virus (DL312) and no treatment as controls. Transgene expression and cell viability were evaluated in transfected cells. Neutral comet assay was performed and the "tail moment," the product of the amount of DNA in the tail and the distance of tail migration, was analyzed for evaluating DNA DSB damage at 24, 48, and 72 hours.


RESULTS
Transgene expression of mutant Nbs1 was confirmed by Western blotting. Ad-Nbs1 gene transfer significantly increased cisplatin-induced cytotoxicity as shown by stunting of 6-day growth curves. Neutral comet analysis revealed that the mean tail moment, indicative of DNA damage, was significantly elevated in cells treated with combined cisplatin and Ad-Nbs1 compared to cisplatin alone in both cell lines.


CONCLUSIONS
Expression of mutant Nbs1 significantly increases cisplatin-induced DNA DSBs and cytotoxicity. The increase in double-strand DNA damage corresponds to the level of cytotoxicity in the different treatment groups and suggests that tumor chemosensitization occurs through augmentation of DNA DSBs.


CONCLUSIONS
Alteration of DNA repair may provide a novel approach to enhancing sensitivity of HNSCC to chemotherapy. Our study supports the potential application of Ad-Nbs1 in combination with cisplatin for treatment of advanced and metastatic HNSCC.
Tran
Hao Mimi
HM
Department of Otolarygology-Head and Neck Surgery, University of Maryland School of Medicine, Baltimore, USA.
Shi
Guoli
G
Li
Gouyan
G
Carney
James P
JP
O'Malley
Bert
B
Li
Daqing
D
D000256:Adenoviridae / Q000235:genetics; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy; D018797:Cell Cycle Proteins / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D002945:Cisplatin / Q000008:administration & dosage / Q000494:pharmacology*; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006258:Head and Neck Neoplasms / Q000188:drug therapy*; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics*; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D002945:Cisplatin