Reduced cisplatin sensitivity of head and neck squamous cell carcinoma cell lines correlates with mutations affecting the COOH-terminal nuclear localization signal of p53.
OBJECTIVE
Head and neck squamous cell carcinomas (HNSCC) are the most frequent malignancies of the upper aerodigestive tract. Cisplatin resistance is a major problem in the treatment of a large number of HNSCC cancer patients. In this study, nine randomly selected HNSCC cell lines were investigated regarding expression, presence of mutations, nucleocytoplasmic distribution of p53, and sensitivity to cisplatin.


METHODS
Protein expression was evaluated by Western blot analysis. The whole open reading frame of p53 was determined by reverse transcription-PCR sequencing. Nucleocytoplasmic distribution was evaluated by confocal laser scanning microscopy. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to test for cisplatin sensitivity.


RESULTS
p53 mutations were found in all nine investigated HNSCC cell lines. Nuclear p53 signal was detected in six cell lines, whereas three cell lines exhibited total loss of nuclear p53 signal. Nuclear signal depended on the presence or absence of the COOH-terminal nuclear localization signal in p53. Cisplatin sensitivity was highly reduced in the group with loss of nuclear p53 signal compared with those with detectable nuclear signal. Transfection of wild-type and mutant p53 into a rat embryonic cell system showed highly reduced activity of the nuclear localization signal mutant p53 protein.


CONCLUSIONS
Taken together, these data suggest that "loss of nuclear p53 signal" correlates with cisplatin resistance in HNSCC. If these results can be validated on a larger number of tumor samples, including fresh tumor tissues, it potentially could help in sparing a subgroup of HNSCC patients the side effects associated with unnecessary chemotherapy by identifying cisplatin nonresponders before chemotherapy induction.
Mandic
Robert
R
Department of Otolaryngology/Head and Neck Surgery, University of Marburg, Marburg, Germany. mandic@med.uni-marburg.de
Schamberger
Chantal J
CJ
Müller
Julia F
JF
Geyer
Matthias
M
Zhu
Li
L
Carey
Thomas E
TE
Grénman
Reidar
R
Dünne
Anja A
AA
Werner
Jochen A
JA
D000328:Adult; D000368:Aged; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D003001:Cloning, Molecular; D004396:Coloring Agents / Q000494:pharmacology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D004591:Electrophoresis, Polyacrylamide Gel; D016158:Genes, p53 / Q000235:genetics; D006258:Head and Neck Neoplasms / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008875:Middle Aged; D008958:Models, Molecular; D009154:Mutation; D019913:Nuclear Localization Signals; D016366:Open Reading Frames; D017434:Protein Structure, Tertiary; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
C493956:CDKN1A protein, human; D004396:Coloring Agents; D050759:Cyclin-Dependent Kinase Inhibitor p21; D019913:Nuclear Localization Signals; D013778:Tetrazolium Salts; D013844:Thiazoles; D016159:Tumor Suppressor Protein p53; C022616:thiazolyl blue; D002945:Cisplatin