Stable formation of mutated p53 multimers in a Chinese hamster cell line causes defective p53 nuclear localization and abrogates its residual function.
We have previously described a methotrexate-resistant cell line (MTX M) characterized by amplified dihydrofolate reductase (DHFR) genes, cytoplasmic p53 localization, and p53 stable tetramers. To investigate the p53 functionality in MTX M, the effect of chemical/physical agents was studied. In MTX M cells, DNA damage did not induce p53 or mdm-2 protein, while in the parental V79 cells, a residual p53 activity was found. cDNA sequencing showed that V79 and MTX M cells share the same mutations, indicating that the complete loss of p53 function in MTX M cells was due to cytoplasmic sequestration of a mutated p53 with residual activity. In Chinese hamster, both p53 and DHFR genes map on short arm of chromosome 2 suggesting that p53 itself might be amplified. However, fluorescence in situ hybridization with a hamster p53 probe showed only a single signal. Thus, the presence of p53 stable tetramers in MTX M cells, although correlated with DNA amplification, could not be the consequence of either p53 or DHFR gene amplification. Expression of a C-terminal human p53 peptide does not induce p53 nuclear accumulation, indicating that the cytoplasmic localization is due to a mechanism different from that already described in cancer cell lines. Treatments with Sodium Butyrate induced beta-tubulin polymerization, but did not apparently organize a normal microtubule network, which is shown to be important for the p53 localization. Our data indicated that in MTX M cells, p53 is sequestered in the cytoplasm by a novel mechanism that abrogates p53 residual function.
Ottaggio
Laura
L
Department of Translational Oncology, Laboratory of Experimental Oncology B, National Institute for Cancer Research (IST), Genova, Italy. laura.ottaggio@istge.it
Campomenosi
Paola
P
Fronza
Gilberto
G
Menichini
Paola
P
Miele
Mariangela
M
Moro
Francesca
F
Viaggi
Silvia
S
Zunino
Annalisa
A
Abbondandolo
Angelo
A
D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D018628:Gene Dosage; D016685:Mitomycin / Q000494:pharmacology; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism*
D002087:Butyrates; D014404:Tubulin; D016159:Tumor Suppressor Protein p53; D016685:Mitomycin; D013762:Tetrahydrofolate Dehydrogenase