[Construction and expression of different mutants of human p53 and their effects on arsenite-induced cell apoptosis].
OBJECTIVE
To construct different mutants of human p53 for expression in eukaryotic cells and investigate the effects of these mutants on stress-induced cell apoptosis.


METHODS
Human p53 cDNA was amplified by PCR and cloned into pcDNA3/HA vector following the routine procedures. The Ser15 and Ser46 of p53 were mutated to Ala and identified by enzyme digestion and PCR, and these mutants were expressed in NIH3T3 cells and detected by Western blotting. After transfection with the plasmids of different p53 mutants, the NIH3T3 cells were double-stained with AnnexinV-FITC and propidium iodide for apoptotic analysis using flow cytometry.


RESULTS
The recombinant plasmids of HA-tagged wild-type p53, HA-p53(WT), and its mutants, HA-p53(S15A) and HA-p53(S46A), were successfully constructed and expressed efficiently in NIH3T3 cells. The apoptotic ratio of p53(WT)-transfected cells induced by arsenite increased and that of p53(S15A)-transfected cells decreased significantly after arsenite stimulation, but no significant changes occurred in the apoptosis of p53(S46A)-transfected cells.


CONCLUSIONS
The phosphorylation on Ser15 of p53 plays an important role in mediating arsenite-induced cell apoptosis.
Liu
Ai-hua
AH
Department of Respiratory Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Gong
Xiao-wei
XW
Wei
Jie
J
Ming
Xiao-yan
XY
Wang
Da-an
DA
Deng
Peng
P
Luo
Shen-qiu
SQ
Jiang
Yong
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018053:Arsenites / Q000494:pharmacology*; D001483:Base Sequence; D005057:Eukaryotic Cells / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
D018053:Arsenites; D016159:Tumor Suppressor Protein p53; C015001:arsenite