[The effect of PKC phosphorylation sites mutation in JWA coding region on TPA-induced MCF-7 cell differentiation].
OBJECTIVE
To investigate the effect of PKC phosphorylation sites mutation in JWA coding region on TPA-induced MCF-7 cell differentiation.


METHODS
Site directed gene mutation was used to construct one or two PKC sites mutations in pEGFP-N1-JWA vectors, and transfected into MCF-7 cells by polyfect reagent, and cell differentiation was characterized by accumulation of lipid droplet as indicated by positive Oil-red-O staining of cells.


RESULTS
All these transfected cell lines, MCF-7-N1(transfected with pEGFP-N1 vector), MCF-7-JWA(transfected with pEGFP-N1-JWA vector), MCF-7-JWA-1(transfected with PKC site 1 mutation pEGFP-N1-JWA vector), MCF-7-JWA-2(transfected with PKC site 2 mutation pEGFP-N1-JWA vector), MCF-7-JWA-1+2 (transfected with both PKC site 1 and 2 mutation pEGFP-N1-JWA vector) were treated with 20 nmol/L TPA for 48 h, and the percentages of positive Oil-red-O staining of cells were 48%, 67%, 69%, 67% and 70% respectively. The percentages of cell differentiation in JWA containing vectors transfected cells treated with TPA were significantly higher those of MCF-7-N1 cells (vector only control). However there were no significant differences between mutated and unmutated cells.


CONCLUSIONS
JWA transfection enhanced MCF-7 cell differentiation induced by TPA significantly, and PKC sites mutation in JWA coding region has no obviously effect on TPA-induced MCF-7 cell differentiation.
Ye
Jian
J
School of Public Health, Nanjing Medical University, Nanjing 210029, China.
Li
Ai-ping
AP
Qiu
Wen
W
Zhou
Jin-hong
JH
Zhou
Jian-wei
JW
D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate