Phorbol ester-potentiated liposomal transfection to monocytic PLB-985 cells.
In order to improve transient gene transfer into PLB-985 cells, we treated cells with 12-tetradecanoylphorbol 13-acetate (TPA) 4 h before transfection, and increased by 540-fold the reporter activity of the firefly luciferase gene transfected by TFL-01, a cationic liposome. Dioctanolyglycerol added before TPA addition inhibited the TPA-dependent increase in activity, suggesting it to be a TPA competitor for PKC binding. H7, staurosporine, GO 6976, and H8, but not GF 109203X and forskolin, inhibited the TPA-dependent increase in reporter activity when added 8 h after TFL-01/gene addition. Forskolin and GF 109203X also inhibited the increase when added before TPA. Therefore, for the potentiation of transfection by the TPA/TFL-01 method, conventional PKC activity with significant but low protein kinase A (PKA) activity are first required, and then a novel PKC activity with significant PKA activity. TPA enhanced the uptake of FITC-labeled phosphorothioated oligonucleotides and their prolonged maintenance by cells, suggesting increased TFL-01-assisted plasmid uptake and its stabilization in TPA-treated PLB-985 cells. This method was used successfully for the sensitive analysis of the promoter function of the gp91(phox) gene, implying the method to be generally useful for promoter analyses of various genes expressed in differentiated human monocytic cells.
Hao
L J
LJ
Department of Host-Defense Biochemistry, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan.
Yang
D
D
Fujii
Y
Y
Yamauchi
A
A
Suzuki
N
N
Kikuchi
H
H
Kaneda
Y
Y
Nakamura
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D017931:DNA Primers; D017930:Genes, Reporter; D006801:Humans; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D017931:DNA Primers; D008081:Liposomes; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate