Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C.
The mechanisms involved in the translocation of exogenously added genetic information through the cellular cytoplasm and into the nucleus are essentially unknown. Several trans-cytoplasmic translocation systems operate within cells to transport information received by the plasma membrane into the nucleus. Protein kinase C may be functionally involved in many of these translocation mechanisms. In order to explore the involvement of protein kinase C activation in the cytoplasmic translocation of DNA, NIH3T3 fibroblasts were transfected using the calcium-phosphate co-precipitation method with a plasmid containing the lacZ gene and treated with tetradecanoylphorbol 12,13-acetate (TPA) or 1,2-dioctanoylglycerol (DiC8). Addition of TPA or DiC8 immediately after glycerol shock resulted in a 5-7-fold increase in the number of cells expressing beta-galactosidase as well as a concomitant increase in the total amount of beta-galactosidase activity in the population during periods of transient and stable expression. TPA added at later times resulted in lesser increases in the efficiency of transfection. In contrast, TPA added at the time of addition of the calcium-phosphate precipitate inhibited transfection. In support of a role for protein kinase C activation in enhancing DNA transfection, the TPA analog 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, was ineffective at enhancing transfection. Furthermore, treatment of cells with the protein kinase C inhibitor sphingosine blocked the TPA-mediated increase in transient and stable expression. The results suggest that protein kinase C activation enhances transfection of exogenous DNA through an as yet unknown mechanism.
Reston
J T
JT
Department of Biomedical Sciences, School of Public Health, Albany, NY.
Gould-Fogerite
S
S
Mannino
R J
RJ
D000818:Animals; D002130:Calcium Phosphates; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation; D015870:Gene Expression / Q000187:drug effects; D051379:Mice; D010703:Phorbol Esters / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection / Q000187:drug effects*; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D002130:Calcium Phosphates; D004075:Diglycerides; D010703:Phorbol Esters; C044295:1,2-dioctanoylglycerol; D004247:DNA; D011493:Protein Kinase C; D001616:beta-Galactosidase; D013755:Tetradecanoylphorbol Acetate