Enhancement of DNA-mediated gene transfer by inhibitors of autophagic-lysosomal function.
A variety of compounds, known to influence the intravesicular transport and degradation of macromolecules, was studied for their effect on the efficiency of DNA-mediated gene transfer (transfection). The efficiency of transfection was measured by transformation of rat 2 thymidine kinase-deficient (tk-) cells by the cloned herpes simplex I thymidine kinase gene (pAGO). When salmon sperm DNA (average molecular weight, 6 X 10(6) D) was used as a carrier, the presence of either 20 mM NH4Cl, 1 microM carbonyl cyanide p-trifluoromethoxy phenyl hydrazone (FCCP), or 5 mM 3-methyl adenine (3-MA) in the medium during incubation of the cells with the DNA-calcium-phosphate (DNA-Ca-Pi) precipitate, enhanced the efficiency of transfection by a factor of 10. If rat thymus DNA (greater than 30 X 10(6) D) was used as a carrier, the transformation efficiency was much higher than with salmon sperm DNA. However, in this case treatment with 3-MA, NH4Cl and FCCP enhanced the transformation frequency by slightly less than a factor of two. 3-MA further increased the transfection frequency if the cells were incubated with the compound after removal of the DNA-Ca-Pi coprecipitate, whereas NH4Cl and FCCP had no such effect. Our results strongly suggest that these inhibitors of intracellular degradation can increase the frequency of transformation by increasing the cytoplasmic levels of exogenous DNA.
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D004247:DNA / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D008247:Lysosomes / Q000187:drug effects*; D008561:Membrane Fusion / Q000187:drug effects; D051381:Rats; D013997:Time Factors; D014162:Transfection / Q000187:drug effects; D014170:Transformation, Genetic / Q000187:drug effects
D000643:Ammonium Chloride; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; C025946:3-methyladenine; D004247:DNA; D000225:Adenine