Effects of microtubule-depolymerizing agents on the transfection of cultured vascular smooth muscle cells: enhanced expression with free drug and especially with drug-gene lipoplexes.

The microtubule-depolymerizing agents colchicine, vinblastine (VB), vincristine, nocodazole, and podophyllotoxin were found to increase dramatically the transfection of cationic phospholipid-DNA (CMV-beta-gal) complexes on cultured vascular smooth muscle cells (VSMCs). Pretreatment of cells with free colchicine before addition of lipoplexes increased transgene expression both in the presence and in the absence of serum. Free vinblastine had similar effects; however, vinblastine was more effective (approximately 30-fold maximal stimulation) when incorporated into the lipoplexes. Under optimal conditions, vincristine, nocodazole, and podophyllotoxin produced 25- and 39-, 31- and 14-, and 26- and 14-fold increases in the absence and presence of serum, respectively. Taxol, which stabilizes microtubules, had no effect on transfection, but it blocked the positive effect of colchicine. Cytochalasin B, which inhibits microfilament polymerization, had no effect on transgene expression. By fluorescence microscopy, normal lipoplexes colocalized with lysosomes. In contrast, there was little, if any, colocalization of VB lipoplexes with lysosomes. Because depolymerization of microtubules induces NF-kappaB-dependent gene expression, the effects of pyrrolidinedithiocarbamate and Nalpha-p-tosyl-L-lysine chloromethyl ketone, inhibitors of NF-kappaB activation, were tested; inhibition of vinblastine stimulation of transfection was 85 and 66%, respectively. Also, immunofluorescence microscopy showed that vinblastine induced the translocation of NF-kappaB from the cytoplasm to the nucleus. It is concluded that microtubule-depolymerizing agents, especially when incorporated into lipoplexes, dramatically increase transfection of VSMCs, probably by two mechanisms: (i) inhibition of transport of lipoplexes to lysosomes and (ii) activation of transcription (via NF-kappaB). There have been some reports on the use of pharmaceutical agents to enhance gene expression, but generally these have involved separate applications of drug and gene. The ability to deliver a drug and a gene in a single therapeutic formulation could have significant clinical implications.