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Gene Therapy and Molecular Biology Vol 11, page 1 Gene Ther Mol Biol Vol 11, 1-14, 2007 Colchicine-mediated focal adhesion formation promotes transient, lipoplex-mediated transfection of A549 cells Research Article Rajesh R. Nair§, David M. Sherry# and Lindsay A. Schwarz* Department of Pharmacological and Pharmaceutical Sciences (RRN & LAS) and College of Optometry (DMS), University of Houston, Houston, TX 77204-5037 __________________________________________________________________________________ *Correspondence: Lindsay A. Schwarz, Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX 77204-5037, USA; Tel: 713-743-1778; Fax: 713-743-1229; E-mail: § Current address: 7777 Knight Road, Dept of Cancer Biology, M.D. Anderson Cancer Center, Houston, TX 77054 # Current address: Department of Cell Biology, College of Medicine, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73104 Key words: lipoplex, cell signaling, focal adhesion kinase, gene transfection Abbreviations: chlorophenol red-b-D-galactopyranoside, (CPRG); focal adhesion kinase, (FAK); focal adhesion-regulated non-kinase, (FRNK); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecytoxyl)-1-propanaminium bromide/cholesterol, (DMRIE/C) Received: 5 October 2006; Revised: 5 January 2007 Accepted: 11 January 2007; electronically published: February 2007 Summary Colchicine, a microtubule-disrupting drug, enhances lipoplex-DNA-mediated transfection. Colchicine-mediated increases in transgene expression are dependent on interference with tubulin polymerization, as pretreatment with paclitaxel, a microtubule-stabilizing agent, significantly inhibited the enhancing effects of colchicine. In addition to its interference with tubulin polymerization, colchicine-treatment activates Rho family GTPases, integrin clustering and the non-receptor tyrosine kinase, focal adhesion kinase (FAK), all known to be involved in formation of focal adhesions. We show that colchicine-mediated enhancement of transgene expression required activation of a Rho GTPase, as Clostridium difficle toxin B inhibited enhancement. Activation of a Rho GTPase lead to engagement of integrins, as the RGD-sequence peptide, an inhibitor of integrin clustering, abrogated colchicine-enhanced transgene expression. Genistein, a tyrosine kinase inhibitor, and cytochalasin D, both capable of inhibiting stress fiber formation, abolished colchicine-induced increases in transgene expression and suppressed focal adhesion formation, suggesting enhanced transgene expression involved stress fiber and focal adhesion formation. FRNK is an endogenous regulator of the tyrosine kinase, focal adhesion kinase (FAK). In A549 cells stably overexpressing the negative regulator, FRNK, colchicine pre-treatment did not enhance transgene expression, suggesting a critical role for FAK. Moreover, PP1, a selective, src-family kinase inhibitor also suppressed the ability of colchicine to enhance transgene expression. We propose that Rho-regulated, integrin clustering stimulates FAK and src kinase activation, formation of both focal adhesions and stress fibers, all of which appear critical to colchicine-mediated enhancement of transgene expression, as transfected by lipoplexes. efficiency. However, exploring the cellular responses that promote uptake, nuclear translocation and expression of transfected genes, should yield strategies for improving transgene expression, as delivered by non-viral systems. Although interactions between liposome-DNA complexes and the cell remain largely unexplained, it is known that liposome-DNA complexes are taken up by endocytosis. After internalization, the transgene DNA within the endosomal vesicles is transported via the I. Introduction While newer, “gutless” adenoviral vectors induce less inflammation, safety issues and questions regarding the efficacy of repetitive therapeutic applications of viral vectors persist (Shayakhmetov et al, 2005; van der Linden et al, 2005). Thus, it is still important to refine and optimize gene delivery by non-viral vectors. The utility of cationic liposomes and other non-viral vectors in gene therapy has been limited due to their low transfection 1


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