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Colchicine-mediated focal adhesion formation promotes transient, lipoplex-mediated transfection of A549 cells
Corresponding author:*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: email@example.com
Department of Pharmacological and Pharmaceutical Sciences (RRN & LAS) and College of Optometry (DMS), University of Houston, Houston, TX 77204-5037
ßCurrent 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
keywords: 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)
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.
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 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 microtubules to the lysosomes. Much of the DNA transfected by lipoplexes is rapidly degraded in the endo-lysosomal compartment. Nevertheless, a fraction of this DNA escapes from endosomal vesicles through an acidification process that is aided by cationic liposomes. Intact transgene DNA is translocated to nucleus where it is transcribed (Zuhorn and Hoekstra, 2002).
The importance of microtubule dynamics in this process was shown by Hasegawa and colleagues using immunocytochemistry to follow lipoplex trafficking. These authors showed that in cells with intact microtubules, fluorescently-labeled transfected DNA colocalizes with lysosomes. However, when cells are treated with nocodazole, an agent that depolymerizes microtubules, or pacitaxel, an agent that stabilizes microtubules, DNA-lysosomal colocalization was not detected. Interestingly, Hasegawa and colleagues showed that, in cells treated with nocodazole or paclitaxel more transfected DNA accumulated in the nucleus over time and was maximal by 4 h after treatment with either agent (Hasegawa et al, 2001).
Agents that depolymerize microtubules, such as colchicine and nocodazole are known to increase the transfection efficiency of cationic liposomes both in vivo and in vitro. Intraperitoneal injection with colchicine increases transgene expression in liver of Gunn rats (2.5-fold) (Chowdhry et al, 1996) as well as in BALB/c mice (2-fold) (Baru et al, 1995). While the mechanisms by which colchicine increases transiently-transfected transgene expression are still unclear, it has been suggested that disruption of microtubules limits the transfer of DNA between endosomes and lysosomes, thus preventing much of the degradation of transfected DNA (Chowdhry et al, 1996). In support of this suggestion, while investigating intracellular transport of transfected DNA, Brisson and colleagues found that inhibiting non-coated pit endocytosis decreased transgene expression but pretreatment of cells with nocodazole, presumed to block late endosome-lysosomal fusion, increased transgene expression by 2- to 3-fold (Brisson et al, 1999). However, Kitson and colleagues found no change in transgene expression in sheep airway epithelia pretreated with nocodazole (Kitson et al, 1999). Furthermore, chloroquine, an agent that prevents acid hydrolysis of lysosomal contents, has been shown to both increase (Baru et al, 1995) or decrease (Brisson et al, 1999) transfection efficiency. Despite the inconsistencies, these findings, nonetheless suggest that interference with microtubule dynamics is critical to improving transient transgene expression when employing non-viral vectors.
In addition to its effects on endo-lysosomal fusion, microtubular disruption also induces cellular signaling events similar to those seen with a variety of growth factors (Bershadsky et al, 1996). These signals ultimately include tyrosine phosphorylation of proteins involved in focal adhesions assembly (Jung et al, 1997; Kirchner et al, 2003). For example, microtubule disruption activates two, focal adhesion-associated tyrosine kinases, c-src and focal adhesion kinase (FAK). Their activation, by colchicine treatment appears to be primarily integrin-dependent and is regulated through the activation of the small GTP-binding protein, Rho A (Enomoto, 1996). Furthermore, FAK activation, subsequent to microtubule disruption, leads to changes in expression of genes such as COX-2 and urokinase-type plasminogen activator gene (Irigoyen and Nagamine, 1999; Subbaramaiah et al, 2000). In light of these reports, we hypothesized that there may be microtubule-mediated cell signaling events, auxiliary to any role microtubules might play in endo-lysosomal fusion, involved in enhancing transient transgene expression.
The present study was undertaken to investigate the involvement of the Rho family of GTPases, integrins and focal adhesions in the enhancement of transgene expression observed in colchicine-treated A549 cells, transiently transfected with DNA-lipoplexes. We show that colchicine-mediated enhancement of transient, transgene expression requires the formation of focal adhesion complexes and activation of tyrosine kinases, c-src and FAK. These events appear to be dependent on the activation of the Rho family of GTPases and the clustering of integrins.
Materials & Methods
A. Chemicals and reagents
Colchicine was purchased from Sigma Chemicals, St. Louis, MO. Genistein, cytochalasin D, PP1, Paclitaxel, RGD and control peptides and Clostridium difficle toxin B were purchased from Calbiochem, San Diego, CA. Dulbecco's minimal essential medium (DMEM), OPTI-MEM, and cationic lipid N-(2-hydroxyethyl) -N,N-dimethyl-2,3-bis(tetradecytoxy)-1-propanaminiumbromide /cholesterol (DMRIE/C) and fetal bovine serum (FBS) were purchased from InVitrogen-Invitrogen/ Life Technologies/GIBCO, Carlsbad, CA. pCMVb-galactosidase (pCMVbgal) containing the bacterial LacZ gene was purchased from Clontech, Palo Alto, CA. pCMVFRNK-HA was generously provided by Dr. H. Sheldon Earp (University of North Carolina, Chapel Hill, NC). Rabbit polyclonal anti-FAK IgG and mouse monoclonal anti-phosphotyrosine IgG2b antibodies were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. The secondary antibodies, goat-anti-rabbit, AlexaFluor-488 was purchased from InVitrogen/Molecular Probes, Carlsbad, CA and the goat-anti-mouse Cy3 was purchased from Jackson ImmunoResearch Laboratories, West Grove, PA.
A549 cells (CCL-185), a human lung adenocarcinoma cell line, representative of the distal respiratory epithelium, were purchased from ATCC, Manassas, VA. Cells were maintained in DMEM plus 15% low endotoxin-defined FBS (GIBCO), 200mM L-glutamine and 50mg/ml gentamycin and incubated in a humidified 370C incubator containing 5% CO2. Cells were trypsinized (0.25% trypsin, 0.53 mM EDTA) and diluted to a cell concentration of 7.5 ¥ 104 cells/ml and either plated in 12- well culture dishes (1ml/well) for b-galactosidase assays or plated on cover slips (22mm, round) (0.4 ml/cover slip) for immunocytochemistry. For western blotting cells were plated at 2.7 X 105 per T25 cell culture flasks. These cell concentrations provide monolayers approximately 30% confluent 24 hours post seeding.
C. Treatment regimen
All treatments with inhibitors were performed before transfection. All inhibitor concentrations were employed at concentrations that did not effect cell viability, as assessed by microscopy and trypan blue exclusion. Cells were treated for 3 hours with indicated concentrations of paclitaxel, for 5 hours with indicated concentrations of genistein, PP1 and cytochalasin D, for 48 hours with indicated concentrations of toxin B and for 24 hours with indicated concentration of RGD-sequence peptide. The dose response for colchicine has been determined previously and the optimal concentration of colchicine of 5 mM was used for a treatment period of 1 hour. After the various treatments, the cells were washed twice with OPTI-MEM before transfection, unless otherwise indicated.
All transfections were performed in OPTI-MEM. Cell monolayer was rinsed with OPTI-MEM prior to transfection with 1 mg pCMVbgal/4 mg DMRIE/cholesterol in 0.5 ml OPTI-MEM. Cells were incubated with DNA-lipid complexes for 2 hrs at 370C in a humidified, 5% CO2 incubator. Transfection complexes were removed and monolayers washed twice with OPTI-MEM and then either immediately subjected to immunocytochemistry and western blotting at 40C or fed with DMEM plus 15% FBS and incubated for 72 hrs for b-galactosidase assay.
E. b-galactosidase assay
Cells were lysed in 10 mM Tris, pH 7.6, 0.1% Triton X-100. Each clarified cell lysate was first assayed for total protein, using bicinchoninic acid assay per the manufacture's instruction (BCA assay, Pierce Chemicals, Rockford, IL). After adjusting samples for equivalent protein concentration in 10 mM Tris, pH 7.6, samples were assayed for b-galactosidase activity as described (CPRG, Roche, Nutley, NJ). A bgal standard curve (b-galactosidase, InVitrogen) was included with each assay and all experimental bgal values were within the linear slope of the standard curve. The b-galactosidase activity was measured in units/ml (U/ml) and the activity of b-galactosidase in cells treated with growth medium before transfection was set as equal to 1. Fold change of all other drug treatment groups was calculated as: (U/ml bgal in drug treated, transfected)/(U/ml bgal in medium-treated, transfected).
Cells were plated on coverslips. Following different drug or inhibitor treatments and transfection, cells were rinsed in PBS. The cells were then fixed with methanol on ice for 5 min, followed by 1 min incubation with NaBH4 (0.5 mg/ml). The cells were then washed three times with PBS and blocked with PBS containing 2% normal goat serum, and 0.1% Triton X-100 (blocking solution) for 45 min. After blocking, the cells were incubated concomitantly with mouse anti-phosphotyrosine IgG (1:40 dilution) for 48 hours followed by rabbit anti-FAK IgG (1:60 dilution) for the last 24 hours, at 40C. The cells were washed three times with PBS, reblocked and incubated for 45 min with anti-mouse-Cy3 (1:500) and anti-rabbit-AlexaFluor 488 (1:400). The cells were examined, in a blinded manner, for fluoresecence (Olympus IX70 epifluorescence microscope).
G. Western blotting
Cells were seeded into T25 cm2 cell culture flasks and then treated with drugs and inhibitors prior to transfection. The flasks containing cells were then placed on ice and lysed in 1 ml of iced cold lysis buffer (1% Triton X-100 and 0.01M Tris, pH 7.6) for 30 min. Flasks were scraped and total protein was determined for the unfractionated lysates using BCA assay. Five mg of total protein was applied to an 8-16% SDS-PAGE gel (Gradipore, Forresttown, Australia) and proteins resolved by electrophoresis, For immunoprecipitations, cell lysates were incubated with polyclonal rabbit anti-FAK antibody overnight. Protein antibody complexes were adsorbed onto protein A beads (Sigma, St. Louis MO) and washed according to manufacturer's instructions. The entire eluted fraction was loaded onto 8-16% SDS-PAGE gels and resolved by electrophoresis. All proteins were transferred to polyvinylidene fluoride membrane (PVDF) (Immuno-BLOT, Bio-Rad Lab, Hercules, CA) and blocked with Tris-buffered saline/Tween-20 (0.05%) containing either 5% non-fat dried milk or membrane blocking solution (Zymed Laboratories, San Francisco, CA). The membranes were probed with indicated antibodies and detected by secondary antibodies coupled to horseradish peroxide and ECL Plus detecting reagent (Amersham Biosciences, Piscataway, NJ). Western protein membranes were stripped and probed with mouse anti-glyceraldehyde phosphodehydrogenase (GAPDH) to normalize for loading differences. For all films, densitometry was performed on exposed x-ray film (Alpha-Innotech, San Leandro, CA).
H. Preparation of stable transformant A549 cells
Cells were plated at 3 ¥ 105 cells/ml in T25 cm2 cell culture flasks. After overnight incubation in growth medium, cells were transfected with 14 mg of pCMV-HA-FRNK plasmid and 2 mg of the selectable vector, pRSVneo, complexed to 64 mg of DMRIE/c in OPTI-MEM. Some cells were transfected with pRSV-neo only and untransfected cells were used as controls. The cells were incubated in growth medium for the first 48 hrs at 370C. After 48 hrs the growth medium was removed and replaced with growth medium containing 1 mg/ml of geneticin (G418, GIBCO). The selection medium was changed periodically changed and G418 was increased to a final concentration of 1.3 mg/ml. The cells surviving selection were expanded and subsequently, individual colonies derived from individual cells were seeded T25 cm2 cell culture flasks containing 1.3 mg/ml of G418. These cells were employed in experiments investigating the role of FAK in colchicine-mediated enhancement of transgene expression.
I. Statistical analysis
All experiments were independently performed three or more times. bgal expression was measured in U/ml of bgalactosidase cell lysates. To obtain fold-change, the bgal U/ml of the treated, transfected lysates were divided by the bgal U/ml in lysates of untreated, transfected controls. The maximal and minimal U/ml for each set of experiments are included in the figure legends as a point of reference. The data were plotted as mean ± SD and analyzed for statistical significance between treatment groups using one-way ANOVA followed by post hoc, Students-Newman-Keuls test (Primer of Biostatistics; Glantz SA, statistical software version 5.0). A P< 0.05 was accepted as statistically significant.
A. Enhancement of transiently transfected reporter gene expression involves cellular mechanisms distinct from endo-lysosomal fusion
We have previously shown that treatment of A549 cells with 5 mM colchicine for 1 h prior to transfection enhances transgene expression by 15- to 30-fold (Nair et al, 2002). While the literature supports the hypothesis that depolymerizing microtubules favorably alters DNA release from the endo-lysosomal pathway, it is difficult to reconcile the observations that both stabilizing and destabilizing microtubules promote the more rapid release of DNA from the endo-lysosomes (Brisson et al, 1999; Kitson et al, 1999). Furthermore, in previous studies we observed that paclitaxel, a compound that stabilizes microtubules, also enhances transgene expression in a variety of cell lines, albeit to modest degree when compared to colchicine (Nair et al, 2002). Thus, it was possible that, in addition to enhancing transfection efficiency strictly through an endo-lysosomal pathway, changes in microtubule dynamics influence other aspects of transgene delivery or expression.
To determine the extent of the contribution of additional colchicine-mediated effects that enhance transgene expression, we transfected A549 cells with DMRIE/C-pCMVbgal complexes for two hours, washed away untransfected complexes and then treated the cells at with 5 mM of colchicine at time 0 (immediately after transfection) or 6, 12, 18 and 24 hours after transfection.
Treatment of cells for a period of 1 hour with 5 mM of colchicine immediately after transfection (t=0) and, at each successive treatment time point produced a significant increase in bgal expression, as compared to bgal expression in untreated, transfected cells (33 ± 5.5- fold increase; Figure 1). Since endo-lysosomal fusion should be complete within the first few hours after transfection (Hasegawa et al, 2001; Rejman et al, 2005), these studies suggested that enhancement in reporter gene expression mediated by colchicine was not exclusively dependent on inhibition of endo-lysosomal fusion. Rather, colchicine treatment mediated other cellular actions that had significant effects on transgene expression.
Paclitaxel stabilizes microtubules through the direct binding of tubulin but at a site that does not interfere with tubulin binding by colchicine (Kumar, 1981). To determine whether or not microtubule disruption, specifically, was important to enhanced transgene expression, A549 cells were treated with paclitaxel prior to treatment with 5 mM of colchicine. Paclitaxel significantly inhibited the ability of colchicine to increase reporter gene expression (colchicine treatment only, 16-fold; paclitaxel plus colchicine treatments, 6-fold). At its highest dose, 10 mM, paclitaxel inhibited the colchicine-induced increase by approximately 63% (data not shown). Detection of additional decreases in transgene expression was limited by paclitaxel's own ability to enhance transgene expression; alone, paclitaxel mediated a 6-fold (+/- 1.4) enhancement of bgal expression (data not shown) (Nair et al, 2002). Thus, the ability of colchicine to enhance transgene expression appears to be dependent, in part, on microtubular disruption.
B. Colchicine-induced enhancement of reporter gene expression requires activation of Rho family of GTPases, integrin clustering and actin stress fiber formation
Colchicine induces integrin clustering and actin stress fiber formation through cellular events that involve Rho A GTPase activation (Enomoto, 1996). Induction of integrin clustering by activated Rho GTPases promotes "outside-in" signaling (Hotchin and Hall, 1995; Clark et al, 1998) known to activate kinase pathways that ultimately alter the cytoskeleton and even gene expression (Schlaepfer et al, 1998; Watanabe et al, 2003). In light of these known activities, specific inhibitors of Rho A activation and events that sequentially follow Rho A activation were employed to determine the involvement of these events in colchicine-mediated enhancement of transgene expression. First, A549 cells were treated with Clostridium difficle toxin B (toxin B) that, through glycosylation of an aspartate residue in the effector region of the Rho GTPases, inactivates GTPase activity in all members of the Rho family (Just et al, 1995). Pretreatment of A549 cells with toxin B alone did not significantly alter transiently transfected bgal transgene expression (Figure 2). A549 cells, pretreated with 5 mM of colchicine only for 1 h prior to transfection, caused a 53-fold (± 6.7) increase in bgal reporter gene expression, as compared with untreated, transfected cells (Figure 2). However, toxin B significantly and dose-dependently inhibited the increase in bgal expression induced by colchicine. These results suggested that activation of a Rho GTPase was involved in the colchicine-mediated enhancement transgene expression.
Next, to determine the role of integrin clustering in the colchicine-mediated enhancement of transient transgene expression, we treated A549 cells with the RGD peptide. While the RGD peptide (H-Gly-Arg-Gly-Asp-Ser-OH) is a ligand for integrin proteins, this peptide also prevents integrin-clustering (Pierschbacher and Rouslathi, 1984). An inactive peptide, (H-Gly-Arg-Ala-Asp-Ser-Pro-OH), and the vehicle, acetic acid, were employed as controls.
For these experiments, A549 cells were treated in suspension to prevent plastic adherence-induced integrin clustering (Ruegg et al, 1992). Cells were treated with either the RGD or the inactive peptides for 30 min at 370C, to allow the binding of the peptides to the integrins. The cell suspension was then plated and incubated in the presence of the RGD peptide for an additional 24 hours. Figure 3 shows that RGD peptide alone, at 1 mM, did not significantly alter bgal transgene expression, as compared to untreated, transfected cells. Colchicine alone, at 5 mM, resulted in a 14-fold increase in bgal transgene expression. This colchicine-mediated increase in transgene expression was similar to that seen in cells treated with acetic acid (the vehicle) or the inactive peptide. In contrast, pretreatment with the active RGD peptide significantly inhibited the colchicine-induced increase in reporter gene expression by 70%, as compared to the controls (Figure 3). These results suggest that microtubule depolymerization, most likely acting through RhoA GTPase, initiated integrin-clustering.
While it is unclear as to whether existing actin stress fibers participate in integrin clustering or whether actin stress fibers form as a result of integrin clustering, colchicine treatment is known to stimulate actin stress fiber formation (Blystone, 2004; Wozniak et al, 2004). To determine the role of actin polymerization in the colchicine-mediated increase in transgene expression, cells were treated with cytochalasin D, an inhibitor of actin polymerization. Cytochalasin D alone, at the doses employed, did not significantly alter bgal expression; colchicine, at 5 mM, increased bgal expression in A549 cells by 15-fold (± 1.1), as compared to untreated, transfected cells (Figure 4). As seen in Figure 4, at its highest dose of 10 mM, cytochalasin D significantly inhibited the colchicine-induced increase in transgene expression by 71%. These results indicated that actin polymerization was critical for the colchicine-mediated increase in transgene expression, as delivered to A549 cells using cationic liposomes.
C. Colchicine-mediated enhancement of reporter gene expression requires src-FAK activation
RhoA-regulated integrin clustering and actin stress fiber formation are associated with focal adhesion formation (Schoenwaelder and Burridge, 1999). In addition to their role in anchoring cells to the extracellular matrix, the assembly of integrin-containing, focal adhesions creates a harbor for the docking of various signaling and accessory proteins, including the tyrosine kinases, FAK and c-src, each of which has been implicated in activation of the MAPK pathway (Schlaepfer et al, 1998; Brunton et al, 2004; Graness et al, 2006). Furthermore, microtubule disruption is known to induce tyrosine phosphorylation of FAK as well as other proteins that participate in the formation of focal adhesions (Bershadsky et al, 1996).
To determine the role of tyrosine phosphorylation in the colchicine-mediated enhancement of reporter gene expression, cells were pretreated with genistein, a broad-spectrum inhibitor of tyrosine kinases. Genistein, itself, did not alter bgal transgene expression, as compared to untreated, transfected cells. As seen in previous experiments, colchicine, at 5 mM increased bgal expression (37-fold ± 6.7), as compared to untreated, transfected cells (Figure 5). When used in combination with colchicine, genistein significantly inhibited the ability of colchicine to induce an increase in transgene expression. At its highest dose of 140 mM, genistein blocked the ability of colchicine to induce an increase in transgene expression by 85% (Figure 5). These results suggested that colchicine, either directly or indirectly activated a tyrosine kinase and that tyrosine phosphorylation of some cellular target was involved in enhancing transgene expression.
Colchicine is known to activate FAK and FAK serves as both a tyrosine kinase as well as a scaffold for proteins that direct downstream cell signaling. Thus, our next experiments focused on the role FAK activation in the enhancement of transgene expression. Endogenous FAK activation is regulated, in part, by the cellular molecule, FAK-related non-tyrosine kinase (FRNK) that inhibits FAK tyrosine phosphorylation and interferes with focal adhesion formation (Richardson and Parsons, 1996). We developed an A549 cell line stably overexpressing FRNK (A549FRNK). A549 cells stably expressing neomycin only, the selectable gene employed in generating A549FRNK, were used as transformation controls (A549NEO). To confirm FRNK overexpression, A549NEO and A549FRNK cells were assessed for FAK immunofluorescence by immunocytochemistry. While FAK-specific puncta resembling focal adhesions were present in colchicine-treated, untransfected, non-transformed A549 or A549NEO, FAK-specific immunofluorescence was undetectable in the A549FRNK cells (data not shown).
Expression of transiently transfected pCMVbgal in untreated A549NEO cells showed bgal levels similar to those seen in untreated, but transiently transfected, non-transformed A549 cells. Although not statistically significant, bgal expression in A549FRNK cells, in the absence of colchicine, was decreased by 30%, as compared to untreated, transfected non-transformed A549 or A549NEO cells (Figure 6). Treatment of non-transformed A549 cells with 5 mM of colchicine prior to transient pCMVbgal transfection produced a 12.5-fold (± 0.47) increase in bgal expression. A similar increase in bgal expression was observed in colchicine-treated, bgal transfected, A549NEO cells. In contrast, in A549FRNK cells, the colchicine-mediated increase in reporter gene expression was blunted, by 80 % (2.6 ± 0.33- fold increase in bgal transgene expression), as compared to the A549NEO cells (Figure 6).
Western blot analysis was performed to determine whether or not overexpression of FRNK would alter tyrosine phosphorylation of FAK. Lysates obtained from colchicine-treated A549FRNK and A549NEO cells were subjected to SDS-PAGE, probed for tyrosine-phosphorylated proteins, then reprobed for FAK, itself and finally probed for GAPDH, as a loading control. In A549NEO cells, colchicine treatment significantly increased the tyrosine phosphorylation of a 125 kD protein (Figure 7, inset), corresponding to FAK (IDVCol 5mM = 4.6 ± 0.2), as compared to untreated, bgal transfected A549NEO cells (T=0, IDVcontrol = 2.1 ± 1.2) (Figure 7). However, in lysates obtained from A549FRNK cells, colchicine treatment did not increase levels of the tyrosine-phosphorylated, 125 kD protein (IDVCol 5mM = 1.8 ± 0.8); band intensity was similar to that obtained in lysates from untreated, bgal transfected A549NEO cells (IDVControl =1.3 ± 0.4). These combined results suggested that tyrosine phosphorylation, specifically of FAK, was important for the colchicine-mediated enhancement in transient transgene expression in A549 cells.
Phosphorylation of FAK has been shown to be important for the recruitment of src-family kinase. Specifically, the phosphorylation on FAK tyrosine residue 397 creates a high affinity binding site recognized by the SH2 domain of src family of kinases (Schaller et al, 1994). In order to elucidate the role of src-kinase in the colchicine-mediated enhancement of reporter gene expression, we utilized with PP1, a more specific inhibitor of src-kinase (Hanke et al, 1996) in subsequent experiments.
As expected, 5mM colchicine, significantly increased bgal expression by 21-fold (± 6.4), as compared to untreated, transfected A549 cells (Figure 8). PP1, alone, at the concentrations employed (2-10 mM), did not alter bgal expression, as compared to untreated, transfected cells. However, when combined with colchicine, PP1 significantly and dose-dependently inhibited the ability of colchicine to mediate an increase in reporter gene expression. The highest dose of PP1 (10 mM,) inhibited the colchicine-mediated increase in bgal expression by 89% (Figure 8). To clarify the relationship between src family tyrosine kinase activity and FAK activation, A549 cells were pretreated with 10 mM of PP1 for 2h prior to treatment with 5 mM colchicine and subsequent pCMVbgal transfection. FAK was immunoprecipitated (IP) with anti-FAK antibody and eluted proteins were resolved by SDS-PAGE and incubated with either anti-phosphotyrosine or anti-FAK. A representative FAK-IP, incubated with anti-phosphotyrosine and anti-FAK immunoblot is presented as an inset in Figure 8 shows that PP1, in addition to blocking colchicine-mediated enhancement of transgene expression, significantly inhibited colchicine-mediated FAK tyrosine phosphorylation. These combined results suggest that colchicine-mediated enhancement in reporter gene expression requires src family kinase activity and that src kinases phosphorylated FAK.
D. Colchicine-induced increase in reporter gene expression involves formation of focal adhesions
In order to determine whether or not focal adhesion formation correlated with colchicine-induced enhancement of reporter gene expression, A549 cells were grown on cover slips and treated with colchicine alone or in combination with genistein prior to transfection and then analyzed by immunocytochemistry, using anti-phosphotyrosine and anti-FAK antibodies.
Dual labeling of untreated, untransfected A549 cells with anti-phosphotyrosine antibody and anti-FAK antibody revealed little or no co-localized staining at the cell periphery, where focal adhesions typically form. A similar lack of colocalized staining at the periphery was observed in genistein-treated, transfected cells (Figure 9). In contrast, colchicine-treated cells displayed dual-stained puncta localized to the cell membrane (Figure 9. panel L). Addition of genistein to colchicine-treated cells completely abolished the appearance of these focal adhesions, indicating that tyrosine phosphorylation was required to produce colchicine-mediated formation of focal adhesions.
This is the first report showing FAK/src activation and focal adhesion formation participate in the enhanced expression of lipoplex-mediated, transiently transfected DNA and offers insight into how efficiency of lipoplex-mediated transfection can be improved.
Although the ability of colchicine to enhance transgene expression has been well documented (Baru et al, 1995; Chowdhry et al, 1996), the mechanism by which microtubule disruption enhances transgene expression was presumed to be due solely to disruption of endo-lysosomal fusion (Chowdhry et al, 1996; Hasegawa et al, 2001; Wang and MacDonald, 2004). However, we show that in A549 cells enhanced transgene expression occurs even when colchicine was added 24 h after transfection, a time by which endo-lysosomal fusion should be complete (Rejman et al, 2005).
Microtubule-disruption also induces activation of Rho A GTPases, and integrin clustering (Bershadsky et al, 1996; Enomoto, 1996; Kirchner et al, 2003; Graness et al, 2006). Clostridium difficile toxin B, an inhibitor of Rho GTPases (Just et al, 1995) inhibited colchicine-mediated enhancement of transient, transgene expression by approximately 65%, indicating Rho GTPase activation was involved in enhanced transgene expression. In these experiments, enhancement of transgene expression by colchicine alone appeared greater (53-fold), relative to other experiments presented in this report (average fold-change, ~22-fold). This greater level of enhanced transgene expression is most likely the result of the longer incubation required with toxin B treatment (48 h). During the incubation, both the untreated and treated A549 cells continued to divide in the experimental cell culture dishes. Thus, at transfection, there were more cells available for transfection than experiments that require a shorter pretreatment incubation.
The mechanism by which microtubular depolymerization causes activation of Rho family of GTPases is not completely understood. However, certain guanine nucleotide exchange factors (GEF), such as GEF-H1, an exchange factor for, rac1 and rho A GTPases, are localized and sequestered on the microtubules (Ren et al, 1998). Microtubule disruption liberates these GEFs where they are free to activate Rho GTPases (Ren et al, 1998).
Rho GTPase activation is seen just prior to integrin clustering. When integrins associate with certain extracellular matrix substrates or activated growth factor receptors, conformational changes within the intracellular portion of the integrin promote the binding of a variety of molecules that direct subsequent signaling cascades (Schlaepfer et al, 1998; Martin et al, 2002). RGD peptide, known to block integrin clustering (Pierschbacher and Rouslathi, 1984), inhibited the colchicine-mediated enhancement of transgene expression by as much as 70%, as compared to cells pretreated with a control peptide, suggesting that either integrin clustering itself or some process associated with integrin clustering was important for enhanced transgene expression.
Keller and colleagues previously reported that they could enhance transient, lipoplex-mediated lymphocyte transfection by allowing a non-adherent, hematopoietic cell line to attach to an adherent cell line prior to transfection with pCMVbgal. Transgene expression within the hematopoeitic cell line was significantly enhanced (Keller et al, 1999). While they did not address the role of integrins in transgene expression, it is tempting to consider that integrins may have also played a role in enhancing transgene expression in this notably difficult-to-transfect, non-adherent cell type. In a more recent report, Perlstein and colleagues examined the role of integrin ligands in transfection of smooth muscle cells (Perlstein et al, 2003). They found that a denatured collagen, a ligand for integrin anb3 the same integrin expressed on A549 cells (Majda et al, 1994), enhanced liposome-mediated transgene expression 10-20 fold (Perlstein et al, 2003).
Clustered integrins provide a scaffold for actin-binding proteins including talin, filamin and a-actinin (Lee and Juliano, 2004). Whether integrins provide an anchor point for the development of actin filaments (Blystone, 2004) or whether polymerized actin fibers direct integrin clustering (Wozniak et al, 2004) is still debatable. Nonetheless, with cellular adhesion, actin stress fibers are formed (Brakebusch and Fassler, 2003). We show that pretreatment of A549 cells with cytochalasin D prior to the addition of colchicine and transfection, diminished colchicine-mediated enhancement of transgene expression by approximately 70%, indicating that stress fiber formation was also involved in enhancing transgene expression.
In contrast to our results, Perlstein and colleagues found that a 24 h treatment of cells with cytochalasin D modestly enhanced (6-fold), rather than blocked transgene expression, a result they attribute to an increase in the amount of cytoplasmic G-actin, known to act as an inhibitor of DNAse I (Perlstein et al, 2003). These authors also showed that jasplakonide, a drug that stabilizes actin polymers, decreased transfection efficiency (Perlstein et al, 2003). However, Brisson and colleagues using cytochalasin B, which also inhibits stress fiber formation, saw little change in transfection efficiency (Brisson et al, 1999). In addition, in our hands neither cytochalasin D nor jasplakonide enhanced pCMVbgal transfection (data not shown). The contradictions between these studies most likely involve differences in cells employed, cell culture substrates (collagen vs. plastic), drug concentration and duration of drug exposure. Nevertheless, our experiments employing RGD, toxin B and cytochalasin D, support a model where activation of Rho GTPases, actin polymerization and clustering of integrins accompany microtubule disruption and play an important role in expression of transiently transfected transgenes.
Downstream of the integrins, signaling critical to enhancement of transgene expression appears to involve tyrosine kinase activation. FAK autophosphorylation of tyr 397 creates a high affinity binding site, recognized by the SH2 domain of src family kinases (Schaller et al, 1994). We show that pretreatment of A549 cells with either genistein, a broad-spectrum tyrosine kinase inhibitor or the more selective PP1, inhibit colchine-mediated enhancement of transgene expression. Notably, colchicine treatment of A549 cells stably expressing FRNK, an endogenous, negative regulator of FAK, failed to enhance transgene expression in these transformants. Lack of enhanced transgene expression correlated with a decrease in colchicine-mediated FAK phosphorylation in A549FRNK cells.
Not only did PP1 dose-dependently decrease the ability of colchicine to augment transgene expression but as shown by FAK immunoprecipitation, PP1 treatment suppressed the degree of FAK tyrosine phosphorylation. These combined results suggest that both src and FAK tyrosine kinases are involved in enhancing transgene expression and that FAK is a target of a src kinase.
How does activation of this src-FAK pathway lead to increased transgene expression? FAK has been implicated, directly or indirectly, with activating expression of endogenous genes. Watanabe and colleagues showed that FAK overexpression induced expression of endogenous monocyte chemotractant protein-1 (MCP-1). Upregulation of MCP-1 was inhibited by genistein and by overexpression of FRNK. (Watanabe et al, 2003).
Irigoyen et al. showed that colchicine-mediated expression of the endogenous urokinase-type plasminogen activator gene involved not only FAK-src activation but subsequently, activation of ERK 1/2 (Irigoyen and Nagamine, 1999). Thus, a potential means whereby FAK increases gene or even transgene expression is via activation of the Ras/MAP kinase cascade. The FAK-src-kinase interaction appears to facilitate the assembly of scaffolding proteins that promote MAP kinase binding and subsequent activation of ERK (Schlaepfer et al, 1998), (Irigoyen and Nagamine, 1999). In support of this signaling pathway, an increase in ERK1/2 phosphorylation was seen in A549 cells pretreated with colchicine (data not shown, Ph. D thesis, R R Nair). Moreover, when vinblastine, another agent that disrupts microtubules, was directly incorporated into cationic liposomes, not only was early lysosomal release of transfected DNA seen but vinblastine also increased NF-kB activation (Wang and MacDonald, 2004). Thus, activation of cellular transcription factors, through a microtubule-mediated stimulation of certain kinases might increase the level of transgene transcription. For example, colchicine is known to increase cyclooxygenase-2 gene transcription via activation of the ERK pathway (Subbaramaiah et al, 2000). Notably, colchicine did not increase actin B gene expression in the A549 cells, indicating that this enhancement does not apply to globally, to every cellular gene (Nair et al, 2002). Further studies will be needed to elucidate these specific mechanisms.
In conclusion, our finding that focal adhesions are involved in the enhancement of transgene expression has important implications for improving the outcome of lipid-mediated gene therapy. As the actual targets within this pathway are identified, specific agents that stably mimic these effects may prove useful for improving lipoplex-mediated, transient transfection.
The authors wish to acknowledge the support of this project by the Department of Pharmacological and Pharmaceutical Sciences at the University of Houston.
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| figure 1
|| Colchicine-mediated enhancement of reporter gene expression occurs independently of endo-lysosomal fusion. Cells were transfected for two hours as described. Transfected cells (TFX) were treated with 5 mM of colchicine (TFX, COL) for an interval of 1 h immediately after transfection (T=0 hours) or 6 h, 12 h, 18 h and 24 h after transfection. Cells were harvested 72 hours post-transfection and supernatants analyzed for bgal activity by CPRG assay, as described. *p<0.05, when compared to untreated, transfected cells (n=3). Average maximal U/ml bgal, 0.023 ± 0.0046; Average control U/ml bgal (fold-increase = 1), 0.00064 ± 0.00008.
| figure 2
|| Rho GTPases participate in colchicine-mediated enhancement of transgene expression in A549 cells. A549 cells were treated with toxin B (4, 8, 12, 16 and 20 ng/ml) in either in growth medium (Medium) or medium plus 5 mM of colchicine (Colchicine, 1 h) for 48 h. Cells were then transfected with pCMVbgal as described. Cells were lysed and supernatants were harvested and bgal activity determined as described. *p<0.05, when compared to untreated, transfected cells; #p<0.05, when compared to colchicine treated, transfected cells (n=4). Average maximal U/ml bgal, 0.012 ± 0.0013; average control U/ml bgal, 0.00022 ± 0.00011. Inset: Effects of toxin B on transfection (U/ml bgal).
| figure 3
|| Colchicine-mediated increases in reporter gene expression involves integrin clustering. Cells were incubated in 0.1% FBS overnight, then treated with 1mM of RGD peptide (H-Gly-Arg-Gly-Asp-Ser –OH) in growth medium (Medium) or with medium containing 5 mM of colchicine (Colchicine, 1 h) for 24 h. Acetic acid only and an inactive peptide (1mM) (H-Gly-Arg-Ala-Asp-Ser-Pro-OH), were employed as controls. Cells were transfected with pCMVbgal as described. Cell supernatants were harvested and bgal levels determined. *p<0.05, when compared with untreated, transfected cells; #p<0.05, when compared with colchicine treated, transfected cells (n=3). Average maximal U/ml bgal, 0.0065 ± 0.006; average control U/ml bgal (fold-change = 1), 0.00043 ± 0.0004.
| figure 4
|| Stress fiber formation is involved in colchicine-mediated enhancement of reporter gene expression in A549 cells. Cells were treated with cytochalasin D (2, 4, 6, 8, and 10 mM) in either growth medium (Medium) or medium containing 5 mM of colchicine (Colchicine, 1 h) for 5 h. Cells were transfected with pCMVbgal as described. Cell supernatants were harvested and bgal levels were determined. *p<0.05, when compared to untreated, transfected cells; #p<0.05, when compared to colchicine-treated, transfected cells (n=3). Average maximal U/ml bgal, 0.00136 ± 0.0013; average control U/ml bgal, 0.00009 ± 0.00004.
| figure 5
|| Colchicine-induced increases in reporter gene expression requires tyrosine phosphorylation. Cells were treated with genistein (60, 80, 100, 120 and 140 mM) either in growth medium (Medium) or with medium containing 5 mM of colchicine (Colchicine, 1 h) for 5 h. Cells were transfected with pCMVbgal as described. Cell supernatants were harvested and bgal levels determined. *p<0.05, when, compared to untreated, transfected cells; #p<0.05, when compared with colchicine treated, transfected cells (n=4). Average maximal U/ml bgal, 0.0048 ± 0.0006; average control U/ml bgal (fold-change = 1), 0.00013 ± 0.00003.
| figure 6
|| Activation of FAK is required for colchicine-induced increase in reporter gene expression in A549 cells. Non-transformed A549 cells (A549 cells), A549 cells stably expressing neomycin only (Neo-stables) and A549 cells stably expressing FRNK plus neomycin (FRNK-stables) were incubated in medium containing 0.1% FBS overnight then treated with either growth medium (Medium) or medium containing 5 mM of colchicine (Colchicine) for 1 h. Cells were transfected with pCMVbgal as described. Cell supernatants were harvested and bgal levels were determined. *p<0.05, when compared to respective untreated, transfected cells; #p<0.05, when compared with colchicine-treated, transfected cells (n=3). Average maximal U/ml bgal, 0.0042 ± 0.0033; average bgal, medium-treated A549 cells, 0.00033 ± 0.00025 (fold-change = 1); average bgal, medium-treated A549, stably expressing pRSVneo, (neo-stables), 0.00035 ± 0.00013 U/ml,(fold-change = 1; average bgal in medium-treated, A549 cells stably expressing FRNK, (FRNK-stables) 0.00020 ± 0.00003 U/ml, (fold-change =1).
| figure 7
|| FAK activation is required for colchicine-induced increase in transgene expression. A549 cells, stably expressing neomycin or FRNK plus neomycin, were incubated overnight in DMEM containing 0.1% FBS, then treated with either medium only, or medium containing either 1 mM or 5 mM of colchicine, for 1 h. (A) Cell lysates were subjected to SDS-PAGE and proteins blotted as described. NEO, untreated A549 cells stably expressing neomycin only; NEO+Col, 1mM, A549 cells stably expressing neomycin, treated with 1 mM of colchicine; NEO+Col, 5mM, A549 cells stably expressing neomycin treated with 5 mM of colchicine; FRNK, untreated A549 cells stably expressing FRNK+neomycin; FRNK Col, 1 mM, A549 cells stably expressing FRNK+neomycin, treated with 1 mM of colchicine; FRNK Col, 5mM, A549 cells stably expressing FRNK+neomycin, treated with 5 mM of colchicine. Blots were analyzed for phosphotyrosine proteins and FAK. GAPDH was used as loading control. (B) Immunoblot band intensities were analyzed by densitometry (integrated density values, IDV) and are represented as mean ± SD of the ratio of the IDV of the 125 kD anti-phosphotyrosine (PY) (125 kD)/ IDV of GAPDH, from 3 independent experiments. *p<0.05, when compared with PY-125 kD/ GAPDH IDV ratio of A549 cells stably expressing FRNK.
| figure 8
|| PP1 decreases colchicine-mediated increase in transgene expression through inhibition of FAK activity. Cells were treated with PP1 (2, 4, 6, 8, and 10 mM) for 5 h before transfection. Colchicine (5 mM, final concentration) was added to one set of PP1-treated cell cultures and all cultures were incubated for an additional h. PP1 treatment was continued during and for 24 hours after transfection. bgal activity was determined in cell supernatants. *p<0.05, as compared to untreated, transfected cells; #p<0.05, as compared to colchicine-treated, transfected cells (n=3). Inset: A549 cells were treated with medium (Control, Transfection), 5 m M colchicine (Colchicine, 1 h), 10 mM PP1 for 5 h (PP1) or 10 mM PP1 (5 h) plus colchicine (5 mM, 1h) (Colchicine+PP1). All cultures except "control" were transfected with pCMVbgal. Cell lysates were immunoprecipitated using anti-FAK antibody and then subjected to SDS-PAGE. Blots were incubated first with anti-phosphotyrosine and after film exposure stripped and incubated with anti-FAK, as loading control.
|| Colchicine induces focal adhesion formation and FAK tyrosine phosphorylation in A549 cells. A549 cells were plated on coverslips and incubated overnight in 0.1% FBS. The cells were treated with medium, 5 mM of colchicine (1h), alone or in combination with 140 mM of genistein (5 h). Cells were transfected and immunocytochemistry performed as described. (A, B, C), untreated, untransfected cells; (D, E, F), untreated, transfected cells; (G, H, I), genistein-treated, transfected cells; (J, K, L), colchicine-treated, transfected cells; (M, N, O), colchicine plus genistein-treated, transfected cells. (A, D, G, J and M ), were stained with anti-FAK; (B, E, H, K and N), are the same cells additionally stained with anti-phosphotyrosine (anti-PY) and finally (C, F, I, L and O) are respective merged images. The panels shown are representative of three independently- performed experiments.