Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation “status” of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.     

RANKL increases migration of human lung cancer cells through intercellular adhesion molecule-1 up-regulation

Invasion of distant tissues by tumor cells is the primary cause of therapeutic failure in the treatment of malignant lung cancer cells. Receptor activator of nuclear factor-κB ligand (RANKL) and its receptor, RANK, play a key role in osteoclastogenesis and tumor metastasis. Intercellular adhesion molecule-1 (ICAM-1, also called CD54), a member of the immunoglobulin supergene family, is an inducible surface glycoprotein that mediates adhesion-dependent cell-to-cell interactions. The effects of RANKL on cell migration and ICAM-1 expression in human lung cancer cells are largely unknown. We found that RANKL directed the migration and increased ICAM-1 expression in human lung cancer (A549) cells. Pretreatment of A549 cells with the MAPK kinase (MEK) inhibitor PD98059 or U0126 inhibited RANKL-mediated migration and ICAM-1 expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, an NF-κB inhibitor (PDTC) and IκB protease inhibitor (TPCK) also inhibited RANKL-mediated cell migration and ICAM-1 up-regulation. Taken together, these results suggest that the RANKL and RANK interaction acts through MEK/ERK, which in turn activates NF-κB, resulting in the activation of ICAM-1 and contributing to the migration of human lung cancer cells.     

The EphB4 receptor-tyrosine kinase promotes the migration of melanoma cells through Rho-mediated actin cytoskeleton reorganization

Several studies have reported the up-regulation of EphB receptor-tyrosine kinases and ephrin-B ligands in a variety of tumors, suggesting a functional relation between EphB/ephrin-B signaling and tumor progression. The ability of the EphB receptors to regulate cell migration and promote angiogenesis likely contributes to tumor progression and metastasis. Here we show that EphB receptors, and especially EphB4, regulate the migration of murine melanoma cells. Highly malignant melanoma cells express the highest levels of EphB4 receptor and migrate faster than less malignant melanoma cells. Furthermore, inhibition of EphB receptor forward signaling by overexpression of a form of EphB4 lacking the cytoplasmic portion or by treatment with competitively acting soluble EphB2-Fc results in slower melanoma cell migration. In contrast, overexpression of active EphB4 significantly enhances cell migration. The effects of EphB4 receptor on cell migration and cell morphology require its kinase activity because the inhibition of EphB4 kinase activity by overexpression of kinase dead EphB4 inhibits cell migration and affects the organization of actin cytoskeleton. Activation of EphB4 receptor with its ligand ephrin-B2-Fc enhances the migratory ability of melanoma cells and increases RhoA activity, whereas inhibiting EphB receptor forward signaling decreases RhoA activity. Moreover, expression of dominant negative RhoA blocks the effects of active EphB4 on cell migration and actin organization. These data suggest that EphB4 forward signaling contributes to the high migratory ability of invasive melanoma cells by influencing RhoA-mediated actin cytoskeleton reorganization.     

Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization

Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells.     

Probe diffusion in solutions of filamentous actin formed in the presence of gelsolin.

Dynamic light scattering was used to characterize the diffusion of monodisperse polystyrene latex spheres (PLS) of different sizes (55-, 105-, and 265-nm radii) in column-purified 0.65 mg/mL actin solutions polymerized with 100 mM KCl in the absence and presence of various amounts of the actin-binding protein gelsolin. The gelsolin and its interaction with actin was initially studied to ensure that the gelsolin could be used to produce filament populations with well-defined mean lengths. Measurements with PLS diffusion probes present showed, in the absence of gelsolin, that the effective local microviscosity in the actin solutions was 5-20 times that of water and that a large fraction of the PLS are trapped within the pores of the actin filament network, as found previously [J. Newman, K. L. Schick, & K. S. Zaner, (1989) Biopolymers 28, 1969-1980]. As the molar ratio of gelsolin to actin was increased, the diffusion coefficients of the PLS approached those in pure water while the degree of PLS trapping went to zero. Measurements of the dependence of the PLS diffusion coefficients on the ratio of actin concentration to the semidilute overlap concentration showed, for the smaller PLS, a transition occurring near the mean global overlap concentration. These results reflect the dissolution of the actin network as the gelsolin concentration is increased and illustrate the role of gelsolin/actin interactions in the control of macromolecular transport within the periphery of cells.     

Overexpression LINC01082 suppresses the proliferation,migration and invasion of colon cancer

The active form of vitamin D, 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃), was reported to improve vascular function in patients with diabetes, yet the underlying mechanisms remain to be fully elucidated. Monoamine oxidase (MAO), a mitochondrial enzyme, with two isoforms (A and B) that generates hydrogen peroxide (H₂O₂) as by-product, has been recently reported to contribute to the pathogenesis of endothelial dysfunction in diabetes. The present study assessed the interaction between vitamin D and MAO in the vascular wall in the setting of type 1 experimental diabetes. To this aim, diabetes was induced in male Wistar rats via a single injection of streptozotocin (STZ, 50 mg/kg, IP) and 1 month later thoracic aortas were harvested and used for organ bath studies and H₂O₂ measurements. MAO expression was assessed by immunohistochemistry and RT-PCR. Endothelial function was evaluated in isolated aortic rings in the absence vs. presence of 1,25(OH)₂D₃ (100 nM, 24 h incubation). In diabetic animals, we found a significant reduction in the endothelial-dependent relaxation to acetylcholine and an increased expression of the MAO-A isoform, respectively. Vitamin D significantly improved vascular function, mitigated oxidative stress and decreased MAO-A expression in diabetic vascular preparations. In conclusion, MAO-A is induced in diabetic aortas and vitamin D can improve diabetes-induced endothelial dysfunction by modulating the MAO-A expression.

     

Alternative splicing of the actin binding domain of human cortactin affects cell migration

Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal "cortactin repeats". Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.     

Increased sensitivity of human colon cancer cells to DNA cross-linking agents after GRP78 up-regulation

We have shown earlier that pre-treatment of V79 Chinese hamster cells with 6-aminonicotinamide (6-AN) or 2-deoxyglucose (2-dG) results in over-expression of the Mr 78,000 glucose-regulated stress protein (GRP78) and the subsequent development of resistance to inhibitors of topoisomerase II. These phenomena also occur in V79-derived cell lines that are deficient in poly(ADP-ribose) (p(ADPR)) metabolism. In contrast, over-expression of GRP78 under the conditions outlined above is found to be associated with hypersensitivity to several clinically-relevant DNA cross-linking agents, namely, 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, and melphalan. We have also previously shown that pre-treatment with 6-AN, an inhibitor of p(ADPR) metabolism, causes an increase in the life span in BCNU-treated mice bearing L1210 tumors. These observations prompted us to examine whether 6-AN pre-treatment can result in the over-expression of GRP78 in human colon cancer cell lines and, if so, whether this increase is associated with sensitization to DNA cross-linking agents outlined above. Following treatment of three colon cancer cell lines, HCT116, SW480, and VACO-8, for 48 h with 0.1 mM 6-AN, cytosolic GRP78 levels were elevated approximately 4.2 times, 8 times, and 2.5 times for each cell line respectively, as measured by Western immunoblotting. To determine sensitivity after GRP78 up-regulation, the cells were washed and grown for 412 h in growth medium devoid of 6-AN, before being treated with DNA cross-linking agents. The 412 h time period allowed p(ADPR) metabolism to return to normal while GRP78 levels remained elevated, thus allowing us to associate GRP78 over-expression with sensitivity to those agents. After treating cells for 1 h with BCNU, cisplatin, or melphalan, cell sensitivity was determined by clonogenic survival assay or a fluorescence-based cytotoxicity assay. Based on changes in IC50 values, 6-AN caused an increase in sensitivity for HCT116, SW480, and VACO-8 cells of 1.5, 2.3, and 1.0 times, respectively, for BCNU, 4.8, 3.8, and 2.6 for cisplatin, and 6.4, 3.7, and 2.2 times for melphalan. Thus, our results show that over-expression of GRP78 in human tumor cell lines is associated with increased sensitivity to clinically useful chemotherapy agents. This sensitization occurred in three different tumor cell lines, each bearing a separate genetic defect associated with altered sensitivity.     

EGF enhances the migration of cancer cells by up-regulation of TRPM7

Ion channels involved in the migration of tumor cells that is required for their invasion and metastasis. In this paper, we describe the interaction of TRPM7 channel and epidermal growth factor (EGF), an important player in cancer development in the migration of lung cancer cells. The TRPM7 currents in A549 cells were first characterized by means of electrophysiology, pharmacology and RNA interference. Removing Ca²⁺ from the extracellular solution not only potentiated a large inward current, but also abolished the outward rectification. 200 μM 2-APB inhibited the outward and the inward TRPM7 currents and at the same time restored the property of outward rectification. EGF greatly enhanced the migration of A549 cells, and also markedly up-regulated the membrane protein expression of TRPM7 and the amplitude of TRPM7 currents. Depressing the function of TRPM7 with RNA interference or pharmacological agents not only reversed the EGF-enhanced migration of A549 cells but also inhibited the basal migration of A549 cells in the absence of EGF. Thus it seems that TRPM7 plays a pivotal role in the migration of A549 cells induced by EGF and thus could be a potential therapeutic target in lung cancers.     

Overexpression of profilin reduces the migration of invasive breast cancer cells

The exact role profilin plays in cell migration is not clear. In this study, we have evaluated the effect of overexpression of profilin on the migration of breast cancer cells. Overexpression was carried out by stably expressing GFP-profilin in BT474 cells. It was observed that even a moderate level of overexpression of profilin significantly impaired the ability of BT474 cells to spread on fibronectin-coated substrate and migrate in response to EGF. GFP-profilin expressing cells also showed increased resistance to detachment in response to trypsin and increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin compared to the parental and GFP-expressing (control) cell lines. These results suggest that perturbation of profilin levels may offer a good strategy for controlling the metastatic potential of breast cancer cells.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

IL-8 promotes cell proliferation and migration through metalloproteinase-cleavage proHB-EGF in human colon carcinoma cells

Interleukin-8 (IL-8) has been reported to promote tumor cell growth in colon cancer cells after binding to its receptors, which are members of the G-protein coupled receptor (GPCR) family. Recent studies demonstrated that stimulation of GPCR can induce shedding of epidermal growth factor (EGF) ligands via activation of a disintegrin and metalloprotease (ADAM), with subsequent transactivation of the EGF receptor (EGFR). In this study, we investigated mechanisms of cell proliferation and migration stimulated by IL-8 in a human colon carcinoma cell line (Caco2). IL-8 increased DNA synthesis of Caco2 in a dose dependent manner and this was inhibited by ADAM, EGFR kinase, and MEK inhibitors. IL-8 transiently induced EGFR tyrosine phosphorylation after 5-90 min and this was completely inhibited by ADAM inhibitor. Neutralizing antibody against HB-EGF as a key ligand for EGFR also blocked transactivation of EGFR and cell proliferation by IL-8. Since IL-8-induced cell migration was further suppressed by the ADAM inhibitor and the HB-EGF neutralizing antibody, our data indicate that IL-8 induces cell proliferation and migration by an ADAM-dependent pathway, and that HB-EGF plays an important role as the major ligand for this pathway.     

Polarization of gelsolin and actin binding protein in kidney epithelial cells

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.     

Vitamin C increases the apoptosis via up-regulation p53 during cisplatin treatment in human colon cancer cells

Vitamin C (VC) is an important antioxidant and enzyme co-factor that works by stimulating the immune system and protecting against infections. It is well known that melanoma cells are more susceptible to VC than any other tumor cells. However, the role of VC in the treatment of colon cancer has not been studied. Cisplatin (CDDP) is a DNA damaging agent and is widely used for treating cancer, while the role of p53 in CDDP-induced cell death has been stressed. Using cell growth assays, morphological methods, Western blotting, flow cytometry, and DNA fragmentation analysis, we measured the expression of p53 level involved in the effect of VC on CDDP-induced apoptosis of HCT116, a human colon cancer cell line. CDDP plus VC treatment resulted in significantly increased apoptosis along with upregulation of p53 compared to untreated cells and/or CDDP-treated cells. These results suggest that VC enhanced CDDP sensitivity and apoptosis via upregulation of p53.     

Hydrogen peroxide stimulates proliferation and migration of human prostate cancer cells through activation of activator protein-1 and up-regulation of the heparin affin regulatory peptide gene

It is becoming increasingly recognized that hydrogen peroxide (HP) plays a role in cell proliferation and migration. In the present study we found that exogenous HP significantly induced human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) seems to be involved in the stimulatory effect of HP, because the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, HP significantly increased HARP mRNA and protein amounts in a concentration- and time-dependent manner. Curcumin and activator protein-1 (AP-1) decoy oligonucleotides abrogated both HP-induced HARP expression and LNCaP cell proliferation and migration. HP increased luciferase activity of the 5'-flanking region of the HARP gene introduced in a reporter gene vector, an effect that was abolished when even one of the two putative AP-1 binding sites of the HARP promoter was mutated. The effect of HP seems to be due to the binding of Fra-1, JunD, and phospho-c-Jun to the HARP promoter. These results support the notion that HARP is important for human prostate cancer cell proliferation and migration, establish the role of AP-1 in the up-regulation of HARP expression by low concentrations of HP, and characterize the AP-1 dimers involved.     

Gelsolin affects the migratory ability of human colon adenocarcinoma and melanoma cells

AimsFormation of different protrusive structures by migrating cells is driven by actin polymerization at the plasma membrane region. Gelsolin is an actin binding protein controlling the length of actin filaments by its severing and capping activity. The main goal of this study was to determine the effect of gelsolin expression on the migration of human colon adenocarcinoma LS180 and melanoma A375 cells.Main methodsColon adenocarcinoma cell line LS180 was stably transfected with plasmid containing human cytoplasmic gelsolin cDNA tagged to enhanced green fluorescence protein (EGFP). Melanoma A375 cells were transfected with siRNAs directed against gelsolin. Real-time PCR and Western blotting were used to determine the level of gelsolin. The ability of actin to inhibit DNase I activity was used to quantify monomeric and total actin level and calculate the state of actin polymerization. Fluorescence confocal microscopy was applied to observe gelsolin and vinculin distribution along with actin cytoskeleton organization.Key findingsIncreased level of gelsolin expression leads to its accumulation at the submembranous region of the cell accompanied by distinct changes in the state of actin polymerization and an increase in the migration of LS180 cells. In addition, LS180 cells overexpressing gelsolin form podosome-like structures as indicated by vinculin redistribution and its colocalization with gelsolin and actin. Downregulation of gelsolin expression in melanoma A375 cells significantly reduces their migratory potential.SignificanceOur experimental data indicate that alterations in the expression level of gelsolin and its subcellular distribution may be directly responsible for determining migration capacity of human cancer cells.     

Lipopolysaccharide from Coxiella burnetii is involved in bacterial phagocytosis, filamentous actin reorganization, and inflammatory responses through Toll-like receptor 4

The role of Toll-like receptors (TLRs) in the recognition of extracellular and facultative intracellular bacteria by the innate immune system has been extensively studied, but their role in the recognition of obligate intracellular organisms remains unknown. Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that specifically inhabits monocytes/macrophages. We showed in this study that C. burnetii LPS is involved in the uptake of virulent organisms by macrophages but not in that of avirulent variants. The uptake of virulent organisms was dependent on TLR4 because it was reduced in macrophages from TLR4(-/-) mice. In addition, LPS was responsible for filamentous actin reorganization induced by virulent C. burnetii, which was prevented in TLR4(-/-) macrophages. In contrast, the intracellular fate of C. burnetii was not affected in TLR4(-/-) macrophages, suggesting that TLR4 does not control the maturation of C. burnetii phagosome and the microbicidal activity of macrophages. These results are consistent with in vivo experiments because the pattern of tissue infection and the clearance of C. burnetii were similar in wild-type and TLR4(-/-) mice. We also showed that the number of granulomas was decreased in the liver of infected TLR4(-/-) mice, and the formation of splenic granulomas was only transient. The impaired formation of granulomas was associated with decreased production of IFN-gamma and TNF. Taken together, these results demonstrate that TLR4 controls early events of C. burnetii infection such as macrophage phagocytosis, granuloma formation, and cytokine production.     

Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric G(i) protein (the Ggamma subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.