Farnesyltransferase inhibitors induce dramatic morphological changes of KNRK cells that are blocked by microtubule interfering agents

Farnesyltransferase inhibitors (FTIs) exhibit the remarkable ability to inhibit transformed phenotypes of a variety of human cancer cell lines and to block the growth of cancer cells in a number of animal model systems. In this paper, we report that the addition of FTI to v-K-ras- transformed NRK cells (KNRK) results in dramatic morphological changes. Within 24 h after the addition of FTI, the round morphology of KNRK cells was changed to an elongated (flattened and spread out) morphology resembling those of untransformed NRK cells. No morphological effects were seen when similar concentrations of FTI were added to NRK cells. Phalloidin staining showed that FTI treatment did not restore the disrupted actin cytoskeleton in KNRK cells. In contrast, FTI addition resulted in the appearance of extensive microtubule networks in KNRK cells. The addition of a low concentration (1.2 nM) of vincristine or vinblastine, agents that interfere with microtubule dynamics, blocked the FTI-induced morphological changes in KNRK cells. In contrast, cytochalasin B, which interferes with actin polymerization, did not block the morphological changes. The FTI-induced morphological changes were associated with a decrease in the percentage of cells in S-phase, and the addition of 1.2 nM vincristine did not have additional effects on cell cycle progression. A higher concentration (12 nM) of vincristine caused synergistic effect with FTI to enrich dramatically KNRK cells in G2/M phase. These results suggest that FTI affects cell morphology and that microtubule dynamics are involved in these processes.     

Role of the actin cytoskeleton on epithelial Na+ channel regulation

The regulatory role of actin filament organization on epithelial Na+ channel activity is reviewed in this report. The actin cytoskeleton, consisting of actin filaments and associated actin-binding proteins, is essential to various cellular events including the maintenance of cell shape, the onset of cell motility, and the distribution and stability of integral membrane proteins. Functional interactions between the actin cytoskeleton and specific membrane transport proteins are, however, not as well understood. Recent studies from our laboratory have determined that dynamic changes in the actin cytoskeletal organization may represent a novel signaling mechanism in the regulation of ion transport in epithelia. This report summarizes work conducted in our laboratory leading to an understanding of the molecular steps associated with the regulatory role of the actin-based cytoskeleton on epithelial Na+ channel function. The basis of this interaction lies on the regulation by actin-binding proteins and adjacent structures, of actin filament organization which in turn, modulates ion channel activity. The scope of this interaction may extend to such relevant cellular events as the vasopressin response in the kidney.     

Depudecin induces morphological reversion of transformed fibroblasts via the inhibition of histone deacetylase

Depudecin is a fungal metabolite that reverts the rounded phenotype of NIH 3T3 fibroblasts transformed with v-ras and v-src oncogenes to the flattened phenotype of the nontransformed parental cells. The mechanism of detransformation induced by this agent had not been determined. Here, we demonstrate that depudecin inhibits histone deacetylase (HDAC) activity effectively both in vivo and in vitro. Depudecin induces similar morphological reversion in v-ras transformed NIH 3T3 cells as do other naturally occurring HDAC inhibitors such as trichostatin A or trapoxin. It competitively inhibits the binding of [3H]trapoxin in vitro and the nuclear binding of a trapoxin-coumarin fluorophore in vivo, suggesting that depudecin shares a nuclear binding protein and site on that protein with trapoxin. Furthermore, depudecin induces hyperacetylation of histones in a dose-dependent manner and at concentrations comparable with that required for detransformation. An in vitro histone deacetylase assay, using purified recombinant HDAC1, reveals that depudecin inhibits 50% of the enzyme activity at a concentration of 4.7 microM. These results demonstrate that depudecin is a novel HDAC inhibitor and suggest that its ability to induce morphological reversion of transformed cells is the result of its HDAC inhibitory activity.     

Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator

The anion-selective channel CFTR (cystic fibrosis transmembrane conductance regulator), whose dysfunction is responsible for the onset of cystic fibrosis, is regulated by cAMP through the activation of protein kinase A (PKA). The nature of this activation process is unknown. In the present study, patch-clamp techniques were applied to both mouse mammary adenocarcinoma cells expressing human epithelial CFTR (CFTR cells) and cultured neonatal rat ventricular myocytes (NRVM), to determine whether CFTR is modulated by the actin cytoskeleton, and whether the actin cytoskeleton may be implicated in the cAMP-stimulated activation of the channel protein. Acute changes in the actin cytoskeleton by addition of cytochalasin D (CD) activated whole-cell currents in CFTR cells and NRVM. Addition of actin to excised, inside-out patches also activated CFTR. A functional characterization of CFTR in either cell type included cAMP-induced, linear whole-cell and single-channel currents in symmetrical Cl-, permeability to ATP, and inhibition by either diphenylamine-carboxylate (DPC) or a monoclonal antibody raised against CFTR. Incubation of CFTR cells and NRVM with CD for over 6 h prevented CFTR activation either by the cAMP pathway under whole-cell conditions or by PKA under excised inside-out conditions. Thus a complete derangement of the actin cytoskeleton prevents the cAMP-dependent activation of CFTR. CFTR activation, however, was restored by subsequent addition of actin. In summary, changes in actin filament organization modulate CFTR channel activity by a mechanism entailing a direct interaction between actin filaments and CFTR.     

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin

The development of human endocrine pancreas has been the subject of many immunohistochemical studies but very little is known at the molecular level. We have determined the patterns of gene expression of glucagon, somatostatin and pancreatic polypeptide during fetal life (16-41 weeks of gestation) using the dot-blot hybridization method. In spite of some dispersion in the mRNA levels, no progressive increase or decrease during this period of fetal life could be found, as previously observed for insulin. In keeping with these molecular data, no increase in immunostaining of the four hormones was observed, but a dispersion of endocrine cells within the exocrine tissue was noticed at 20 weeks of gestation followed by a clear differentiation of the Langerhans islets at 31 weeks. Interestingly, the mRNA levels of the four hormones were always higher in the fetal pancreas than in the adult pancreas.     

A cell-free system to study regulation of focal adhesions and of the connected actin cytoskeleton

Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca2+ concentrations induce quasi-reversible diffusion of beta1 integrins out of focal adhesions, whereas low Ca2+ concentrations induce irreversible recruitment of beta1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated beta1 receptors show that the cytoplasmic portion of beta1 is required for low Ca2+-induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified alpha-actinin colocalizes and redistributes with beta1 receptors on ventral plasma membranes depleted of actin, implicating binding of alpha-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.     

Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton

Hepatic stellate cell activation, thought to play a key role in fibrosis of the liver, is characterized by changes in cellular morphology. The intracellular signals regulating morphological alterations associated with stellate cell activation are uncertain. The ras-like guanosine triphosphate-binding protein, rho, has recently emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The aim of this study was to test the hypothesis that rho signaling pathways direct activation-associated morphological changes in stellate cells by regulating the actin cytoskeleton. The morphology and actin cytoskeleton of primary rat hepatic stellate cells were studied with phase contrast, differential interference contrast, and epifluorescence microscopy. Immunohistochemistry and immunoblot analysis were used to examine rho expression and activity, respectively. Quiescent and activated stellate cells were investigated in the absence and presence of C3 transferase, a bacterial toxin that specifically inhibits rho. Stellate cell activation was characterized by the development of prominent intracellular fibers, and the loss of dendrite-like processes and perinuclear retinoid droplets. Moreover, activation was accompanied by the formation of prominent actin stress fibers and focal adhesions. Both rho expression and activity were demonstrated in stellate cells. C3 transferase blocked and reversed, both activation-associated morphological alterations and activation-associated changes in the actin cytoskeleton, in quiescent and activated stellate cells, respectively. These results indicate that rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton.     

Receptor-mediated folate uptake is positively regulated by disruption of the actin cytoskeleton

Receptor-mediated folate uptake is initiated by binding of ligand to a glycosyl phosphatidylinositol-anchored protein, folate receptor alpha (FR alpha). This receptor is expressed in a limited number of normal tissues but is overexpressed in a large number of epithelial malignancies. FR alpha synthesis, at least in part, is regulated by endogenous folate and by hormones in some cells, but much less is known about the control of function. Recently, we showed that phorbol 12-myristate 13-acetate increases the rate of receptor cycling, increases the rate of folate delivery, and causes the majority of the receptor to reside on the cell surface in nonmalignant cells in vitro (C. M. Lewis et al., Biochim. Biophys. Acta, 1401: 157-169, 1998). However, based upon effects (or lack of effects) of specific inhibitors of protein kinase C, the mechanism of action of phorbol 12-myristate 13-acetate is not likely via protein kinase C. Because exo- and endocytosis are controlled by the actin cytoskeleton, we tested cytochalasin D and latrunculin B, actin-disrupting agents, on FR alpha-mediated folate uptake. Disruption of the actin cytoskeleton reversibly increases the proportion of receptors on the cell surface and increases the rate of 5-methyltetrahydrofolate delivery. Disrupting microtubules with nocodazole had no effect. The increased rate of folate delivery caused by cytochalasin D is not observed in FR-negative cell lines. Although we have not yet identified the upstream effectors, likely candidates include small G-proteins such as rho, which are known to cause actin polymerization. In addition to identifying the machinery for receptor-mediated folate uptake, it may be important to integrate this new data into studies of FR alpha as a tumor antigen for imaging or delivering molecules via anti-FR antibodies or compounds coupled to folic acid.     

Identification of a novel mechanism of regulation of the adherens junction by E1A, Rac1, and cortical actin filaments that contributes to tumor progression

Transformation progression toward more malignant behavior often results from a loss of epithelial cell behavior, especially cell-cell adhesion. E1A cooperates with ras to transform primary epithelial cells such that they maintain epithelial cell differentiation, including the proper localization of adherens junctions (AJs). Second exon mutants of E1A 12S cooperate with ras to produce a more aggressively transformed phenotype, termed hypertransformation, that includes the loss of adhesion. Such hypertransformation can also be achieved by the addition of activated Rac1 to cells expressing wild-type E1A and ras, suggesting that actin reorganization may be important for the hypertransformed phenotype. Primary epithelial cells expressing hypertransforming mutants of E1A or V12Rac1 exhibit the loss of cortical actin filaments. In these cells, AJ complexes do not incorporate alpha-catenin, fail to associate with the cytoskeleton, and fail to localize to the plasma membrane, resulting in the destabilization of the AJ components and a loss of function. Loss of these epithelial cell characteristics predisposes these cells to a more malignant phenotype due to the loss of cell-cell adhesion. Taken together, these results suggest a novel mechanism of regulation of AJ function in tumor progression that involves the correct targeting of the AJ components, and this is affected by the status of cortical actin, which can be differentially affected by E1A or Rac1.     

Regulation of the actin cytoskeleton by thrombin in human endothelial cells: role of Rho proteins in endothelial barrier function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.     

Effects of rhizopodin and latrunculin B on the morphology and on the actin cytoskeleton of mammalian cells

The effects of the novel myxobacterial compound rhizopodin on mammalian cells were studied and compared with those of latrunculin B. Both substances induced adherently growing L929 mouse fibroblasts and PtK2 potoroo kidney cells to produce long, narrow, branched extensions or runners. Rhizopodin was more efficient than latrunculin B in that respect (minimal inhibitory concentration with L929 cells 5 nM vs 50 nM), and, in contrast to latrunculin B, its effects were permanent. Rhizopodin-treated cells became much larger than normal cells and were multinucleate, yet stayed alive and biochemically active for several weeks. Latrunculin B-treated cells returned to a quasi-normal state within 3-4 days. But latrunculin B acted faster, with the first effects becoming visible almost immediately upon the addition of the drug, while the first rhizopodin effects were seen 10 min later. Both substances caused reorganization of the actin cytoskeleton. When 100 nM rhizopodin was added to PtK2 cells, the stress fibers began to decay after just 10 min and had disappeared completely after about 3 h. Later there was a gradual restitution of F-actin. Long F-actin fibers were seen within the runners, and only there; in fact, these fibers may be responsible for the development and extension of the runners. The microtubuli network adjusted itself to the new cell morphology, but was not directly impaired by the compound.     

Calcium and protein kinase C regulate the actin cytoskeleton in the synaptic terminal of retinal bipolar cells

The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 +/- 0.1 in the dark to 2.1 +/- 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark. Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K+, the actin network in the synaptic pedicle extended up to 2.5 micrometer from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca2+ influx through L-type Ca2+ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca2+ influx and accelerated F-actin breakdown on removal of Ca2+. To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca2+ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.     

Capping of the barbed ends of actin filaments by a high-affinity profilin-actin complex

Profilin, a ubiquitous 12 to 15-kDa protein, serves many functions, including sequestering monomeric actin, accelerating nucleotide exchange on actin monomers, decreasing the critical concentration of the barbed end of actin filaments, and promoting actin polymerization when barbed ends are free. Most previous studies have focused on profilin itself rather than its complex with actin. A high-affinity profilin-actin complex (here called profilactin) can be isolated from a poly-(L)-proline (PLP) column by sequential elution with 3 M and 7 M urea. Profilactin inhibited the elongation rate of pyrenyl-G-actin from filament seeds in a concentration- and time-dependent manner. Much greater inhibition of elongation was observed with spectrin-F-actin than gelsolin-F-actin seeds, suggesting that the major effect of profilactin was due to capping the barbed ends of actin filaments. Its dissociation constant for binding to filament ends was 0.3 microM; the on- and off-rate constants were estimated to be 1.7 x 10(3) M-1 s-1 and 4.5 x 10(-4) s-1, respectively. Purified profilin (obtained by repetitive applications to a PLP column and assessed by silver-stained polyacylamide gels) did not slow the elongation rate of pyrenyl-G-actin from filament seeds. Capping protein could not be detected by Western blotting in the profilactin preparation, but low concentrations of gelsolin did contaminate our preparation. However, prolonged incubation with either calcium or EGTA did not affect capping activity, implying that contaminating gelsolin-actin complexes were not primarily responsible for the observed capping activity. Reapplication of the profilactin preparation to PLP-coupled Sepharose removed both profilin and actin and concurrently eliminated its capping activity. Profilactin that was reapplied to uncoupled Sepharose retained its capping activity. Phosphatidylinositol-4,5-bisphosphate (PIP2) was the most potent phosphoinositol in reducing the capping activity of profilactin. Dissociation of the tight profilactin complex may serve as a unique mechanism by which profilin helps regulate actin filament growth.     

G protein beta subunit-null mutants are impaired in phagocytosis and chemotaxis due to inappropriate regulation of the actin cytoskeleton

Chemotaxis and phagocytosis are basically similar in cells of the immune system and in Dictyostelium amebae. Deletion of the unique G protein beta subunit in D. discoideum impaired phagocytosis but had little effect on fluid-phase endocytosis, cytokinesis, or random motility. Constitutive expression of wild-type beta subunit restored phagocytosis and normal development. Chemoattractants released by cells or bacteria trigger typical transient actin polymerization responses in wild-type cells. In beta subunit-null cells, and in a series of beta subunit point mutants, these responses were impaired to a degree that correlated with the defect in phagocytosis. Image analysis of green fluorescent protein-actin transfected cells showed that beta subunit- null cells were defective in reshaping the actin network into a phagocytic cup, and eventually a phagosome, in response to particle attachment. Our results indicate that signaling through heterotrimeric G proteins is required for regulating the actin cytoskeleton during phagocytic uptake, as previously shown for chemotaxis. Inhibitors of phospholipase C and intracellular Ca2+ mobilization inhibited phagocytosis, suggesting the possible involvement of these effectors in the process.     

Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase

The Rho subfamily of low molecular weight GTPases have been implicated in a variety of cellular functions that include reorganization of the actin cytoskeleton and stress-induced activation of the c-Jun kinase. The downstream targets that mediate the effects of Cdc42 on the actin cytoskeleton have yet to be fully identified. We have used the transient transfection of COS-7 cells with epitope-tagged Cdc42 to identify candidate signaling partners for this GTPase and identified the IQGAP protein as a major in vivo target for activated Cdc42. Epidermal growth factor stimulation of serum-starved COS-7 cells promoted the formation of a Cdc42-IQGAP complex, indicating that growth factors can increase the pool of activated Cdc42. Activated HA-Cdc42 co-localized with IQGAP or F-actin in vivo, whereas cells transfected with dominant-negative forms of Cdc42 (Cdc42(T17N)) showed predominantly dispersed distributions for both HA-Cdc42 and endogenous IQGAP. In detergent lysates from COS-7 cells transiently transfected with different forms of Cdc42, or from stably transfected CHO cells, the induction of actin polymerization by phalloidin resulted in the incorporation of both IQGAP and Cdc42 into actin-containing complexes. Taken together, these findings are consistent with a model whereby IQGAP serves as a target for GTP-bound Cdc42 providing a direct link between the activated GTPase and the actin cytoskeleton.     

Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p

Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex. Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis. By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast. The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p. Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37 degreesC. Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches. Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants. In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p. Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p.