The different effects of type I and IV collagens on the survival and proliferation of cultured BSC-1 cells

The effects of type I and IV collagens on the survival and proliferation of cells were investigated to clarify a possible involvement of the substratum in the regulation of cell function. BSC-1 cells attached, spread and sustained their viability in the absence of calf serum on culture dishes coated with type IV collagen, but were unable to spread and survive on untreated culture dishes. The effects of adding type IV collagen in solution were similar to those of type IV coating. The fraction of the solution of type IV collagen with molecular mass of more than 100 kDa enhanced spreading and survival of cells, but the fraction of less than 100 kDa did not. Type I collagen did not support cell viability in the absence of calf serum. Moreover, coating of culture dishes with type I collagen, but not with type IV collagen, inhibited DNA synthesis and cell proliferation in the presence of calf serum. The cells grown on type I collagen were long, thin and spindle-shaped, and their stress fibers were not well developed, but the cells grown on type IV collagen, as well as those grown on untreated culture dishes, were polygonal in shape with well-developed stress fibers. These results indicate that the interactions of BSC-1 cells with the substratum, when it is derived from type I and IV collagens, differentially modulate the survival and proliferation of BSC-1 cells.     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

Survival of 3T3-L1 cells induced by fibroblast growth factor depends on cell density and adhesion to the substratum

The effects of cell contacts and the attachment of cells to the substratum on growth-factor-induced survival of 3T3-L1 cells were investigated to clarify their involvement in the maintenance of cell viability. When 3T3-L1 cells in low-density cultures or in high-density cultures were harvested with EDTA solution and cultured in the absence of calf serum, almost all cells from the low-density cultures lost viability 24 h later. However, about 15% of the cells harvested from high-density cultures survived for 24 h in the absence of calf serum. Addition of calf serum also enhanced the survival of cells from high-density cultures to a much greater extent than that of cells from low-density cultures. Addition of fibroblast growth factor enhanced the survival of cells, especially in the case of cells from high-density cultures. However, epidermal growth factor and platelet-derived growth factor failed to enhance survival. Coating of cultures dishes with vitronectin slightly enhanced cell survival. Addition of fibroblast growth factor markedly enhanced the survival of cells on the dishes coated with vitronectin or with fibronectin, but not on the dishes coated with heat-denatured bovine serum albumin. These results suggest that fibroblast growth factor promotes survival of 3T3-L1 cells, depending on cell to-cell contacts during prior culture and on the adhesion of cells to the substratum.     

Shark cartilage extract interferes with cell adhesion and induces reorganization of focal adhesions in cultured endothelial cells

In this study, we examined the effects of shark cartilage extract on the attachment and spreading properties and the focal adhesion structure of cultured bovine pulmonary artery endothelial cells. Treatment with cartilage extract resulted in cell detachment from the substratum. Immunofluorescence staining of those treated cells that remained attached showed that, instead of being present in both central and peripheral focal adhesions as in control cells, both integrin alpha(v)beta(3) and vinculin were found only in peripheral focal adhesion and thinner actin filament bundles were seen. In addition to causing cell detachment, cartilage extract partially inhibited the initial adherence of the cells to the substratum in a dose-dependent manner. Integrin alpha(v)beta(3) and vinculin staining of these cells also showed a peripheral focal adhesion distribution pattern. Vitronectin induced cell spreading in the absence of serum, but was blocked by simultaneous incubation with cartilage extract, which was shown to inhibit both integrin alpha(v)beta(3) and vinculin recruitment to focal adhesion and the formation of stress fibers. Dot binding assays showed that these inhibitory effects on cell attachment and spreading were not due to direct binding of cartilage extract components to integrin alpha(v)beta(3) or vitronectin. Shark cartilage chondroitin sulfate had no inhibitory effect on either cell attachment or spreading of endothelial cells. These results show that the inhibitory effects of cartilage extract on cell attachment and spreading are mediated by modification of the organization of focal adhesion proteins.     

12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodamine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. The enhancement of binding of the beads by TPA was suppressed by addition of an adhesion-inhibitory peptide (Gly-Arg-Gly-Asp-Ser-Pro). Treatment with TPA did not enhance nonspecific binding of beads coated with heat-denatured bovine serum albumin. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.     

Cell shape-dependent Control of Ca2+ influx and cell cycle progression in Swiss 3T3 fibroblasts

The ability of adherent cells such as fibroblasts to enter the cell cycle and progress to S phase is strictly dependent on the extent to which individual cells can attach to and spread on a substratum. Here we have used microengineered adhesive islands of 22 and 45 mum diameter surrounded by a nonadhesive substratum of polyhydroxyl methacrylate to accurately control the extent to which individual Swiss 3T3 fibroblasts may spread. The effect of cell shape on mitogen-evoked Ca2+ signaling events that accompany entry into the cell cycle was investigated. In unrestricted cells, the mitogens bombesin and fetal calf serum evoked a typical biphasic change in the cytoplasmic free Ca2+ concentration. However, when the spreading of individual cells was restricted, such that progression to S phase was substantially reduced, both bombesin and fetal calf serum caused a rapid transient rise in the cytoplasmic free Ca2+ concentration but failed to elicit the normal sustained influx of Ca2+ that follows Ca2+ release. As expected, restricting cell spreading led to the loss of actin stress fibers and the formation of a ring of cortical actin. Restricting cell shape did not appear to influence mitogen-receptor interactions, nor did it influence the presence of focal adhesions. Because Ca2+ signaling is an essential component of mitogen responses, these findings implicate Ca2+ influx as a necessary component of cell shape-dependent control of the cell cycle.     

Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells

The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites.     

Loss of focal contacts accompanies the density dependent inhibition of cell growth

The formation of focal contacts by fibroblast-like 3T3 and 9390 cells and endothelial XTH-2 cells was examined with the use of reflection interference contrast microscopy at different cell densities. It was found that cells density-inhibited in growth loose all focal contacts with the substratum. This observation explains the disappearance of stress fibers in density-inhibited cells, and also offers an explanation for the mechanism of density-dependent inhibition of proliferation of anchorage-dependent cells.     

The significance of alpha 5 beta 1 integrin dependent and independent actin cytoskelton organization in cell transformation and survival

Cell-matrix and cell-cell interactions are important physiological determinants of cell growth, survival and transformation. Cell adhesion to the extra cellular matrix (ECM) via integrins also crucially influences the organization of the cytoskeleton. It triggers a cascade of intracellular biochemical events, which regulate cell viability and growth. We have studied the relationship between cell attachment to the substratum and cytoskeletal organization and cell survival and transformation. Our results demonstrate that in the absence of attachment to the substratum, adhesion-dependent fibroblasts exhibit rapid loss of viability. However, a small percentage of cells survive even after remaining non-adherent for 16h. The adherent and non-adherent cells differ from one another both morphologically and physiologically. The latter show a loss of alpha5beta1 integrin expression on their surface and bind non-specifically to the substratum and ECM, thereby activating certain pathways more efficiently than adherent cells. We have also shown that non-adherent cells grow faster and have worse cytoskeletal organization after attachment to the substratum, and do not form focal adhesions or actin stress fibres. Hence, our data suggests that rat fibroblasts in prolonged suspension exhibit some properties that are comparable to cells undergoing transformation, by adapting integrin-dependent or independent signalling pathways for their survival.     

Focal Adhesion and Stress Fiber Formation Is Regulated by Tyrosine Phosphatase Activity

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.     

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha_s1-casein, lactoferrin (Lf), and alpha-lactalhumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf scrum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 μg/ml medium, which was 2–3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for eryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin. and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.     

Effects of a tumour promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on expression of differentiated phenotype in the chick retinal pigmented epithelial cells and on their interactions with the native basement membrane and with artificial substrata

Chick retinal pigmented epithelial (RPE) cells grown in vitro on basement membrane matrices from the Engelbreth-Holm-Swarm tumour (BM-matrigel) do not spread, and they maintain their differentiated phenotype, most notably the heavy pigmentation. Maintenance of the differentiated phenotype by RPE cells on BM-matrigel is promoted not only by the biochemical composition of the gel but also by its mechanical properties, i.e., its low rigidity prevents cell spreading. In this report, RPE cells on BM-matrigel were treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to promote the transformed phenotype and diminish cell traction. In contrast to most cell types TPA treatment induced RPE cells to increase their spread area. TPA promoted RPE cell spreading on BM-matrigel and changed the spatial organization of actin and actin-associated proteins in the cytoskeleton-ECM linkage complexes, uncoupling actin from its extracellular counterpart. TPA did not affect other components of the cytoskeleton in RPE cells. TPA also affected labile adhesions i.e., focal contacts and adherens junctions in statu nascendi, but preformed, stable adherens junctions were resistant to TPA. TPA enhanced proliferation, blocked melanogenesis and thus inhibited differentiation of RPE cells grown on either artificial substrata or their natural basement membrane.     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.     

The serum dependence of baby hamster kidney cell attachment to a substratum

Normal attachment and spreading of baby hamster kidney cells onto a non-living substratum requires the presence of a specific serum component adsorbed to the substratum surface and Ca²⁺ ions in the medium. In the absence of the adsorbed serum factor or Ca²⁺ ions cells attach but do not spread. Thus, although the initial rate of BHK cell attachment is faster in serum-free medium than serum-containing medium, no cell spreading occurs in serum-free medium. Adsorption of serum onto the substratum results in a lag phase in the time course of cell attachment which can be eliminated by blocking the negatively charged groups of the serum components adsorbed to the substratum surface; blocking positively charged groups or free sulfhydryl groups of the adsorbed serum components is without effect. The requirement for serum components can be substituted for by adsorbing molecules such as concanavalin A or polycationic ferritin to the substratum surface; however, only ConA results in normal morphology of cell spreading. The data are discussed in terms of a non-electrostatic direct cell-substratum binding model of cell attachment.     

Intercellular contacts and the organization of actin filaments in cultured epithelial FL cells are altered by growth on type I collagen and treatment with 12-O-tetradecanoylphorbol-13-acetate through the modulation of interactions between cells and the substratum

We have investigated the effects of various types of collagen and a tumor-promoting phorbol ester on intercellular contacts and the organization of actin in human amnion epithelial FL cells and mouse fibroblast 3T3-A31 cells. Our purpose was to investigate how modulation of interactions between cells and the substratum leads to alterations in intercellular contacts and organization of actin filaments. When cells were cultured on dishes coated with a solution containing type I collagen, but not type IV, changes were induced in the morphology of FL cells and their intercellular contacts. Type I collagen also caused changes in the organization of their actin filaments, although no such effects were observed with 3T3-A31 cells. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) caused morphological changes, dissociation of groups of cells, and reorganization of actin filaments in cultures of FL and 3T3-A31 cells. It also disrupted the sites of adhesion of FL cells to the substratum. Both type I collagen and TPA rapidly induced spreading of FL cells in the absence of serum. However, cis-hydroxyproline, known to inhibit secretion of collagen, did not suppress the TPA-induced dissociation of groups of FL cells. These results suggest that the interactions with type I collagen of epithelial FL cells, but not of fibroblastic 3T3-A31 cells, tend to disorganize cellular morphology, intercellular contacts, and actin filaments in ways similar to, but not directly related to, the effects of TPA.     

Adhesiveness and proliferation of epithelial cells are differentially modulated by activation and inhibition of protein kinase C in a substratum-dependent manner

In the present study, we have examined the regulation of attachment, onset of proliferation and the subsequent growth, in vitro, of chick retinal pigmented epithelial (RPE) cells as a function of the nature of the substratum and of either the activation or inhibition of protein kinase C (PKC). The RPE cells have an adhesive preference for protein carpets which contain laminin. This preference disappears gradually with time in culture. The adhesion of RPE cells to fibronectin is shown to be a receptor-mediated process which involves the RGD recognition signal. This study also demonstrates that a PKC activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), affects RPE cell adhesion in a substratum-dependent manner. Exposure of RPE cells to TPA lowers the cell attachment efficacy to ECM protein substrata but does not affect cell attachment to plastic. The onset of cell proliferation is accelerated by TPA on all of the substrata tested. The minimal duration of an effective TPA pulse exerting a long-lasting influence on RPE cell proliferation is between 1.5 and 3.5 hr. Stimulation of cell proliferation by TPA in long-term cultures is independent of the nature of the growth substratum. The acceleration of the onset of cell proliferation by TPA is sensitive to 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), an inhibitor of conventional PKC, and thus appears to be dependent on the activation of conventional PKC. H7 also affects cell-cell contacts, causing an alteration in the shape (“squaring”) of RPE cells packed into large colonies. Conversely, the effects of TPA on both the attachment and the long-term proliferation of RPE cells are not dependent a conventional PKC isotype, since H7 cannot abolish the influence of TPA on either process. We conclude that the effect of TPA on long-term proliferation of RPE cells is either dependent on a novel PKC isotype or independent of PKC. © 1993 Wiley-Liss, Inc.     

Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.     

Visualization of cellular focal contacts using a monoclonal antibody to 80 kD serum protein adsorbed on the substratum

We have obtained a monoclonal antibody to 80 kD protein of calf serum; this protein easily and uniformly adsorbs on glass from serum-containing media. Indirect immunofluorescence staining of chick and mouse embryo fibroblasts cultured in the presence of calf serum, fixed with formaldehyde and permeabilized with Triton X-100, revealed black non-fluorescent strips and dots under the ventral cell surface, whereas all other parts of the substratum under and between cells were highly fluorescent. The distribution of non-fluorescent regions coincided with the distributed of focal contacts of cells with the substratum, revealed by interference reflection microscopy, as well as with the distribution of vinculin-containing plaques. The dark regions were also associated with the ends of microfilament bundles revealed by immunofluorescence with an anti-actin antibody. Thus, non-fluorescent regions seen after anti-80 kD staining are parts of the substratum under the focal contacts. Visualization of focal contacts with anti-80 kD provides very contrasting and high resolution pictures. Evidence is presented that 80 kD protein is adsorbed to glass in the areas of focal contacts, but the antibodies used for staining cannot penetrate these contacts.     

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.