Etoposide and adriamycin but not genistein can activate the checkpoint kinase Chk2 independently of ATM/ATR.

We have investigated the effects of three unrelated topoisomerase 2 inhibitors, genistein, adriamycin, and etoposide, on phosphorylation/activation of the checkpoint kinase Chk2 in normal or ATM-deficient (ATM-) human fibroblasts and in cells overexpressing a catalytically inactive ATR kinase. We demonstrate that genistein activates Chk2 in a strictly ATM-dependent manner, whereas etoposide and adriamycin can trigger Chk2 activation in long-term cultures of ATM- cells. Moreover, these two latter genotoxic compounds were found to activate Chk2 in fibroblasts expressing the dominant negative form of ATR. We also report a significant decrease in the accumulation in G2-phase of ATM- cells when genistein did not activate Chk2. In conclusion, our results strongly support that activation of Chk2 could be dependent on the type and/or extent of DNA damage and under the control of either an ATM-dependent or an ATM and, maybe, an ATR-independent pathway.

The p42/p44 mitogen-activated protein kinase activation triggers p27Kip1 degradation independently of CDK2/cyclin E in NIH 3T3 cells.

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.

Effects of structurally related flavonoids on cell cycle progression of human melanoma cells: regulation of cyclin-dependent kinases CDK2 and CDK1.

We have investigated the effects of a series of flavonoids on cell proliferation and cell cycle distribution in human melanoma cells OCM-1. Among the compounds that potently inhibited OCM-1 cell proliferation, we show that the presence of a hydroxyl group at the 3'-position of the ring B in quercetin and luteolin, correlated to a G1 cell cycle arrest while its absence in kaempferol and apigenin correlated to a G2 block. Genistein with a hydroxyl at 5-position of the ring A arrested cells in G2 while daidzein which lacks it, induced an accumulation of cells in G1. We demonstrate that flavonoids, which induced a cell cycle block in G1, inhibited the activity of CDK2 by 40-60%. By contrast, those which caused an accumulation of cells in G2/M were without effect. On the other hand, while quercetin, daidzein and luteolin did not alter the activity of CDK1, kaempferol, apigenin and genistein inhibited this kinase by 50-70%. We demonstrate that the up-regulation of the CDK inhibitors p27(KIP1) and p21(CIP1) is likely responsible for the inhibition of CDK2 while inhibition of CDK1 was rather due to the phosphorylation of the kinase on Tyr15 residue.

Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase.

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.

Cisplatin and UV radiation induce activation of the stress-activated protein kinase p38gamma in human melanoma cells.

The p38 alpha mitogen-activated protein kinase has been implicated in the cellular response to genotoxic agents. Here we show that another p38 family member is also activated in response to cisplatin exposure in human melanoma cells. We identified this isoform as p38gamma based on its recognition by specific antibodies and because, in contrast to p38alpha, its activity was not affected by SB203580. We also found that etoposide caused a much more discrete phosphorylation of both p38alpha and p38gamma than either cisplatin or UV treatment. These results indicate that genotoxic stresses activate several p38 isoforms whose implication in the cellular response might depend on the type of DNA damage.

Cyclin-dependent kinase inhibitory protein expression in human choroidal melanoma tumors.

PURPOSE: Recent studies have demonstrated the close link between oncogenesis and cell cycle machinery. Cyclin-dependent kinase inhibitory proteins (CKIs) have been shown to play a critical role in the regulation of cell cycle progression. Alteration of CKI levels and/or functions could be implicated in cell transformation. The three CKIs-p16, p21, and p27-were investigated in human uveal melanoma tumors, and an attempt was made to correlate their levels with clinicopathologic parameters, as well as to p53 and Ki-67 (Mib-1) protein levels. METHODS: Immunochemistry was performed on 32 formalin-fixed, paraffin-embedded specimens of malignant choroidal melanoma. Immunoblot was performed to confirm the immunochemistry study. Prognostic histologic markers such as cell typing, pigmentation, larger tumor dimension, mitotic figures, nucleolar size, scleral invasion, and optic nerve head invasion were reported. RESULTS: Nuclear positivity for p16 was observed in 11 tumors (34%) without any association with clinicopathologic parameters. Tumor cells positive for p21 were detected in 12 choroidal melanomas (37%). Unexpectedly, a positive relationship was seen between p21 and scleral invasion (P: = 0.008). Nuclear positivity for p27 was observed in nine tumors (28%). An inverse correlation was observed between the number of mitotic figures and p27 immunoreactivity (P: = 0.03), as well as between Mib-1 positivity and p27 expression (P: = 0.02). Western blot assays of tumor extracts confirmed overexpression of p21 and p27. CONCLUSIONS: The results suggest that p21 and p27 may be involved in tumorigenesis in choroidal melanoma.

p21CIP1 is dispensable for the G2 arrest caused by genistein in human melanoma cells.

We have investigated the effect of genistein on cell cycle distribution of the human choroidal melanoma cell line OCM-1. We report that this isoflavonoid arrested cells in G2. This effect was correlated with the induction of the CDK inhibitor p21CIP1. However, while CDK1 activity was markedly reduced following genistein treatment, CDK2 activity was not affected. This was in agreement with the absence of G1 arrest that we observed but caused some doubt about the functionality of p21CIP1. Attempts to demonstrate mutation or post-translational modification of p21CIP1 from OCM-1 cells were unsuccessful. In fact, the level of p21CIP1 induced by genistein was shown to be insufficient to cause CDK2 inhibition. The role of p21CIP1 in the inhibition of CDK1 was questionable, as we demonstrated that genistein impaired Tyr15 dephosphorylation of CDK1 and because CDK1-cyclin B1 complexes from treated cells could be reactivated upon exposure to CDC25 phosphatase. Finally, we report that p21CIP1 was not absolutely required for the genistein-induced G2 arrest, as the isoflavone caused at least partial G2 arrest in p21-deficient Rat-1 fibroblasts as well as in p21-/- mouse embryo fibroblasts.

Distinct Chk2 activation pathways are triggered by genistein and DNA-damaging agents in human melanoma cells.

Genistein, a natural isoflavone found in soybeans, exerts a number of biological actions suggesting that it may have a role in cancer prevention. We have previously shown that it potently inhibits OCM-1 melanoma cell proliferation by inducing a G(2) cell cycle arrest. Here we show that genistein exerts this effect by impairing the Cdc25C-dependent Tyr-15 dephosphorylation of Cdk1, as the overexpression of this phosphatase allows the cells to escape G(2) arrest and enter an abnormal chromatin condensation stage. Caffeine totally overrides the genistein-induced G(2) arrest, whereas the block caused by etoposide is not bypassed and that caused by adriamycin is only partially abolished. We also report that genistein activates the checkpoint kinase Chk2 as efficiently as the two genotoxic agents and that caffeine may counteract the activation of Chk2 by genistein but not by etoposide. In contrast, caffeine abolishes the accumulation of p53 caused by all the compounds. Wortmannin does not suppress the Chk2 activation in any situation, suggesting that the ataxia telangiectasia-mutated kinase is not involved in this regulation. Finally, unlike etoposide and adriamycin, genistein induces only a weak response in terms of DNA damage in OCM-1 cells. Taken together, these results suggest that the G(2) checkpoints activated by genistein and the two genotoxic agents involve different pathways.

Retrovirus-mediated transfer of a suicide gene into lens epithelial cells in vitro and in an experimental model of posterior capsule opacification.

PURPOSE: The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification and was found to be an important feature contributing to opacification of the posterior capsule. We investigated the feasibility of killing the residual lens epithelial cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, a well-studied suicide gene, into rabbit lens epithelial cells followed by ganciclovir (GCV) treatment. METHODS: The capacity of retroviral vectors to transfer genes into rabbit lens epithelial cells was determined either in vitro (culture of rabbit lens epithelial cells) or in vivo (experimental model of PCO in rabbits) using cDNA encoding the beta-galactosidase (LacZ) reporter gene. To evaluate the efficiency of suicide gene therapy (infection with retroviral vectors encoding the HSV-tk gene followed by GCV treatment) we determined the sensitivity of HSV-tk infected lens epithelial cells to different concentrations of GCV in vitro. Then, in an experimental model of PCO, rabbits were treated with HSV-tk retroviral vectors at the end of the surgery and they received repeated intracameral and intravitreal injections of GCV at the concentration determined by the in vitro experiments. RESULTS: Infection efficiency using LacZ retroviral vectors was about 29% in vitro and 10% in vivo. After infection of the HSV-tk cDNA in vitro, the cell killing effect of GCV was evaluated. A significant enhancement (four- to five-fold) of the cell sensitivity to GCV was shown in FLY-DFGtk as compared with mock infected (P < 0.01) cells even without selection of the HSV-tk positive cells. The GCV concentration leading to 50% reduction in cell number (IC50) was 50 microg/ml. In vivo infection with a HSV-tk vector led to the tk gene transfer into lens epithelial cells. Despite this local HSV-tk gene expression, we could not prevent capsule opacification. CONCLUSIONS: Lens epithelial cells were successfully infected both in vitro and in vivo by beta-galactosidase and HSV-tk genes via retroviral vectors. In vitro infected lens epithelial cells displayed a strong sensitivity to GCV treatment. In vivo, we could not prevent capsule opacification in the rabbit model, very likely due to the limited level of the HSV-tk gene expression. However, our results suggest that virus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification with a more powerful vector.

Adenovirus-mediated suicide gene transduction: feasibility in lens epithelium and in prevention of posterior capsule opacification in rabbits.

The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification, and has been found to be an important feature contributing to opacification of the posterior capsule. Adenoviral vector-mediated transfer is a suitable method for transducing the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating cells, allowing for the selective killing of these cells by ganciclovir (GCV) treatment. To determine the potential of gene transduction for lens epithelial cells, we studied the transduction of rabbit lens epithelial cells with adenoviral vectors containing either the Escherichia coli beta-galactosidase (lacZ) gene or the HSV-tk gene in vitro and in vivo in an experimental model of PCO. The efficiency of lacZ gene transfer in rabbit lens epithelial cells was at least 95% both in vitro and in vivo. In vivo transduction with HSV-tk adenoviral vector followed by GCV treatment significantly inhibited the development of PCO (p<0.001). These results suggest that adenoviral vector-mediated transfer of HSV-tk into the proliferating lens epithelial cells is feasible and may provide a novel therapeutic strategy for PCO.

Phosphorylation of the myristoylated protein kinase C substrate MARCKS by the cyclin E-cyclin-dependent kinase 2 complex in vitro.

The myristoylated alanine-rich C-kinase substrate (MARCKS) purified from brain was recently characterized as a proline-directed kinase(s) substrate in vivo [Taniguchi, Manenti, Suzuki and Titani (1994) J. Biol. Chem. 269, 18299-18302]. Here we have investigated the phosphorylation of MARCKS by various cyclin-dependent kinases (Cdks) in vitro. We established that Cdk2, Cdk4 and, to a smaller extent, Cdk1 that have been immunoprecipitated from cellular extracts phosphorylate MARCKS. Comparison of MARCKS phosphorylation by protein kinase C (PKC) and by the purified cyclin E-Cdk2 complex suggested that two residues were phosphorylated by Cdk2 under these conditions. To identify these sites, Cdk2-phosphorylated MARCKS was digested with lysyl endoprotease and analysed by electrospray MS. Comparison with the digests obtained from the unphosphorylated protein demonstrated that two peptides, Gly12-Lys30 and Ala138-Lys152, were phosphorylated by cyclin E-Cdk2. The identity of these peptides was confirmed by automatic Edman degradation. On the basis of the consensus phosphorylation sequence described for Cdk2, and on MS/MS analysis of the Ala138-Lys152 peptide, we concluded that Ser27, one of the phosphorylation sites identified in vivo, and Thr150 were the Cdk2 targets in vitro. None of the other sites described in vivo were phosphorylated in these conditions. Interestingly, a preliminary phosphorylation of MARCKS by PKC improved the initial rate of phosphorylation by Cdk2 without modifying the number of sites concerned. In contrast, phosphorylation of MARCKS by Cdk2 did not significantly affect further phosphorylation by PKC.

[Cyclin-dependent kinase inhibitory proteins in normal and transformed choroidal melanocytes]. / Les inhibiteurs des CDK-cyclines dans les mélanocytes choroïdiens normaux et transformés.

PURPOSE: Recent studies have demonstrated the close link between oncogenesis and cell cycle machinery. Cyclin dependent kinase inhibitory proteins (Ckis) have been shown to be implicated in cancer progression. We investigated the levels of the different regulatory inhibitory proteins involved in the G1 progression and G1/S in choroidal melanomas. METHODS: Immunoblotting, immunoprecipitation and immunohistochemistry were performed on human choroidal cell lines and human choroïdal tumors. RESULTS: Our findings suggested a lack of expression of Cdk inhibitor p21 in two of three melanoma cell lines and a striking underexpression of p27 in the three transformed cell lines. The p16 level was found to be almost the same in both normal and transformed cells, a loss of p16-Cdk4 interaction was observed in two of the three melanoma cell lines. In immunohistochemistry, nuclear positivity for p16 was observed in six tumors. Nuclear positivity for p21 was observed in five tumors. All of theses tumors had scleral invasion (p = 0.003). Nuclear positivity for p27 was observed in only two tumors. CONCLUSION: Our results demonstrate that immunoreactivity for p16, p21 and p27 could be implicated in progression of melanoma tumors. More cases are required to further clarify this issue.

Regulation by transforming growth factor-beta 1 of G1 cyclin-dependent kinases in human retinal epithelial cells.

Transforming growth factor beta (TGFbeta) is a potent inhibitor of epithelial cell proliferation, delaying or arresting cell cycle progression in mid-late G1. In long-term life span cells this growth inhibitory action has been attributed to regulatory events on both the levels and activities of G1 cyclin-dependent kinases (CDKs). CDK inhibitors have been shown to play important role in the TGFbeta-induced inhibition of G1 CDKs. In this work, we have investigated the effect of TGFbeta1 on both cell proliferation and G1 CDK activities in primary cultures of human retinal pigment epithelial (RPE) cells. We show that TGFbeta1 exerts a partial inhibitory effect on RPE cell proliferation by causing a significant increase of the RPE cell number in G1. TGFbeta1 induces an up-regulation of the CDK inhibitor p15(INK4B)with its subsequent association to CDK4, and a decline in CDK4 protein level. In parallel, we have observed a decline of p27(KIP1)associated to CDK4 and a significant increase of the inhibitor associated to CDK2. Finally, we show that TGFbeta1 reduces both CDK4 and CDK2 enzymatic activities. The fact that TGFbeta exerts only partial inhibitions on G1 CDKs and cell cycle progression in RPE cells suggests a propensity of these cells to escape from the anti-proliferative action of the cytokine, a phenomenon which could be reinforced during the development of proliferative vitreoretinopathy.

Differential expression of G1 cyclins and cyclin-dependent kinase inhibitors in normal and transformed melanocytes.

PURPOSE: To investigate the levels of the different regulatory proteins involved in the G1 progression and G1/S transition in normal and transformed human choroidal melanocytes (CM). METHODS: Three choroidal melanoma cell lines and three CM cultures were used. The purity of the CM cultures was assessed by different approaches, including morphologic study, specific immunostaining, cell proliferation behavior, and transforming growth factor-beta1 responsiveness. The cell cycle protein levels were evaluated by specific immunoblotting of total extracts obtained from the different cell lines. RESULTS: Alterations were observed in the expression of cylins D1 and E in the transformed cells, whereas the amounts of the cyclin-dependent kinases (CDKs) CDK2 and CDK4 were almost identical in both cell types. Although the expression of cyclin H was slightly increased in transformed cells, neither the CDK7 level nor the CDK7 and cyclin H localizations were altered when compared with those in normal CM. The results suggest the absence of the CDK inhibitor (CKI) p21 in two of the three melanoma cell lines and, as a main feature, a striking underexpression of p27 in the three transformed cell lines. Finally, although the p16 level was almost the same in normal and transformed cells, a loss of p16-CDK4 interaction was observed in two of the three melanoma cell lines. CONCLUSIONS: Deregulated expression of G1 cyclins and CKIs and alteration in the interaction of CKIs with CDKs may be implicated in the neoplastic transformation of human ocular melanocytes to malignant melanoma cells.

Overexpression of the myristoylated alanine-rich C kinase substrate in human choroidal melanoma cells affects cell proliferation.

Reduced expression of the myristoylated alanine-rich C kinase substrate (MARCKS) has been described in various cell lines after oncogenic or chemical transformation, leading to the question of whether this protein may be involved in cell proliferation. Here we compare the expression of MARCKS in human tumor-derived choroidal melanoma cells (OCM-1) and in primary cultures of normal choroidal melanocytes. We found an important down-regulation of the protein in the melanoma cell line. Stable transfection of these cells with the cDNA coding for MARCKS led to the selection of several clones expressing variable levels of the protein. Proliferation experiments performed with four of these clones revealed that cell growth was reduced by 35-40% when compared with control cells. Upon serum starvation, cell proliferation was almost abolished when the expression level of MARCKS was high, whereas it was only partially reduced in the controls. MARCKS overexpression induced a higher percentage of cells in the G0-G1 phase of the cell cycle upon serum starvation, as well as the inhibition of colony formation in soft agar. Finally, the expression of the CDK inhibitor p27 was increased in the cells presenting a high level of MARCKS protein. Altogether, these data suggest that the expression of this protein kinase C substrate affects the proliferation and partially reverts the transformed phenotype of the OCM-1 cells.

Phosphatidylinositol 3-kinase inhibitors block aortic smooth muscle cell proliferation in mid-late G1 phase: effect on cyclin-dependent kinase 2 and the inhibitory protein p27KIP1.

In the present study, we investigated the involvement of phosphatidylinositol 3-kinase (PI 3-kinase) activity in the progression of vascular smooth muscle cells (VSMCs) throughout the G1 phase of cell cycle. Addition of two selective inhibitors of PI 3-kinase, LY 294002 or wortmannin, to quiescent VSMCs prevented serum-induced DNA synthesis in a dose-dependent manner with IC50 of 8.7 +/- 2.0 microM and 53.9 +/- 8.5 nM, respectively. Time course studies revealed that the two PI 3-kinase inhibitors blocked VSMC proliferation in mid-late G1 phase, about 6 h before the G1/S transition. This G1 growth arrest was due, at least in part, to the reduction of the CDK2 associated kinase activity resulting mainly from the upregulation of the inhibitory protein p27KIP1.

G1 phase arrest by the phosphatidylinositol 3-kinase inhibitor LY 294002 is correlated to up-regulation of p27Kip1 and inhibition of G1 CDKs in choroidal melanoma cells.

We have investigated the effect of the flavonoid derivative LY 294002, a potent and selective phosphatidylinositol 3-kinase inhibitor, on cell cycle progression in human choroidal melanoma cells. We demonstrate that LY 294002 induces a specific G1 block in asynchronously growing cells leading to an almost complete inhibition of cell proliferation after three days of treatment. When melanoma cells are released from a nocodazole-induced G2/M block, LY 294002 is shown to delay and greatly restrain the G1/S transition. The inhibitor is able to exert its action as long as it is added during the G1 progression and before the cells enter in S phase. We report that the LY 294002-induced G1 arrest is closely correlated to inhibition of CDK4 and CDK2 activities leading to the impairment of pRb phosphorylation which normally occurs during G1 progression. While the inhibition of CDK4 may be attributed at least in part to the decline in CDK4 protein level, CDK2 activity reduction is rather due to the up-regulation of the CDK inhibitor p27Kip1 and to its increased association to CDK2.

Interleukin-6 in tear fluid after photorefractive keratectomy and its effects on keratocytes in culture.

PURPOSE: To investigate the participation of interleukin-6 (IL-6) after photorefractive keratectomy (PRK) and its possible roles and sources in corneal wound healing. METHODS: IL-6, levels were measured in the tear fluids of patients before and after PRK and in conditioned media of human corneal epithelial cells and keratocytes. Its effects on total collagen and collagen-type synthesis by keratocytes were studied with a 3H-proline incorporation assay and Northern blot analysis. Zymography was used to evaluate the metalloproteinase content in the conditioned medium of IL-6-stimulated keratocytes. RESULTS: IL-6 is present in the tear fluid samples after photorefractive keratectomy, possibly synthesized by epithelial cells and keratocytes. CONCLUSIONS: IL-6 stimulates collagen synthesis in general and collagen type I in particular. Furthermore, it reduces the production of MMP-2, the latent form of the metalloproteinase, by cultured keratocytes. The results suggest that IL-6 might be regarded as a mediator involved in corneal healing after excimer laser.

Specific proteolytic cleavage of the myristoylated alanine-rich C kinase substrate between Asn 147 and Glu 148 also occurs in brain.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a major ubiquitous substrate of protein kinase C. The expression of the protein is regulated during cell cycle progression and cell proliferation. Specific proteolytic cleavage of the protein between Asn 147 and Glu 148 was described recently in cultured cells, and the corresponding proteolytic activity was observed in various tissue extracts except for brain. We purified a 40 kDa fragment of MARCKS from bovine brain that we characterized as the C-terminal specific fragment found in other tissues. The identification of the fragment was achieved by in vitro phosphorylation by protein kinase C, calcium-dependent interaction with calmodulin, mass spectrometric analysis, and N-terminal sequencing. These data suggest that specific proteolytic cleavage of MARCKS also occurs in brain and may be a general mechanism of down-regulation of the protein.

The major myristoylated PKC substrate (MARCKS) is involved in cell spreading, tyrosine phosphorylation of paxillin, and focal contact formation.

The expression of the myristoylated PKC substrate MARCKS is reduced in tumor-derived choroidal melanoma cells (OCM-1). We transfected the OCM-1 cells with MARCKS cDNA and we selected clones with stable overexpression of the protein. Tyrosine phosphorylation of paxillin, a biochemical marker of focal contact formation, was conserved upon serum starvation when MARCKS was overexpressed, while it was almost abolished in the control cells. Immunofluorescent labelling of paxillin and vinculin, another component of focal contact, revealed that these structures were conserved upon serum starvation when MARCKS was overexpressed but not in the control cells. Furthermore, the cell morphology was affected by the ectopic expression of MARCKS, leading to increased spreading and formation of membrane processes. These data suggest the involvement of MARCKS in cell spreading and focal contact formation.