Fig. 4 Downregulation of RhoA GTP-loading is necessary but not sufficient for cortical actin rearrangement in dormant cells. Cells on fibronectin-coated cover slips in medium containing FGF-2 10 ng/ml (A. and B.) or lacking FGF-2 (C. and D.) were transiently transfected with 10:1 ratios of the three see more RhoA vectors and the GFP vector or with the GFP vector alone and stained with rhodamine phalloidin (red) and DAPI (blue nuclear staining). Cortical actin was identified and quantitated in the GFP-transfected green

cells only. a Cortical distribution of F-actin was observed in GFP only- and RhoA 19N (dominant negative)-transfected dormant cells (arrows), but was markedly diminished in dormant PCI-34051 mw cells transfected with RhoA63L (constitutively active) or RhoA wild type (RhoAWT). These latter two transfectants also induced the appearance of stress fibers. Cells were photographed at 400 x magnification. b Quantitative assessment of the percentage of cells with >50% cortical distribution demonstrates a statistically significant increase in cortical actin

in dormant cells compared with growing cells (*p < 0.01), between GFP- and RhoA63L-transfected dormant cells (**p < 0.001) and between GFP- and RhoAWT-transfected dormant cells (***p < 0.02) (Student’s t test). Error bars are + standard deviations. All other differences were not statistically significant. c Transfection of growing cells with dominant negative RhoA19N did not induce either the dormant phenotype or actin rearrangement. Transfection with either constitutively active RhoA63L or wild type RhoA also did not affect cortical actin (not shown). D. Statistical comparison of cell distributions with cortical actin was not affected in growing cells by dominant negative RhoA19N, nor by the other vectors (not shown) Activation of Focal Adhesion kinase in Dormant Cells

is Associated with Membrane Localization of the GTP Activating Protein GRAF We investigated whether focal adhesion kinase (FAK) was affected in dormant cells as part of the re-differentiation process. Integrin-mediated cell adhesion activates FAK and results in focal adhesion complex formation, initiation of stress fiber formation and motility [34]. The cellular levels and activation state of FAK are increased Montelukast Sodium in breast cancer progression [35–39]. In this context however, we found that instead of inactivation with dormancy, FAK became membrane localized and activated in the dormant cells. The percentage of cells staining for peripheral, activated Y397 find more phospho-FAK increased from 16.5 + 8.6% of growing cells to 83.1 + 12.6% of dormant cells (p < 0.005) (Fig. 5). This activation depended on binding of integrin α5β1, as integrin α5β1 blocking antibody or fibronectin blocking peptide P1 incubated with dormant cells decreased the percentage of cells with peripherally staining activated FAK to 15.9 + 2.9% (p < 0.001) and 32.2 + 9.5% (p < 0.01), respectively.