The integrity of cell-cell contacts such as adherens junctions (AJ) and tight junctions (TJ) is essential for the function of epithelia. During carcinogenesis, the increased motility and invasiveness of tumor cells reflect the loss of characteristic epithelial features, including cell adhesion. While ␤-catenin, a component of AJ, plays a well characterized dual role in cell adhesion and signal transduction leading to epithelial cell transformation, little is known about possible roles of tight junction components in signaling processes. Here we show that mutants of the TJ protein zonula occludens protein-1 (ZO-1), which encode the PDZ domains (ZO-1 PDZ) but no longer localize at the plasma membrane, induce a dramatic epithelial to mesenchymal transition ( An essential role of epithelial monolayers is the formation of cellular barriers that allow the generation and maintenance of compartments important for the physiological function of organs. In epithelial monolayers, individual cells are joined to each other by specialized structures including gap junctions, desmosomes, adherens junctions (AJ) 1 and tight junctions (TJ).In addition to mediating cell-cell adhesion, TJ regulate the paracellular diffusion across epithelial monolayers and the maintenance of the asymmetric distribution of proteins and lipids to the apical and basolateral plasma membrane domains of epithelial cells (1-3). The tight junction protein zonula occludens protein 1 (ZO-1) is part of a multi-protein complex and binds directly to the integral TJ proteins occludin and to members of the claudin family (4), thereby linking the TJ to the cytoskeleton via a direct or indirect interaction with actin (5). ZO-1 belongs to the membrane-associated guanylate kinase (MAGUK) protein family and contains three PDZ domains (6), an Src homology 3 (SH3) domain, a guanylate kinase (GUK) homology domain, and a proline rich C-terminal region (see Fig. 1). Since occludin lacks PDZ-binding motifs, binding between occludin and ZO-1 probably does not involve the PDZ domains. The function of the GUK domain, which lacks kinase activity in the MAGUK proteins analyzed so far, is not known but has been suggested to be important for binding of ZO-1 to occludin (5). ZO-2 and ZO-3, two additional members of the MAGUK protein family present in TJ, show extensive homology to each other and to ZO-1. ZO-3 interacts with ZO-1 and the cytoplasmic C-terminal tail of occludin, but does not bind ZO-2 (7). ZO-2 binds directly to ZO-1 and occludin. Actin cosedimentation studies showed that ZO-2, ZO-3, and occludin all interact directly with F-actin in vitro and colocalize with actin aggregates at cell boarders in cytocholasin D-treated MDCK cells. The suggested model at the moment is that two independent complexes comprising ZO-1-ZO-2 and ZO-1-ZO-3 exist (rather than a three-member complex, ZO-1-ZO-2-ZO-3), and that these complexes link the tight junction to the actin cytoskeleton (8). Several other proteins have been described to interact with ZO-1, but the domains involved in binding...

Con¯uent 3T3-L1 preadipocytes dierentiate to adipocytes in the presence of insulin, dexamethasone, and isobutylmethylxanthine (IDI). A transient increase of DNA synthesis is induced in 3T3-L1 cells 18 h after addition of IDI, followed by an arrest in the G1 phase of the cell cycle. Growth arrested cells express the protooncogene c-myc and the gene for the CCAAT/enhancer binding protein (C/EBPa) between day 2 and 5. While cMyc is strongly implicated in cell proliferation, C/EBPa is a dierentiation-speci®c transcription factor with antiproliferative activity. Here we have characterized the cell cycle arrest in dierentiating 3T3-L1 cells. Arrested cells express the Cdk inhibitors p21 and p27, but, at the same time, show hyperphosphorylation of Rb and expression of the E2F-regulated thymidine kinase gene. The addition of new serum to arrested cells resulted in cyclin A expression and Cdk2 activity, but not in DNA synthesis. Simian virus 40 large tumor antigen (LTAg) is a potent mitogen. The mutant LTAg-K1, de®cient in binding of pocket proteins and unable to induce DNA synthesis in serum-starved 3T3-L1 cells, eciently induced DNA synthesis in dierentiating 3T3-L1 cells. This indicates that pocket proteins are probably not involved in the control of the cell cycle arrest during 3T3-L1 cell dierentiation. Our data suggest that the dierentiationspeci®c cell cycle block in 3T3-L1 cells is resistant to high levels of c-Myc, inactivation of pocket proteins, upregulation of cyclin A levels, and Cdk2 activation, but can be abolished by a function of LTAg that is independent of binding to pocket proteins.

Zonula occludens protein 1 (ZO-1) is a cytosolic tight junction protein that tethers transmembrane proteins such as occludin, claudin and junctional adhesion molecule to the actin cytoskeleton. The interaction between ZO-1 and claudin or junctional adhesion molecule occurs via the amino-terminal PSD95/Dlg/ZO-1 (PDZ) domains in ZO-1. A yeast two-hybrid screen to search for proteins that interact with the PDZ domains of ZO-1 identified connexin (Cx) 45. Cx45 interacts with the PDZ domains of ZO-1 and ZO-3, but not ZO-2, via a short C-terminal PDZ binding motif (SVWI). In transfected epithelial Madin^Darby canine kidney cells, Cx45 co-localizes with endogenous ZO-1 at or near tight junctions and co-precipitation experiments show that Cx45 and ZO-1 directly interact. Inactivating the C-terminal PDZ-binding motif in Cx45 affects its co-precipitation and co-localization with ZO-1. The growing number of connexins (i.e. Cx43 and Cx45) that can associate with ZO proteins indicate that ZO proteins may play a more general role in organizing gap junctions and/or in recruiting signaling molecules that regulate intercellular communication. ß

BACKGROUNDGlioblastoma commonly is characterized by hypoxia and acidosis and the histologic features of tissue necrosis and neovascularization. Current approaches of adjuvant radiochemotherapy for patients with glioblastoma have only a modest impact on the natural course of this disease.METHODSThe authors examined the effects of acidosis on growth and response to irradiation and chemotherapy in cultured human malignant glioma cells.RESULTSThe authors found that mild acidosis (pH 7.0) inhibited the growth of cell lines that retained wild type p53 activity but did not inhibit the growth of cell lines that were devoid of p53 function. Transfer of a dominant‐negative p53 gene into p53 wild type cells failed to override the acidosis‐conferred growth arrest, suggesting that loss of p53 activity per se does not mediate escape from acidosis‐induced growth inhibition. Moderate acidosis (pH 6.6) inhibited the growth of all cell lines. Acidosis‐mediated growth arrest was not associated with a specific type of cell cycle arrest, e.g., in G0/G1 or G2/M phase. Acidosis did not result in consistent changes in radiosensitivity; however, it enhanced the cytotoxic effects of lomustine but conferred protection from topotecan, vincristine, teniposide, and cisplatin cytotoxicity. Lomustine exhibited enhanced stability at low pH, providing a putative mechanism for the enhanced cytotoxic effects of lomustine in acidotic conditions. Decreased sensitivity to the other drugs did not result from altered multidrug resistance drug transport activity.CONCLUSIONSTaken together, the current results suggest that tissue acidosis may be an important determinant of glioma cell responses to adjuvant radiochemotherapy. The superior activity of nitrosoureas, such as lomustine, compared with other agents in patients with glioblastoma may result in part from prolonged drug stability in an acidotic microenvironment. Cancer 2002;95:1113–9. © 2002 American Cancer Society.DOI 10.1002/cncr.10767

Using immobilized GST-Raf-1 as bait, we have isolated the intermediate filament protein vimentin as a Raf-1-associated protein. Vimentin coimmunoprecipitated and colocalized with Raf-1 in fibroblasts. Vimentin was not a Raf-1 substrate, but was phosphorylated by Raf-1-associated vimentin kinases. We provide evidence for at least two Raf-1-associated vimentin kinases and identified one as casein kinase 2. They are regulated by Raf-1, since the activation status of Raf-1 correlated with the phosphorylation of vimentin. Vimentin phosphorylation by Raf-1 preparations interfered with its polymerization in vitro. A subset of tryptic vimentin phosphopeptides induced by Raf-1 in vitro matched the vimentin phosphopeptides isolated from v-raf-transfected cells labeled with orthophosphoric acid, indicating that Raf-1 also induces vimentin phosphorylation in intact cells. In NIH 3T3 fibroblasts, the selective activation of an estrogen-regulated Raf-1 mutant induced a rearrangement and depolymerization of the reticular vimentin scaffold similar to the changes elicited by serum treatment. The rearrangement of the vimentin network occurred independently of the MEK/ERK pathway. These data identify a new branch point in Raf-1 signaling, which links Raf-1 to changes in the cytoskeletal architecture.