Modulation of Wnt3a-mediated nuclear beta-catenin accumulation and activation by integrin-linked kinase in mammalian cells.

The Wnt gene family encodes secreted signaling molecules that play important roles in tumorgenesis and embryogenesis. The canonical Wnt signaling pathway regulates target gene expression via the stabilization and nuclear translocation of the cytoplasmic pool of beta-catenin. The activation of integrin-linked kinase (ILK) is also known to regulate the stabilization and subsequent nuclear translocation of beta-catenin in several epithelial cell models. We now report that molecular and pharmacological inhibition of ILK activity in mammalian cells directly modulates Wnt signaling by suppressing the stabilization and nuclear translocation of beta-catenin, as well as beta-catenin/Lef-mediated transcription. Inhibition of ILK activity, but not phosphatidylinositol-3 kinase (PI3K) or MEK activities suppresses nuclear beta-catenin stabilization in cells stably expressing Wnt3a as well as in cells exposed to either Wnt3a conditioned media or purified Wnt3a. Furthermore, ILK inhibition reverses the Wnt3a-induced suppression of beta-catenin phosphorylation that accompanies beta-catenin stabilization. In addition, we show that ILK can be identified in a complex with Wnt pathway components such as adenomatous polyposis coli and GSK-3. Upon treatment of L cells with Wnt3a-CM, glycogen synthase kinase-3 (GSK-3beta) becomes highly phosphorylated on Ser 9, which is completely abolished upon inhibition of ILK activity. However, acute exposure of L cells to purified Wnt3a does not result in the stimulation of GSK-3beta Ser 9 phosphorylation, despite beta-catenin stabilization. Together our data demonstrate that ILK activity can modulate acute Wnt3a mediated beta-catenin phosphorylation, stabilization and nuclear activation in a PI3K-independent manner, as well as the more prolonged PI3K-dependent secondary effects of Wnt signaling on GSK-3 phosphorylation. Finally, we suggest that a novel small molecule inhibitor of ILK, QLT-0267, may be a useful tool in the regulation of pathological Wnt signaling.

Identification of genes differentially expressed in V79 cells grown as multicell spheroids.

PURPOSE: Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids. MATERIALS AND METHODS: Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay. RESULTS: Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calcium binding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide. CONCLUSIONS: Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.

Changes in subcellular distribution of topoisomerase IIalpha correlate with etoposide resistance in multicell spheroids and xenograft tumors.

The outer cells of Chinese hamster V79 spheroids are about 10 times more resistant than monolayers to DNA damage and cell killing by the topoisomerase (topo) II inhibitor etoposide. Although the amount and catalytic activity of topo IIalpha are identical for monolayers or the outer cells of spheroids, and the cell proliferation rate is the same, our previous results indicated that phosphorylation of topo IIalpha is at least 10 times higher in V79 monolayers than in spheroids. Because phosphorylation of topo IIalpha has been associated with nuclear translocation, we examined subcellular distribution of Topo IIalpha in monolayers, spheroids, and xenograft tumors using immunohistochemistry. Topo IIalpha was located predominantly in the nucleus of V79, human SiHa, and rat C6 monolayers but was found mainly in the cytoplasm of the proliferating outer cells of spheroids formed from these cell lines. Conversely, the outer cells of WiDr human colon carcinoma spheroids showed predominantly nuclear localization of topo IIalpha, and only WiDr cells showed no increase in resistance to etoposide when grown as spheroids. Cells sorted from xenografts resembled the spheroids in terms of sensitivity to etoposide and location of topo IIalpha. When the outer cells of V79 spheroids were returned to monolayer growth, the rate of redistribution of topo IIalpha to the nucleus occurred with similar kinetics as the increase in sensitivity to killing by etoposide. Removal and return of individual outer V79 spheroid cells to suspension culture resulted in the translocation of topo IIalpha to the nucleus for the first 24 h, accompanied by an increase in sensitivity to DNA damage by etoposide. Therefore, the cytoplasmic topo IIalpha distribution in outer spheroid cells and tumors appears to correlate not with morphological changes associated with growth in suspension but rather with the presence of neighboring, noncycling cells.

Chaotic dynamics of coupled transverse-longitudinal plasma oscillations in magnetized plasmas.

The propagation of intense electromagnetic waves in cold magnetized plasma is tackled through a relativistic hydrodynamic approach. The analysis of coupled transverse-longitudinal plasma oscillations is performed for traveling plane waves. When these waves propagate perpendicularly to a static magnetic field, the model is describable in terms of a nonlinear dynamical system with 2 degrees of freedom. A constant of motion is obtained and the powerful classical mechanics methods can be used. A new class of solutions, i.e., the chaotic solutions, is discovered by the Poincaré surface of sections. As a result, coupled transverse-longitudinal plasma oscillations become aperiodically modulated.

Controlling Hamiltonian chaos via Gaussian curvature.

We present a method allowing one to partly stabilize some chaotic Hamiltonians which have two degrees of freedom. The purpose of the method is to avoid the regions of V(q(1),q(2)) where its Gaussian curvature becomes negative. We show the stabilization of the Hénon-Heiles system, over a wide area, for the critical energy E=1/6. Total energy of the system varies only by a few percent.

Cell fusion studies to examine the mechanism for etoposide resistance in Chinese hamster V79 spheroids.

When exposed to etoposide, the outer cells from Chinese hamster V79 spheroids are about 10 times more resistant to DNA strand breaks and cell killing than V79 cells grown as monolayers. Previous results have shown that the outer cells of both spheroids and monolayers grow at the same rate and contain the same amount and activity of the target enzyme, topoisomerase II. In order to examine possible mechanisms for this resistance, cell fusion studies were conducted with fluorescent dye-tagged monolayer and spheroid cells. Fused cells were exposed for 30 min to 1.2 microg/ml etoposide and then separated using fluorescence-activated cell sorting into binucleate cells consisting of two monolayer cells, two spheroid cells, or a mixed doublet consisting of one cell of each type. Individual sorted cell doublets were examined for the presence of etoposide-induced DNA strand breaks using the alkaline comet assay. As expected, doublets of monolayer cells were sensitive to etoposide and doublets of spheroid cells were resistant. However, mixed doublets were as resistant to DNA damage by etoposide as spheroid doublets. In comparison, when etoposide- or adriamycin-resistant V79 monolayer cells were fused to the parent monolayer cells, the expected intermediate sensitivity to etoposide was observed for the mixed doublets. We conclude that etoposide resistance associated with the outer cells of spheroids can be "transferred" to produce resistance in monolayer cells. Rapid changes in phosphorylation that can affect topoisomerase II activity or localization, or that can alter chromatin structure, are suggested as possible mechanisms of resistance. In support of this hypothesis, topo IIalpha phosphorylation was at least 10 times greater in monolayers than in the outer cell layer of spheroids.