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Contains fulltext : 135917.pdf (Publisher’s version ) (Closed access) Many patients with glioma harbor specific mutations in the isocitrate dehydrogenase gene IDH1 that associate with a relatively better prognosis. IDH1-mutated tumors produce the oncometabolite 2-hydroxyglutarate. Because IDH1 also regulates several pathways leading to lipid synthesis, we hypothesized that IDH1-mutant tumors have an altered phospholipid metabolite profile that would impinge on tumor pathobiology. To investigate this hypothesis, we performed (31)P-MRS imaging in mouse xenograft models of four human gliomas, one of which harbored the IDH1-R132H mutation. (31)P-MR spectra from the IDH1-mutant tumor displayed a pattern distinct from that of the three IDH1 wild-type tumors, characterized by decreased levels of phosphoethanolamine and increased levels of glycerophosphocholine. This spectral profile was confirmed by ex vivo analysis of tumor extracts, and it was also observed in human surgical biopsies of IDH1-mutated tumors by (31)P high-resolution magic angle spinning spectroscopy. The specificity of this profile for the IDH1-R132H mutation was established by in vitro (31)P-NMR of extracts of cells overexpressing IDH1 or IDH1-R132H. Overall, our results provide evidence that the IDH1-R132H mutation alters phospholipid metabolism in gliomas involving phosphoethanolamine and glycerophosphocholine. These new noninvasive biomarkers can assist in the identification of the mutation and in research toward novel treatments that target aberrant metabolism in IDH1-mutant glioma. Cancer Res; 74(17); 4898-907. (c)2014 AACR.

Contains fulltext : 97603.pdf (Publisher’s version ) (Closed access) DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Item does not contain fulltext Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real-time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so-called barrier-assay to define the contribution of fibronectin (FN) and Collagen-I (Col-I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)-induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col-I stimulated migration, but FN-induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule-organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536-1552, 2006. (c) 2006 Wiley-Liss, Inc.

Contains fulltext : 50157.pdf (Publisher’s version ) (Closed access) In motile fibroblasts, stable microtubules (MTs) are oriented toward the leading edge of cells. How these polarized MT arrays are established and maintained, and the cellular processes they control, have been the subject of many investigations. Several MT "plus-end-tracking proteins," or +TIPs, have been proposed to regulate selective MT stabilization, including the CLASPs, a complex of CLIP-170, IQGAP1, activated Cdc42 or Rac1, a complex of APC, EB1, and mDia1, and the actin-MT crosslinking factor ACF7. By using mouse embryonic fibroblasts (MEFs) in a wound-healing assay, we show here that CLASP2 is required for the formation of a stable, polarized MT array but that CLIP-170 and an APC-EB1 interaction are not essential. Persistent motility is also hampered in CLASP2-deficient MEFs. We find that ACF7 regulates cortical CLASP localization in HeLa cells, indicating it acts upstream of CLASP2. Fluorescence-based approaches show that GFP-CLASP2 is immobilized in a bimodal manner in regions near cell edges. Our results suggest that the regional immobilization of CLASP2 allows MT stabilization and promotes directionally persistent motility in fibroblasts.

Contains fulltext : 109674.pdf (Publisher’s version ) (Closed access) TRPM7 encodes a Ca(2+)-permeable nonselective cation channel with kinase activity. TRPM7 has been implicated in control of cell adhesion and migration, but whether TRPM7 activity contributes to cancer progression has not been established. Here we report that high levels of TRPM7 expression independently predict poor outcome in breast cancer patients and that it is functionally required for metastasis formation in a mouse xenograft model of human breast cancer. Mechanistic investigation revealed that TRPM7 regulated myosin II-based cellular tension, thereby modifying focal adhesion number, cell-cell adhesion and polarized cell movement. Our findings therefore suggest that TRPM7 is part of a mechanosensory complex adopted by cancer cells to drive metastasis formation. Cancer Res; 72(16); 4250-61. (c)2012 AACR.

Contains fulltext : 76049.pdf (Publisher’s version ) (Open Access) BACKGROUND: Creatine Kinases (CK) catalyze the reversible transfer of high-energy phosphate groups between ATP and phosphocreatine, thereby playing a storage and distribution role in cellular energetics. Brain-type CK (CK-B) deficiency is coupled to loss of function in neural cell circuits, altered bone-remodeling by osteoclasts and complement-mediated phagocytotic activity of macrophages, processes sharing dependency on actomyosin dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for direct coupling between CK-B and actomyosin activities in cortical microdomains of astrocytes and fibroblasts during spreading and migration. CK-B transiently accumulates in membrane ruffles and ablation of CK-B activity affects spreading and migration performance. Complementation experiments in CK-B-deficient fibroblasts, using new strategies to force protein relocalization from cytosol to cortical sites at membranes, confirmed the contribution of compartmentalized CK-B to cell morphogenetic dynamics. CONCLUSION/SIGNIFICANCE: Our results provide evidence that local cytoskeletal dynamics during cell motility is coupled to on-site availability of ATP generated by CK-B.

Item does not contain fulltext Tumour necrosis factor-alpha (TNF) has been used in the clinic for more than 10 years in an isolated limb perfusion (ILP). However, intra-tumoural expression of TNF receptor-1 (TNF-R1) and TNF-R1 upregulating factors are unknown. We determined the expression of TNF-R1, proEMAP and endothelial monocyte-activating polypeptide-II (EMAP-II) before and after ILP and evaluated this against clinical response. Tumour biopsies were taken before and after ILP of patients (n = 27) with advanced sarcoma or metastatic melanoma. Biopsies were randomly analysed by western blotting for proEMAP/EMAP-II and TNF-R1 expression. Appropriate melanoma biopsies were stained for EMAP-II, TNF-R1, CD31 and CD68. For melanomas we found that an up-regulation of EMAP-II, in contrast to proEMAP or TNF-R1, directly after ILP significantly correlated with a complete tumour response. No correlation was found for sarcoma patients. In a comparative analysis we found that the overall proEMAP and EMAP-II expression was higher in melanoma as compared to sarcoma cases and measurements in cell lines revealed high proEMAP expression by melanoma cells. We report high EMAP-II expression by endothelial cells and association with macrophages. In addition, macrophages are recruited to vessel-remnants after ILP. An upregulation of EMAP-II directly after ILP of melanoma patients correlates with and might predict a complete response to TNF-based ILP. The association of macrophages with EMAP-II expression and vascular damage suggests a role for EMAP-II in regulating the TNF-based anti-tumour effects observed with an ILP. Analysis of EMAP-II expression in melanoma biopsies should be implemented in the ILP procedure.