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Objective. The goal of this work was to evaluate the involvement of gonadotropins in the regulation of adhesion of human epithelial ovarian carcinoma. We studied two pathways that were previously implicated in the metastatic implantation of ovarian carcinoma to the peritoneum, namely hyaluronan-CD44 and RGD-integrin mediated adhesion. Methods. Two cell lines derived from human epithelial ovarian carcinoma (MLS and OC238) were stimulated with luteinizing hormone (LH) and/or follicle stimulating hormone (FSH). Expression of CD44 was evaluated by Western blotting. Expression of αv-integrins was studied by RT-PCR and Northern blot. Integrin and CD44 mediated adhesion of the cells was analyzed using culture plates coated either with a thrombin derived RGD containing peptide or fibronectin for integrin mediated adhesion or with hyaluronan for CD44 mediated adhesion. Results. MLS cells stimulated with either LH or FSH showed increased adhesion to culture plates coated with hyaluronan, as well as to culture plates coated with fibronectin or with a thrombin derived RGD containing peptide. In these cells, gonadotropin stimulation led to induced expression of the integrin subunit α v and CD44, the cell surface hyaluronan receptor. On the other hand, OC238 cells showed no expression of the integrin subunit α v and no hormonal effect on the expression of CD44. Accordingly, adhesion of OC238 cells on either RGD or CD44 was not affected by hormonal stimulation. Conclusions. Elevated levels of gonadotropins may in some cases facilitate peritoneal metastatic dissemination of ovarian cancer by increasing cell adhesion, the first essential step in the invasion process.

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.

Multiple cell types have an inherent ability to contract the extracellular matrix to which they are attached and grow on. Cells exert contractile forces on a compliant substrate, increase the tension, and deform it. Numerous intracellular as well as environmental factors are involved in determination of cellular contractility, which can be precisely measured by atomic force microscopy, laser tweezers, or other complex apparatus. These, however, are far from being standard equipment in most cell biology labs. Fibrin gels provide a simple and affordable alternative for evaluation of changes in cell contractility by either quantitation of end-point gel contraction or in a dynamic mode by time-lapse imaging. They also provide a flexible system in which the physical properties, such as density and compliance, as well as their biochemical composition can easily be altered to suit the special requirements of various cell types and experimental models.

Preface Contributors I. Molecular Biology Retroviral delivery of ECM genes Vitali Alexeev and Olga Igoucheva Tissue-Specific KO of ECM Proteins Mara Brancaccio, Emila Turco and Emilio Hirsch Recombinant Collagen Trimers from Insect Cells and Yeast- Johanna Myllyharju Eukaryotic Expression and Purification of Recombinant Extracellular Matrix Proteins Carrying the Strep II Tag Neil Smyth, Uwe Odenthal, Barbara Merkl, and Mats Paulsson Tissue Recombinants to Study Extracellular Matrix Targeting to Basement Membranes Patricia Simon-Assmann, Anne-Laure Bolcato-Bellemin, Annick Klein and Michele Kedinger Preparation of recombinant fibronectin fragments for functional and structural studies David Staunton, Christopher J Millard, Radu Aricescu and Iain D Campbell II. Biochemical and biophysical analysis Quantitative Determination of Collagen Cross-links Nicholas C Avery, Trevor J Sims and Allen J Bailey ECM Macromolecules: Height-mapping and Nano-mechanics Using Atomic Force Microscopy Nigel W. Hodson, Cay M. Kielty and Michael J. Sherratt Atomic Force Microscopy Measurements of Intermolecular Binding Forces- Gradimir N. Misevic, Yannis Karamanos and Nikola J. Misevic Mass-Mapping of ECM Macromolecules by Scanning Transmission Electron Microscopy Michael J. Sherratt, Helen K. Graham, Cay M. Kielty and David F. Holmes Chemical Microscopy of Biological Samples by Dynamic Mode Secondary Ion Mass Spectrometry (SIMS) Gradimir N. Misevic, Bernard Rasser, Vic Norris, Cedric Derue, David Gibouin, Fabrice Lefebvre, Marie-Claire Verdus, Anthony Delaune, Guillaume Legent and CamilleRipoll ECM Macromolecules: Rotary Shadowing and Transmission Electron Microscopy Michael J. Sherratt, Roger S. Meadows, Helen K. Graham, Cay M. Kielty and David F. Holmes Using Self-Assembled Monolayers to Pattern ECM Proteins and Cells on Substrates Emanuele Ostuni, George M. Whitesides, Donald E. Ingber and Christopher S. Chen Solid Phase Assays for Studying ECM Protein-Protein Interactions Paul A Mould III. Cell biology assays Cell Adhesion Assays Martin J. Humphries ECM Degradation Assays for Analyzing Local Cell Invasion Vira V. Artym, Kenneth M. Yamada, Susette C. Mueller Fluorescence-Based Assays for In Vitro Analysis of Cell Adhesion and Migration Paola Spessotto, Katia Lacrima, Pier Andrea Nicolosi, Eliana Pivetta, Martina Scapolan and Roberto Perris Fibrin Gel Model for Assessment of Cellular Contractility Sharona Even-Ram Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis ( Labeling fibronectin fibrillogenesis) Roumen Pankov and Albena Momchilova Stromagenesis During Tumorigenesis: Characterization of Tumor-associated Fibroblasts and Stroma-derived 3D Matrices Remedios Castello-Cros and Edna Cukierman IV. Organ models ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor /matrix regulation of gene expression during submandibular gland development Ivan T. Rebustini and Matthew P. Hoffman Analyzing Cell-ECM Interactions in Adult Mammary Gland by Transplantation of Embryonic Mammary Tissue from Knockout Mice Teresa C. M. Klinowska and Charles H. Streuli V. Tissue

Directional migration moves cells rapidly between points, whereas random migration allows cells to explore their local environments. We describe a Rac1 mechanism for determining whether cell patterns of migration are intrinsically random or directionally persistent. Rac activity promoted the formation of peripheral lamellae that mediated random migration. Decreasing Rac activity suppressed peripheral lamellae and switched the cell migration patterns of fibroblasts and epithelial cells from random to directionally persistent. In three-dimensional rather than traditional two-dimensional cell culture, cells had a lower level of Rac activity that was associated with rapid, directional migration. In contrast to the directed migration of chemotaxis, this intrinsic directional persistence of migration was not mediated by phosphatidylinositol 3′-kinase lipid signaling. Total Rac1 activity can therefore provide a regulatory switch between patterns of cell migration by a mechanism distinct from chemotaxis.

Integrins are receptors for extracellular matrix proteins that engage in reciprocal crosstalk with growth factor receptors. Recent work identifies a unique mechanism for the regulation of growth factor receptor phosphorylation by integrins, indicating multiple ways of achieving cooperation between these major signalling systems.