Purification of vitronectin.

This unit describes the purification of extracellular vitronectin from plasma or serum by using heparin-affinity chromatography. First, the plasma is depleted of fibronectin plus other heparin- and Sepharose-binding proteins and treated with urea to activate the heparin-binding activity of vitronectin, which is subsequently bound to a heparin affinity column and eluted. The resulting vitronectin should be ∼ 98% pure.

Purification of fibronectin.

This unit describes the purification of the multifunctional adhesive glycoprotein fibronectin from plasma or of cell-derived fibronectin from cell surfaces and from conditioned medium. Fibronectin can be used in cell adhesion and migration assays, and can be obtained in relatively high purity using simple affinity chromatography techniques.

p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion.

Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to the spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV, as measured by electric cell-substrate impedance sensing.

CYLD inhibits melanoma growth and progression through suppression of the JNK/AP-1 and β1-integrin signaling pathways.

The molecular mechanisms mediating cylindromatosis (CYLD) tumor suppressor function appear to be manifold. Here, we demonstrate that, in contrast to the increased levels of phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CYLD was decreased in a majority of the melanoma cell lines and tissues examined. Exogenous expression of CYLD but not its catalytically deficient mutant markedly inhibited melanoma cell proliferation and migration in vitro and subcutaneous tumor growth in vivo.

A novel role for the T-box transcription factor Tbx1 as a negative regulator of tumor cell growth in mice.

The T-box transcription factor, Tbx1, an important regulatory gene in development, is highly expressed in hair follicle (HF) stem cells in adult mice. Because mouse models of skin carcinogenesis have demonstrated that HF stem cells are a carcinogen target population and contribute significantly to tumor development, we investigated whether Tbx1 plays a role in skin carcinogenesis. We first assessed Tbx1 expression levels in mouse skin tumors, and found down-regulation in all tumors examined.

Lysine 63-linked ubiquitination is important for arachidonic acid-induced cellular adhesion and migration.

Arachidonic acid, a dietary cis-polyunsaturated fatty acid, stimulates adhesion and migration of human cancer cells on the extracellular matrix by activation of intracellular signaling pathways. Polyubiquitin chains bearing linkages through different lysine residues convey distinct structural and functional information that is important for signal transduction. We investigated whether ubiquitination was required for arachidonic acid-induced cellular adhesion and migration of MDA-MB-435 cells on collagen type IV.

BCL2 interaction with actin in vitro may inhibit cell motility by enhancing actin polymerization.

In addition to its well-defined role as an antagonist in apoptosis, we propose that BCL2 may act as an intracellular suppressor of cell motility and adhesion under certain conditions. Our evidence shows that, when over-expressed in both cancer and non-cancer cells, BCL2 can form a complex with actin and gelsolin that functions to decrease gelsolin-severing activity to increase actin polymerization, and, thus, suppress cell adhesive processes. The linkage between increased BCL2 and increased actin polymerization on the one hand, and suppression of cell adhesion, spreading, and motility on the other hand, is a novel observation that may provide a plausible explanation for why BCL2 over-expression in some tumors is correlated with improved patient survival.

BCL2 inhibits cell adhesion, spreading, and motility by enhancing actin polymerization.

BCL2 is best known as a multifunctional anti-apoptotic protein. However, little is known about its role in cell-adhesive and motility events. Here, we show that BCL2 may play a role in the regulation of cell adhesion, spreading, and motility.

Cell-surface nucleolin is a signal transducing P-selectin binding protein for human colon carcinoma cells.

We have previously shown that P-selectin binding to Colo-320 human colon carcinoma cells induces specific activation of the alpha(5)beta(1) integrin with a concomitant increase of cell adhesion and spreading on fibronectin substrates in a phosphatidylinositol 3-kinase (PI3-K) and p38 MAPK-dependent manner. Here, we identified by affinity chromatography and characterized nucleolin as a P-selectin receptor on Colo-320 cells. Nucleolin mAb D3 significantly decreases the Colo-320 cell adhesion to immobilized P-selectin-IgG-Fc.

P-selectin activates integrin-mediated colon carcinoma cell adhesion to fibronectin.

During hematogenous cancer metastasis, tumor cells separate from a primary mass, enter the bloodstream, disperse throughout the body, migrate across vessel walls, and generate distant colonies. The later steps of metastasis superficially resemble leukocyte extravasation, a process initiated by selectin-mediated cell tethering to the blood vessel wall followed by integrin-mediated arrest and transendothelial migration. Some cancer cells express P-selectin ligands and attach to immobilized P-selectin, suggesting that these cells can arrest in blood vessels using sequential selectin- and integrin-mediated adhesion, as do leukocytes.

Aberrant N-glycosylation of beta1 integrin causes reduced alpha5beta1 integrin clustering and stimulates cell migration.

Altered expression of cell surface N-linked oligosaccharides is associatedwith the oncogenic transformation of many types of animal cells. One of the most common forms of glycosylation in transformed cells and human tumors is the highly elevated beta1,6 branching of N-linked oligosaccharides caused by increased transcription of N-acetylglucosaminyltransferase V (GnT-V). To characterize the effects of increased beta1,6 branching on cell-matrix adhesion-mediated phenotypes, human fibrosarcoma HT1080 cells were transfected with retroviral systems encoding GnT-V that used both noninducible and tetracycline-inducible promoters.