When released into or formed in the extracellular space, adenosine acts as an autacoid via interaction with four types of G protein 1 -coupled receptors, termed A 1 -, A 2A -, A 2B -, and A 3 -adenosine receptor. These receptors are encoded by distinct genes and can be differentiated based on their affinities for adenosine analogues and methylxanthine antagonists (1, 2). In addition, the receptors can be classified based on their mechanism of signal transduction; A 1 -and A 3 -adenosine receptors interact with pertussis toxin-sensitive G proteins of the G i and G o family (3, 4, 5), whereas A 2A -and A 2B -adenosine receptors stimulate adenylyl cyclase via G s (6, 7).Adenosine is ubiquitously released by hypoxic tissues in large amounts; the nucleoside has therefore been proposed as one of the angiogenic factors that link the altered metabolism in oxygen-deprived cells to the formation of new capillaries (8).Earlier observations suggested that adenosine acts as an endothelial mitogen in vivo (9, 10). The mitogenic effect of adenosine has been verified in cultured endothelial cells derived from several vascular beds (11-13). In human endothelial cells, the proliferative response is mediated by the A 2A -adenosine receptor, an effect mimicked by stimulation of the endothelial ␤ 2 -adrenergic receptor (14). However, the mechanism by which adenosine analogues promote endothelial cell growth is not clear; there is, in particular, the apparent paradox that persistent stimulation of the signaling cascade composed of G s , adenylyl cyclase, and protein kinase A, which is downstream of A 2A -adenosine receptor, inhibits endothelial cell proliferation (14 -16). In the present work, we have therefore searched for additional effector mechanisms. We report that, in human endothelial cells, the A 2A -adenosine receptor stimulates the mitogen-activated protein kinase; this activation is independent of G s , G i , and typical protein kinase C isoforms but is associated with activation of p21 ras . EXPERIMENTAL PROCEDURESMaterials-Cell culture media were from Life Technologies, Inc., and cell culture dishes were from Greiner (Krems, Austria). [␥-32 P]ATP and [ 32 P]orthophosphate were from DuPont NEN. Cholera toxin, CPA, (Ϫ)isoproterenol, 8-Br-cAMP, collagenase (type IV), TPA, protein ASepharose, rabbit anti-rat IgG, forskolin, and epidermal growth factor (EGF) were obtained from Sigma, pertussis toxin was from Peninsula Laboratories (St. Helens, UK); bFGF, guanine nucleotides, and adenosine deaminase were from Boehringer Mannheim (FRG); XAC was from Research Biochemicals (Natick, MA), GF109203X was from Calbiochem, molecular weight standards (covering the range from 14 to 97 kDa) and reagents for SDS-polyacrylamide gel electrophoresis were from Bio-Rad. PD 098059, an inhibitor of MAP kinase kinase 1 (17), was from New England BioLabs (Beverly, MA). NECA and CGS 21680 were

To date, cancer is still the second most prevalent cause of death after cardiovascular diseases in the industrialized word, whereby the primary cause of cancer is not attributed to primary tumor formation, but rather to the growth of metastases at distant organ sites. For several years it was considered that the well-known phenomenon of organ-specific spreading of tumor cells is mostly a mechanical process either directed passively due to size constraints (mechanical trapping theory) or due to a fertile environment provided by the organ in which tumor cells can proliferate (seed and soil hypothesis). Both mechanisms strongly depend on the adhesive properties of tumor cells either to endothelial cells and/or cancer cells, which are facilitated by a variety of cell adhesion molecules including carbohydrates and integrins. Within the past years it became evident that the organ-specific metastatic spreading of tumor cells does not only rely on heterotypic and homotypic adhesive interactions, but also on the interplay of chemokines and their appropriate receptors. Moreover, the identification of cancer stem cells in various tumor tissues has opened new questions. Cancer stem cells possess self-renewal, differentiation, and tumor-initiating capacities. Thus these cells are ideal candidates to be the seed of a secondary tumor. In the present review we will give a brief overview about the complex process of organ-specific metastasis formation depending on the interplay of adhesion molecules, chemokines, and the putative role of cancer stem cells in metastasis formation.

The abilities of tumor cells to extravasate from the blood vessel system and to migrate through the connective tissue are prerequisites in metastasis formation. Both processes are chiefly mediated by integrins, which mediate both cell-cell and cell-matrix interactions. We investigated the role of integrin subunits in the adhesion, extravasation and migration of the highly invasive human bladder carcinoma cell line T24. Here we show that inhibition of the beta(1)-integrin subunit using the specific beta(1)-integrin blocking antibody 4B4 significantly reduces the adhesion to HUVEC and transmigratory activity of T24 cells. The blockade of the beta(1)-integrin subunit also resulted in a significantly reduced locomotory activity of T24 cells. A detailed cell migration analysis on a single cell level revealed that blockade of the beta(1)-integrin subunit leads to an altered migration pattern of single cells but does not influence migration per se. Migration parameters such as time active, velocity and distance migrated were significantly reduced as compared to untreated control cells. Our observations strongly suggest a central role for the beta(1)-integrin subunit in forming the cell-cell and cell-matrix bonds necessary for adhesion, extravasation and migration.

Realtime laser scanning confocal microscopy leads to a better understanding of the complex and dynamic cell-to-cell and cell-to-matrix interactions during the extravasation process.