The integrin ␤ 1 subunit can form a heterodimer with 12 different ␣ subunits. According to the present model, the expression level of any ␣␤ complex is regulated by the availability of the specific ␣ subunit, whereas ␤ 1 subunit is constantly present in a large excess. The expression of several heterodimers containing the ␣ V subunit seems to be regulated by an identical mechanism. The fact that many cells express ␣ V ␤ 1 heterodimer, and that this fibronectin/vitronectin receptor may be selectively regulated, compromises the present model of the regulation of ␤ 1 and ␣ V integrins. We have tried to solve this problem by assuming that distinct ␣␤ heterodimers are formed with different tendency. To test the hypothesis, we analyzed WM-266-4 melanoma cells transfected with a cDNA construct coding for an intracellular single-chain anti-␣ V integrin antibody. We could see 70 -80% reduction in the cell surface expression of ␣ V subunit. However, the only one of the ␣ V integrins reduced on the cell surface was ␣ V ␤ 1 . This suggests that the cell surface expression level of ␣ V ␤ 1 is dependent on the number of ␣ V subunits available after the formation of other ␣ V -containing heterodimers. Thus, there seems to be a hierarchy in the complex formation between ␣ V and its different ␤-partners. These observations explain how ␣ V ␤ 1 can be specifically regulated without concomitant changes in the expression of other ␣ V or ␤ 1 integrins.The members of the integrin family that form receptors for various extracellular matrix proteins can be divided into two major subgroups according to the subunits present in the receptors. Integrin ␤ 1 can form a complex with 12 different ␣ subunits. The ␤ 1 -containing heterodimers are receptors for collagens, laminins, tenascins, and fibronectin. The other subset, the ␣ V integrins, are fibronectin and vitronectin receptors, some of which also have the ability to bind various other matrix and plasma proteins.In many cells the two promiscuous subunits, ␣ V and ␤ 1 , can form a heterodimer with each other. ␣ V ␤ 1 was originally described as a fibronectin or vitronectin receptor (1, 2). It may also have a capacity for binding to osteopontin and to the latent form of transforming growth factor-␤ (3, 4). Some viruses, including parechovirus 1, adenovirus, and foot-and-mouth disease virus, use ␣ V ␤ 1 as their cellular receptor (5-7). The tissue distribution of ␣ V ␤ 1 is mostly unknown because of the lack of a specific antibody against ␣ V ␤ 1 complex. For the same reason the function of ␣ V ␤ 1 integrin in many human cell types is unknown, or the published information is based on cell transfections or on the use of combinations of function blocking antibodies against different ␣ V and ␤ 1 integrins. In ␣ V -transfected Chinese hamster ovary cells, ␣ V ␤ 1 integrin has been found to function as a fibronectin receptor while not supporting cell migration on fibronectin (8). On the other hand, in squamous carcinoma cells derived from head and neck tumors, ␣ V ␤ 1 integrin contributes to migr...

The expression of integrin ␣V subunit on 4 melanomaderived cell lines (A2058, SK-mel-5, WM-115 and WM-266-4) was analyzed. WM-115 cells, which originate from a primary tumor, were negative, whereas all 3 metastasis-derived lines had high levels of ␣V. To study ␣V integrins in the survival of melanoma cells, we developed a novel strategy that is exempt from extracellular inhibitors of ligand binding, which can activate integrin signaling and have integrin-independent effects on apoptosis. A recombinant adenovirus was used to transfer cDNA coding for a single-chain intracellular anti-␣V integrin antibody into the melanoma cells. Anti-␣V integrin adenovirus effectively inhibited the cell surface expression of ␣V integrins. In cell culture experiments, the depletion of ␣V integrins detached cells from extracellular matrix and induced apoptosis. Moreover, it prevented WM-266-4 cells from forming tumors in severe combined immunodeficiency mice but it could not prevent the growth of tumors that were formed by ␣V-negative WM-115 cells. Our results indicate that in primary melanomas there are cells that survive without ␣V integrins, whereas during the progression of disease cells can develop a dependency on these receptors. Furthermore, the data oppose the possibility that in melanoma cells apoptosis could occur due to direct activation of caspases by ligand-free ␣V integrins on the cell surface. © 2004 Wiley-Liss, Inc. Key words: integrin; melanoma; intracellular single-chain antibody; adenovirusThe ␣V integrin can form a heterodimeric adhesion receptor with 5 different integrin ␤ subunits, namely, ␤1, ␤3, ␤5, ␤6 and ␤8. These receptors bind to various extracellular matrix proteins such as fibronectin, vitronectin and osteopontin. In adult individuals, relatively few cell types express significant amounts of ␣V, and genetic data from mouse experiments indicate that most organs develop properly even in the absence of all ␣V integrins. 1 In human melanoma, the expression of ␣V together with its partner ␤3 has been connected to tumor growth, disease progression and poor prognosis. 2-8 Additionally, ␣V integrins may play a role in the progression of ovarian carcinoma, cervical tumors, breast cancer and squamous cell carcinoma, 9 -13 and it has also been connected to the formation of bone metastasis in prostate cancer. 14 Based on the analysis of knockout mice, it is obvious that in vivo, most normal cell types can survive without ␣V integrins. 1 In cancer cells, the inhibition of ␣V function has been associated with apoptosis, and appropriate ligand binding may be essential for the function of ␣V integrins during cancer progression. For example, the secretion of osteopontin, a ligand for ␣V␤3, by melanocytes has been reported to promote antiapoptotic signaling. 15 Furthermore, after disruption of cell-matrix contacts, unbound ␣V integrins may promote apoptosis in human melanoma and human intestinal carcinoma cells. 16,17 It has also been reported that in T24-E carcinoma cells, death is stimulated by attachment to the extrac...

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