Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.

The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.

Modulation of p53 protein expression during cellular transformation with simian virus 40.

We analyzed the relation of metabolic stabilization of the p53 protein during cellular transformation by simian virus 40 (SV40) to (i) expression of the transformed phenotype and (ii) expression of the large tumor antigen (large T). Analysis of SV40-tsA28-mutant-transformed rat cells (tsA28.3 cells) showed that both p53 complexed to large T and free p53 (W. Deppert and M. Haug, Mol. Cell. Biol. 6:2233-2240, 1986) were metabolically stable when the cells were cultured at 32 degrees C and expressed large T and the transformed phenotype. At the nonpermissive temperature (39 degrees C), large-T expression is shut off in these cells and they revert to the normal phenotype. In such cells, p53 was metabolically unstable, like p53 in untransformed cells. To determine whether metabolic stabilization of p53 is directly controlled by large T, we next analyzed the metabolic stability of complexed and free p53 in SV40 abortively infected normal BALB/c mouse 3T3 cells. We found that neither p53 in complex with large T nor free p53 was metabolically stable. However, both forms of p53 were stabilized in SV40-transformed cells which had been developed in parallel from SV40 abortively infected cultures. Our results indicate that neither formation of a complex of p53 with large T nor large-T expression as such is sufficient for a significant metabolic stabilization of p53. Therefore, we suggest that metabolic stabilization of p53 during cellular transformation with SV40 is mediated by a cellular process and probably is the consequence of the large-T-induced transformed phenotype.

Cooperation of SV40 large T antigen and the cellular protein p53 in maintenance of cell transformation.

We analysed large T antigen expression and metabolic stabilisation of the cellular protein p53 in cells of a matched pair of SV40 tsA mutant (tsA58) N-type or A-type transformants, respectively. At the permissive growth temperature (32 degrees C), cells of both transformants, like SV40 wild-type transformed cells, were phenotypically transformed and expressed large T antigen, as well as metabolically stable p53 (both complexed and free p53). At the nonpermissive growth temperature (39 degrees C), cells of the N-type transformant reverted to a normal phenotype, whereas cells of the A-type transformant still displayed a transformed phenotype. Under these growth conditions, the mutant large T antigens in both cell types were no longer able to complex p53 (both in vivo and in vitro), but the metabolic stabilities of the free p53 in these cells correlated with their phenotypes: p53 in cells of the N-type transformant was rapidly degraded, whereas it was metabolically stable in cells of the A-type transformant. This difference in p53 stability correlated with an in vivo functional difference between the mutant large T antigens at the nonpermissive growth temperature: large T antigen in cells of the N-type transformant no longer stably associated with the cellular chromatin and the nuclear matrix, but accumulated in the nucleoplasm. In contrast, large T antigen in cells of the A-type transformant at least partially had retained this ability. Maintenance of SV40 cell transformation thus seems to require both a functional large T antigen and a metabolically stabilised p53.

Identification of a melanoma progression antigen as integrin VLA-2.

The expression of the integrin receptors VLA-1, -2, -3, and -6 was studied in normal cultured melanocytes and in five melanoma cell lines. Normal melanocytes synthesized VLA-3, but did not reveal detectable levels of VLA-1, -2, and -6. All melanoma cell lines, however, expressed VLA-2, -3, and -6. VLA-1 was synthesized by two of five melanoma lines. In parallel, we had analyzed the expression of four previously characterized melanoma cell surface antigens. One of them (antigen A.1.43), which is associated with tumor progression of human melanoma, revealed a striking similarity to VLA-2. In sequential immunoprecipitation experiments, we show that A.1.43 is identical with the integrin VLA-2, a cell surface receptor for collagen, laminin, and fibronectin.

Integrins of normal human epidermis: differential expression, synthesis and molecular structure.

The expression of integrin cell surface receptors in normal skin and their synthesis and molecular structure in keratinocyte cultures was investigated. The reactivity of four different polypeptides of the integrin family (alpha 2-, alpha 3-, alpha 6- and beta 1-chains) was demonstrated in the basal cell layer of normal epidermis. Studies of labelled keratinocyte cell lines showed that the polypeptides were expressed as alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins. Only the alpha 6 beta 4 integrin showed polarization towards the basement membrane attachment site of basal layer keratinocytes, and was preferentially expressed at microvillous projections. In contrast, alpha 2 beta 1 and alpha 3 beta 1 integrins were equally expressed throughout the basal cell plasma membrane.