SELECTIVE DOWN-REGULATION OF INTEGRIN RECEPTORS IN SPHEROIDS OF SQUAMOUS-CELL CARCINOMA

Integrin expression in squamous cell carcinomas have been reported to he down-regulated in vivo. Because integrins are molecules whose functions include the reorganization of cytoskeleton in response to the surrounding extracellular matrix, we have examined the expression of grins in the transformed cell line A431 when grown as multicellular spheroids or as monolayers. The spheroids were grown to sizes of approximately 100 mum and approximately 600 mum. Since larger A431 spheroids require epidermal growth factor for growth, we also investigated the effect of this growth factor on the expression of integrins in cells grown as monolayers or as small spheroids. Immunostaining studies using monoclonal antibodies specific for alpha6, beta1, and beta4 subunits revealed a strong staining pattern in the periphery of the spheroids. The interior cells of the spheroids showed a moderate, positive reaction with the beta1 antibody but significantly reduced from that at the periphery. Anti-alpha2 antibody, on the other hand, revealed a uniform staining around the cells throughout the spheroids. Western blot analyses confirmed an overall diminution of alpha6 and beta1 protein levels in the spheroids compared with monolayers. Northern blot analyses showed that the low expression of integrin subunits alpha6, beta1, and beta4 in spheroids was due to a reduction in mRNA transcripts. Northern blot analyses, however, showed no significant change in the expressions of alpha2, alpha5, or beta5 mRNA. Conversely, the expression of av was slightly reduced in spheroids. Epidermal growth factor increased the mRNA expression of alpha2, alpha6, beta1, and beta4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal growth factor had no detectable effect on the expression of av or beta5. These results mimic the pattern of expression found in vivo and indicate that cell-cell contact and the microenvironments of cells within a spheroid regulate the expression and distribution of a subset of integrin molecules.