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 funct ions include the reorganization of cytoskeleton in response to the sur rounding extracellular matrix, we have examined the expression of grin s in the transformed cell line A431 when grown as multicellular sphero ids or as monolayers. The spheroids were grown to sizes of approximate ly 100 mum and approximately 600 mum. Since larger A431 spheroids requ ire epidermal growth factor for growth, we also investigated the effec t of this growth factor on the expression of integrins in cells grown as monolayers or as small spheroids. Immunostaining studies using mono clonal antibodies specific for alpha6, beta1, and beta4 subunits revea led a strong staining pattern in the periphery of the spheroids. The i nterior cells of the spheroids showed a moderate, positive reaction wi th the beta1 antibody but significantly reduced from that at the perip hery. Anti-alpha2 antibody, on the other hand, revealed a uniform stai ning 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 s lightly reduced in spheroids. Epidermal growth factor increased the mR NA expression of alpha2, alpha6, beta1, and beta4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal gr owth factor had no detectable effect on the expression of av or beta5. These results mimic the pattern of expression found in vivo and indic ate that cell-cell contact and the microenvironments of cells within a spheroid regulate the expression and distribution of a subset of inte grin molecules.