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Selective down-regulation of integrin receptors in spheroids of squamous cell carcinoma.

Integrin expression in squamous cell carcinomas have been reported to be 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 integrins in the transformed cell line A431 when grown as multicellular spheroids or as monolayers. The spheroids were grown to sizes of approximately 100 microns and approximately 600 microns. 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 alpha 6, beta 1, and beta 4 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 beta 1 antibody but significantly reduced from that at the periphery. Anti-alpha 2 antibody, on the other hand, revealed a uniform staining around the cells throughout the spheroids. Western blot analyses confirmed an overall diminution of alpha 6 and beta 1 protein levels in the spheroids compared with monolayers. Northern blot analyses showed that the low expression of integrin subunits alpha 6, beta 1, and beta 4 in spheroids was due to a reduction in mRNA transcripts. Northern blot analyses, however, showed no significant change in the expressions of alpha 2, alpha 5, or beta 5 mRNA. Conversely, the expression of alpha v was slightly reduced in spheroids. Epidermal growth factor increased the mRNA expression of alpha 2, alpha 6, beta 1, and beta 4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal growth factor had no detectable effect on the expression of alpha v or beta 5. 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.