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.

[1]  T. Hunter.  Growth factors: The epidermal growth factor receptor gene and its product , 1984, Nature.

[2]  G. Stamp,et al.  Low expression of β1, α2 and α3 subunits of VLA integrins in malignant mammary tumours , 1991 .

[3]  B. Teicher,et al.  Acquired multicellular-mediated resistance to alkylating agents in cancer. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[4]  K. Burridge,et al.  An interaction between alpha-actinin and the beta 1 integrin subunit in vitro , 1990, The Journal of cell biology.

[5]  A. Sonnenberg,et al.  Laminin receptor on platelets is the integrin VLA-6 , 1988, Nature.

[6]  R. Sutherland.  Cell and environment interactions in tumor microregions: the multicell spheroid model. , 1988, Science.

[7]  B. Obrink,et al.  Cell-cell contacts mediated by E-cadherin (uvomorulin) restrict invasive behavior of L-cells , 1991, The Journal of cell biology.

[8]  R. Kramer,et al.  Basic FGF and TGF-beta differentially modulate integrin expression of human microvascular endothelial cells. , 1992, Experimental cell research.

[9]  R. Knüchel,et al.  Importance of tyrosine phosphatases in the effects of cell‐cell contact and microenvironments on EGF‐stimulated tyrosine phosphorylation , 1992, Journal of cellular physiology.

[10]  E. Ruoslahti,et al.  Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor , 1985, Cell.

[11]  M. Lampugnani,et al.  The role of integrins in the maintenance of endothelial monolayer integrity , 1991, The Journal of cell biology.

[12]  A. Sonnenberg,et al.  Association of the VLA alpha 6 subunit with a novel protein. A possible alternative to the common VLA beta 1 subunit on certain cell lines. , 1989, The Journal of biological chemistry.

[13]  W. Fiers,et al.  Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role , 1991, Cell.

[14]  P. Richman,et al.  Co‐ordinate expression of the alpha‐6 integrin laminin receptor sub‐unit and laminin in breast cancer , 1991, The Journal of pathology.

[15]  W. Bodmer,et al.  Low expression of collagen receptors in moderate and poorly differentiated colorectal adenocarcinomas. , 1990, British Journal of Cancer.

[16]  M. Beckerle,et al.  Interaction of plasma membrane fibronectin receptor with talin—a transmembrane linkage , 1986, Nature.

[17]  S. Santoro,et al.  Decreased expression of integrin adhesive protein receptors in adenocarcinoma of the breast. , 1990, The American journal of pathology.

[18]  V. Quaranta,et al.  Immunohistochemical localization of integrins in the normal, hyperplastic, and neoplastic breast. Correlations with their functions as receptors and cell adhesion molecules. , 1991, The American journal of pathology.

[19]  R. Clark.  Fibronectin matrix deposition and fibronectin receptor expression in healing and normal skin. , 1990, The Journal of investigative dermatology.

[20]  M. Takeichi,et al.  Cadherin cell adhesion receptors as a morphogenetic regulator. , 1991, Science.

[21]  C. Klein,et al.  Integrin VLA-3: ultrastructural localization at cell-cell contact sites of human cell cultures , 1989, The Journal of cell biology.