Waleh N S, Gallo J, Grant T D, Murphy B J, Kramer R H, Sutherland R M
Cellular and Molecular Biology Laboratory, SRI International, Menlo Park, California 94025.
Cancer Res. 1994 Feb 1;54(3):838-43.
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.
据报道，整合素在鳞状细胞癌中的表达在体内下调。由于整合素是一类分子，其功能包括响应周围细胞外基质而重组细胞骨架，因此我们研究了转化细胞系A431在形成多细胞球体或单层细胞时整合素的表达情况。球体生长至约100微米和约600微米大小。由于较大的A431球体生长需要表皮生长因子，我们还研究了这种生长因子对单层细胞或小球体细胞中整合素表达的影响。使用针对α6、β1和β4亚基的单克隆抗体进行的免疫染色研究显示，球体周边有强烈的染色模式。球体内部细胞与β1抗体呈中度阳性反应，但明显低于周边细胞。另一方面，抗α2抗体显示整个球体细胞周围染色均匀。蛋白质印迹分析证实，与单层细胞相比，球体中α6和β1蛋白水平总体降低。Northern印迹分析表明，球体中整合素亚基α6、β1和β4的低表达是由于mRNA转录物减少所致。然而，Northern印迹分析显示α2、α5或β5 mRNA的表达没有显著变化。相反，球体中αv的表达略有降低。表皮生长因子增加了单层细胞或球体细胞中α2、α6、β1和β4整合素亚基的mRNA表达，而表皮生长因子对αv或β5的表达没有可检测到的影响。这些结果模拟了体内发现的表达模式，表明细胞间接触和球体中细胞的微环境调节了一部分整合素分子的表达和分布。
