Modulation of Cyclin D1 Expression by Integrin Signals in Oral Squamous Carcinoma Cells

Cyclin D1过表达在口腔鳞状细胞癌（oral squamous cell carcinoma， OSCC）中较为常见，且与OSCC患者的生存密切相关。我们用差异PCR技术检测了细胞周期蛋白D1基因的扩增。然而，检查的任何OSCC细胞系均未显示细胞周期蛋白D1基因扩增。Western blot检测磷酸化GSK-3β的表达，并与细胞周期蛋白D1在OSCC细胞中的表达进行比较。有趣的是，磷酸化的GSK-3β的表达与cyclin D1的表达呈负相关，这表明缺乏磷酸化的GSK-3β会导致OSCC细胞内cyclin D1的积累。接下来，我们测试了cyclin D1在不同培养条件下的表达，如在含10% fbs的培养基或无血清培养基中培养，单层培养细胞或多细胞聚集体（MCA），以及在特定的细胞外基质蛋白包被表面或非包被表面，我们发现在这些培养条件下cyclin D1水平没有差异。此外，通过免疫荧光染色研究细胞周期蛋白D1、GSK-3、Akt和Erk在单层细胞和MCAs中的定位。结果表明，两种不同的培养条件和两种不同的蛋白之间均存在弥漫性阳性反应。在本项目中，我们无法提供明确的证据证明整合素信号参与了OSCC细胞系中cyclin D1的表达。在OSCC中GSK-3β功能的调节机制有待进一步研究。

Cyclin D1 overexpression is frequently detected in oral squamous cell carcinomas (OSCC) and it is closely associated with the survival of OSCC patients. We examined cyclin D1 gene amplification with a panel of OSCC cell lines by the differential PCR technique. Any OSCC cell lines examined, however, did not show gene amplification of cyclin D1. Then, the expression of phosphorylated GSK-3β was examined by Western blotting, and was compared with cyclin D1 expression in OSCC cells. Interestingly, the expression of phosphorylated GSK-3β was reversely correlated with cyclin D1 expression, suggesting that lack of phosphorylated GSK-3β causes the intracellular accumulation of cyclin D1 in OSCC cells.Next, we tested cyclin D1 expression in various culture conditions, such as culturing with 10% FBS-containing medium or serum-free medium, as monolayer culture cells or multicellular aggregates (MCA), and on specific extracellular matrix proteins-coated surfaces or non-coated surface, and we found no differences in cyclin D1 level under these culture conditions. Furthermore, localizations of cyclin D1, GSK-3, Akt and Erk were investigated in monolayer cells and MCAs by immunofluorescent stainings. Results showed diffuse positive reactions without any differences between these proteins and between both the culture conditions.In this project, we could not present definite evidence of involvement of integrin signals in cyclin D1 expression in OSCC cell lines. Further studies are required to clarify the mechanism of modulation of GSK-3β functionality in OSCC.