Phenotypical Changes Associated with Exogenous Expression of p16^<INK4a>

Abstract

Cells exogenously expressing p16^<INK4a> reduce proliferative activity by suppressing cyclin dependent kinase activity. However it remains unclear how phenotypic changes are associated with exogenous p16 expression. The human glioblastoma cell line U87MG was doubly transfected with plasmids pVgRXR and pIND horboring the wild-type p16 gene. The cells that express p16 protein in an eckdysoneinducible manner were used in this study. Phenotypic changes of cell characteristics associated with p16 expression such as cell shapes and cell adhesion on extracellular matrix (ECM) proteins were studied microscopically. Furthermore, the cell responses for extra-cellular growth stimuli such as EGF were also studied. In this study, changes in cell characteristics were observed as follows ; (1) The cells expressing p16 protein became larger and flatter than p16-negative cells; (2) these p16 protein showed an increased formation of actin-stress fibers, a finding assessed by TRITC-labeled phalloidin staining and vinculin accumulation in the focal adhesion contacts ; (3) cell adhesion to ECM proteins such as laminin, fibronectin, and type IV collagen significantly increased, and an increased expression of integrin α5 and αv was observed in the p16 positive cell, and p16 expression was also associated with a decrease in Rac protein ; (4) phosphorylation of ERK and expression of c-fos following the of EGF-stimulation was significantly reduced in the p16 positive cell, compared with that of p16-negative cells. The results suggest that the deletion or mutation of p16 gene during malignant progression of glioma may induce histopathological changes since changes in integrins may modulate cell adhesion and crosstalk between various surrounding cells and tumor cells in vivo. Additionally, mutational change in the p16 gene may result in an increased response to extracellular growth stimuli such as EGF.