RhoA activation stimulates IEC-6 cell proliferation by decreasing p21Wafl1/Cip1 and increasing Cdk2 expression in a polyamine dependent manner

Abstract

Background & Aims: Although RhoA plays an important role m ceil proliferation and in Ras transformation in fibroblasts and mammary epithelial cells; its role in intestinal epithelial cells is still unknown. In our previous study, we showed that polyamine depletion (DFMO treatment) strongly inhibits proliferation of intestinal epithelial cells (IEC-6). In this report, we examined the effects of active RhoA and the mechanism underlying 1EC-6 cell proliferation, contact inhibition, and focus formation. We also examined whether polyamine depletion inhibits cell proliferation m the presence of constitutively active RhoA. Methods: Constitutively active RhoA and vector transfected 1EC-6 cell lines were used. Cells were grown in the presence or absence of DFMO, an irreversible inhibitor of omithine decarboxylase (ODC). Growth characteristics were assessed by microscopic observation, cell counting, and ceil cycle analysis by flow cytometry. Cell cycle regulators were evaluated by western blot, functional assay, and real-time PCR. Results: RhoA transfection significantly increased the rate of cell proliferation. These cells also demonstrated attenuated contact inhibition and conspicuous loci when they were fully confluent, increased proliferation was accompanied by decreased p21Wafl/Cipl, increased Cdk2 expression and activity. The inhibition of p21Wafl/Cipl was independent of p53. There was no activation of the Ras-Raf-Mek-Erk pathway in the RhoA transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on 1EC-6 cell proliferation and focus formation, decreased Cdk2 expression and activity independent of p21Wafl/Cipl. Conclusions: First, RhoA activation decreases p21, increase~ Cdk2 protein and Cdk2 activity, leading to the stimulation of intestinal epithelial cell proliferation and transformation; second, polyamine depletion totally prevents stimulating effect of RhoA on proliferation by decreasing Cdk2 expression and activity.