Neoplastic Hepatocyte Growth Associated with Ccylin D1 Redistribution from the Cytoplasm to the Nucleus in Mouse Hepatocarcinogenesis

Proc Amer Assoc Cancer Res, Volume 47, 2006 4908 In chemically-induced mouse hepatocarcinogenesis, colonies of carcinogen-altered hepatocytes emerge in early stage, which later progress to adenomas and finally to hepatocellular carcinomas (HCC). Analyses of cell cycle proteins revealed that cyclin D1, cyclin E, p27, cdk2 and cdk4 are overexpressed in the neoplastic hepatocytes from early stage and that cyclin D1 is mainly localized in their cytoplasm. When the animals bearing the hepatic lesions were subjected to partial hepatectomy, cyclin D1 was shifted from the cytoplasm to the nucleus in association with proliferation of the neoplastic hepatocytes, but it returned to the cytoplasm in association with termination of proliferation. When the cells derived from mouse HCC were cultured in the medium containing growth factors (insulin plus EGF)/10% FBS, cyclin D1 was also localized in the nucleus in association with cell proliferation, while in the absence of growth factors/FBS it was localized in the cytoplasm without cell proliferation. To understand the intracellular signals necessary for the nuclear shift of cyclin D1, we treated the cells with various inhibitors to signal transduction. Inhibition of phosphatidyl-inositol-3-phosphate kinase (PI3K) by Ly294002 prevented both cyclin D1 nuclear shift and cell proliferation. Knockdown of phosphatase and tensin homolog (PTEN) by siRNA activated the PI3K pathway, but it induced neither cyclin D1 nuclear shift nor cell proliferation, suggesting that the PI3K activation is necessary but not sufficient for the cyclin D1 nuclear shift. Although MEK inhibition by PD098059 or mTOR inhibition by rapamycin less affected both on the cyclin D1 nuclear shift and cell proliferation, it decreased the cyclin D1 expression. Finally, because the proteins bound to cyclin D1 may determined the cyclin D1 localization, we analysed the cyclin D1 complex in the proliferating and quiescent states. Although p27, cdk4 and calmodulin were detected in the cyclin D1 immunoprecipitates from both the quiescent and proliferating cells, Hsc70 and SSeCKS were detected only in those from the quiescent cells, and p21 only in those from the proliferating cells. These observations indicate that 1) the nuclear/cytoplasmic cyclin D1 localization plays an important role in proliferation/quiescence of neoplastic hepatocytes, 2) the PI3K pathway may be mainly responsible to relocalization of cyclin D1 to the nucleus, while the MAPK pathway may be mainly responsible to the cyclin D1 abundance, and 3) the nuclear/cytoplasmic localization of cyclin D1 may be dependent on the proteins bound to cyclin D1.