Interferon-alpha delays S-phase progression in human hepatocellular carcinoma cells via inhibition of specific cyclin-dependent kinases

The potential antiproliferative effects of interferon-alpha (IFN-alpha) in the treatment ofhepatocellular carcinoma (HCC) are controversial, and the g rowth inhibitory mechanisms remain poorly understood. Therefore, the curren t study was designed to delineate the molecular mechanisms responsible for direct antiproliferative actions of IFN-alpha in HCC cells. IFN-alpha recep tor expression and signal transduction were examined by RT-PCR, immunopreci pitation, Western analysis, and transient transactivation assays. Effects o f IFN-alpha on cell growth and cell-cycle distribution were evaluated based on cell numbers and flow cytometry, Composition and activity of cyclin-dep endent kinase complexes were determined by immunoblotting and histone-H1-ki nase assays, Expression of IFN-alpha receptors was found in all 3 HCC cell lines. IFN-alpha binding initiated phosphorylation of Jak1 and Tyk2 kinases leading to Stat1/Stat2, activation, nuclear translocation, and transactiva tion of an ISRE-luciferase reporter gene construct. IFN-alpha treatment res ulted in a time-and dose-dependent reduction of proliferation. Cell cycle a nalysis of G1-synchronized, IFN-alpha -treated HCC cells revealed a substan tial delay in S-phase progression but no alteration of G1/S-phase transitio n or evidence of apoptotic cell death. Reflecting the time course of S-phas e accumulation, cell cycle-dependent induction of Cyclin A and Cyclin B was impaired, resulting in reduced activity of Cdk2 and Cdc2 kinases, Furtherm ore, Cdc25C was selectively downregulated. IFN-alpha treatment inhibits gro wth of HCC cells by specifically delaying S-phase progression, most likely because of inhibition of Cyclin A induction, resulting in decreased activit y of the associated Cdk2 and Cdc2 kinases.