Downregulation of p21(waf/cip-1) mediates apoptosis of human hepatocellular carcinoma cells in response to interferon-gamma.
There is no effective treatment for advanced hepatocellular carcinoma (HCC). We therefore explored the molecular mechanisms of interferon-gamma (IFN-gamma)-mediated growth regulation in human HCC cell lines. IFN-gamma receptor expression, signal transduction, and regulation of effectors were examined by RT-PCR, immunoprecipitation, immunoblotting, and reporter gene assays. Growth and apoptosis were determined based on cell numbers, cell cycle analyses, kinase assays, DNA fragmentation, and PARP cleavage. HCC cell lines express functionally intact IFN-gamma receptors and downstream effectors. IFN-gamma profoundly inhibited growth of HCC cells via two different mechanisms: inhibition of G1 cell cycle progression and induction of apoptosis. Analyses in SK-Hep-1 cells revealed a deficient cyclin D induction in IFN-gamma-treated cells, resulting in reduced activity of CDK4 and CDK2 kinases and pRB hypophosphorylation. In contrast, apoptosis prevailed in IFN-gamma-treated HepG2 cultures. A survey of apoptosis relevant IFN-gamma effectors including IRF-1, caspase-1, caspase-3, and p21(waf/cip-1) documented a dramatic transcriptional downregulation of p21(waf/cip-1) exclusively in apoptosis-susceptible HepG2 cells. Reconstitution of p21(waf/cip-1) rescued HepG2 cells from IFN-gamma-induced apoptosis, indicating that p21(waf/cip-1) reduction was required for apoptosis execution. Inversely, downregulation of p21(waf/cip-1) sensitized SK-Hep-1 cells to IFN-gamma-induced apoptosis. Thus, downregulation of p21(waf/cip-1) in HCC cells functions as a novel, critical determinant of alternative growth inhibitory pathways in response to IFN-gamma.
Detjen
Katharina M
KM
Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Universitätsklinikum Charité, Campus Virchow-Klinikum, Humboldt Universität zu Berlin, Germany.
Murphy
Derek
D
Welzel
Martina
M
Farwig
Katrin
K
Wiedenmann
Bertram
B
Rosewicz
Stefan
S
D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D042846:CDC2-CDC28 Kinases; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D056746:Cyclin G; D056766:Cyclin G1; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000187:drug effects; D016213:Cyclins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects; D011518:Proto-Oncogene Proteins; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
C534702:CCNG1 protein, human; C493956:CDKN1A protein, human; D056746:Cyclin G; D056766:Cyclin G1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D007371:Interferon-gamma; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495900:CDK4 protein, human; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases