title: Modulation of retinoblastoma and retinoblastoma-related proteins in regenerating rat liver and primary hepatocytes.
abstract: Protein expression of the retinoblastoma (Rb) tumor suppressor gene product was examined by immunoblot analysis of nuclei isolated from regenerating rat liver after 70% partial hepatectomy (PH). Levels were almost undetectable in quiescent 0-h livers but increased 15- to 60-fold 3 to 24 h post-PH, 105-fold at 30 h, and 20- to 50-fold at 60 to 72 h post-PH. Expression returned to near baseline levels at 18, 42, and 48 h post-PH. A similar pattern of Rb protein expression in the regenerating liver was observed by indirect immunofluorescence microscopy, with peak nuclear expression at 30 h post-PH. Rb-related proteins with apparent molecular masses of 300, 156, and 74 kDa were detected in regenerating liver using mAbs to the Rb protein. Their expression increased 6- to 8-fold during regeneration, and only p156 returned to baseline levels at 60 h post-PH. Rb and its related proteins were detected in cultured primary hepatocytes, and although total protein levels did not change appreciably, there was a dramatic shift from cytosol into nuclei through 96 h. The half-life of the Rb protein was determined to be 1.9 h in regenerating liver and 2.2 h in cultured primary hepatocytes. Rb protein abundance in synchronized HuH-7 human hepatoma cells was cell cycle dependent and exhibited peak nuclear expression during S phase. Rb protein was detected primarily in its hyperphosphorylated state during liver regeneration and through the cell cycle of the HuH-7 cells. In vivo administration of transforming growth factor beta 1, an inhibitor of DNA synthesis in regenerating liver, resulted in reduced expression of Rb as well as its protein partners, cell cycle-dependent kinase 4 and cyclin E. The results suggest that in the regenerating rat liver and in synchronized HuH-7 cells, expression of Rb protein is modulated in a cell cycle-dependent fashion, remains primarily in a hyperphosphorylated state, and exhibits a relatively short half-life. The inhibition of Rb protein expression by transforming growth factor beta 1 may be linked to its simultaneous suppression of cell cycle-dependent kinase 4 and cyclin E protein levels.
authors.affiliation: Department of Medicine, University of Minnesota Medical School, Minneapolis 55455, USA.
authors.forename: G
authors.initials: G
authors.lastname: Fan
authors.forename: R
authors.initials: R
authors.lastname: Xu
authors.forename: M W
authors.initials: MW
authors.lastname: Wessendorf
authors.forename: X
authors.initials: X
authors.lastname: Ma
authors.forename: B T
authors.initials: BT
authors.lastname: Kren
authors.forename: C J
authors.initials: CJ
authors.lastname: Steer
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D006528:Carcinoma, Hepatocellular; D002453:Cell Cycle / Q000502:physiology; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000502:physiology; D016213:Cyclins / Q000502:physiology; D004247:DNA / Q000096:biosynthesis; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology*; D008115:Liver Regeneration / Q000502:physiology*; D008297:Male; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D012175:Retinoblastoma / Q000503:physiopathology*; D016160:Retinoblastoma Protein / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000502:physiology
chemical_list: D016213:Cyclins; D011518:Proto-Oncogene Proteins; D016160:Retinoblastoma Protein; D016212:Transforming Growth Factor beta; D004247:DNA; C495900:CDK4 protein, human; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases