title: The ATPase activity of reptin is required for its effects on tumor cell growth and viability in hepatocellular carcinoma.
abstract: Reptin is overexpressed in most human hepatocellular carcinomas. Reptin is involved in chromatin remodeling, transcription regulation, or supramolecular complexes assembly. Its silencing leads to growth arrest and apoptosis in cultured hepatocellular carcinoma cells and stops hepatocellular carcinoma progression in xenografts. Reptin has an ATPase activity linked to Walker A and B domains. It is unclear whether every Reptin function depends on its ATPase activity. Here, we expressed Walker B ATPase-dead mutants (D299N or E300G) in hepatocellular carcinoma cells in the presence of endogenous Reptin. Then, we silenced endogenous Reptin and substituted it with siRNA-resistant wild-type (WT) or Flag-Reptin mutants. There was a significant decrease in cell growth when expressing either mutant in the presence of endogenous Reptin, revealing a dominant negative effect of the ATPase dead mutants on hepatocellular carcinoma cell growth. Substitution of endogenous Reptin by WT Flag-Reptin rescued cell growth of HuH7. On the other hand, substitution by Flag-Reptin D299N or E300G led to cell growth arrest. Similar results were seen with Hep3B cells. Reptin silencing in HuH7 cells led to an increased apoptotic cell death, which was prevented by WT Flag-Reptin but not by the D299N mutant. These data show that Reptin functions relevant for cancer are dependent on its ATPase activity, and suggest that antagonists of Reptin ATPase activity may be useful as anticancer agents.
authors.affiliation: INSERM U1053, Université Bordeaux Segalen, 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
authors.forename: Aude
authors.initials: A
authors.lastname: Grigoletto
authors.forename: Véronique
authors.initials: V
authors.lastname: Neaud
authors.forename: Nathalie
authors.initials: N
authors.lastname: Allain-Courtois
authors.forename: Patrick
authors.initials: P
authors.lastname: Lestienne
authors.forename: Jean
authors.initials: J
authors.lastname: Rosenbaum
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D017209:Apoptosis / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D048708:Cell Growth Processes / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D004265:DNA Helicases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009154:Mutation; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C122079:RUVBL2 protein, human