INSERM U1053, Université Bordeaux Segalen, 146 rue Léo Saignat, 33076 Bordeaux cedex, France.
Mol Cancer Res. 2013 Feb;11(2):133-9. doi: 10.1158/1541-7786.MCR-12-0455. Epub 2012 Dec 10.
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.
Reptin 在大多数人类肝细胞癌中过表达。Reptin 参与染色质重塑、转录调控或超分子复合物组装。其沉默导致培养的肝癌细胞生长停滞和凋亡，并阻止异种移植物中的肝癌进展。Reptin 具有与 Walker A 和 B 结构域相关的 ATP 酶活性。尚不清楚 Reptin 的每个功能是否都依赖于其 ATP 酶活性。在这里，我们在存在内源性 Reptin 的情况下在肝癌细胞中表达 Walker B ATPase 失活突变体（D299N 或 E300G）。然后，我们沉默内源性 Reptin 并用 siRNA 抗性野生型（WT）或 Flag-Reptin 突变体取代它。在存在内源性 Reptin 的情况下表达任一种突变体时，细胞生长明显下降，表明 ATPase 失活突变体对肝癌细胞生长具有显性负效应。WT Flag-Reptin 取代内源性 Reptin 挽救了 HuH7 的细胞生长。另一方面，用 Flag-Reptin D299N 或 E300G 取代内源性 Reptin 会导致细胞生长停滞。在 Hep3B 细胞中也观察到类似的结果。HuH7 细胞中 Reptin 的沉默导致凋亡细胞死亡增加，WT Flag-Reptin 可阻止这种增加，但 D299N 突变体则不能。这些数据表明，与癌症相关的 Reptin 功能依赖于其 ATP 酶活性，并表明 Reptin ATP 酶活性的拮抗剂可能可用作抗癌剂。
