Abstract
Transcription factor hepatocyte nuclear factor 1-beta (HNF-1β) enhances checkpoint kinase 1 (Chk1) activation and promotes G2/M cell cycle progression in ovarian clear cell carcinoma (CCC) following exposure to diverse genotoxic agents including bleomycin. However, the underlying mechanism leading to checkpoint activation of HNF-1β still remains largely unknown. To clarify the effects of HNF-1β on cell cycle checkpoints, human CCC cell lines were transfected with siRNAs targeting HNF-1β, Claspin, USP28, or a control vector. Ubiquitination and stabilization of Claspin protein by HNF-1β was assessed by immunoprecipitation. Loss-of-function studies using RNAi-mediated gene silencing indicated that HNF-1β facilitated the Claspin expression after treatment with a genotoxic agent bleomycin, resulting in accumulation of phosphorylated Chk1 (p-Chk1) and promotion of survival in CCC cell lines. This study showed for the first time that USP28, a de-ubiquitinase crucial for Claspin expression, is one target gene of HNF-1β. Knockdown of endogenous USP28 suppressed the Claspin expression and p-Chk1 activation and cell viability. Our findings identify a novel pathway of the HNF-1β―USP28―Claspin―Chk1 axis in checkpoint signal amplification in response to DNA damage. Targeting this pathway may represent a putative, novel, anticancer strategy in ovarian CCC.
Highlights
Clear cell carcinoma (CCC) of the ovary is intrinsically chemoresistant to platinum-based antineoplastic agents, which results in treatment failure [1]
Endogenous HNF-1β induces up-regulation of p-checkpoint kinase 1 (Chk1) expression in clear cell carcinoma TOV21G and KOC-7c cells exposed to bleomycin [7]
HNF-1β-induced upregulation of phosphorylated Chk1 (p-Chk1) protein expression only occurs after a genotoxic DNA damage and no effect is found in the absence of DNA damage, suggesting that the observed effects of HNF-1β are strictly activated by DNA damaging
Summary
Clear cell carcinoma (CCC) of the ovary is intrinsically chemoresistant to platinum-based antineoplastic agents, which results in treatment failure [1]. The implications of genotoxic stress could be important in the understanding on the mechanism of drug resistance. Heme and iron species in the contents of tumor cysts was associated with genotoxicities such as oxidative stress and DNA mutations [2, 3]. The DNA damage response involves the control of DNA damage checkpoints and DNA repair mechanisms [4]. In response to DNA damage, the protein kinases. ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3-related protein) phosphorylate checkpoint kinases 1 and 2 (Chk and Chk2) for signaling to cell cycle checkpoint [5]. The checkpoint proteins become activated to arrest cell division until all DNA damages are repaired
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