Abstract

BackgroundThe Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Eukaryotic DNA topoisomerases, such as DNA topoisomerase 1 (TOPO1) and DNA topoisomerase 2 (TOPO2), are the essential DNA metabolism regulators that usually overexpressed in cancer cells, and multiple proteins have been reported to regulate the enzyme activity and the clinical efficacy of their inhibitors. The present study unraveled the interaction of YB-1 with TOPO1, and further investigated the related function and potential mechanisms during the interaction.MethodsThe direct association of TOPO1 with specific domain of YB-1 was explored by co-immunoprecipitation and GST pull-down assays. The interaction function was further clarified by DNA relaxation assays, co-immunoprecipitation and WST-8 assays with in vitro gain- and loss- of function models.ResultsWe found that YB-1 interacts directly with TOPO1 (but not with TOPO2) and promotes TOPO1 catalytic activity. Interactions between YB-1 and TOPO1 increased when cancer cells were treated with the TOPO1 inhibitor, camptothecin (CPT), but not with the TOPO2 inhibitor, adriamycin (ADM). Furthermore, we found that the interaction is prevented by pretreatment with the antioxidant agent, N-acetyl cysteine, and that YB-1 downregulation renders cells resistant to CPT.ConclusionsOur findings suggest that nuclear YB-1 serves as an intracellular promoter of TOPO1 catalytic activity that enhances CPT sensitivity through its direct interaction with TOPO1.

Highlights

  • The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells

  • Previous studies have shown that YB-1 enhances cellular resistance to genotoxic stress through its direct or indirect interactions with several DNA replication and repair proteins [2,4], and that oxidative DNA damage plays an important part in initiation of the repair process [5]

  • YB-1 binds to topoisomerase 1 (TOPO1) via its cold shock and C terminal domains To determine whether TOPO1 interacted with YB-1 in mammalian cells, we performed co-immunoprecipitation experiment

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Summary

Introduction

The Y-box binding protein 1 (YB-1) possesses pleiotropic functions through its interactions with various cellular proteins, and its high expression levels make it a potential useful prognostic biomarker for cancer cells. Previous studies have shown that YB-1 enhances cellular resistance to genotoxic stress through its direct or indirect interactions with several DNA replication and repair proteins [2,4], and that oxidative DNA damage plays an important part in initiation of the repair process [5]. DNA topoisomerases are ubiquitous nuclear enzymes that catalyze conformational changes in a double-stranded helix DNA through breakage and rejoining reactions [13]. The activity of these enzymes is essential for various DNA-related processes, such as replication, transcription, chromosome condensation and de-condensation [14].

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