TOX4 and its binding partners recognize DNA adducts generated by platinum anticancer drugs
Research highlights
► TOX4/PNUTS/WDR82 complex interacts with DNA damaged by anticancer platinum drugs. ► SPRi shows that TOX4 affinity for platinum adducts is weaker than that of HMGB1. ► Plasmid probes and proteomics allow identification of damaged DNA interactome.
Introduction
Cisplatin was the first member of platinum-based compounds approved as an anticancer drug in 1978 [1]. This molecule is still widely used to treat patients suffering from tumoral diseases such as testicular, upper aero-digestive tract, lung or ovarian cancers. In order to circumvent adverse side effects or to enlarge the panel of targeted tumors, further research led to the development and approbation of carboplatin and oxaliplatin, the latter being often used in chemotherapeutic regimen to treat colorectal tumors. Hundreds of other platinating agents are still in the pipelines at research or clinical trial stages, including compounds such as satraplatin or picoplatin [2]. Following intracellular activation, the main mechanism of action of these molecules consists in forming DNA lesions such as intra- and interstrand crosslinks (ICL)1, the most common being 1,2-d(GpG) and 1,2-d(ApG) intrastrand adducts [3]. These damages impair transcription and replication, and generally cause a cell-cycle arrest to provide a time frame for DNA repair. When the damage extent exceeds repair capacities, apoptosis is induced. These cellular and molecular events involve a wide range of proteins that interact with DNA lesions or with subsequent DNA distortions and constitute the “platinated DNA interactome”. These include damage-response proteins (DRP) but also other factors whose ordinary targets are not DNA lesions.
Xeroderma pigmentosum complementation group C (XPC) is an important member of this subproteome as it binds to platinum adducts during the recognition step of the global genome repair subpathway of nucleotide excision repair (NER). Among the other well-studied members also stand the high mobility group (HMG) proteins, whose affinity for platinum adducts has been known for years [4]. Indeed, HMGB1 was one of the first proteins identified as being able to interact with platinated DNA [5]. The biological repercussions of HMGB1 binding are still controversial, as some published works demonstrated a subsequent facilitation of DNA repair whereas others suggest a shielding effect towards NER factors [6]. A second example is hUBF. This ribosomal RNA transcription factor is hijacked from its natural binding site by cisplatin lesions, for which it demonstrates a strong affinity [7]. Lists of other proteins already characterized as capable of recognizing DNA platinum adducts are accessible in dedicated reviews [8]. Discovery of these protein–DNA interactions already brought crucial data for the understanding of platinating agents pharmacology. In order to establish the cartography of damaged DNA-interacting proteins, protocols using proteomic tools were recently designed for DNA lesions such as double-strand breaks [9], abasic sites [10], oxidative damage [11] and DNA crosslinks generated by platinating agents [12], [13]. The aim of our work was to optimize one of these approaches combining a ligand fishing system and high throughput proteomics, to allow us to trap and identify new proteins interacting with DNA alterations. Our tool is made of plasmids damaged by one of three different platinum drugs (cisplatin, oxaliplatin or the satraplatin metabolite JM118) and immobilized onto magnetic beads. This system was exposed to HeLa (cervical cancer cell line) and MDA-MB231 (breast cancer cell line) nuclear extracts, and retained proteins were identified by proteomics. This strategy allowed selection of relevant candidates that were validated by immunoassays and SPRi. Identified proteins may improve our understanding of molecular and cellular responses to this particular type of anticancer drugs.
Section snippets
Protein sources
Two different protein sources were used in our study. HeLa nuclear extracts were purchased from CIL Biotech (Mons, Belgium). MDA-MB231 extracts were prepared in our laboratory. This cell line was a gift from Prof. Philippe Becuwe (University of Nancy, France). Cells were grown in RPMI 1640 medium supplemented with 2 mM l-glutamine, 10% (v/v) fetal calf serum and 800 μg/ml geneticin (Invitrogen) at 37 °C under 5% CO2. Cells were passaged or harvested at ∼80% confluence. Nuclear extracts were
Ligand fishing
The list of proteins isolated from HeLa nuclear extracts by our ligand fishing approach was obtained after proper optimizations of crucial parameters including (i) levels of platinum DNA damage within grafted probes (1,2-(d(GpG) and 1,2-d(ApG) crosslinks levels were respectively of ≈22–35 and ≈2–7 lesions/plasmid depending on the platinating agent), (ii) plasmidic probes concentration/beads quantity ratio (iii) beads blocking and (iv) methods and buffers used to wash the trap and to release
Discussion
Proteomics methods applied to the identification of proteins recognizing DNA lesions constitute a recent research area. Various approaches have been developed to study these groups of proteins. Regarding lesions induced by platinating agents, Stansfield et al. [27] used protein microarrays incubated with cisplatin-modified fluorescent ODNs. Using this tool, the authors identified new interactions such as an association between cisplatin-modified DNA and Aurora kinase A. The most common strategy
Acknowledgments
We thank R Bombera for his help in the optimization of SPRi, C Saint-Pierre for ODN controls and JL Ravanat for his expertise in mass spectrometry. We are also thankful to Agennix for providing JM118. This work was supported by a grant from “La Ligue contre le Cancer, comité de la Drôme”.
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