Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage

Carter, Rachel J, Nickson, Catherine M ORCID: 0000-0003-4611-2852, Thompson, James M, Kacperek, Andrzej, Hill, Mark A and Parsons, Jason L ORCID: 0000-0002-5052-1125 (2019) Characterisation of deubiquitylating enzymes in the cellular response to high-LET ionising radiation and complex DNA damage. International Journal of Radiation: Oncology - Biology - Physics, 104 (3). pp. 656-665.

Access the full-text of this item by clicking on the Open Access link.

Text Carter et al Final.docx - Author Accepted Manuscript Download (1MB)

Abstract

<h4>Purpose</h4>Ionizing radiation, particular high-linear energy transfer (LET) radiation, can induce complex DNA damage (CDD) wherein 2 or more DNA lesions are induced in close proximity, which contributes significantly to the cell killing effects. However, knowledge of the enzymes and mechanisms involved in coordinating the recognition and processing of CDD in cellular DNA are currently lacking.<h4>Methods and materials</h4>A small interfering RNA screen of deubiquitylation enzymes was conducted in HeLa cells irradiated with high-LET α-particles or protons, versus low-LET protons and x-rays, and cell survival was monitored by clonogenic assays. Candidates whose depletion led to decreased cell survival specifically in response to high-LET radiation were validated in both HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells, and the association with CDD repair was confirmed using an enzyme modified neutral comet assay.<h4>Results</h4>Depletion of USP6 decreased cell survival specifically after high-LET α-particles and protons, but not low-LET protons or x-rays. USP6 depletion caused cell cycle arrest and a deficiency in CDD repair mediated through instability of poly(ADP-ribose) polymerase-1 (PARP-1) protein. Increased radiosensitivity of cells to high-LET protons as a consequence of defective CDD repair was furthermore mimicked using the PARP inhibitor olaparib, and through PARP-1 small interfering RNA.<h4>Conclusions</h4>USP6 controls cell survival in response to high-LET radiation by stabilizing PARP-1 protein levels, which is essential for CDD repair. We also describe synergy between CDD induced by high-LET protons and PARP inhibition, or PARP-1 depletion, in effective cancer cell killing.

Item Type:	Article
Uncontrolled Keywords:	Cell Line, Tumor, Hela Cells, Humans, Carcinoma, Squamous Cell, Oropharyngeal Neoplasms, DNA Damage, Protons, Piperazines, Phthalazines, Ubiquitin Thiolesterase, Proto-Oncogene Proteins, RNA, Small Interfering, Cell Survival, DNA Repair, Linear Energy Transfer, Alpha Particles, Radiation, Ionizing, Radiation Tolerance, Cell Cycle Checkpoints, Poly(ADP-ribose) Polymerase Inhibitors, Poly (ADP-Ribose) Polymerase-1
Depositing User:	Symplectic Admin
Date Deposited:	08 Mar 2019 10:48
Last Modified:	18 Feb 2023 06:16
DOI:	10.1016/j.ijrobp.2019.02.053
Open Access URL:	https://doi.org/10.1016/j.ijrobp.2019.02.053
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3033936