Characterisation of Deubiquitylating Enzymes in the Cellular Response to High-LET Ionizing Radiation and Complex DNA Damage

Rachel J Carter, Catherine M Nickson, James M Thompson, Andrzej Kacperek, Mark A Hill, Jason L Parsons

Research output: Contribution to journalArticlepeer-review

Abstract

PURPOSE: 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.

METHODS AND MATERIALS: 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.

RESULTS: 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.

CONCLUSIONS: 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.

Original languageEnglish
Pages (from-to)656-665
Number of pages10
JournalInternational Journal of Radiation: Oncology - Biology - Physics
Volume104
Issue number3
DOIs
Publication statusPublished - 1 Jul 2019

Bibliographical note

Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

Keywords

  • Alpha Particles
  • Carcinoma, Squamous Cell
  • Cell Cycle Checkpoints
  • Cell Line, Tumor
  • Cell Survival
  • DNA Damage
  • DNA Repair
  • HeLa Cells
  • Humans
  • Linear Energy Transfer
  • Oropharyngeal Neoplasms
  • Phthalazines/pharmacology
  • Piperazines/pharmacology
  • Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors
  • Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
  • Proto-Oncogene Proteins/deficiency
  • Protons
  • RNA, Small Interfering
  • Radiation Tolerance
  • Radiation, Ionizing
  • Ubiquitin Thiolesterase/deficiency

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