TP53 modulates radiotherapy fraction size sensitivity in normal and malignant cells

Selvakumar Anbalagan,Anna Wilkins,Penny A Jeggo,Kevin J Harrington,Navita Somaiah,Jessica A Downs,Cecilia Ström,Kai Rothkamm,David Mcbay,John R Yarnold

doi:10.1038/s41598-021-86681-6

Abstract

Recent clinical trials in breast and prostate cancer have established that fewer, larger daily doses (fractions) of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis. Understanding cell and molecular mechanisms determining fraction size sensitivity is essential to fully exploit this therapeutic variable for patient benefit. The hypothesis under test in this study is that fraction size sensitivity is dependent on the presence of wild-type (WT) p53 and intact non-homologous end-joining (NHEJ). Using single or split-doses of radiation in a range of normal and malignant cells, split-dose recovery was determined using colony-survival assays. Both normal and tumour cells with WT p53 demonstrated significant split-dose recovery, whereas Li-Fraumeni fibroblasts and tumour cells with defective G1/S checkpoint had a large S/G2 component and lost the sparing effect of smaller fractions. There was lack of split-dose recovery in NHEJ-deficient cells and DNA-PKcs inhibitor increased sensitivity to split-doses in glioma cells. Furthermore, siRNA knockdown of p53 in fibroblasts reduced split-dose recovery. In summary, cells defective in p53 are less sensitive to radiotherapy fraction size and lack of split-dose recovery in DNA ligase IV and DNA-PKcs mutant cells suggests the dependence of fraction size sensitivity on intact NHEJ.

Highlights

Recent clinical trials in breast and prostate cancer have established that fewer, larger daily doses of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis
We explore the cellular basis of fractionation sensitivity in a range of normal human wild-type (WT)/mutant and malignant cell lines to test the hypothesis that fraction size sensitivity is modulated by TP53 and depends on error-prone non-homologous end-joining (NHEJ) of radiation-induced DNA double-strand breaks (DSB) in G0/1 phase of the cell cycle
We show for the first time in a range of normal and malignant human cell lines that the sparing effect of small fractions on cell survival is dependent on functional p53 and that sparing is lost/reduced if p53 is mutated or transiently knocked down

Summary

Introduction

Recent clinical trials in breast and prostate cancer have established that fewer, larger daily doses (fractions) of radiotherapy are safe and effective, but these do not represent personalised dosing on a patient-by-patient basis. We postulate that the low fidelity DSB repair characteristic of non-homologous end-joining (NHEJ) explains the increased sensitivity to fraction size in dose-limiting lateresponding normal tissues These tissues including brain, lung, heart, liver, kidney and musculoskeletal tissues are characterised by very low proliferative indices with cells predominantly in G0 phase of the cell cycle, for which NHEJ is the dominant, if not exclusive, DSB repair system in humans[17,18,19]. In cancers such as breast and prostate with low proliferative indices[20,21] Against this background, we explore the cellular basis of fractionation sensitivity in a range of normal human wild-type (WT)/mutant and malignant cell lines to test the hypothesis that fraction size sensitivity is modulated by TP53 and depends on error-prone NHEJ of radiation-induced DNA DSBs in G0/1 phase of the cell cycle

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Conclusion