期刊
FRONTIERS IN PUBLIC HEALTH
卷 9, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fpubh.2021.675095
关键词
chromatid breaks; chromosomal radiosensitivity; G2-M checkpoint; DDR inhibitors; G2-assay
资金
- DAAD [57339330]
- German Federal Ministry of Education and Research [BMBF-02NUK037B, 02NUK043B]
- project NCSRD -INRASTES research activities in the framework of the national RIS3 - Operational Programme Competitiveness, Entrepreneurship and Innovation (NSRF 2014-2020) [MIS 5002559]
- European Union (European Regional Development Fund)
Although technological advances in radiation oncology have improved the delivery of radiation dose and reduced side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) and overcome tumor resistance. Effective signaling pathways enable cell cycle arrest for DNA repair, and key kinases like ATM, ATR, and Chk1 play a crucial role in this process. Establishing a cytogenetic assay to evaluate the potency of DDR inhibitors for radiosensitization is important for assessing treatment efficacy.
While technological advances in radiation oncology have led to a more precise delivery of radiation dose and a decreased risk of side effects, there is still a need to better understand the mechanisms underlying DNA damage response (DDR) at the DNA and cytogenetic levels, and to overcome tumor resistance. To maintain genomic stability, cells have developed sophisticated signaling pathways enabling cell cycle arrest to facilitate DNA repair via the DDR-related kinases and their downstream targets, so that DNA damage or DNA replication stress induced by genotoxic therapies can be resolved. ATM, ATR, and Chk1 kinases are key mediators in DDR activation and crucial factors in treatment resistance. It is of importance, therefore, as an alternative to the conventional clonogenic assay, to establish a cytogenetic assay enabling reliable and time-efficient results in evaluating the potency of DDR inhibitors for radiosensitization. Toward this goal, the present study aims at the development and optimization of a chromosomal radiosensitivity assay using the DDR and G2-checkpoint inhibitors as a novel modification compared to the classical G2-assay. Also, it aims at investigating the strengths of this assay for rapid radiosensitivity assessments in cultured cells, and potentially, in tumor cells obtained from biopsies. Specifically, exponentially growing RPE and 82-6 hTERT human cells are irradiated during the G2/M-phase transition in the presence or absence of Caffeine, VE-821, and UCN-1 inhibitors of ATM/ATR, ATR, and Chk1, respectively, and the induced chromatid breaks are used to evaluate cell radiosensitivity and their potency for radiosensitization. The increased yield of chromatid breaks in the presence of DDR inhibitors, which underpins radiosensitization, is similar to that observed in cells from highly radiosensitive AT-patients, and is considered here as 100% radiosensitive internal control. The results highlight the potential of our modified G2-assay using VE-821 to evaluate cell radiosensitivity, the efficacy of DDR inhibitors in radiosensitization, and reinforce the concept that ATM, ATR, and Chk1 represent attractive anticancer drug targets in radiation oncology.
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