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. 2021 May 28:9:675095.
doi: 10.3389/fpubh.2021.675095. eCollection 2021.

G2/M Checkpoint Abrogation With Selective Inhibitors Results in Increased Chromatid Breaks and Radiosensitization of 82-6 hTERT and RPE Human Cells

Aggeliki Nikolakopoulou  1   2 , Aashish Soni  3 , Martha Habibi  1   2 , Pantelis Karaiskos  2 , Gabriel Pantelias  1 , Georgia I Terzoudi  1 , George Iliakis  3
Affiliations

G2/M Checkpoint Abrogation With Selective Inhibitors Results in Increased Chromatid Breaks and Radiosensitization of 82-6 hTERT and RPE Human Cells

Aggeliki Nikolakopoulou et al. Front Public Health. .

Abstract

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.

Keywords: DDR inhibitors; G2-M checkpoint; G2-assay; chromatid breaks; chromosomal radiosensitivity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Representative examples of CBs in RPE metaphases treated with VE-821 or left untreated. (A) CBs in a cell analyzed 1 h after exposure to 1 Gy. (B) As in A for a cell also exposed to VE-821 1 h before IR. Note the increase in the yields of CBs.
Figure 2
Figure 2
(A) Yield of CBs in 82-6 hTERT cells, treated with Caffeine or left untreated, following exposure to 0.5 Gy. (B) As in (A) for cells exposed to 1 Gy. The cells were harvested at 1 and 2 h after IR. Both graphs also show the yield of chromatid breaks in the unirradiated control groups (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05).
Figure 3
Figure 3
(A) Yield of chromatid breaks in the 82-6 hTERT cells, treated with VE-821 or left untreated, following exposure to 0.5 Gy. (B) As in (A) for cells exposed to 1 Gy. Other details as in Figure 2 (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05).
Figure 4
Figure 4
(A) Yield of chromatid breaks in the 82-6 hTERT cells, treated with UCN-01, or left untreated, following exposure to 0.5 Gy. (B) As in (A) after exposure to 1 Gy. Other details as in Figure 2 (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05).
Figure 5
Figure 5
(A) Yield of chromatid breaks in the RPE cells, treated with Caffeine or left untreated, after exposure to 0.5 Gy. (B) As in (A) for cells exposed to 1 Gy. Other details as in Figure 2 (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05).
Figure 6
Figure 6
(A) Yield of CBs in the RPE cells, treated with VE-821 or left untreated, following exposure to 0.5 Gy. (B) As in (A) for cells exposed to 1 Gy. Cells were harvested at 1 and 2 h after IR (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05).
Figure 7
Figure 7
(A) Yield of chromatid breaks in the RPE cells, treated with UCN-01 or left untreated, following exposure to 0.5 Gy. (B) As in (A) for cells exposed to 1 Gy. Other details as in Figure 2 (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05).
Figure 8
Figure 8
Yield of chromatid breaks in the 82-6 hTERT cells pre-treated with Caffeine, VE-821, UCN-01 or left untreated following exposure to 1 Gy (Mean ± SD based on three independent experiments; statistically significance criterion: p ≤ 0.05). VE-821 significantly increased the number of CB compared to Caffeine or UCN-01 (p < 0.01).
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References

    1. Huang RX, Zhou PK. DNA damage response signaling pathways and targets for radiotherapy sensitization in cancer. Signal Transduct Target Ther. (2020) 5:60. 10.1038/s41392-020-0150-x - DOI - PMC - PubMed
    1. Iliakis G, Mladenov E, Mladenova V. Necessities in the processing of dna double strand breaks and their effects on genomic instability and cancer. Cancers. (2019) 11:1671. 10.3390/cancers11111671 - DOI - PMC - PubMed
    1. Schipler A, Iliakis G. DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice. Nucleic Acids Res. (2013) 41:7589–605. 10.1093/nar/gkt556 - DOI - PMC - PubMed
    1. Nogueira A, Fernandes M, Catarino R, Medeiros R. RAD52 functions in homologous recombination and its importance on genomic integrity maintenance and cancer therapy. Cancers (Basel). (2019) 11:1622. 10.3390/cancers11111622 - DOI - PMC - PubMed
    1. Bakhoum SF, Kabeche L, Compton DA, Powell SN, Bastians H. Mitotic DNA damage response: at the crossroads of structural and numerical cancer chromosome instabilities. Trends Cancer. (2017) 3:225–34. 10.1016/j.trecan.2017.02.001 - DOI - PMC - PubMed

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