Induction of DNA Damage and G2 Cell Cycle Arrest by Diepoxybutane through the Activation of the Chk1-Dependent Pathway in Mouse Germ Cells

10066 Background: TR is an alkylating agent approved in Europe for the treatment of advanced STS as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair mechanism, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity combining it with other anti-cancer drugs. PARP-1 inhibitors disable DNA base-excision repair mechanism causing the accumulation of DSBs and look like a reasonable TR partner to be explored. We focused our in vitro studies on the effects of the combination of TR with the PARP-1 inhibitor Olaparib (OL). Methods: We explored the activity of TR-OL combination against a panel of different histotypes of STS cell lines, evaluating cell viability after 72h treatment with escalating doses of TR (0-2 nM), OL (0-20 µM), and their constant combination. Following colony formation, cell cycle, apoptosis (annexin V+/PI+) and DNA damage (phospho-histone H2AX - Ser139) were checked. Results: The TR-OL combination strongly affects STS cell viability, showing synergism (Combination Index < 1, based on Chou–Talalay method) on 8 out of 13 cell lines tested. We observed a strong synergism, as a massive reduction of colony growth (402.91, MES-SA, and DMR-SN-8.4.98 lines) induced by the combination if compared with each single agent (78% vs. ~27% : p<0.05). OL potentiates the S/G2 cell-cycle arrest caused by TR at 48h (Control= 29%, TR= 33.4%, OL= 31.5%, TR-OL= 80.9%), and induces a strong increase of the apoptotic cells at 72h (Control= 17.4%, TR= 28.3%, OL= 33.5%, TR-OL= 56.8%). Furthermore, the TR-OL synergism on DNA damage is confirmed by a significant increase of the DSBs marker (Control= 8.7%, TR= 59.5%, OL= 23.8%, TR-OL= 76.5%). Conclusions: These results validate the biological rationale to combine TR and PARP-1 inhibitors in STS and suggest assessing this drug combination in the clinical setting.

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NRF2 preserves genomic integrity by facilitating ATR activation and G2 cell cycle arrest

Nucleic Acids Research ◽

10.1093/nar/gkaa631 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9109-9123 ◽

Cited By ~ 2

Author(s):

Xiaohui Sun ◽

Yan Wang ◽

Kaihua Ji ◽

Yang Liu ◽

Yangyang Kong ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Cycle Arrest ◽

Cellular Response ◽

Genome Stability ◽

Related Factor ◽

Dna Double Strand Breaks ◽

Ros Scavenging ◽

Cycle Arrest ◽

G2 Cell Cycle Arrest

Abstract Nuclear factor erythroid 2-related factor 2 (NRF2) is a well-characterized transcription factor that protects cells against oxidative and electrophilic stresses. Emerging evidence has suggested that NRF2 protects cells against DNA damage by mechanisms other than antioxidation, yet the mechanism remains poorly understood. Here, we demonstrate that knockout of NRF2 in cells results in hypersensitivity to ionizing radiation (IR) in the presence or absence of reactive oxygen species (ROS). Under ROS scavenging conditions, induction of DNA double-strand breaks (DSBs) increases the NRF2 protein level and recruits NRF2 to DNA damage sites where it interacts with ATR, resulting in activation of the ATR–CHK1–CDC2 signaling pathway. In turn, this leads to G2 cell cycle arrest and the promotion of homologous recombination repair of DSBs, thereby preserving genome stability. The inhibition of NRF2 by brusatol increased the radiosensitivity of tumor cells in xenografts by perturbing ATR and CHK1 activation. Collectively, our results reveal a novel function of NRF2 as an ATR activator in the regulation of the cellular response to DSBs. This shift in perspective should help furnish a more complete understanding of the function of NRF2 and the DNA damage response.

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