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  • APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells.

APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells.

Frontiers in cell and developmental biology (2021-11-20)
Md Akram Hossain, Yunfeng Lin, Garrett Driscoll, Jia Li, Anne McMahon, Joshua Matos, Haichao Zhao, Daisuke Tsuchimoto, Yusaku Nakabeppu, Jianjun Zhao, Shan Yan
ABSTRACT

The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3'-5' exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Celastrol, Celastrus scandens, A cell-permeable dienone-phenolic triterpene compound that exhibits antioxidant and anti-inflammatory properties.
Sigma-Aldrich
Etoposide, A cell-permeable derivative of podophyllotoxin that acts as a topoisomerase II inhibitor (IC₅₀ = 59.2 µM) has major activity against a number of tumors, including germ cell neoplasms, small cell lung cancer, and malignant lymphoma.
Sigma-Aldrich
Anti-phospho Histone H2A.X (Ser139), clone JBW301, Alexa Fluor 488 Conjugate Antibody, clone JBW301, from mouse, ALEXA FLUOR 488
Sigma-Aldrich
Gemcitabine hydrochloride, ≥98% (HPLC)
Sigma-Aldrich
Camptothecin, Camptotheca acuminata, A cell-permeable DNA topoisomerase I inhibitor.