Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Moeglin, Eric; Desplancq, Dominique; Conic, Sascha; Oulad‐Abdelghani, Mustapha; Stoessel, Audrey; Chiper, Manuela; Vigneron, Marc; Didier, Pascal; Tora, Làszlò; Weiss, Étienne

doi:10.3390/cancers11030355

Cited by 68 publications

(56 citation statements)

References 56 publications

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“…Liver tumor sections were embedded in paraffin before being fixed in 8% buffered formalin solution for 12 h. Then the paraffin-embedded hepatic tumor tissues were analyzed for any expression of Bax, PUMA and p53 by immunohistochemistry as previously described [23]. Tumor tissue sections were incubated with primary anti-PUMA, anti-Bax from Cell Signaling Technology (Boston, MA, USA) and anti-P53 antibodies from Abcam (Boston, MA, USA).…”

Section: Methodsmentioning

confidence: 99%

RETRACTED: Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation

Hong

et al. 2019

Cells

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The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown strong and explicit cancer cell-selectivity, which results in little toxicity toward normal tissues, and has been recognized as a potential, relatively safe anticancer agent. However, several cancers are resistant to the apoptosis induced by TRAIL. A recent study found that shikonin b (alkannin, 5,8-dihydroxy-2-[(1S)-1-hydroxy-4-methylpent-3-en-1-yl]naphthalene-1,4-dione) might induce apoptosis in TRAIL-resistant cholangiocarcinoma cells through reactive oxygen species (ROS)-mediated caspases activation. However, the strong cytotoxic activity has limited its potential as an anticancer drug. Thus, the current study intends to discover novel shikonin derivatives which can sensitize the liver cancer cell to TRAIL-induced apoptosis while exhibiting little toxicity toward the normal hepatic cell. The trypan blue exclusion assay, western blot assay, 4′,6-diamidino-2-phenylindole (DAPI) staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay as well as the ‘comet’ assay, were used to study the underlying mechanisms of cell death and to search for any mechanisms of an enhancement of TRAIL-mediated apoptosis in the presence of ASH. Herein, we demonstrated that non-cytotoxic doses of acetylshikonin (ASH), one of the shikonin derivatives, in combination with TRAIL, could promote apoptosis in HepG2 cells. Further studies showed that application of ASH in a non-cytotoxic dose (2.5 μM) could increase intracellular ROS production and induce DNA damage, which might trigger a cell intrinsic apoptosis pathway in the TRAIL-resistant HepG2 cell. Combination treatment with a non-cytotoxic dose of ASH and TRAIL activated caspase and increased the cleavage of PARP-1 in the HepG2 cell. However, when intracellular ROS production was suppressed by N-acetyl-l-cysteine (NAC), the synergistic effects of ASH and TRAIL on hepatocellular carcinoma (HCC) cell apoptosis was abolished. Furthermore, NAC could alleviate p53 and the p53 upregulated modulator of apoptosis (PUMA) expression induced by TRAIL and ASH. Small (or short) interfering RNA (siRNA) targeting PUMA or p53 significantly reversed ASH-mediated sensitization to TRAIL-induced apoptosis. In addition, Bax gene deficiency also abolished ASH-induced TRAIL sensitization. An orthotopical HCC implantation mice model further confirmed that co-treated ASH overcomes TRAIL resistance in HCC cells without exhibiting potent toxicity in vivo. In conclusion, the above data suggested that ROS could induce DNA damage and activating p53/PUMA/Bax signaling, and thus, this resulted in the permeabilization of mitochondrial outer membrane and activating caspases as well as sensitizing the HCC cell to apoptosis induced by TRAIL and ASH treatment.

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Section: Methodsmentioning

confidence: 99%

RETRACTED: Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation

Hong

et al. 2019

Cells

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“…57,58 In addition, a recent study has linked DNA-PKcs to replication stress-induced, pan-nuclear H2AX phosphorylation in S-phase cells. 59 To better understand the mechanism behind the enhanced, olaparib-induced H2AX phosphorylation in ATM-deficient cells, A549-CRISPR-control, A549-CRISPR-DNA-PKcs and A549-CRISPR-ATM cells were treated with 4 µM olaparib for 1-4 days as in Fig. 3, and analysed for γ-H2AX foci using immunofluorescence.…”

Section: Resultsmentioning

confidence: 99%

“… 57 , 58 In addition, a recent study has linked DNA-PKcs to replication stress-induced, pan-nuclear H2AX phosphorylation in S-phase cells. 59 …”

Section: Resultsmentioning

confidence: 99%

Combined poly-ADP ribose polymerase and ataxia-telangiectasia mutated/Rad3-related inhibition targets ataxia-telangiectasia mutated-deficient lung cancer cells

et al. 2019

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BACKGROUND: Up to 40% of lung adenocarcinoma have been reported to lack ataxia-telangiectasia mutated (ATM) protein expression. We asked whether ATM-deficient lung cancer cell lines are sensitive to poly-ADP ribose polymerase (PARP) inhibitors and determined the mechanism of action of olaparib in ATM-deficient A549 cells. METHODS: We analysed drug sensitivity data for olaparib and talazoparib in lung adenocarcinoma cell lines from the Genomics of Drug Sensitivity in Cancer (GDSC) project. We deleted ATM from A549 lung adenocarcinoma cells using CRISPR/Cas9 and determined the effects of olaparib and the ATM/Rad3-related (ATR) inhibitor VE-821 on cell viability. RESULTS: IC 50 values for both olaparib and talazoparib positively correlated with ATM mRNA levels and gene amplification status in lung adenocarcinoma cell lines. ATM mutation was associated with a significant decrease in the IC 50 for olaparib while a similar trend was observed for talazoparib. A549 cells with deletion of ATM were sensitive to ionising radiation and olaparib. Olaparib induced phosphorylation of DNA damage markers and reversible G2 arrest in ATM-deficient cells, while the combination of olaparib and VE-821 induced cell death.

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“…TOPK depletion in combination with HU treatment was also associated with an increase in cells displaying diffuse pan-nuclear γH2AX staining. Nuclear-wide formation of γH2AX occurs during catastrophic replication stress, and invariably precedes irreversible cell death [27].…”

Section: Topk Promotes Radioresistance Via S-phasemediated Dna Damagementioning

confidence: 99%

Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress

et al. 2020

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T-LAK-originated protein kinase (TOPK) overexpression is a feature of multiple cancers, yet is absent from most phenotypically normal tissues. As such, TOPK expression profiling and the development of TOPK-targeting pharmaceutical agents have raised hopes for its future potential in the development of targeted therapeutics. Results presented in this paper confirm the value of TOPK as a potential target for the treatment of solid tumours, and demonstrate the efficacy of a TOPK inhibitor (OTS964) when used in combination with radiation treatment. Using H460 and Calu-6 lung cancer xenograft models, we show that pharmaceutical inhibition of TOPK potentiates the efficacy of fractionated irradiation. Furthermore, we provide in vitro evidence that TOPK plays a hitherto unknown role during S phase, showing that TOPK depletion increases fork stalling and collapse under conditions of replication stress and exogenous DNA damage. Transient knockdown of TOPK was shown to impair recovery from fork stalling and to increase the formation of replication-associated single-stranded DNA foci in H460 lung cancer cells. We also show that TOPK interacts directly with CHK1 and Cdc25c, two key players in the checkpoint signalling pathway activated after replication fork collapse. This study thus provides novel insights into the mechanism by which TOPK activity supports the survival of cancer cells, facilitating checkpoint signalling in response to replication stress and DNA damage.

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Uniform Widespread Nuclear Phosphorylation of Histone H2AX Is an Indicator of Lethal DNA Replication Stress

Cited by 68 publications

References 56 publications

RETRACTED: Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation

RETRACTED: Acetylshikonin Sensitizes Hepatocellular Carcinoma Cells to Apoptosis through ROS-Mediated Caspase Activation

Combined poly-ADP ribose polymerase and ataxia-telangiectasia mutated/Rad3-related inhibition targets ataxia-telangiectasia mutated-deficient lung cancer cells

Targeting TOPK sensitises tumour cells to radiation-induced damage by enhancing replication stress

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