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1.
BMC Cancer ; 18(1): 423, 2018 04 16.
Article in English | MEDLINE | ID: mdl-29661172

ABSTRACT

BACKGROUND: Targeted therapies are based on exploiting cancer-cell-specific genetic features or phenotypic traits to selectively kill cancer cells while leaving normal cells unaffected. Oxidative stress is a cancer hallmark phenotype. Given that free nucleotide pools are particularly vulnerable to oxidation, the nucleotide pool sanitising enzyme, MTH1, is potentially conditionally essential in cancer cells. However, findings from previous MTH1 studies have been contradictory, meaning the relevance of MTH1 in cancer is still to be determined. Here we ascertained the role of MTH1 specifically in lung cancer cell maintenance, and the potential of MTH1 inhibition as a targeted therapy strategy to improve lung cancer treatments. METHODS: Using siRNA-mediated knockdown or small-molecule inhibition, we tested the genotoxic and cytotoxic effects of MTH1 deficiency on H23 (p53-mutated), H522 (p53-mutated) and A549 (wildtype p53) non-small cell lung cancer cell lines relative to normal MRC-5 lung fibroblasts. We also assessed if MTH1 inhibition augments current therapies. RESULTS: MTH1 knockdown increased levels of oxidatively damaged DNA and DNA damage signaling alterations in all lung cancer cell lines but not normal fibroblasts, despite no detectable differences in reactive oxygen species levels between any cell lines. Furthermore, MTH1 knockdown reduced H23 cell proliferation. However, unexpectedly, it did not induce apoptosis in any cell line or enhance the effects of gemcitabine, cisplatin or radiation in combination treatments. Contrastingly, TH287 and TH588 MTH1 inhibitors induced apoptosis in H23 and H522 cells, but only increased oxidative DNA damage levels in H23, indicating that they kill cells independently of DNA oxidation and seemingly via MTH1-distinct mechanisms. CONCLUSIONS: MTH1 has a NSCLC-specific p53-independent role for suppressing DNA oxidation and genomic instability, though surprisingly the basis of this may not be reactive-oxygen-species-associated oxidative stress. Despite this, overall our cell viability data indicates that targeting MTH1 will likely not be an across-the-board effective NSCLC therapeutic strategy; rather it induces non-cytotoxic DNA damage that could promote cancer heterogeneity and evolution.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA Repair Enzymes/genetics , Phosphoric Monoester Hydrolases/genetics , A549 Cells , Apoptosis/drug effects , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , DNA Damage/drug effects , DNA Repair Enzymes/deficiency , Gene Expression Regulation, Neoplastic/drug effects , Humans , Oxidation-Reduction , Oxidative Stress/drug effects , Phosphoric Monoester Hydrolases/deficiency , Pyrimidines/pharmacology , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Small Molecule Libraries/pharmacology
2.
Free Radic Biol Med ; 51(3): 719-25, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21658444

ABSTRACT

Single-cell gel electrophoresis (comet assay) is one of the most common methods used to measure oxidatively damaged DNA in peripheral blood mononuclear cells (PBMC), as a biomarker of oxidative stress in vivo. However, storage, extraction, and assay workup of blood samples are associated with a risk of artifactual formation of damage. Previous reports using this approach to study DNA damage in PBMC have, for the most part, required the isolation of PBMC before immediate analysis or freezing in cryopreservative. This is very time-consuming and a significant drain on human resources. Here, we report the successful storage of whole blood in ~250 µl volumes, at -80°C, without cryopreservative, for up to 1 month without artifactual formation of DNA damage. Furthermore, this blood is amenable for direct use in both the alkaline and the enzyme-modified comet assay, without the need for prior isolation of PBMC. In contrast, storage of larger volumes (e.g., 5 ml) of whole blood leads to an increase in damage with longer term storage even at -80°C, unless a cryopreservative is present. Our "small volume" approach may be suitable for archived blood samples, facilitating analysis of biobanks when prior isolation of PBMC has not been performed.


Subject(s)
Biomarkers/analysis , Cryopreservation , DNA Damage/genetics , DNA/metabolism , Leukocytes, Mononuclear/metabolism , Blood Specimen Collection , Cell Line , Comet Assay/methods , Comet Assay/trends , DNA Damage/immunology , Humans , Leukocytes, Mononuclear/pathology , Oxidative Stress
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