ABSTRACT
Mutations in the Werner syndrome protein (WRN), a caretaker of the genome, result in Werner syndrome, which is characterized by premature aging phenotypes and cancer predisposition. Methylseleninic acid (MSeA) can activate DNA damage responses and is a superior compound to suppress tumorigenesis in mouse models of cancer. To test the hypothesis that targeting WRN can potentiate selenium toxicity in cancer cells, isogenic WRN small hairpin RNA (shRNA) and control shRNA U-2 OS osteosarcoma cells were treated with MSeA for 2d, followed by recovery for up to 7d. WRN deficiency sensitized U-2 OS cells to MSeA-induced necrotic death. Co-treatment with the ataxia-telangiectasia mutated (ATM) kinase inhibitor KU55933 desensitized the control shRNA cells, but not WRN shRNA cells, to MSeA treatment. WRN did not affect MSeA-induced ATM phosphorylation on Ser-1981 or H2A.X phosphorylation on Ser-139, but promoted recovery from the MSeA-induced DNA damage. Taken together, WRN protects U-2 OS osteosarcoma cells against MSeA-induced cytotoxicity, suggesting that oxidative DNA repair pathway is a promising target for improving the efficacy of selenium on tumor suppression.
Subject(s)
DNA Damage , Drug Resistance, Neoplasm/genetics , Exodeoxyribonucleases/metabolism , Organoselenium Compounds/pharmacology , Osteosarcoma/metabolism , RecQ Helicases/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/antagonists & inhibitors , Cell Line, Tumor , DNA-Binding Proteins/antagonists & inhibitors , Exodeoxyribonucleases/genetics , Gene Knockdown Techniques , Histones/metabolism , Humans , Mice , Morpholines/pharmacology , Necrosis , Osteosarcoma/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrones/pharmacology , RecQ Helicases/genetics , Tumor Suppressor Proteins/antagonists & inhibitors , Werner Syndrome HelicaseABSTRACT
OLFM4 was identified initially as a gene highly induced in myeloid stem cells by G-CSF treatment. A bioinformatics method using a global meta-analysis of microarray data predicted that OLFM4 would be associated with specific granules in human neutrophils. Subcellular fractionation of peripheral blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression of OLFM4 protein.
Subject(s)
Granulocyte Colony-Stimulating Factor/metabolism , Neutrophils/classification , Neutrophils/metabolism , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Differentiation/genetics , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/pharmacology , Humans , Myeloid Cells/drug effects , Myeloid Cells/metabolism , Neutrophils/drug effects , Protein Transport/physiology , RNA, Messenger/metabolismABSTRACT
8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1)-initiated DNA base excision repair (BER). Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH), by chromosome orientation-FISH (CO-FISH), and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/-)) mouse tissues and primary embryonic fibroblasts (MEFs) cultivated in hypoxia condition (3% oxygen), whereas telomere shortening was detected in Ogg1(-/-) mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen) or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/-) mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/-) mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/-) MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity in mammals.
