A novel R229Q OGG1 polymorphism results in a thermolabile enzyme that sensitizes KG-1 leukemia cells to DNA damaging agents
Introduction
Reactive oxygen species (ROS) produce numerous forms of DNA damage, including 7,8-dihydro-8-oxoguanine (8-oxoG), a major mutagenic modification in DNA. 8-OxoG can mispair with adenine during DNA replication and, if left unrepaired, result in the fixation of G:C to T:A transversion mutations [1]. Such transversions are common mutations at three major mutational hotspots in the p53 gene in lung and head and neck cancers [2]. The primary mammalian enzyme for removing 8-oxoG from DNA is 8-oxoguanine-DNA glycosylase (OGG1) [3]. In human tumor cells, the OGG1 locus is frequently missing or mutated and several cancer cell lines studied show decreased 8-oxoguanine excision activity, suggesting that in some cases, 8-oxoG repair capacity may be associated with cancer incidence. The importance of OGG1 as a tumor suppressor gene has been highlighted by numerous association studies linking single nucleotide polymorphisms (SNPs) in the OGG1 gene with elevated risk for lung, prostate, and orolaryngeal cancers [4]. A single amino acid change in polymorphic S326C OGG1 has recently been shown to affect intracellular localization and alter repair activity, substrate specificity, molecular stoichiometry, and stimulation of the enzyme by AP-endonuclease 1 [5], [6]. A decrease in 8-oxoG excision activity resulting from OGG1 deficiency or polymorphisms could be anticipated to lead to increased genomic 8-oxoG and promote the accumulation of ROS-induced mutations which may promote genomic instability and carcinogenesis.
An increase in 8-oxoG in DNA was recently reported for a model human leukemia cell line, KG-1, which was found to have a homozygous mutation in the OGG1 gene that results in the substitution of arginine 229 for glutamine (R229Q) [7] in the expressed enzyme. KG-1 cells have elevated levels of genomic 8-oxoG and are sensitive to killing via apoptosis when challenged with 8-hydroxydeoxyguanosine nucleoside (8-oxodG) or ionizing radiation exposure [7], [8], [9]. Cellular extracts of KG-1 have markedly reduced 8-oxoG excision capacity when compared with leukemic cells expressing wild-type OGG1 [7], thus it was proposed that decreased repair of 8-oxoG and cellular sensitivity to ionizing radiation and 8-oxodG in KG-1 cells may be consequences of the R229Q mutation resulting in a dysfunctional OGG1 protein [7], [8], [9]. We characterized the enzymatic activity of R229Q and determined the effect of R229Q expression on KG-1 survival following exposure to DNA damaging agents. Our results show that R229Q OGG1 is highly thermolabile and rapidly inactivated at physiological temperatures both in vitro and in vivo. Expression of both nuclear and mitochondrial R229Q OGG1 sensitized KG-1 cells to killing via an apoptotic pathway following exposure to menadione and 8-oxodG, thus R229Q promotes apoptosis following ROS and oxidized nucleoside exposure. Initially reported as a unique somatic mutation in KG-1 cells [7], we report that the R229Q allele is a documented polymorphism in human populations. With the significant incidence of the allele in the population, our observations of OGG1 structural destabilization and increased cell killing following induction of oxidative DNA damage resulting from the R229Q polymorphism suggest that the variant may be a potential marker for cancer susceptibility.
Section snippets
Cell culture
Cells were obtained from ATCC and maintained in the recommended growth medium (Gibco) in 5% CO2 at 37 °C. KG-1 cells were transfected with Nucleofector R transfection reagents (Amaxa) according to the manufacturer's instructions. HeLa cells were transfected using Fugene 6 transfection reagent (Roche) according to the manufacturer's instructions. Nuclear extracts were prepared using NE-PER extraction reagents (Pierce). Mitochondria were prepared using a Mitochondria Isolation Kit for Mammalian
R229Q OGG1 is thermolabile in vitro and in vivo
Since amino acid substitutions in proteins can often result in thermolability, we examined the effect of preincubating purified R229Q OGG1 and the wild-type enzyme (Fig. 1A) at 37 °C prior to measuring excision activity. Surprisingly, purified R229Q OGG1 had 8-oxoG excision activity comparable to wild-type OGG1 (Fig. 2A). However, R229Q OGG1 was highly sensitive to heat inactivation and nearly completely inactivated by a 30 min preincubation at 37 °C (Fig. 2B). Therefore, the R229Q substitution
Discussion
Our observations of R229Q thermolability in vitro and in vivo explain previous reports of low 8-oxoguanine excision activity in KG-1 cells. Interestingly, thermolability of R229Q (Fig. 2B and C) is not the first reported instance of a thermolabile mammalian OGG1. SAMP1 mice, which exhibit premature senescence, shortened lifespan, and elevated levels of 8-oxoG in DNA, have a homozygous R304W mutation in OGG1, which results in a thermolabile OGG1 protein that is devoid of enzymatic activity [11].
Conflict of interest
None.
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, National Institute on Aging.
References (23)
Involvement of mammalian OGG1(MMH) in excision of the 8-hydroxyguanine residue in DNA
Free Radic Biol Med
(2002)- et al.
Radiation sensitivity depends on OGG1 activity status in human leukemia cell lines
Free Radic Biol Med
(2002) - et al.
Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria
J Biol Chem
(2002) - et al.
Thermolabile 8-hydroxyguanine DNA glycosylase with low activity in senescence-accelerated mice due to a single-base mutation
Free Radic Biol Med
(1999) - et al.
Efficiency, specificity and DNA polymerase-dependence of translesion replication across the oxidative DNA lesion 8-oxoguanine in human cells
Mutat Res
(2002) - et al.
The novel DNA glycosylase, NEIL1, protects mammalian cells from radiation-mediated cell death
DNA Repair (Amst)
(2003) - et al.
Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells
Biochem Pharmacol
(1992) - et al.
Voltammetric determination of γ radiation-induced DNA damage
Anal Biochem
(2006) - et al.
Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis
Mutat Res
(2006) - et al.
Base excision repair by hNTH1 and hOGG1: a two edged sword in the processing of DNA damage in gamma-irradiated human cells
DNA Repair (Amst)
(2006)
Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG
Nature
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2012, Free Radical Biology and MedicineCitation Excerpt :In another study, we used KG-1 cells, which express a temperature-sensitive, functionally inactive OGG1 variant (OGG1R229Q) [32]. OGG1R229Q regains its 8-oxoG excision activity at 25 °C and it is comparable to wild-type OGG1 [32]. Thus we utilized these cells to test whether endogenously generated 8-oxoG forms 8-oxoG*, which oxidizes H2DCF to DCF.