Prdx1 deficiency in mice promotes tissue specific loss of heterozygosity mediated by deficiency in DNA repair and increased oxidative stress

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Abstract

The loss of the H2O2 scavenger protein encoded by Prdx1 in mice leads to an elevation of reactive oxygen species (ROS) and tumorigenesis of different tissues. Loss of heterozygosity (LOH) mutations could initiate tumorigenesis through loss of tumor suppressor gene function in heterozygous somatic cells. A connection between the severity of ROS and the frequency of LOH mutations in vivo has not been established. Therefore, in this study, we characterized in vivo LOH in ear fibroblasts and splenic T cells of 3–4 month old Prdx1 deficient mice. We found that the loss of Prdx1 significantly elevates ROS amounts in T cells and fibroblasts. The basal amounts of ROS were higher in fibroblasts than in T cells, probably due to a less robust Prdx1 peroxidase activity in the former. Using Aprt as a LOH reporter, we observed an elevation in LOH mutation frequency in fibroblasts, but not in T cells, of Prdx1−/− mice compared to Prdx1+/+ mice. The majority of the LOH mutations in both cell types were derived from mitotic recombination (MR) events. Interestingly, Mlh1, which is known to suppress MR between divergent sequences, was found to be significantly down-regulated in fibroblasts of Prdx1−/− mice. Therefore, the combination of elevated ROS amounts and down-regulation of Mlh1 may have contributed to the elevation of MR in fibroblasts of Prdx1−/− mice. We conclude that each tissue may have a distinct mechanism through which Prdx1 deficiency promotes tumorigenesis.

Highlights

► Significant elevation in ROS levels in Prdx1−/− T cells and ear fibroblasts. ► Reduced Prdx1 peroxidase activity in fibroblasts compared to T cells. ► Significant elevation in LOH mutant frequency only in Prdx1 deficient fibroblasts. ► Significant reduction in Mlh1 expression in Prdx1−/− fibroblasts.

Introduction

Reactive oxygen species (ROS), specifically, superoxide radicals (O2), are produced as the by-products of mitochondrial oxidative phosphorylation and various metabolic processes. The superoxide radicals are dismutated to hydrogen peroxide (H2O2) by a family of superoxide dismutase (Sod) enzymes functional in different sub-cellular compartments [1]. H2O2 is then converted to H2O and O2 by different families of peroxidases: catalase, glutathione peroxidases (Gpxs) and peroxiredoxins (Prdxs). Prdx1 is easily over-oxidized due to its Cys51 that during catalysis exists as thiolate anion, whereas the other cysteines (Cys17, Cys80 and Cys172) remain protonated at neutral pH. The thiolate Cys51 is very unstable and highly reactive with any accessible thiol to form either a disulfide or to further oxidize to sulfinic or sulfonic acid structures [2], which are believed to possess peroxidase-inactive chaperone functions (reviewed in [3]). The switch to chaperone functionality leads to catalase activity that scavenges more of the cellular H2O2. Overall, the peroxidases sequentially maintain homeostatic amounts of H2O2, which curtails the formation of highly reactive hydroxyl (OHradical dot) radicals (through the Fenton reaction).

Elevated amounts of intracellular ROS (oxidative stress) produced as a result of an exogenous stressor (e.g. H2O2 or irradiation) can damage DNA, protein and lipids through oxidation. At least 100 different types of oxidative DNA lesions are known to form, which include base modifications (e.g. 8-oxo-guanine (8-oxoG), thymidine glycol and 8-hydroxycytosine), single-strand breaks (SSBs), double-strand breaks (DSBs) and interstrand cross-links [4]. For example, H2O2 treatment of human T-lymphocytes led to increased Hprt mutant frequency with a majority of mutations classified as transitions at GC base pairs [5]. Additionally, oxidative DNA damage could stem from compromised ROS scavenging such as in the case of Prdx1 deficient mouse embryonic fibroblasts (MEFs) that exhibited increased ROS amounts compared to wild-type MEFs and an elevated number of 8-oxoG DNA lesions [6]. Moreover, there was also greater oxidation of hemoglobin in Prdx1 deficient erythrocytes as evidenced by the appearance of Heinz bodies and eventual hemolytic anemia. A separate study involving adult Prdx1−/− mice found increased levels of various types of oxidative DNA lesions in the brain, spleen and liver [7]. In addition to causing DNA base damage, elevated ROS could also lead to increased frequency of loss of heterozygosity (LOH) mutations primarily derived from mitotic recombination (MR) events that originated in response to the need for DNA DSB repair [8], [9].

LOH is frequently a rate-limiting step in tumorigenesis [10] and increased frequency of LOH mutations is positively correlated with increased cancer incidence such as that which is observed in Prdx1-deficient mice [6]. We have previously utilized Aprt+/− mice as a model system to study in vivo LOH [11], [12]. Cells that have undergone in vivo LOH that includes Aprt are recovered as cell colonies in vitro by virtue of their resistance to 2,6-diaminopurine (DAP). The recovered colonies can then be analyzed to determine the mutational mechanism that produced the in vivo LOH. In order to determine whether LOH is elevated in vivo by ROS, which may consequently contribute to increased tumorigenesis, we measured the spontaneous LOH mutant frequencies in ear fibroblasts and splenic T cells of Prdx1 deficient mice. We observed that while ROS amounts are increased in both fibroblasts and T cells derived from Prdx1−/− mice, they are much higher in fibroblasts than in T cells regardless of Prdx1 functional status. Correspondingly, significant elevations in Aprt LOH mutant frequencies were found only in Prdx1+/− and Prdx1−/− fibroblasts.

Section snippets

Mice breeding

129XC57 Prdx1+/− and Prdx1−/− male mixed strain mice were as described [6]. These mice were backcrossed to 129 strain and C57 strain, respectively, for four generations. We utilized “speed congenics” by analyzing distribution of microsatellite markers along chromosome 8 to determine the appropriate mice for mating for the subsequent generation until pure strain 129 or C57 mice were generated. The N4 129 Prdx1+/− mice were then crossed with 129 Aprt+/− mice to generate N5 129 Prdx1+/− Aprt+/−

Increased ROS amounts in ear fibroblasts and splenic T cells of Prdx1−/− mice

We previously showed that loss of Prdx1 in MEFs led to increased amounts of ROS [6]; therefore, we wanted to determine if it had a similar impact in splenic T cells and in ear fibroblasts. ROS amounts were quantified by measuring the amount of intracellular CM-H2DCFDA that is cleaved by intracellular esterases and made fluorescent under oxidizing conditions [15]. Indeed, significantly higher amounts of ROS were observed with the complete loss of Prdx1 in both T cells and fibroblasts; ROS

Discussion

We showed that the elevated ROS amounts in Prdx1−/− fibroblasts directly correlated to increased LOH mutant frequency, which we speculate is a direct consequence of an increased need for repair of DSBs through an increased accumulation of highly reactive OHradical dot radicals [4] that occurs with the loss of Prdx1 [7]. Repair of DSBs could happen either by homologous recombination (HR) or non-homologous end-joining (NHEJ). A previous study found that mammalian cells in S-phase compared to cells in G1

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

We would like to thank Jennifer Schulte for performing the Prdx1 Western blot analyses. This work was supported by grants from the National Institutes of Health [grant numbers ES011633 and P30ES05022].

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