The Mammalian circadian clock gene per2 modulates cell death in response to oxidative stress - PubMed (original) (raw)
The Mammalian circadian clock gene per2 modulates cell death in response to oxidative stress
Maria Chiara Magnone et al. Front Neurol. 2015.
Abstract
Living in the earth's oxygenated environment forced organisms to develop strategies to cope with the damaging effects of molecular oxygen known as reactive oxygen species (ROS). Here, we show that Per2, a molecular component of the mammalian circadian clock, is involved in regulating a cell's response to oxidative stress. Mouse embryonic fibroblasts (MEFs) containing a mutation in the Per2 gene are more resistant to cytotoxic effects mediated by ROS than wild-type cells, which is paralleled by an altered regulation of bcl-2 expression in Per2 mutant MEFs. The elevated survival rate and alteration of NADH/NAD(+) ratio in the mutant cells is reversed by introduction of the wild-type Per2 gene. Interestingly, clock synchronized cells display a time dependent sensitivity to paraquat, a ROS inducing agent. Our observations indicate that the circadian clock is involved in regulating the fate of a cell to survive or to die in response to oxidative stress, which could have implications for cancer development and the aging process.
Keywords: SIN-1; UV; adenovirus; apoptosis; bcl-2; p53; paraquat; plumbagin.
Figures
Figure 1
Mutation in the clock gene Per2 leads to altered response of asynchronous cells to oxidative stress. (A) Mouse embryonic fibroblasts (MEFs) of wild type (wt, solid line) and Per2 mutant mice (Per2Brdm1, hatched line) display differences in cytotoxicity in response to various amounts of paraquat. LDH released into the medium versus LDH in living cells was measured after 24 h of paraquat treatment to determine cytotoxicity (n = 4–5 MEF preparations per genotype, *p < 0.05 two-way-ANOVA with subsequent Bonferroni test). Cell death due to plating was deducted (wt = 7 ± 0.8%, Per2Brdm1 = 7.4 ± 1%, n = 3) (B) _N_-acetyl cystein (NAC), a radical scavenger, reverses paraquat mediated cytotoxicity. About 100 mM NAC was added to MEFs and subsequently the cells were treated with 600 μM paraquat. Top: photomicrographs of cells. Scale bar = 200 μm. Bottom: quantification of viability using the crystal violet method. (C) Wild type (wt, white bar) and Per2 mutant (Per2Brdm1, black bar) MEFs display differences in cytotoxicity in response to ultra-violet (UV) treatment (120 mJ/cm2), in response to plumbagin (1.5 μM) (D), and in response to SIN-1 (2 mM) (E) (*p < 0.05).
Figure 2
Time dependent sensitivity of synchronized wild type and Per2Brdm1 mutant MEFs to paraquat and altered oxidative state in Per2Brdm1 mutant MEFs. (A) Cells were synchronized with dexamethasone and treated with paraquat after the indicated times. LDH released into the medium versus LDH in living cells was measured after 24 h of paraquat treatment to determine cytotoxicity. Cytotoxicity of paraquat in both genotypes is time dependent and significantly reduced in Per2Brdm1 mutant MEFs (*p < 0.05, n = 3). The panel below shows clock synchronization of MEFs monitored by expression of Cry1 relative to the histone H2Az. Wild-type cells show a significant 24 h cycling (*p < 0.05, n = 3) whereas Per2Brdm1 mutant MEFs display altered Cry1 expression. Significant differences in Cry1 expression in these cells at 6 and 18 h are observed (*p < 0.05, n = 3). (B) Rescue of sensitivity toward paraquat in Per2Brdm1 mutant MEFs 6 h after dexamethasone treatment by introduction of the wild-type Per2 gene into Per2Brdm1 mutant MEFs using adenovirus (Ad-Per2) (*p < 0.05, n = 3). (C) NADH/NAD+ ratio is elevated in Per2Brdm1 mutant MEFs (*p < 0.05, n = 3). This ratio is normalized to wild-type levels by introduction of the wild-type Per2 gene (Ad-Per2) but not GFP (Ad-GFP) using adenovirus (n = 3).
Figure 3
Endogenous reactive oxygen species (ROS) and sensitivity to H2O2 are similar in wild type and Per2Brdm1 mutant MEFs but mitochondrial function is different between the two genotypes. (A) Enzymatic activities of aconitase, total superoxide dismutase (SOD), and lactate dehydrogenase (LDH) in wild type and Per2Brdm1 mutant MEFs reveal no differences in the amounts of endogenous ROS between the two genotypes (n = 3). (B) The sensitivity to H2O2 is comparable between wild type and Per2Brdm1 mutant MEFs pointing to similar cell membrane sensitivity of the two genotypes to this compound (n = 3). Cell death due to plating was deducted (wt = 9.2 ± 2%, Per2Brdm1 = 7.4 ± 2.4%, n = 3) (C) Activity of complex I (NADH ubiquinone oxidoreductase) of the oxidative chain is significantly reduced in Per2Brdm1 mutant compared to wild-type MEFs suggesting altered mitochondrial function in Per2Brdm1 mutant MEFs (*p < 0.05, n = 5). (D) Assessment of intracellular ROS levels after 6 h of paraquat treatment using H2DCFDA. ROS mediated conversion to DCF was measured and values were normalized to untreated wild-type MEFs (*p < 0.05, n = 4, unpaired _t_-test).
Figure 4
Amount of mitochondrial DNA in MEFs, apoptosis after paraquat treatment and bcl-2 expression. (A) Labeling of mitochondria with MitoTraker®Green FM. No difference in number of mitochondria can be detected between wild type and Per2Brdm1 mutant MEFs (scale bar = 100 μm). (B) Top panel: PCR amplification of a 2.3 kb fragment of mitochondrial DNA (mtDNA) is compared to amplification of a 0.3 kb fragment of nuclear DNA (histone H2Az) after 15, 20, 30, and 35 cycles. Lane 1 = wild type, lane 2 = Per2Brdm1 mutant, lane 3 = no DNA. Similar amounts of mtDNA relative to histone H2Az DNA are observed in both genotypes. Bottom panel: quantification of amplified mtDNA normalized to histone H2Az DNA after 30 and 35 cycles (n = 3). (C) Staining of wild type and Per2Brdm1 mutant MEFs with annexin V (green) and propidium iodide (orange) 10 and 16 h after paraquat treatment at low (left panel) and high (right panel) magnification. Scale bar = 100 μm. (D) Example of apoptosis GEArrays after hybridization with labeled cDNA probes of untreated wild-type and Per2Brdm1 mutant MEFs. Three arrays per genotype were hybridized and genes significantly changed in expression are marked by colored squares, bcl-2 (red) and fadd (green). Controls (two bottom rows) were PUC18 plasmid, blank spots (non-specific binding), Ppia and Rpl13a as positive controls and two housekeeping genes for normalization (b-actin and Gapdh). (E) Northern blot of mRNA isolated from livers of wild-type and Per2Brdm1 mutant mice sacrificed at the indicated time points. (F) Expression of bcl-2 in wild type and Per2Brdm1 mutant MEFs before and after paraquat treatment (n = 3, *p < 0.05).
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