The circadian mutation PER2(S662G) is linked to cell cycle progression and tumorigenesis

Abstract

Circadian oscillation and cell cycle progression are the two most essential rhythmic events present in almost all organisms. Circadian rhythms keep track of time and provide temporal regulation with a period of about 24 h. The cell cycle is optimized for growth and division, but not for time keeping. Circadian gated cell divisions are observed in nearly all organisms. However, the implications of this coupling to the physiology of mammals are unknown. A mutation (S662G) in the clock protein PERIOD2 (PER2) is responsible for familial advanced sleep phase syndrome in which sleep onset occurs in the early evening and wakefulness occurs in the early morning. Here, we provide evidence that the PER2(S662) mutation leads to enhanced resistance to X-ray-induced apoptosis and increased E1A- and RAS-mediated oncogenic transformation. Accordingly, the PER2(S662) mutation affects tumorigenesis in cancer-sensitized p53(R172H/+) mice. Finally, analyzing the clock-controlled cell cycle genes p21, c-Myc, Cyclin D1 and p27, we found that the relative phases between p21 and Cyclin D expression profiles have been changed significantly in these Per2 allele mutant mouse embryonic fibroblasts. This key role of the Per2-mediated phase alteration of p21 provides what we believe to be a novel mechanism in understanding cell cycle progression, its plasticity and its resistance to interference.

Figure 1

The PER2^S662 mutation leads to decreased levels of apoptosis. (a) Wild-type, Per2^−/−, PER2^S662G:Per2^−/− and PER2^S662D:Per2^−/− mice were irradiated with 6 Gy X-rays at ZT 0 (0800 hours) and ZT 9 (1700 hours). Spleen cells were isolated 6 h after irradiation, stained with propidium iodide (PI) and analyzed by flow cytometry. One representative experiment is shown. Ap, apoptotic percentage. (b) Percentages of apoptotic cells measured by flow cytometry are shown in the histograms. Experiments were repeated at least three times with consistent results and at least two mice were used per group for each time (N=3 for each genotype; ^***P<0.001; one-way ANOVA followed by Dunnett's test)

Figure 2

Generation and characterization of PER2 BAC transgenic mice. (a) Generation of different copy BAC transgenic mice. (b) Transgene copy number was estimated by Southern blot by comparing with copy standards. Experiments were repeated two times with consistent results and one mouse was used per line. (c) The relative transgene copy number was further estimated by q-PCR. (d–f) The mRNA expression level was assessed by qRT-PCR in liver tissues (d), spleen tissues (e) and non-synchronized MEFs (f) of different BAC transgenic mice as indicated genotypes. Experiments were repeated independently at least three times with consistent results and at least two replicates were used per time point. ‘→' denotes the band of PER2 and ‘*' denotes a unspecific band. (g) PER2 protein levels in spleen tissues of H line and PER2^S662D. Experiments were repeated three times with consistent results, and one mouse for each time point was used for western blot. (h) Voluntary locomotor activities were recorded as wheel-running activity. BAC transgenic mice were subjected first to LD (light and dark cycle) and then released into DD (dark and dark cycle). Representative actograms from low- (23.64±0.11, n=6), middle- (24.34±0.18, n=7) and high-copy BAC transgenic mice (25.07±0.17, n=9). Alternating white and dark bars indicate the LD cycles during entrainment before release in DD

Figure 3

High-dose PER2 results in resistance to apoptosis induced by X-rays. (a) Low-, middle- and high-copy PER2 mice were irradiated with 6 Gy X-rays at ZT 9. Spleen cells were isolated 6 h after irradiation and the DNA content was analyzed by flow cytometry. One representative experiment is shown. (b) Percentages of apoptotic cells detected by flow cytometry from low-, middle- and high-copy BAC transgenic spleens (^***P<0.001; one-way ANOVA followed by Dunnett's test)

Figure 4

Dysregulation of PER2 increases formation of oncogenic E1A–RAS-mediated foci in MEFs. (a) and (c) Passage 0 MEFs with the indicated genotypes were infected with retroviruses encoding E1A–RAS. Transfections with empty virus vector were used as negative controls. Two representative culture plates are shown per genotype from at least two repeated independent experiments. (b) and (d) The numbers of foci per 60-mm plate in the assays are expressed as means±S.D. (N=6 for each group; one-way ANOVA followed by Dunnett's test)

Figure 5

PER2^S662 mutations increased tumor incidence in the p53^R172H/+ background. (a) Overall survival. (b) Male survival. Survival curves (Kaplan–Meier representation) of the indicated mouse cohorts. n=number of mice per genotype. The survival curves are plotted with respect to the number of weeks using SPSS software. Statistical significance was assessed using the log-rank test and is indicated (P-value). Alterations in median life span of the indicated genotypes relative to control (increase as plus or decrease as minus) are labeled

Figure 6

Female-specific neural tube defects in p53^R172H/R172H mice. (a) p53^R172H/R172H female embryos develop exencephaly, which results from a failure of neural tube defects. This is a represent from 13.5-day mouse embryo from p53^R172H/+ and p53^R172H/+ crosses. Two p53^R172H/R172H females all develop exencephaly (arrow). (b) HE staining for female embryo neural tubes with exencephaly (arrow)

Figure 7

The relative phases of cell cycle genes are shifted by PER2 mutations. Wild-type and different PER2 mutant MEFs were synchronized by a dexamethasone treatment, and RNA was extracted at 3 h intervals stating 10 h after the shock. p21, Cyclin D1 and Nr1d1 expression analysis were done by q-PCR. Experiments were repeated independently at least three times with consistent results. Plotted values are the mean values±S.D. Cosinor analysis was performed on mRNA profiles using SPSS 17.0. The amplitude and acrophase obtained from cosinor analysis were further analyzed by Students t-test. The null hypothesis was rejected at P<0.05