High expression of the circadian gene mPer 2 diminishes the radiosensitivity of NIH 3 T 3 cells

Period2 is a core circadian gene, which not only maintains the circadian rhythm of cells but also regulates some organic functions. We investigated the effects of mPeriod2 (mPer2) expression on radiosensitivity in normal mouse cells exposed to 60Coγ-rays. NIH 3T3 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous mPer2 expression or transfected with pcDNA3.1(+)-mPer2 and irradiated with 60Co-γ-rays, and then analyzed by several methods such as flow cytometry, colony formation assay, RT-PCR, and immunohistochemistry. Flow cytometry and colony formation assay revealed that irradiated NIH 3T3 cells expressing high levels of mPer2 showed a lower death rate (TPA: 24 h 4.3% vs 12 h 6.8% and control 9.4%; transfection: pcDNA3.1-mPer2 3.7% vs pcDNA3.1 11.3% and control 8.2%), more proliferation and clonogenic survival (TPA: 121.7 ± 6.51 vs 66.0 ± 3.51 and 67.7 ± 7.37; transfection: 121.7 ± 6.50 vs 65.3 ± 3.51 and 69.0 ± 4.58) both when treated with TPA and transfected with mPer2. RT-PCR analysis showed an increased expression of bax, bcl-2, p53, cmyc, mre11, and nbs1, and an increased proportionality of bcl-2/bax in the irradiated cells at peak mPer2 expression compared with cells at trough mPer2 expression and control cells. However, no significant difference in rad50 expression was observed among the three groups of cells. Immunohistochemistry also showed increased protein levels of P53, BAX and proliferating cell nuclear antigen in irradiated cells with peak mPer2 levels. Thus, high expression of the circadian gene mPer2 may reduce the radiosensitivity of NIH 3T3 cells. For this effect, mPer2 may directly or indirectly regulate the expressions of cell proliferationand apoptosis-related genes and DNA repair-related genes.


Introduction
Correspondence: Z.R. Wang, Health Ministry Key Laboratory of Chronobiology, West China Medical Center, Sichuan University, No.17 Section 3 South Renming Road, Chengdu, 610041, China.Fax: +86-028-8550-3204.E-mail: wangzhengrong@126.comRadiobiology studies have shown that ionizing radiation is a DNA-damaging agent inducing cell death, gene mutations and chromosome aberrations even at low doses (1).Most of these reactions are induced by hydroxyl radicals (indirect effects) and by one-electron oxidation (direct effects) resulting from exposure to ionizing radiation (2).Cell DNA molecules are important targets of radiation injury (3,4).Activation of the damage checkpoint occurs in response to many types of genomic lesions, including double-strand DNA breaks, single-strand DNA breaks and chemical modification of DNA by UV and γ irradiation (5,6).The cell cycle potentially stalls at several phases to provide ample time for the cell to repair DNA lesions before the S-phase (G1 arrest) and/or mitosis (G2 arrest).When cells recognize DNA injuries, especially double-strand DNA breaks, they activate the DNA damage checkpoint and repair the damage.When cells fail to fully repair the disordered DNA they activate the apoptotic cell death pathways.This fine tuning of the balance between DNA repair and apoptosis may be mediated by the DNA-binding properties of the related proteins and by their transactivation of gene transcription such as the P53 protein (7)(8)(9).
Circadian genes constitute the physiological basis of circadian clocks, which control the daily oscillations of biochemical, physiological, and behavioral processes of most organisms and enable these processes to occur at appropriate times of day (10)(11)(12).Recently, it has been shown that circadian genes not only play an important www.bjournal.com.brBraz J Med Biol Res 42 (10) 2009 role in controlling circadian rhythms, but also participate in other physiological and pathological activities, such as drug dependence, tumor development and radiation response (13)(14)(15).The Period2 (Per2) gene, an indispensable component of the circadian clock, not only modulates circadian oscillations, but also regulates other organic functions.mPeriod2 (mPer2) gene-deficient mice are cancer prone.After γ radiation, these mice showed a marked increase in tumor development and reduced thymocyte apoptosis.Temporal expression of the genes involved in cell cycle regulation and tumor suppression, such as Cyclin D1, Cyclin A, Mdm-2, and Gadd45, were reportedly altered in mPer2 mutant mice (16).The mPer2 gene may play an important role in tumor suppression by regulating DNA damage-response pathways.
It previous studies from our laboratory, Zhang et al. (17) observed that high expression of the circadian gene mPer2 might reduce the radiosensitivity of irradiated mouse tumor cells such as LLC and EMT6 cells.In the present study, we focus on the effects of the circadian gene mPer2 on irradiated normal cells such as NIH 3T3 cells.We treated NIH 3T3 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce endogenous circadian mPer2 expression, or established mPer2-overexpressing cells by transfecting pcDNA3.1(+)-mPer2into NIH 3T3 cells.We then exposed the cells to 60 Co-γ-rays, assessed the effects of mPer2 on cell death, proliferation and clonogenic survival after radiation, and explored the possible mechanism involved.We trust that the present study will offer a new theory for radiochronotherapy and provide a new way to protect normal cells against radiation injury.

Induction of rhythm by TPA treatment
TPA, the classic protein kinase C activator, was purchased from Promega (USA).We treated NIH 3T3 cells with 100 nM TPA to induce endogenous circadian mPer2 expression.At time zero, TPA was added to the medium, which was replaced with serum-free DMEM after 2 h.TPA treatment without serum can trigger the induction of the circadian oscillation of expression of some genes, including mPer2, with an approximate period of 24 h in NIH 3T3 cells.And the trough level of mPer2 mRNA occurs 12 h after TPA treatment and peaks 24 h after TPA treatment (18).Cells were then divided into three radiotreatment groups: a) control group (cells irradiated without TPA treatment), b) TPA 12-h group (cells irradiated at 12 h after TPA treatment with trough mPer2 level), and c) TPA 24-h group (cells irradiated at 24 h after TPA treatment with peak mPer2 level).

Cell transfection
The eukaryotic expression vector pcDNA3.1(+)-mPer2containing a cDNA copy of mPer2 (GenBank No. NM_011066) was used in this study.The mPer2 gene was confirmed as being in frame with no mutations by DNA sequencing.The cells were transfected with the indicated plasmids using the lipofectamine 2000 transfection reagent (Invitrogen, USA).Cell lysates were prepared 48 h later for the examination of protein expression and radiotreatment.The cells were divided into three groups: a) control group (cells without transfection), b) pcDNA3.1 group (cells transfected with the empty vector pcDNA3.1),and c) pcDNA3.1-mPer2 group (cells transfected with pcDNA3.1-mPer2).

Antibodies
Mouse antibodies against mPER2, P53, BAX and proliferating cell nuclear antigen (PCNA) were purchased from Sigma (USA).Rabbit anti-goat IgG and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (USA).

Western blot analysis
At 48 h after transfection, cells were lysed with cold RIPA lysis buffer (Sigma-Aldrich, USA) containing protease inhibitors, and proteins were collected by centrifugation.Protein concentrations were determined by the bicinchoninic acid assay (Pierce, USA) and transferred electro-phoretically to a polyvinylidene difluoride membrane (Pierce).Detection was carried out using an enhanced chemiluminescence reagent (Pierce).

Radiotreatment
All cell groups were irradiated with γ-rays, with a total absorbed dose of 4 Gy, using a 60 Co teletherapy machine (Phoenix, Japan).Cells were exposed at a dose rate of 115.38 cGy/min in an exposure field of 25 x 25 cm, with spacing of 80 cm.Cells were then processed for flow cytometry and colony formation assay to test cell death, proliferation and clonogenic survival.

Flow cytometry
To determine the expression of mPER2 protein, the cells were harvested at 48 h after transfection, fixed in 70% ethanol for 30 min at 4°C, and incubated with 0.1% saponin for 20 min.They were then incubated with primary antibodies (Sigma) at 1:200 dilution for 30 min and with fluoresceinisothiocyanate-conjugated secondary antibodies (Chemicon, USA) at 1:150 dilution for 30 min, and analyzed by flow cytometry (Beckman Coulter Elite ESP, USA).
Six hours after radiotreatment, all cells were digested with trypsin, harvested, washed with phosphate-buffered saline (PBS), fixed in 70% ethanol for 30 min at 4°C, treated with 50 μg/mL RNase A (Sigma), stained with 50 μg/mL propidium iodide for 20 min at 4°C without light, and analyzed by flow cytometry using an instrument equipped with a 488-nm argon laser for the determination of DNA synthesis and cell cycle status.Data were collected in linear mode and analyzed with the Multicycle Software (Beckman Coulter, USA).Apoptotic cells with degraded DNA appear as cells with hypodiploid DNA content and are represented in socalled "sub-G1" peaks on DNA histograms.Four distinct phases were recognized by flow cytometry in a proliferating cell population, including the G0/G1, S-(DNA synthesis phase), G2 and M-phases (mitosis).

Colony formation assay
Six hours after radiotreatment, all cells were digested with trypsin, statically cultured in DMEM in the cell culture incubator (SANYO, Japan) for 14 days, washed with PBS, fixed with methanol, stained for 15 min and washed again.The preparation was then photographed with the Omegapic formatter and analyzed.

Immunohistochemistry
Six hours after irradiation, the cells treated with TPA were used for immunohistochemistry.The test was carried out using the avidin-biotin complex method.The numbers of P53-, BAX-and PCNA-positive and -negative NIH 3T3 cells were determined in four random fields at 100X and 400X magnification, and the percentages of positive cells were calculated.

Statistical analysis
Data are reported as means ± SD.One-way ANOVA was used to compare difference among groups and P values of less than 0.05 were considered to be statistically significant.

The effects of radiation on NIH 3T3 cells treated with TPA
Parental NIH 3T3 cells normally produce very low and barely detectable levels of mPer2.In the present study, NIH 3T3 cells, TPA treatment was used to induce endogenous mPer2 expression and to trigger the induction of the circadian oscillation of expression of various clock and clock-related genes, including mPer2.The expression level of mPer2 mRNA oscillated within an approximate period of 24 h, with the trough occurring after 12 h and the peak after 24 h of TPA treatment.

Role of radiation in NIH 3T3 cells with up-regulated mPer2
We established mPer2-overexpressing cells by transfecting pcDNA3.1(+)-mPer2into NIH 3T3 cells.Successful transfection of the mPer2 gene using the lipofectamine 2000 reagent was confirmed by Western blotting (Figure 4A).It was also evident by flow cytometry that the fluorescence intensity of mPER2 protein expression in pcDNA3.1(+)-mPer2-transfectedcells was  higher than in pcDNA3.1-transfectedcells and in control cells without transfection (Figure 4B).

Discussion
Circadian clocks are molecular time-keeping mechanisms that reside in a diverse range of cell types in a variety of organisms.The primary role of these cell-autonomous clocks is to maintain their own approximately 24-h molecular rhythms and to drive the rhythmic expression of genes involved in physiology, metabolism and behavior (19,20).The circadian clock is composed of multiple, single-cell circadian oscillators, which, when synchronized, generate coordinated circadian outputs that regulate overt rhythms.Eight clock genes involved in the interacting transcriptional-/ translational-feedback loops that compose the molecular clockwork have been cloned (21).
Parental NIH 3T3 fibroblasts normally produce very low and barely detectable levels of circadian genes.TPA treatment is as effective as serum shock in triggering the induction of circadian gene expression in cultured cells, including mPer2.As a core circadian gene, mPer2 not only maintains the circadian rhythm of cells, but also sustains the normal cell cycle.After TPA treatment, parental NIH 3T3 cells present circadian oscillation of mPer2 expression within an approximate period of 24 h, with the trough at zeitgeber time 12 (ZT12)and the peak at ZT24 (18,22,23).
In the present study, we treated NIH 3T3 cells with TPA to induce endogenous mPer2 expression, and cells were irradiated at ZT12 with trough mPer2 level and at ZT24 with peak mPer2 level.The death, proliferation and clonogenic survival of irradiated cells were determined by flow cytom- A, Western blot analysis of mPER2 expression.The mPER2 expression of mPer2 group cells was significantly higher than that of pcDNA3.1 group cells and of control cells.B, Flow cytometric analysis of mPER2 expression.The fluorescence intensity of mPER2 expression in mPer2 group cells was also significantly higher than that of pcDNA3.1 group cells and of control cells (P < 0.01, one-way ANOVA).).Control: cells irradiated without 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; 12 h: cells irradiated 12 h after TPA treatment with trough mPer2 level; 24 h: cells irradiated 24 h after TPA treatment with peak mPer2 level.The mRNA levels of mouse bax, bcl-2, p53, c-myc, mre11, and nbs1 were higher in 24-h group cells than in 12-h group cells and control cells.In particular, the expression of c-myc and bcl-2 was obviously up-regulated, but there was no significant difference in rad50 level among the three groups of cells.
etry and colony formation assay.The results of the present study showed that high expression of mPer2 might reduce cell death and enhance cell proliferation of irradiated NIH 3T3 cells.However, after TPA treatment, parental NIH 3T3 cells presented circadian expression not only of mPer2, but also of other clock and clock-related genes such as mPer1 and DBP (18).Therefore, the radiobiological effects may result from mPer2 or other clock and clock-related genes.In order to assess the effects of mPer2 expression on the irradiated cells, we transfected pcDNA3.1(+)-mPer2into NIH 3T3 cells.The results showed that overexpression of mPER2 in NIH 3T3 cells resulted in reduced cell death and enhanced cell proliferation after radiation, suggesting that overexpressed mPer2 may diminish the radiosensitivity of NIH 3T3 cells.
Fu et al. (16) reported that the loss of mPer2 function resulted in increased tumor development and deficiencies in response to DNA damage in mice, suggesting that the mPer2 gene functions in tumor suppression by regulating DNA damage-responsive pathways.Compared with wildtype mice, mPer2 mutant mice showed a neoplastic growth phenotype and an increased sensitivity to γ radiation in thymocytes.Zhang et al. (17) also found that high expression of the circadian gene mPer2 might diminish radiosensitivity of irradiated mouse tumor cells.LLC and EMT6 cells with high mPer2 expression exposed to 60 Co-γ-rays presented reduced DNA damage, increased survival and clone-forming rate, which suggested that mPer2 might protect cells against the radiation injury of γ-rays and increase the survival rate of tumor cells.The present study demonstrated that high expression of mPer2 could diminish the radiosensitivity of NIH 3T3 cells, with reduced cell death, enhanced cell proliferation and increased clonogenic survival.Moreover, tumor cells and NIH 3T3 cells were irradiated at different times after TPA induction, i.e., at 12 h (trough time of mPer2 expression) and at 24 h (peak time) after induction, whereas LLC cells were irradiated at 18 h (trough time) and 30 h (peak time) after induction.The present research combined with the previous research of Fu et al. (16) and Zhang et al. (17) would contribute to protecting normal cells against radiation injury in cancer radiotherapy.We propose that radiotherapy could be used against cancer at a certain time of Per2 expression for the best ratio of tumor suppression and normal tissue protection, which can kill tumor cells and protect normal cells maximally.However, our hypothesis needs further study.
Without radiation, overexpression of mPER2 results in reduced proliferation and rapid apoptosis of tumor cells, but not of NIH 3T3 cells, suggesting that mPer2 may play an important role in tumor suppression by inducing apoptotic cell death (24).However, irradiated NIH 3T3 cells with mPER2 overexpression showed reduced cell death, suggesting that mPer2 may affect the radiation-induced cell death by other ways.
In response to ionizing radiation, cells immediately activate a series of biochemical pathways that promote cell survival while maintaining genetic integrity.The main cellular defense system against ionizing radiation exposure is composed of two distinct types of biochemical pathways, i.e., the DNA damage cell cycle checkpoint pathways and the DNA repair pathways (25).Arrest of replicative DNA synthesis after DNA damage is thought to occur to provide ample time for the cell to repair DNA lesions before the Sphase (G1 arrest) and/or mitosis (G2 arrest).The RAD50-MRE11-NBS1 (MRN) complex plays an important role in the repair of DNA damage caused by radiation.The complex practically participates in all repair mechanisms, especially homologous recombination and non-homologous end-joining, which are the most important mechanisms in the repair of DNA double-strand breaks (26).
In the present study, the levels of rad50 expression of the various cell groups were not different, in agreement with reports showing that rad50 expression is persistent and stable (27).As the core part of the MRN complex, MRE11 has not only nuclease activity, but also a connective effect on RAD50 and NBS1, and its expression changes rapidly after radiation (28).NBS1 is also an important part of the MRN complex (29).The irradiated NIH 3T3 cells expressing high levels of mPer2 showed an increased expression of mre1l and nbs1, which suggested that mPer2 could enhance the function of DNA damage repair MRN complex.
c-Myc, a proto-oncogene, plays an important role in both cell proliferation and apoptosis (30,31).Circadian regulators may target genes that are controlled by c-myc.c-Myc itself is also controlled by the circadian clock, and the level of c-myc mRNA oscillated in 24-h light/dark cycles in wild-type mouse livers, peaking at ZT14 (16).Gamma radiation may increase the expression of c-myc, whose overexpression could drive cells to progress through the cell cycle in the presence of genomic DNA damage in order to improve the efficiency of DNA repair (32).It has also been reported that c-myc directly regulates the transcription of the nbs1 gene involved in DNA double-strand break repair (33).The present study showed that c-myc expression increased in irradiated NIH 3T3 cells with high expression of mPer2, thus suppressing cell death and enhancing cell repair.
BCL-2 and BAX are members of the BCL-2 family that is a key regulator of the mitochondrial response to apoptotic signals in the intrinsic pathway (34,35).Bcl-2 is an antiapoptotic gene acting as a potent suppressor of apoptosis by blocking the release of cytochrome c, whereas bax is a proapoptotic gene with oppose functions acting as a promoter of cell death.The ratio of antiapoptotic-toproapoptotic molecules determines the response to a death signal (36).In the present study, the high expression of mPer2 increased the BCL-2/BAX ratio, thus suppressing cell death and enhancing cell growth.
Wild-type P53 protein levels rise dramatically after exposure to ionizing radiation.This rise results from as yet undefined changes in the post-transcriptional modifications undergone by the P53 protein such as phosphorylation, binding to other proteins, or oligomerization.At subsequent end points of DNA-damage, a prolonged half-life was observed as well as increased DNA-binding activity of the P53 protein and enhanced transcriptional transactivation activity driven by this protein (37,38).P53 can keep genomic stability by mediating apoptosis and DNA repair (7)(8)(9)39) and can induce a transient arrest of the cell cycle at G1, so that the cells will have time to repair damaged DNA.Activated p53 can also eliminate cells through mechanisms involving prolonged arrest at G1 or apoptosis.Our study showed an increased expression of p53 in irradiated NIH 3T3 cells at peak mPer2 expression, with decreased cell death.This indicated that a high expression of mPER2 up-regulated P53 expression, which could enhance DNA repair.
PCNA is a protein that acts as a processivity factor for DNA polymerase delta in eukaryotic cells.PCNA is an essential factor in cell proliferation and can be used as an index to evaluate cell proliferation (40).Since DNA polymerase delta is involved in resynthesis of excised damaged DNA strands during DNA repair, PCNA is important for both DNA synthesis and DNA repair.PCNA is also involved in the DNA damage tolerance pathway known as post-replication repair.In the present study, the irradiated cells with high expression of mPer2 showed enhanced cell proliferation, and the increase in PCNA staining after DNA damage could be a result of DNA repair.
After radiation, the high expression of mPer2 in NIH 3T3 cells results in reduced cell death and enhanced cell proliferation and clonogenic survival, which means that high expression of the circadian gene mPer2 diminishes radiosensitivity of NIH 3T3 cells.A high expression of mPer2 may up-regulate the expression of apoptosis-related genes, with an increased proportionality of bcl-2/bax, and may reduce cell death and enhance cell proliferation to diminish radiosensitivity.Moreover, mPer2 may also up-regulate DNA repair-related genes to increase DNA-repair efficiency after radiation to diminish radiosensitivity.Future research should be focused on studying the detailed mechanisms by which the circadian clock controls genes related to radiosensitivity.

Figure 1 .
Figure 1.Apoptosis in NIH 3T3 cells irradiated after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment.Control: cells irradiated without TPA treatment; 12 h: cells irradiated 12 h after TPA treatment; 24 h: cells irradiated 24 h after TPA treatment.The percentage of sub-G1 cells undergoing apoptosis is indicated by the bar.The cells of the TPA 24-h group had a significantly lower apoptotic peak than the cells of the TPA 12-h group and of control cells (P < 0.01, one-way ANOVA).

Figure 2 .
Figure 2. The cell cycle in NIH 3T3 cells irradiated after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment.Control: cells irradiated without TPA treatment; 12 h: cells irradiated 12 h after TPA treatment; 24 h: cells irradiated 24 h after TPA treatment.G0/G1, S and G2/M phases are indicated.The S-phase fraction of the TPA 24-h group was significantly higher than that of the TPA 12-h group and of control cells (P < 0.01, one-way ANOVA).

Figure 3 .
Figure 3. Colony formation of 12-O-tetradecanoylphorbol-13acetate (TPA)-treated NIH 3T3 cells after irradiation.A, Control: cells irradiated without TPA treatment; 12 h: cells irradiated 12 h after TPA treatment; 24 h: cells irradiated 24 h after TPA treatment.B, High expression of mPER2 dramatically increased the clonogenic survival of irradiated NIH 3T3 cells.The colony-forming efficiency of TPA 24-h group cells was significantly higher than that of TPA 12-h group cells and of control cells (P < 0.01, one-way ANOVA).

Figure 4 .
Figure 4. Detection of mPER2 expression by Western blot analysis and flow cytometry.Control: cells without transfection; pcDNA3.1:cells transfected with pcDNA3.1;mPer2: cells transfected with pcDNA3.1-mPer2.A, Western blot analysis of mPER2 expression.The mPER2 expression of mPer2 group cells was significantly higher than that of pcDNA3.1 group cells and of control cells.B, Flow cytometric analysis of mPER2 expression.The fluorescence intensity of mPER2 expression in mPer2 group cells was also significantly higher than that of pcDNA3.1 group cells and of control cells (P < 0.01, one-way ANOVA).

Figure 7 .
Figure 7. Colony formation of transfected NIH 3T3 cells after irradiation.A, Control: cells without transfection; pcDNA3.1:cells transfected with pcDNA3.1;mPer2: cells transfected with pcDNA3.1-mPer2.B, mPER2 dramatically increased the clonogenic survival of irradiated NIH 3T3 cells.The colony-forming efficiency of mPer2 overexpressing cells was significantly higher than that of empty vector cells and of control cells (P < 0.01, one-way ANOVA).

Figure 5 .
Figure 5. Apoptosis in NIH 3T3 cells irradiated after transfection.Control: cells without transfection; pcDNA3.1:cells transfected with pcDNA3.1;mPer2: cells transfected with pcDNA3.1-mPer2.The percentage of sub-G1 cells undergoing apoptosis is indicated by the bar.The apoptotic peak of mPer2 overexpressing cells was lower than that of empty vector cells and of control cells (P < 0.01, one-way ANOVA).

Figure 8 .
Figure 8. RT-PCR analysis of mouse bax, bcl-2, p53, c-myc, rad50, mre11, and nbs1 genes (RT-PCR electropherogram and integral optical density ratio results).Control: cells irradiated without 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment; 12 h: cells irradiated 12 h after TPA treatment with trough mPer2 level; 24 h: cells irradiated 24 h after TPA treatment with peak mPer2 level.The mRNA levels of mouse bax, bcl-2, p53, c-myc, mre11, and nbs1 were higher in 24-h group cells than in 12-h group cells and control cells.In particular, the expression of c-myc and bcl-2 was obviously up-regulated, but there was no significant difference in rad50 level among the three groups of cells.