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The Period2 gene, an indispensable component of the circadian clock, not only modulates circadian oscillations, but also regulates organic function. We examined whether overexpression of the mouse Period2 gene (mPer2) in tumor cells influences cell growth and induces apoptosis. Overexpression of PERIOD2 in the mouse Lewis lung carcinoma cell line (LLC) and mammary carcinoma cell line (EMT6) results in reduced cellular proliferation and rapid apoptosis, but not in NIH 3T3 cells. Overexpressed mPER2 also altered the expression of apoptosis-related genes. The mRNA and protein levels of c-Myc, Bcl-XL and Bcl-2 were downregulated, whereas the expression of p53 and bax was upregulated in mPER2-overexpressing LLC cells compared with control cells transferred with empty plasmid. Our results suggest that the circadian gene mPeriod2 may play an important role in tumor suppression by inducing apoptotic cell death, which is attributable to enhanced pro-apoptotis signaling and attenuated anti-apoptosis processes. (Cancer Sci 2006; 97: 589–596)
Circadian rhythms, which are daily oscillations regulated by an endogenous clock,(1–3) enable organisms to adapt to daily environmental changes such as light, temperature and social communication, and serve to synchronize multiple molecular, biochemical, physiological and behavioral processes.(4–6) Recent studies suggest that the circadian system is not only required for proper growth control, but is also involved in the circadian regulation of cell proliferation and apoptosis.(7–9) It has been reported that 2–10% of all mammalian genes are clock-controlled.(4,5,10–12) Most of these show tissue- or organ-specific expression and are involved in organ function. Only a small set of clock-controlled genes are expressed in multiple organs. Among them are genes that encode key regulators of cell cycle progression.(10,11) Deregulation of the circadian clock may disturb the expression of clock-controlled genes and can have a profound influence on organ function.
It is now documented that alterations in circadian rhythm can be associated with cancers in both animal and human tumors.(13,14) Up to now, at least eight core circadian clock genes have been identified in mammals and humans,(2,15) and the Period2 gene is regarded as an indispensable component of the circadian clock.(16) Gene targeting studies have demonstrated that the deletion of mPer2 induces arrhythmicity at both the behavioral and molecular levels.(17) Mice without mPER2 function have a transient rhythm with a shortened period length of 22 h. A majority of mutant mice lose the persistence of circadian rhythm when placed in free-running conditions.(18) The mice deficient in the mPer2 gene were cancer-prone. After γ radiation, these mice showed a marked increase in tumor development and reduced apoptosis in thymocytes. 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.(19)
The relationship between mPer2 and cancer still lacks direct demonstration. In the present study, we transferred pcDNA3.1(+)-mPer2 into LLC cells, EMT6 cells and NIH 3T3 cells, assessed the effect of mPER2 overexpression on these cells and explored the possible signaling pathways leading to apoptotic cell death. Results show that overexpression of mPER2 may induce tumor cell apoptosis by the p53-mediated mitochondrial signaling pathway.
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- Materials and Methods
In the present study, the overexpression of mPER2 in mouse LLC and EMT6 cells was first described. As a core circadian gene, mPer2 has the function of not only maintaining the circadian rhythm of cells, but also sustaining the normal cell cycle. mPer2 exhibited a significant growth-inhibitory effect on LLC and EMT6 cells, as evaluated by MTT assay, cell growth curves and cell colony-forming assay. Several lines of evidence, including flow cytometry, DNA laddering and ultramicrostructure, consistently documented cell apoptosis in LLC and EMT6 cells. However, mPer2 overexpression neither inhibited cell growth nor induced apoptosis in NIH 3T3 cells. The inhibition of endogenous mPer2 expression by antisense oligonucleotides attenuated serum starvation-induced cell death.
The results of the present study suggest that overexpressed mPER2 may alter the expression of apoptosis-related genes. Recent studies have reported that approximately 7% of all clock-controlled genes identified in rodents regulate either cell proliferation or apoptosis. These clock-controlled genes involved in regulating the cell cycle and apoptosis include c-Myc and the tumor suppressor p53, as well as genes that encode caspases, cyclins and transcription factors.(10,11,19,22) The rhythmic expression of several cyclins, as well as that of the tumor suppressor p53, are synchronized with the circadian oscillation patterns of Per1 and Bmal1 expression in human oral mucosa.(23,24)
The tumor suppressor p53 is a transcription factor that is activated in response to DNA damage or oncogenic transformation. Loss of p53 in many cancers leads to impaired cell cycle regulation, genomic instability and inhibition of apoptosis.(25–27) p53 can induce a transient arrest in G1 in cells, thus the cells have time to repair damaged DNA. Activated p53 can also eliminate cells through mechanisms involving prolonged arrest in G1 or apoptosis. The elimination of damaged, stressed or abnormally proliferating cells by p53 is considered to be the principal means by which p53 mediates tumor suppression. The levels of p53 mRNA and protein in mPER2-overexpressing LLC cells tended to be higher than in the vector control cells. This indicated that overexpressed mPER2 induced an increase in p53 levels, which may contribute, at least in part, to apoptosis in mPER2-overexpressing cells.
The c-MYC oncoprotein is a transcription factor that promotes cell growth and proliferation, as well as apoptosis under certain conditions. The c-Myc P1 promoter is suppressed directly by the core circadian regulator Bmal1, indicating that c-Myc is a clock-controlled gene.(24) The expression of c-Myc mRNA shows a significant increase in all mouse tissues studied in Per2 mutants. In the present study, there was an apparent decrease in c-MYC mRNA and protein levels in mPER2-overexpressing cells than in vector control cells. Downregulation of c-Myc is one possible mechanism by which overexpression of mPER2 could promote apoptosis. The overexpression of mPER2 induces Bmal1 expression throughout 12:12 L : D cycles, and then increases intracellular levels of Bmal1–Npas2 or Bmal1–Clock proteins, and repression of c-Myc. c-Myc is demonstrated to be repressed by p53 in a number of mouse and human cell lines and mouse tissues. They provide evidence that p53 binds to the c-Myc promoter in vivo and represses the promoter through a mechanism that involves histone deacetylation.(28) Whether mPER2 downregulates c-Myc expression in a p53-dependent manner or not warrants further investigation.
Recent studies suggest that cytosolic p53 may interact directly with members of the Bcl-2 family of apoptosis regulators, thereby triggering mitochondrial outer membrane permeabilization and apoptosis. The Bcl-2 family of proteins is a key regulator of the mitochondrial response to apoptotic signals in the intrinsic pathway. The Bcl-2 gene family comprises more than 20 different members that regulate apoptosis either positively or negatively.(29,30) It includes antiapoptotic Bcl-2 members, such as Bcl-2, Bcl-XL, Bcl-W and Mcl-1, acting as potent suppressors of apoptosis by blocking the release of cytochrome c. Whereas proapoptotic members, such as Bax, Bak, Bad, Bcl-Xs, Bid, Bik, Bim and Hrk, act as promoters that have opposing functions and promote cell death, the pro-apoptotic proteins Bax, Bak and Bid translocate to the mitochondria with apoptotic stimuli. The ratio of antiapoptotic-to-proapoptotic molecules determines the response to a death signal.
In the present study, we checked the expression of BCL-2, BAX, BCL-XL and the ratio of BAX to BCL-XL. As shown in Fig. 6, the levels of BAX mRNA and protein were increased significantly in mPER2-overexpressing cells compared with vector control cells. In contrast, we showed an apparent decrease in the mRNA and protein levels of BCL-2 in mPER2-overexpressing cells compared with vector control cells. The mRNA and protein levels of BCL-XL were decreased significantly in mPER2-overexpressing cells compared with vector control cells. The mean values for the Bax : Bcl-XL mRNA ratios were higher in mPER2-overexpressing cells than in vector control cells. Western blot analysis also demonstrated an increased ratio of BAX to BCL-XL in mPER2-overexpressing cells, compared with control cells. These may be involved in the effect of mPER2 on apoptosis.
In summary, overexpression of mPER2 results in reduced cellular proliferation and rapid apoptosis in tumor cells, but not in NIH 3T3 cells. Overexpressed mPER2 alters the expression of apoptosis-related genes and regulates the expression of proteins involved directly or indirectly in apoptosis. In addition to p53-dependent apoptosis, there may be other mechanisms by which overexpressed mPER2 induces apoptosis. The exact character of the tumor-associated death pathway activated by mPER2 still needs to be studied. An important clue to the nature of this apoptotic pathway may come from results showing that mPER2 preferentially kills tumorigenic cells but not non-tumorigenic NIH 3T3 cells, indicating that tumor-specific client proteins with which mPER2 interacts may be required for initiating the programmed cell death. Other circadian regulators may also play a similar role in tumor suppression. Cancer should no longer be treated as a local disorder, but a general disorder regulated by the circadian clock. Future research should be focused on studying the detailed mechanisms by which the circadian clock controls genes related to cell proliferation and apoptosis.