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[Cancer Research 65, 6828-6834, August 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Cryptochrome, Circadian Cycle, Cell Cycle Checkpoints, and Cancer

Michele A. Gauger and Aziz Sancar

Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina

Requests for reprints: Aziz Sancar, Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, 314 Mary Ellen Jones Building, CB 7260, Chapel Hill, NC 27599. Phone: 919-962-0115; Fax: 919-843-8627; E-mail: aziz_sancar{at}med.unc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been reported that disruption of the circadian clock may lead to increased risk of breast cancer in humans and to a high rate or ionizing radiation–induced tumors and mortality in mice. Cryptochrome 1 and cryptochrome 2 proteins are core components of the mammalian circadian clock and mice mutated in both genes are arrhythmic. We tested Cry1–/–Cry2–/– mice and fibroblasts derived from these mice for radiation-induced cancer and killing and DNA damage checkpoints and killing, respectively. We find that the mutant mice are indistinguishable from the wild-type controls with respect to radiation-induced morbidity and mortality. Similarly, the Cry1–/–Cry2–/– mutant fibroblasts are indistinguishable from the wild-type controls with respect to their sensitivity to ionizing radiation and UV radiation and ionizing radiation–induced DNA damage checkpoint response. Our data suggest that disruption of the circadian clock in itself does not compromise mammalian DNA repair and DNA damage checkpoints and does not predispose mice to spontaneous and ionizing radiation–induced cancers. We conclude that the effect of circadian clock disruption on cellular response to DNA damage and cancer predisposition in mice may depend on the mechanism by which the clock is disrupted.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The circadian clock and cell cycle are two global regulatory systems in most eukaryotic organisms. It has been known for some time that disruption of the circadian rhythm by genetic or environmental factors causes a variety of disorders in humans, such as sleep disturbances, seasonal affective disorder, and jet lag. Disruption of cell cycle regulation causes cancer. Recent epidemiologic studies have raised the possibility that disruption of the circadian clock may also increase cancer risk in humans (1) and adversely affect prognosis in cancer patients (2). In particular, it was reported that women working nightshift exhibited a significant increase in breast cancer risk (3, 4). Similarly, it was reported that cancer patients with altered circadian rhythm had poorer survival relative to patients with normal rhythm (5).

These epidemiologic studies were complemented by studies with mouse model systems. In one study, transplantation of an osteosarcoma or a pancreatic adenocarcinoma into mice with ablations to the master circadian clock, the suprachiasmatic nuclei, caused accelerated tumor growth rate relative to animals with intact suprachiasmatic nuclei (6). In a second study, it was found that mice that were rendered arrhythmic by repeat 8-hour advance of the light-dark cycle every 2 days exhibited faster rates of implanted tumor growth relative to control mice maintained under a light/dark 12-hour/12-hour cycle (7).

Finally, the circadian rhythm was disrupted in mice by targeted mutations of the core clock genes that engender the molecular clock not only in the suprachiasmatic nuclei but in all peripheral organs and the effects of this disruption on cell growth and spontaneous and ionizing radiation–induced tumor incidence were analyzed. The core clock proteins are Clock and Bmal1 that act as transcriptional activators of the Cryptochrome (Cry), Period (Per), and Bmal1 genes and the Cry1, Cry2, Per1, and Per2 proteins that function as transcriptional repressors of the Clock-Bmal1-driven genes (refs. 8, 9; Fig. 1). The effect of these core clock proteins is modulated by additional proteins such as Rev-Erb{alpha} and CK1{varepsilon} to generate a rather precise molecular oscillator with ~24-hour periodicity. This periodicity is transmitted to the clock-controlled genes (CCG) that constitute about 10% of the expressed genes in a given tissue to generate rhythmic outputs at the physiologic and behavioral levels (10). The molecular mechanism of the mammalian circadian clock has been elucidated in considerable detail in recent years, making it possible to investigate the interfacing of this global regulatory pathway with other global regulatory systems, such as cell cycle checkpoints, at a mechanistic level. One such study found that in mice with a Per2 mutation, c-Myc transcription was up-regulated and p53 was down-regulated; consequently, these animals had increased incidence of spontaneous and ionizing radiation–induced lymphomas and an increased rate of mortality after ionizing radiation (11). Another study reported that in cryptochromeless mice, Wee1 antimitotic kinase was elevated and, consequently, liver regeneration in these mice following partial hepatectomy was delayed relative to wild-type controls (12). Finally, we have recently found that the mammalian Timeless protein, which is considered to be a clock protein according to some studies (13) but not others (14), binds to DNA damage checkpoint proteins ATR and Chk1 and is essential for the DNA damage checkpoint response (15).



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  		Figure 1. Model for circadian clock in mammals. Clock and Bmal1 are transcriptional activators that make up the Clock-Bmal1 complex, which activates the transcription of Cry, Per, and Bmal1 genes. The Cry (Cry1 and Cry2) and Per (Per1 and Per2) proteins are transcriptional repressors that interfere with the activity of Clock-Bmal1 and down-regulate transcription of genes controlled by these factors. In addition, Per and Cry proteins stimulate the transcription of Bmal1 by an ill-defined mechanism. CCGs, which include Wee1 and c-myc, are regulated by the clock proteins but do not affect the activity of the clock proteins or the transcription of the clock genes. Wee1 expression is positively regulated by Clock-Bmal1, and c-Myc expression is repressed by this complex. Hence, in the absence of Cry or Per, Wee1 expression is expected to be upregulated due to lack of inhibition of Clock-Bmal1 by Cry and Per. It has been reported that c-myc transcription is elevated in Per2 mutant mice because of a reduced level of Bmal1, which represses the c-myc promoter.

 
Taken together, the epidemiologic data, the data from mouse model systems, and that from cell-based assays have led to an emerging consensus that the circadian cycle and cell cycle are tightly coupled and that disruption of the circadian cycle by any means would disrupt cell cycle checkpoints as well, causing animals with circadian clock disruption to be more prone to spontaneous and DNA damage-induced cancers (1–4). Within this conceptual framework, then, we wished to examine the effect of circadian clock disruption caused by cryptochrome knockout on DNA damage checkpoints and on predisposition to spontaneous and ionizing radiation–induced cancers. We find that clock disruption by cryptochrome knockout does not measurably affect DNA damage checkpoints and does not cause mice to be more susceptible to cancer. We conclude that circadian clock disruption per se does not prime cells to cancerous transformation, presumably due to the presence of homeostatic mechanisms, in addition to the circadian rhythm, that regulate cell cycle and cellular responses to DNA damage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, ionizing radiation treatment, and survival. Cry1–/–Cry2–/– mice (16, 17) were backcrossed with C57BL/6J six times to obtain Cry mutant mice in essentially the same genetic background as wild-type control C57BL/6J mice obtained from The Jackson Laboratory (Bar Harbor, ME). The mice were maintained on a light/dark 12:12 schedule under ambient room lighting at an ambient temperature of 21°C to 23°C and 50% to 70% humidity. For irradiation treatment, a cesium-137 radiation source emitting {gamma}-rays at a rate of 0.82 Gy/min was used. Twenty-four wild-type and 27 Cry1–/–Cry2–/– mice were treated at 8 weeks of age with a single dose of 4 Gy at zeitgeiber time (ZT) 10. (By convention, ZT0 is the time of lights-on and ZT12 is the time of lights-off).

Fibroblast cell lines, growth rate measurement, and UV and ionizing radiation survival. Dermal fibroblast cell lines were isolated as described (18) using skin biopsies from wild-type and Cry1–/–Cry2–/– mice. Fibroblasts underwent spontaneous immortalization.

Cells were grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gemini, Woodland, CA) and 100 units/mL penicillin and 100 µg/mL streptomycin (Life Technologies). Cells were maintained in an incubator at 37°C under 5% CO2. For growth rate measurements, cells were plated in 150-mm plates at low density to ensure continued proliferation throughout the duration of the experiment. Cells were trypsinized and counted at the indicated time points using a hemocytometer.

Cell survival to radiation was determined by clonogenic assay. Wild-type and Cry1–/–Cry2–/– fibroblasts were plated at low density to ensure the formation of ~200 colonies per 100-mm plate in the absence of radiation treatment. Following plating, cells were incubated in growth medium for 10 to 14 hours and treated with either UV or ionizing radiation of appropriate doses. UV treatment at the indicated doses was done using a GE germicidal lamp emitting mainly at 254 nm. Cells were washed with PBS, irradiated with UV at a fluence rate of 0.65 J/m2s in the absence of growth medium, and new growth medium was added after treatment. For ionizing radiation treatment, cells in growth medium were irradiated from a cesium-137 radiation source at a rate of 0.82 Gy/min. After radiation treatment, cells were incubated for 9 to 10 days until colonies were readily visible. Cells were fixed for 20 minutes in 3:1 methanol/acetic acid, rinsed with water, and stained with Giemsa stain. Colonies containing >50 cells were scored.

Flow cytometry. Fibroblasts were grown in DMEM and plated to achieve a density of one to two million cells at the time of experiment. Cells were treated with ionizing radiation as described above. At the indicated time posttreatment, cells were trypsinized and fixed in 70% ethanol. DNA content analysis was done using propidium iodide staining and a Beckton Dickinson FACScan analytic flow cytometer. Data acquisition and representation was done with Cicero Software (Cytomation, Inc., Fort Collins, CO).

Western blotting. Standard Western blotting procedures were used for Wee1, c-Myc, and Bmal1 proteins from wild-type and Cry mutant mouse liver extracts and fibroblast cell lysates. For some of the Western blots, we used livers from Cry mutant mice in an rd/rd background because of the ready availability of these animals (18). The rd mutation does not affect the molecular clock (8, 9). Anti-Wee1 rabbit polyclonal antibody (H-300, Santa Cruz Biotechnology, Santa Cruz, CA) and anti-cMyc mouse monoclonal antibody (9E10, Santa Cruz Biotechnology) were used to probe for Wee1 and c-Myc, respectively. Anti-Bmal1 guinea pig polyclonal antibody (19) was a kind gift of Dr. Choogon Lee (Florida State University). Anti-IgG rabbit and mouse antibodies (Amersham Biosciences Group, Piscataway, NJ) and anti-IgG guinea pig antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were used for secondary antibody blotting.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of Bmal1, Wee1, and c-Myc in cryptochromeless mice and fibroblasts. In the conventional clock model, Clock and Bmal1 constitute the positive regulatory branch, whereas the Crys and Pers make up the negative branch of the autoregulatory oscillatory circuit (Fig. 1). In addition, Crys and Pers seem to stimulate transcription of Bmal1 by an ill-defined mechanism (8, 9). Consequently, Per and Cry mutants often exhibit similar molecular and behavioral phenotypes. However, it must be noted that there are subtle but significant differences between Cry and Per mutant mice. Hence, it is not possible to predict the responses of Cry or Per mutant cells and mice to a certain treatment based on responses observed with their Per or Cry mutant counterparts, respectively.

Recently, it was reported that in Cry mutant mice, the level of antimitotic Wee1 kinase was elevated in the liver (12) and that in Per2 mutant mice, c-myc transcription was up-regulated (11). Both genes are CCGs that contain multiple E-boxes in their promoters, which are recognized by the Clock-Bmal1 complex. In the case of wee1, the binding of Clock-Bmal1 to the promoter stimulates transcription; in the case of c-myc, binding of the heterodimer (or of the NPas2-Bmal1 complex) inhibits transcription. The elevation of Wee1 in the Cry mutant was ascribed to the lack of inhibition of Clock-Bmal1 by cryptochrome (12, 20). Up-regulation of c-myc transcription in the Per2 mutant was ascribed to the reduced level of Bmal1 because Per2, in addition to its inhibitory effect on the Clock-Bmal1 complex, stimulates transcription of the Bmal1 gene (11, 21, 22). Thus, to begin to investigate the effect of cryptochrome knockout on cellular and organismic response to DNA damage, we wished first to determine the expression of Bmal1, c-Myc, and Wee1 in Cry mutant mice and fibroblasts.

Bmal1 is expressed very highly and at comparable levels in wild-type and Cry mutant fibroblasts (Fig. 2A). However, in comparing Bmal1 expression in the liver, we find that Bmal1 is expressed with a circadian periodicity in wild-type liver but at a reduced and nonoscillating level in the Cry mutant (Fig. 2B), in agreement with a previous report (19). The level of c-Myc has not been previously analyzed in Cry mutant mice. However, it was reported that c-Myc transcription is up-regulated in Per2 mutant mice, presumably due to a reduced Bmal1 level (11). Because Bmal1 is reduced in the Cry mutant, we expected to observe elevated c-Myc levels in Cry mutant mice and possibly fibroblasts as well. Figure 2C-D shows that c-Myc is expressed in mutant and wild-type fibroblasts at comparable levels and at statistically indistinguishable and nonoscillating levels in the livers of mutant and wild-type mice. These results differ from those of the previous study, which reported that reduction in Bmal1 levels as a consequence of Per2 mutation causes a substantial increase in c-Myc activity (11). However, in that study the c-Myc RNA but not protein level was measured. Regardless of the cause of the discrepancy between the two studies, it seems that a decrease in the Bmal1 transcriptional regulator does not necessarily lead to increased c-Myc protein in the mouse liver or fibroblasts.



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  		Figure 2. Effect of cryptochrome on Bmal1 and c-Myc expression in mouse fibroblasts and mouse liver. A and B, Bmal1 expression analysis. A, (200 µg) from wild-type (WT) or Cry mutant fibroblasts were analyzed for Bmal1 expression by Western blotting using actin as a loading control. B, liver extracts from wild-type (WT) and Cry mutant mice were prepared at the indicated zeitgeiber times (ZT0 = lights on, ZT12 = lights off) and analyzed for Bmal1 expression using actin as a loading control. It has been reported that in the liver of wild-type mice, Bmal1 levels are lowest at ZT6 and highest at ZT18. C and D, c-Myc expression analysis. Extracts (200 µg) from fibroblasts (A) or mouse liver (B) were probed for Wee1 and actin by Western blotting. Top, Western blot; bottom, quantitative analysis of Western blot. Averages of three independent experiments, including the one shown in top. Columns, average (n = 3); bars, SD.

 
Because it has been reported that Cry and Per regulate Wee1 (12) and c-Myc transcription through their effects on Bmal1 activity and Bmal1 transcription, respectively, we reasoned that the reduced Bmal1 in the Cry mutant would be accompanied by elevated Wee1. Figure 3A-B shows that Wee1 is indeed elevated in Cry mutant fibroblasts and liver, respectively, relative to the wild-type control and the moderately elevated (relative to the low level of Wee1 in wild-type mice) Wee1 in Cry mutant liver does not oscillate, in agreement with previous reports (12, 20).



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  		Figure 3. Effect of cryptochrome on Wee1 expression and growth rate of mouse fibroblasts. A and B, Wee1 expression analysis. Extracts (200 µg) from fibroblasts (A) or mouse liver (B) were probed for Wee1 and actin by Western blotting. Liver extracts were prepared at the indicated zeitgeiber times. C, growth kinetics of immortalized fibroblasts from wild-type ({bullet}) and Cry1–/–Cry2–/– ({blacktriangleup}). Cells were plated at densities of ~104 cells per dish and cells were counted at the indicated time points. Points, n = 3; bars, SD.

 
DNA damage checkpoints and DNA repair in cryptochromeless fibroblasts. Wee1 is a cell cycle kinase that plays a key role in the G2-M transition. Ongoing DNA replication or the presence of DNA damage activate Wee1, which then phosphorylates Y14 of the Cdc2 mitotic kinase, causing its inactivation and delay of mitosis or arrest of the cell cycle at the G2-M interface (23, 24). It is conceivable that elevated Wee1 in Cry mutant mice phosphorylates Cdc2 at an increased rate even in nonstressed cells, slowing down the G2-M transition and the overall growth rate. Indeed, it was reported that 3 days after partial hepatectomy, the regenerating liver of cryptochromeless mice contained fewer mitotic figures and lagged in weight recovery by about 15% relative to the wild-type control (12). Based on these considerations, then, we expected cryptochromeless fibroblasts to grow more slowly than wild-type fibroblasts and to arrest for a longer period at the G2-M boundary after DNA damage, possibly remaining at this interface indefinitely and thus undergoing replicative death. Interestingly, we did not observe either of these two effects. First, cryptochromeless fibroblasts grew at a rate indistinguishable from wild-type fibroblasts under standard growth conditions (Fig. 3C). Second, and more importantly, after 8 Gy of ionizing radiation, the cryptochromeless fibroblasts seem to arrest at G2-M and to recover from this arrest at the same rate as wild-type fibroblasts (Table 1). It is likely that multiple factors contribute to the difference between the reported effect of cryptochrome on liver regeneration rate and our results with tissue culture. It is also possible that the tissue culture conditions do not possess the requisite sensitivity to detect a 15% difference in growth rate. Regardless of these considerations, however, it is safe to conclude that the ~3-fold increase in Wee1 level in cryptochromeless cells does not cause a comparable decrease in cell growth rate and it has no detectable effect on the DNA damage checkpoint response.


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  		Table 1. G2-M checkpoint in wild-type and Cry mutant fibroblasts after ionizing radiation treatments

 
As a further test of DNA damage checkpoint response in the absence of a functional clock, we determined the survival of cryptochromeless fibroblasts to UV and ionizing radiation. Figure 4 shows that the Cry mutant is indistinguishable from wild type in its response to both genotoxicants. As most checkpoint defects increase cellular sensitivity to genotoxicants (23, 24) these results further confirm the conclusion based on Table 1 that in the absence of cryptochrome, DNA damage checkpoints are normal. Of equal significance, the data in Fig. 4 also reveals that nucleotide excision repair (as measured by UV resistance) and double-strand break repair (as measured by resistance to ionizing radiation) are unaffected by loss of the clock. Thus, we conclude that, in the absence of a circadian clock, both DNA damage checkpoints and the two major DNA repair pathways for repairing base damage and backbone breaks operate essentially normally.



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  		Figure 4. Radiation survival of wild-type and cryptochrome mutant fibroblasts. A, UV survival; B, ionizing radiation (IR) survival. {bullet}, wild-type; {blacktriangleup}, Cry mutant. Points, averages of three independent experiments (n = 3); bars, SD.

 
Ionizing radiation–induced morbidity and mortality in cryptochromeless/clockless mice. Per2 mutant mice lack circadian rhythm and were reported to be more sensitive to the acute and chronic effects of ionizing radiation relative to wild-type controls (11). The acute effects included hair graying, hair loss, and skin ulcerations. Chronic effects were an ~10-fold increase in incidence of lymphomas relative to wild-type controls and a comparable increase in mortality within 70 weeks following treatment with 4 Gy of ionizing radiation. The increased incidence of lymphomas and higher mortality rate were ascribed in part to the elevated c-Myc and in part to a general dysregulation of cell cycle genes as a result of clock disruption.

To determine whether or not clock disruption by any means has similar effects on ionizing radiation–induced morbidity and mortality, we irradiated 8-week-old Cry1–/–Cry2–/– mice and wild-type controls with 4 Gy of ionizing radiation at ZT10 and followed their survival for 90 weeks. The results obtained differed from those obtained with Per2 mutant mice. First, we did not observe a difference in the timing and intensity of hair graying and loss between Cry mutant and wild-type mice (data not shown). Second, and most significantly, over the 90-week observation period there was no significant difference in the mortality of irradiated Cry mutant mice and wild-type mice (Fig. 5). Moreover, in contrast to the similarly treated Per2 mutant mice, we did not detect overt lymphomas in irradiated Cry mutant animals. The irradiated mice died from a variety of causes including genitourinary prolapses and infections, paralysis, and seizures that necessitated euthanasia, and in some cases from indeterminable causes. Importantly, however, there was no detectable difference between the causes of death of cryptochromeless and wild-type animals. These results suggest that clock disruption per se does not make mice hypersensitive to the acute effects of ionizing radiation nor does it predispose them to increased incidence of spontaneous or ionizing radiation–induced cancers or mortality from any other cause. The significance of these findings is discussed below.



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  		Figure 5. Ionizing radiation survival of wild-type (solid line) and Cry1–/–Cry2–/– (broken line) mice. Eight-week-old mice were exposed to 4 Gy at ZT10 and were observed for 90 weeks. Survival is plotted according to the Kaplan-Meier method.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Circadian cycle and cell cycle. The circadian cycle and cell cycle are two global regulatory mechanisms that directly or indirectly influence all biochemical reactions in cells. Hence, it is logical to assume that disruption of one would cause dysregulation of the other with adverse consequences for the cell (25–27). Recent advances in understanding the circadian clock at the molecular level have provided the opportunity to approach the circadian cycle cell cycle connection from a mechanistic perspective. Thus, it was reported that the core circadian clock machinery affects cell cycle progression in proliferating cells by controlling the expression of Wee1, a kinase that regulates the activity of Cdc2 and hence the G2-M transition (12). Another study concluded that Per2 mutant mice had constitutively elevated levels of expression of the cell growth/proliferation gene c-Myc and reduced expression of p53, which plays a critical role in the G1-S checkpoint (11). Finally, we have recently found that the mammalian Tim plays a direct role in cell cycle checkpoints including the intra-S and replication checkpoints (15). Although the role of the mammalian Tim in the circadian clock is a matter of some debate (13, 14), these studies in aggregate have given credence to the general view that circadian disruption by whatever means may lead to a failure of cell cycle checkpoints (28). However, this view needs to be critically tested experimentally. In particular, it remains to be determined if proteins that have been presumed to be exclusively clock proteins such as Clock and Bmal1, which work as partners in the positive branch of the clock, and Cry and Per, which work as partners in the negative branch, also perform functions outside of the clock and unique to each protein. In the case of mammalian Tim, it is clear that the protein participates in cell cycle control independent of its role in the clock (15). If the so-called canonical clock proteins Clock, Bmal1, Cry, and Per have unique cellular functions outside of the clock machinery, then the mutants of these proteins are expected to exhibit some unique features as well. An additional possibility is that different clock mutants disturb the circadian cycle in different ways, leaving a different pattern of circadian genes "on" and "off" depending on when the clock stops. Although the molecular clock does not "stop" at a given time but rather falls into a stable equilibrium that terminates the cycles in the abundance of its components, one can imagine that leaving certain circadian genes "on" or "off" might cause a susceptibility to cancer or not, without necessarily making cancer susceptibility caused by clock disruption specific to a particular clock gene. Moreover, cell cycle and carcinogenic transformation are also regulated by homeostatic mechanisms and therefore a potentially cell cycle–disruptive effect of a clock gene mutation might be mitigated or completely alleviated by such compensatory mechanisms. Consequently, a priori it cannot be known whether all circadian disruptions will have the same effect on cell cycle and DNA damage checkpoints and whether their potential cell cycle-disruptive effects will necessarily lead to actual disruption.

Cell cycle checkpoints in the absence of circadian rhythm. In this study, we analyzed the growth properties, cell cycle checkpoints, and DNA repair capacity of Cry1–/–Cry2–/– fibroblasts and the susceptibility to ionizing radiation–induced cancer and mortality of Cry1–/–Cry2–/– mice. Based on published reports of Cry and Per mutations on cellular growth (12) and damage response (11), we were expecting the Cry mutants to be defective in DNA damage checkpoints and to exhibit increased ionizing radiation–induced morbidity and mortality for the reasons outlined below.

First, current clock models presume that Cry and Per function as heterodimers and because it has been reported that in Per2 mutant mice, c-Myc is up-regulated, p53 is down-regulated, and there is a general cell cycle dysregulation (11), we expected that the Cry mutant fibroblasts would exhibit some cell cycle checkpoint defects and that the Cry mutant mice, like the Per2 mutants, would be cancer prone. We find that in Cry mutant livers, Bmal1 expression, as in the case of the Per2 mutant, is reduced. However, in an apparent contrast to the Per2 mutant mice, the c-Myc level is not elevated in either Cry fibroblasts or mice, indicating that Pers and Crys affect c-Myc expression differently. It must be noted, however, that in the Per2 mutant mice the c-Myc RNA but not the protein level was measured. It is conceivable that even in the Per2 mutant mice the elevated level of c-Myc mRNA is not accompanied by elevated c-Myc protein and that the increased incidence of spontaneous and ionizing radiation–induced lymphomas reported in these animals was caused by an unknown effect of Per2 on cell growth and proliferation.

Second, it was reported (12) that Wee1 kinase, which inhibits the G2-M transition, is elevated in Cry mutant mice and evidence was presented suggesting that after partial hepatectomy, the liver of Cry mutant mice regenerates more slowly than that of wild-type controls, presumably because of inhibition of mitosis by elevated Wee1. In agreement with previous reports (12, 20), we find that Wee1 is elevated in Cry mutant fibroblasts and liver and other tissues of Cry mutant mice. However, despite this elevation in Wee1 level the Cry mutant fibroblasts grow at a rate indistinguishable from the wild-type controls. It seems that exponentially growing cultures of mutant fibroblasts had fewer mitotic figures than wild-type (data not shown). We assume that a slight delay in mitotic entry was compensated by faster progression through other phases of the cell cycle such that there was no change in overall growth rate relative to the control. Importantly, the mutant cells did not exhibit an amplified checkpoint response to DNA damage and, consequently, their kinetics of checkpoint-induced inhibition of cell cycle progression through G2-M was indistinguishable from wild-type controls. This again indicates the presence of compensatory mechanisms that ensure normal checkpoint response even in the presence of elevated Wee1. Our results seem contradictory to the report indicating slower recovery of liver mass in Cry mutant mice after partial hepatectomy (12). However, it is possible that the apparent discrepancy may stem from differences in stress responses induced by DNA damaging agents, as opposed to partial hepatectomy, and the nature of the cell types analyzed in the two studies.

Circadian disruption and cancer predisposition. Epidemiologic studies have suggested that circadian disruption may contribute to cancer incidence (1) and adversely affect the course of the disease (2). A prospective study with Per2 mutant mice seems to have provided a molecular explanation for the connection between circadian rhythm disruption and cancer predisposition. Our work indicates that disruption of the clock does not necessarily predispose mice to cancer. The cancer predisposition of Per2 mutant mice was ascribed, in large part, to decreased Bmal1 expression and the consequent increase in c-Myc expression. Bmal1 expression is reduced in both Per2 and Cry mutant mice (ref. 19; this work); hence, it remains to be proven that the increased c-Myc transcription reported in Per2 mutant mice is a direct consequence of Bmal1 reduction, which, in the form of either Bmal1-NPas2 or Bmal1-Clock heterodimer, represses c-Myc transcription (11). Whatever the cause of elevation of c-Myc transcription in Per2 mutants, we do not observe a measurable change in the c-Myc protein level in Cry mutant mice; hence, it is possible that the absence of Per2 makes mice cancer-prone not by overexpression of c-Myc but through an unknown mechanism. It must be noted, however, that the ionizing radiation–induced mortality of the wild-type mice in our study was the same as that of the Cry mutant and, importantly, it was significantly higher than that of the wild-type control mice used in the Per2 mutant mouse study. It is possible that the genetic background (C57BL/6J in our study and C57/SV129 in the Per2 study) affects the susceptibility of even "wild-type" mice to both ionizing radiation–induced cancers and ionizing radiation–induced mortality. In our study, both the Cry mutant and the wild-type control mice were in C57BL/6J background; therefore, we suggest that the lack of difference in morbidity and mortality between the wild-type and Cry mutant mice is most likely because circadian clock disruption by eliminating Cry does not affect cell cycle checkpoints, DNA repair, or apoptosis in a way that would result in increased mutations, reduced apoptosis, and eventually cancer.

It should be of interest to find out how mutations in other clock genes, in particular Clock and Bmal1, affect the incidence of spontaneous and ionizing radiation–induced cancers. While this article was in preparation, a report was published showing that the sensitivity of mice to the acute effects (weight loss and death) of high doses of cyclophosphamide, an alkylating anticancer drug, was strongly dependent on the circadian time of drug delivery (29) and that the Cry mutant used in our study was resistant to the acute effects of cyclophosphamide at all times of the day. Clearly, further studies are needed to explain the apparent resistance of Cry mutant mice to the acute effects of cyclophosphamide. Regardless of the precise mechanism of the resistance, the results of the study on the acute effects of a DNA damaging agent and our study on the long-term effects of ionizing radiation are, in general, in agreement in demonstrating that clock disruption per se does not make mice more susceptible to the acute or chronic effects of DNA-damaging agents.


   Acknowledgments
 
Grant support: NIH grants GM31082 and GM32833, NIEHS grant ES11659, and DOD grant W81XWH-04-1-0387.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Choogon Lee for his generous gift of Bmal1 antibodies, Dr. Christopher P. Selby (Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC) for providing the Cry mutant mice, Dr. Terry Van Dyke for helpful advice on mouse cancer biology, and Drs. Keziban Ünsal-Kaçmaz and Laura Lindsey-Boltz for useful comments on the article.

Received 4/ 1/05. Revised 5/ 2/05. Accepted 5/13/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Stevens RG. Circadian disruption and breast cancer: from melatonin to clock genes. Epidemiology 2005;16:254–8.[CrossRef][Medline]
  2. Mormont LC, Lévi F. Circadian-system alterations during cancer processes: a review. Int J Cancer 1997;70:241–7.[CrossRef][Medline]
  3. Davis S, Mirick DK, Stevens RG. Night shift work, light at night, and risk of breast cancer. J Natl Cancer Inst 2001;93:1557–62.[Abstract/Free Full Text]
  4. Schernhammer ES, Laden F, Speizer FE, et al. Rotating night shifts and risk of breast cancer in women participating in the nurses' health study. J Natl Cancer Inst 2001;93:1563–8.[Abstract/Free Full Text]
  5. Mormont MC, Waterhouse J, Bleuzen P, et al. Marked 24-h rest/activity rhythms are associated with better quality of life, better response, and longer survival in patients with metastatic colorectal cancer and good performance status. Clin Cancer Res 2000;6:3038–45.[Abstract/Free Full Text]
  6. Filipski E, King VM, Li X, et al. Host circadian clock as a control point in tumor progression. J Natl Cancer Inst 2002;94:690–7.[Abstract/Free Full Text]
  7. Filipski E, Delaunay F, King VM, et al. Effects of chronic jet lag on tumor progression in mice. Cancer Res 2004;64:7879–85.[Abstract/Free Full Text]
  8. Young MW, Kay SA. Time zones: a comparative genetics of circadian clocks. Nat Rev Genet 2001;2:702–15.[CrossRef][Medline]
  9. Reppert SM, Weaver DR. Coordination of circadian timing in mammals. Nature 2002;418:935–41.[CrossRef][Medline]
  10. Delaunay F, Laudet V. Circadian clock and microarrays: mammalian genome gets rhythm. Trends Genet 2002;18:595–7.[CrossRef][Medline]
  11. Fu L, Pelicano H, Liu J, Huang P, Lee C. The circadian gene Period 2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 2002;111:41–50.[CrossRef][Medline]
  12. Matsuo T, Yamaguchi S, Mitsui S, Emi A, Shimoda F, Okamura H. Control mechanism of the circadian clock for timing of cell division in vivo. Science 2003;302:255–9.[Abstract/Free Full Text]
  13. Barnes JW, Tischkau SA, Barnes JA, et al. Requirement of mammalian Timeless for circadian rhythmicity. Science 2003;302:439–42.[Abstract/Free Full Text]
  14. Gotter AL, Manganaro T, Weaver DR, et al. A time-less function for mouse timeless. Nat Neurosci 2000;3:755–6.[CrossRef][Medline]
  15. Ünsal-Kaçmaz K, Mullen TE, Kaufmann WK, Sancar A. Coupling of human circadian and cell cycles by the Timeless protein. Mol Cell Biol 2005;25:3109–16.[Abstract/Free Full Text]
  16. Selby CP, Thompson C, Schmitz TM, Van Gelder RN, Sancar A. Functional redundancy of cryptochromes and classical photoreceptors for nonvisual ocular photoreception in mice. Proc Natl Acad Sci U S A 2000;97:14697–702.[Abstract/Free Full Text]
  17. Vitaterna MH, Selby CP, Todo T, et al. Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc Natl Acad Sci U S A 1999;96:12114–9.[Abstract/Free Full Text]
  18. Thompson CL, Selby CP, Partch CL, et al. Further evidence for the role of cryptochromes in retinohypothalamic photoreception/phototransduction. Mol Brain Res 2004;122:158–66.[Medline]
  19. Lee C, Etchegaray JP, Cagampang FR, Loudon AS, Reppert SM. Posttranslational mechanisms regulate the mammalian circadian clock. Cell 2001;107:855–67.[CrossRef][Medline]
  20. Oishi K, Miyazaki K, Kadota K, et al. Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes. J Biol Chem 2003;278:41519–27.[Abstract/Free Full Text]
  21. Hogenesch JB, Panda S, Kay S, Takahashi JS. Circadian transcriptional output in the SCN and liver of the mouse. Novartis Found Symp 2003;253:171–80.[Medline]
  22. Shearman LP, Sriram S, Weaver DR, et al. Interacting molecular loops in the mammalian circadian clock. Science 2000;288:1013–9.[Abstract/Free Full Text]
  23. Nyberg KA, Michelson RJ, Putnam CW, Weinert TA. Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 2002;36:617–56.[CrossRef][Medline]
  24. Sancar A, Lindsey-Boltz LA, Ünsal-Kaçmaz K, Linn S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem 2004;73:39–85.[CrossRef][Medline]
  25. Bjarnason GA, Jordan R. Circadian variation of cell proliferation and cell cycle protein expression in man: clinical implications. Prog Cell Cycle Res 2000;4:193–206.[Medline]
  26. Nagoshi E, Saini C, Bauer C, Laroche T, Naef F, Schibler U. Circadian gene expression in individual fibroblasts: cell-autonomous and self-sustained oscillators pass time to daughter cells. Cell 2004;119:693–705.[Medline]
  27. Welsh DK, Yoo S, Liu AC, Takahashi JS, Kay SA. Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression. Curr Biol 2004;14:2289–95.[CrossRef][Medline]
  28. Fu L, Lee CC. The circadian clock: pacemaker and tumor suppressor. Nat Rev Cancer 2003;3:350–61.[CrossRef][Medline]
  29. Gorbacheva VY, Kondratov RV, Zhang R, et al. Circadian sensitivity to the chemotherapeutic agent cyclophosphamide depends on the functional status of the CLOCK/BMAL1 transactivation complex. Proc Natl Acad Sci U S A 2005;102:3407–12.[Abstract/Free Full Text]



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