An association between clock genes and clock‐controlled cell cycle genes in murine colorectal tumors

Disruption of circadian machinery appears to be associated with the acceleration of tumor development. To evaluate the function of the circadian clock during neoplastic transformation, the daily profiles of the core clock genes Per1, Per2, Rev‐Erbα and Bmal1, the clock‐controlled gene Dbp and the clock‐controlled cell cycle genes Wee1, c‐Myc and p21 were detected by real‐time RT‐PCR in chemically induced primary colorectal tumors, the surrounding normal tissue and in the liver. The circadian rhythmicity of Per1, Per2, Rev‐Erbα and Dbp was significantly reduced in tumor compared with healthy colon and the rhythmicity of Bmal1 was completely abolished. Interestingly, the circadian expression of Per1, Per2, Rev‐Erbα and Dbp persisted in the colonic tissue surrounding the tumor but the rhythmic expression of Bmal1 was also abolished. Daily profiles of Wee1, c‐Myc and p21 did not exhibit any rhythmicity either in tumors or in the colon of healthy animals. The absence of diurnal rhythmicity of cell cycle genes was partially associated with ageing, because young healthy mice showed rhythmicity in the core clock genes as well as in the Wee1 and p21. In the liver of tumor‐bearing mice the clock gene rhythms were temporally shifted. The data suggest that the circadian regulation is distorted in colonic neoplastic tissue and that the gene‐specific disruption may be also observed in the non‐neoplastic tissues. These findings reinforce the role of peripheral circadian clockwork disruption for carcinogenesis and tumor progression.     

Critical cholangiocarcinogenesis control by cryptochrome clock genes

A coordinated network of molecular circadian clocks in individual cells generates 24‐hr rhythms in liver metabolism and proliferation. Circadian disruption through chronic jet lag or Per2 clock gene mutation was shown to accelerate hepatocarcinoma development in mice. As divergent effects were reported for clock genes Per and Cry regarding xenobiotic toxicity, we questioned the role of Cry1 and Cry2 in liver carcinogenesis. Male WT and Cry1^?/?Cry2^?/? mice (C57Bl/6 background) were chronically exposed to diethylnitrosamine (DEN) at ZT11. Rest‐activity and body temperature rhythms were monitored using an implanted radiotransmitter. Serum aspartate and alanine aminotransferases (AST and ALT) were determined on four occasions during the progression stage. After 7 months, serum alkaline phosphatases (ALP) were determined, and livers were sampled for microscopic tumor nodule counting and histopathology. Five months after initiation of DEN treatment, we found that Cry1^?/?Cry2^?/? mice developed severe liver dysplasia, as evident from the increased AST, ALT and ALP levels, as compared to WT mice. DEN exposure induced primary liver cancers in nearly fivefold as many Cry1^?/?Cry2^?/? mice as compared to WT mice (p = 0.01). Microscopic study revealed no difference in the average number of hepatocarcinomas and a nearly eightfold increase in the average number of cholangiocarcinomas in Cry1^?/?Cry2^?/? mice, as compared to WT mice. This study validated the hypothesis that molecular circadian clock disruption dramatically increased chemically induced liver carcinogenesis. In addition, the pronounced shift toward cholangiocarcinoma in DEN exposed Cry1^?/?Cry2^?/? mice revealed a critical role of the Cry clock genes in bile duct carcinogenesis.     

Time‐dependent changes in proliferation,DNA damage and clock gene expression in hepatocellular carcinoma and healthy liver of a transgenic mouse model

Hepatocellular carcinoma (HCC) is highly resistant to anticancer therapy and novel therapeutic strategies are needed. Chronotherapy may become a promising approach because it may improve the efficacy of antimitotic radiation and chemotherapy by considering timing of treatment. To date little is known about time‐of‐day dependent changes of proliferation and DNA damage in HCC. Using transgenic c‐myc/transforming growth factor (TGFα) mice as HCC animal model, we immunohistochemically demonstrated Ki67 as marker for proliferation and γ‐H2AX as marker for DNA damage in HCC and surrounding healthy liver (HL). Core clock genes (Per1, Per2, Cry1, Cry2, Bmal 1, Rev‐erbα and Clock) were examined by qPCR. Data were obtained from samples collected ex vivo at four different time points and from organotypic slice cultures (OSC). Significant differences were found between HCC and HL. In HCC, the number of Ki67 immunoreactive cells showed two peaks (ex vivo: ZT06 middle of day and ZT18 middle of night; OSC: CT04 and CT16). In ex vivo samples, the number of γ‐H2AX positive cells in HCC peaked at ZT18 (middle of the night), while in OSC their number remained high during subjective day and night. In both HCC and HL, clock gene expression showed a time‐of‐day dependent expression ex vivo but no changes in OSC. The expression of Per2 and Cry1 was significantly lower in HCC than in HL. Our data support the concept of chronotherapy of HCC. OSC may become useful to test novel cancer therapies.     

Down regulation of circadian clock gene Period 2 accelerates breast cancer growth by altering its daily growth rhythm

Purpose Per2, a core circadian clock gene, has tumor suppressor properties and is mutated or down regulated in human breast cancers. We have manipulated the expression of this gene in vitro and in vivo to more fully understand how the Per2 clock gene product affects cancer growth. Methods We used siRNA and shRNA to down regulate Per2 expression in vitro and in vivo and measured cancer cell proliferation, tumor growth rate and several molecular pathways relevant to cancer growth and their circadian organizations. All statistical tests were two-sided. Results Down regulation of functional Per2 gene expression increases Cyclin D and Cyclin E levels and doubles in vitro breast cancer cell proliferation (P < 0.05). Down regulation of Per2 also accelerates in vivo tumor growth and doubles the daily amplitude of the tumor growth rhythm (P < 0.05). Conclusions The clock gene Per2 exerts its tumor suppressor function in a circadian time dependent manner. Therefore, Per2 and perhaps other clock genes represent a new class of potential therapeutic targets whose manipulation will modulate cancer growth and cancer cell proliferation.     

Daily coordination of cancer growth and circadian clock gene expression

Background.Circadian coordination in mammals is accomplished, in part, by coordinate, rhythmic expression of a series of circadian clock genes in the central clock within the suprachiasmatic nuclei (SCN) of the hypothalamus. These same genes are also rhythmically expressed each day within each peripheral tissue.Methods.We measured tumor size, tumor cell cyclin E protein, tumor cell mitotic index, and circadian clock gene expression in liver and tumor cells at six equispaced times of day in individual mice of a 12-h light, 12-h dark schedule.Results.We demonstrate that C3HFeJ/HeB mice with transplanted syngeneic mammary tumor maintain largely normal circadian sleep/activity patterns, and that the rate of tumor growth is highly rhythmic during each day. Two daily 2.5-fold peaks in cancer cell cyclin E protein, a marker of DNA synthesis, are followed by two daily up-to-3-fold peaks in cancer cell mitosis (one minor, and one major peak). These peaks are, in turn, followed by two prominent daily peaks in tumor growth rate occurring during mid-sleep and the second, during mid-activity. These data indicate that all therapeutic targets relevant to tumor growth and tumor cell proliferation are ordered in tumor cells within each day. The daily expression patterns of the circadian clock genes Bmal1, mPer1, and mPer2, remain normally circadian coordinated in the livers of these tumor bearing mice. Bmal1 gene expression remains circadian rhythmic in cancer cells, although damped in amplitude, with a similar circadian pattern to that in normal hepatocytes. However, tumor cell mPer1 and mPer2 gene expression patterns fail to maintain statistically significant daily rhythms.Conclusion. We conclude that, if core circadian clock gene expression is essential to gate tumor cell proliferation within each day, then there may be substantial redundancy in this timing system. Alternatively, the daily ordering of tumor cell clock gene expression may not be essential to the daily gating of cancer cell DNA synthesis, mitosis and growth. This would indicate that host central SCN-mediated neuro–humoro-behavioral controls and/or daily light-induced changes in melatonin or peripherally-induced rhythms such as those resulting from feeding, may be adequate for the daily coordination of cancer cell expression of proliferation related therapeutic targets.     

Correlated downregulation of estrogen receptor beta and the circadian clock gene Per1 in human colorectal cancer

There is a growing body of evidence that disturbed circadian clock gene expression is associated with tumor development and tumor progression. Based on our initial experiments demonstrating decreased period 1 (Per1) expression in colon cancer, we evaluated clock gene and estrogen receptor (ER) alpha/beta expression in colon cancer cells of primary colorectal tumors and adjacent normal colon mucosa (NM) by real‐time RT‐PCR. Analysis of gene expression in G₂ and G₃ colorectal tumors revealed a decrease of Per1 mRNA compared with paired NM (G₂: 0.52‐fold; P = n.s. and G₃: 0.48‐fold; P = 0.03). A significant gender specific difference of Per1 expression was observed in G₂ tumors as compared with NM (female: 0.38‐fold; P = 0.004 vs. male: 0.73‐fold; P = n.s.). Expression of CLOCK was significantly elevated in G₂ tumors of male patients (1.63‐fold, P = 0.01). The expression of ER‐beta was significantly decreased in G₂ and G₃ tumors (G₂: 0.32‐fold; P = 0.003 and 0.27; P = 0.001). No significant gender specific differences of ER‐beta reduction in tumors were observed. A significant correlation between the decrease of Per1 and ER‐beta in colorectal tumors (r = 0.61; P < 0.001) was found. No changes in gene expression were detected for ER‐alpha and Per2. Our data demonstrate a correlated decrease of Per1 and ER‐beta in colorectal tumors, mediated probably by epigenetic mechanisms. The observed gender differences in the expression of CLOCK and Per1 in G₂ tumors might suggest a gender‐specific, distinctive role of the cellular clock in colorectal tumorigenesis. © 2009 Wiley‐Liss, Inc.     

Disruption of Circadian Coordination and Malignant Growth

Altered circadian rhythms predicted for poor survival in patients with metastatic colorectal or breast cancer. An increased incidence of cancers has been reported in flying attendants and in women working predominantly at night. To explore the contribution of circadian structure to tumor growth we ablated the 24-h rest-activity cycle and markedly altered the rhythms in body temperature, serum corticosterone and lymphocyte count in mice by complete stereotaxic destruction of the suprachiasmatic nuclei (SCN) or by subjecting the mice to experimental chronic jet-lag. Such disruption of circadian coordination significantly accelerated malignant growth in two transplantable tumor models, Glasgow osteosarcoma and Pancreatic adenocarcinoma. The mRNA expression of clock genes per2 and reverb-α in controls displayed significant circadian rhythms in the liver (Cosinor, p=0.006 and p=0.003, respectively) and in the tumor (p=0.04 and p<0.001, respectively). Both rhythms were suppressed in the liver and in the tumor of jet lagged mice. This functional disturbance of molecular clock resulted in down regulation of p53 and overexpression of c-Myc, two effects which may favor cancer growth. Conclusions:These results indicate that circadian system could play an important role in malignant growth control. This should be taken into consideration in cancer prevention and therapy.     

Tumor Suppression by the Mammalian Period Genes

The Period (Per) genes are key circadian rhythm regulators in mammals. Expression of the mouse Per (mPer) genes have diurnal pattern in the suprachiamstic nuclei and in peripheral tissues. Genetic ablation mPER1 and mPER2 function results in a complete loss of circadian rhythm control based on wheel running activity in mice. In addition, these animals also display apparent premature aging and significant increase in neoplastic and hyperplastic phenotypes. When challenged by γ-radiation, mPer2 deficient mice response by rapid hair graying, are deficient in p53-mediated apoptosis in thymocytes and have robust tumor occurrences. Our studies have demonstrated that the circadian clock function is very important for cell cycle, DNA damage response and tumor suppression in vivo. Temporal expression of genes involved in cell cycle regulation and tumor suppression, such as c-Myc, Cyclin D1, Cyclin A, Mdm-2 and Gadd45α is deregulated in mPer2 mutant mice. In addition, genetic studies have demonstrated that many key regulators of cell cycle and growth control are also important circadian clock regulators confirming the critical role of circadian function in organismal homeostasis. Recently studies of human breast and endometrial cancers revealed that the loss and deregulation of PERIOD proteins is common in the tumor cells.     

Inhibition of tumorigenesis by intratumoral delivery of the circadian gene mPer2 in C57BL/6 mice

Biological clocks are intrinsic time-keeping systems that regulate behavior and physiological functions in most living organisms. Previous works suggested a possible link between the endogenous circadian clock and cell cycle regulation. The mammalian Period-2 gene (mPer2), an important component of the circadian clock mechanism, is recently demonstrated to play an important role in repressing tumor growth. In this study, we found that polyethylenimine-mediated intratumoral Per2 gene delivery had significant antitumor effects in C57BL/6 mice transplanted with Lewis lung carcinoma. Our data illustrated that the Per2 gene delivery inhibited PCNA expression and induced apoptosis. Our results support the emerging role of the circadian clock in critical aspects of tumorigenesis. These findings underscore the potential use of Per2 gene delivery as a novel therapeutic intervention for the treatment of malignant tumors.     

缺氧诱导因子-3α（HIF-3α）对肝癌细胞生物钟基因表达的影响

目的:研究缺氧诱导因子-3α(HIF-3α)对肝癌细胞生物钟基因表达的影响。方法:将HIF-3α过表达的慢病毒载体感染HepG2细胞，构建稳定过表达HIF-3α肝癌细胞系，同时通过siRNA干扰HepG2细胞中HIF-3α的表达。然后通过Real-time PCR和Western Blot检测HIF-3α感染前后肝癌细胞中生物钟基因Clock、Bmal1、NPAS2、Per1、Per2、Per3、Timeless、Cry1、Cry2、REV-ERBA、Rora、CKIε的表达水平。结果:Real-time PCR结果显示HIF-3α使Per3、CKIε mRNA表达升高(P均<0.05)，Bmal1、Per1、Per2、Cry1、REV-ERBA、Rora mRNA表达降低(P均<0.05)，HIF-3α对Clock、NPAS2、Timeless、Cry2 mRNA的表达无明显影响(P均>0.05)。Western Blot结果与PCR结果相一致。结论:HIF-3α可引起肝癌细胞生物钟基因的紊乱，可能是肝癌生物钟基因表达异常的原因之一。     

Elevated mPer1 gene expression in tumor stroma imaged through bioluminescence

The tumor stroma has significant effects on cancer cell growth and metastasis. Interactions between cancer and stromal cells shape tumor progression through poorly understood mechanisms. One factor regulating tumor growth is the circadian timing system that generates daily physiological rhythms throughout the body. Clock genes such as mPer1 serve in molecular timing events of circadian oscillators and when mutated can disrupt circadian rhythms and accelerate tumor growth. Stimulation of mPer1 by cytokines suggests that the timing of circadian oscillators may be altered by these tumor‐derived signals. To explore tumor and stromal interactions, the pattern of mPer1 expression was imaged in tumors generated through subcutaneous injection of Lewis lung carcinoma (LLC) cells. Several imaging studies have used bioluminescent cancer cell lines expressing firefly luciferase to image tumor growth in live mice. In contrast, this study used non‐bioluminescent cancer cells to produce tumors within transgenic mice expressing luciferase controlled by the mPer1 gene promoter. Bioluminescence originated only in host cells and was significantly elevated throughout the tumor stroma. It was detected through the skin of live mice or by imaging the tumor directly. No effects on the circadian timing system were detected during three weeks of tumor growth according to wheel‐running rhythms. Similarly, no effects on mPer1 expression outside the tumor were found. These results suggest that mPer1 activity may play a localized role in the interactions between cancer and stromal cells. The effects might be exploited clinically by targeting the circadian clock genes of stromal cells.     

Clinical impact of c‐MET expression and genetic mutational status in colorectal cancer patients after liver resection

c‐MET is implicated in the pathogenesis and growth of a wide variety of human malignancies, including colorectal cancer (CRC). The aim of the present study was to clarify the association between c‐MET expression and tumor recurrence in CRC patients after curative liver resection, and to evaluate concordance in c‐MET expression and various mutations of KRAS, BRAF and PIK3CA between primary CRC and paired liver metastases. A cohort of patients was tested for c‐MET immunoreactivity (i.e. immunohistochemistry [IHC]) and KRAS, BRAF and PIK3CA mutations. Analyses were performed both on primary tumors and paired liver metastases, and the association between IHC and mutations results were assessed. A total of 108 patients were eligible. A total of 53% of patients underwent simultaneous resection of primary tumors and metastases, and the others underwent metachronous resection. Levels of concordance between primary tumors and metastases were 65.7%, 87.7%, 100% and 95.2% for c‐MET, KRAS, BRAF and PIK3CA, respectively. High levels of c‐MET expression (c‐MET‐high) in the primary tumors were observed in 52% of patients. Relapse‐free survival was significantly shorter for patients with c‐MET‐high primary tumors (9.7 months) than for those with c‐MET‐low primary tumors (21.1 months) (P = 0.013). These results suggest that a high level of genetic concordance in KRAS, BRAF and PIK3CA between primary tumors and liver metastases, and c‐MET‐high in the primary tumors were associated with shorter relapse‐free survival after hepatic metastasectomy.     

Expression of the Circadian Clock Genes Per1 and Per2 in Sporadic and Familial Breast Tumors

There is a growing body of evidence implicating aberrant circadian clock expression in the development of cancer. Based on our initial experiments identifying a putative interaction between BRCA1 and the clock proteins Per1 and Per2, as well as the reported involvement of the circadian clock in the development of cancer, we have performed an expression analysis of the circadian clock genes Per1 and Per2 in both sporadic and familial primary breast tumors and normal breast tissues using real-time polymerase chain reaction. Significantly decreased levels of Per1 were observed between sporadic tumors and normal samples (P < .00001), as well as a further significant decrease between familial and sporadic breast tumors for both Per1 (P < .00001) and Per2 (P < .00001). Decreased Per1 was also associated with estrogen receptor negativity (53% vs 15%, P = .04). These results suggest a role for both Per1 and Per2 in normal breast function and show for the first time that deregulation of the circadian clock may be an important factor in the development of familial breast cancer. Aberrant expression of circadian clock genes could have important consequences on the transactivation of downstream targets that control the cell cycle and on the ability of cells to undergo apoptosis, potentially promoting carcinogenesis.     

Daily timed meals dissociate circadian rhythms in hepatoma and healthy host liver

Dividing cells, including human cancers, organize processes necessary for their duplication according to circadian time. Recent evidence has shown that disruption of central regulation of circadian rhythms can increase the rate at which a variety of cancers develop in rodents. To study circadian rhythms in liver tumors, we have chemically induced hepatocellular carcinoma in transgenic rats bearing a luciferase reporter gene attached to the promoter of a core circadian clock gene (Period 1). We explanted normal liver cells and hepatomas, placed them into short-term culture, and precisely measured their molecular clock function by recording light output. Results show that isolated hepatocellular carcinoma is capable of generating circadian rhythms in vitro. Temporally restricting food availability to either day or night altered the phase of the rhythms in both healthy and malignant tissue. However, the hepatomas were much less sensitive to this signal resulting in markedly different phase relationships between host and tumor tissue as a function of mealtime. These data support the conclusion that hepatoma is differentially sensitive to circadian timing signals, although it maintains the circadian organization of the nonmalignant cells from which it arose. Because circadian clocks are known to modulate the sensitivity of many therapeutic cytotoxic targets, controlling meal-timing might be used to increase the efficacy of treatment. Specifically, meal and treatment schedules could be designed that take advantage of coincident times of greatest tumor sensitivity and lowest sensitivity of host tissue to damage.     

h‐Prune is associated with poor prognosis and epithelial–mesenchymal transition in patients with colorectal liver metastases

The prognosis of patients with colorectal liver metastases (CRLM) remains low despite advances in chemotherapy and surgery. The expression of h‐prune (human homolog of Drosophila prune protein; HGNC13420), an exopolyphosphatase, is correlated with progression and aggressiveness in several cancers and promotes migration and invasion. We investigated the role of h‐prune in CRLM. To investigate the role of h‐prune, immunohistochemical analysis for h‐prune was performed in 87 surgically resected specimens of CRLM obtained between 2001 and 2009 at the Hiroshima University Hospital. Immunohistochemical analysis revealed positive staining for h‐prune in 24 (28%) cases. The overall survival rate was significantly lower in h‐prune‐positive cases than in h‐prune‐negative cases (p = 0.003). Multivariate analysis showed that h‐prune positivity was the only independent factor related to poor overall survival of patients after curative hepatectomy of CRLM. In vitro and in vivo, h‐prune‐knocked‐down and h‐prune‐overexpressing cells were analyzed. In vitro, h‐prune was associated with increased cell motility and upregulation of epithelial–mesenchymal transition (EMT) markers. In a mouse model, h‐prune was associated with invasion of the tumor and distant metastases. In summary, h‐prune expression is a useful marker to identify high‐risk patients for resectable colorectal liver metastasis. h‐Prune expression is necessary for cancer cell motility and EMT and is associated with liver and lung metastasis in colorectal cancer cells. h‐Prune could be a new prognostic marker and molecular target for CRLM.     

Gene expression of 5-fluorouracil metabolic enzymes in primary colorectal cancer and corresponding liver metastasis

Purpose Expression of thymidylate synthase (TS) and the 5-fluorouracil (5-FU) metabolic enzymes, including dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), thymidine phosphorylase (TP), and uridine phosphorylase (UP), has been reported to be associated with the sensitivity to 5-FU-based chemotherapy in colorectal cancer. We evaluated the correlation of the expression of these genes between primary tumors and corresponding liver metastases.Method The mRNA levels of TS, DPD, OPRT, TP, and UP were measured by real-time quantitative RT-PCR in samples from 23 consecutive patients with both primary colorectal adenocarcinoma and liver metastasis.Results The DPD, OPRT, TP, and UP mRNA levels were significantly higher in liver metastases than in primary tumor (expression in relation to that of -actin mRNA: 0.42 vs 0.16, P=0.00053; 1.4 vs 0.92, P=0.016; 23 vs 11, P=0.00014; 0.36 vs 0.25, P=0.0026; respectively). However, the TS mRNA level did not differ significantly between liver metastases than primary tumor (0.20 vs 0.16, P=0.28). No correlation was observed for any gene between primary tumor and liver metastases. In both primary tumor and liver metastasis, the TS mRNA levels correlated significantly with the OPRT mRNA level (primary r _S=0.83, P=0.00000081; liver metastasis r _S=0.49, P=0.017), while the DPD mRNA level correlated significantly with the TP mRNA level r _S=0.81, P=0.0000024; r _S=0.63, P=0.0014; respectively).Conclusions The differential gene expression of 5-FU metabolic enzymes between primary colorectal cancer and corresponding liver metastases should be taken into consideration when estimating the sensitivity to 5-FU-based chemotherapy in colorectal cancer. The gene expression of TS and OPRT, which are involved in de novo pyrimidine synthesis, and that of DPD and TP, may be coregulated.     

Tissue inhibitor of matrix metalloproteinase‐1 expression in colorectal cancer liver metastases is associated with vascular structures

Metastatic growth by colorectal cancer cells in the liver requires the ability of the cancer cells to interact with the new microenvironment. This interaction results in three histological growth patterns of liver metastases: desmoplastic, pushing, and replacement. In primary colorectal cancer several proteases, involved in the degradation of extracellular matrix components, are up‐regulated. In liver metastases, their expression is growth pattern dependent. Tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) is a strong prognostic marker in plasma from colorectal cancer patients, with significant higher levels in patients with metastatic disease. We therefore wanted to determine the expression pattern of TIMP‐1 in primary colorectal cancers and their matching liver metastases. TIMP‐1 mRNA was primarily seen in α‐smooth‐muscle actin (α‐SMA)‐positive cells. In all primary tumors and liver metastases with desmoplastic growth pattern, TIMP‐1 mRNA was primarily found in α‐SMA‐positive myofibroblasts located at the invasive front. Some α‐SMA‐positive cells with TIMP‐1 mRNA were located adjacent to CD34‐positive endothelial cells, identifying them as pericytes. This indicates that TIMP‐1 in primary tumors and liver metastases with desmoplastic growth pattern has dual functions; being an MMP‐inhibitor at the cancer periphery and involved in tumor‐induced angiogenesis in the pericytes. In the liver metastases with pushing or replacement growth patterns, TIMP‐1 was primarily expressed by activated hepatic stellate cells at the metastasis/liver parenchyma interface. These cells were located adjacent to CD34‐positive endothelial cells, suggesting a function in tumor‐induced angiogenesis. We therefore conclude that TIMP‐1 expression is growth pattern dependent in colorectal cancer liver metastases. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.     

Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer

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.