Host circadian clock as a control point in tumor progression

BACKGROUND: The circadian timing system controlled by the suprachiasmatic nuclei (SCN) of the hypothalamus regulates daily rhythms of motor activity and adrenocortical secretion. An alteration in these rhythms is associated with poor survival of patients with metastatic colorectal or breast cancer. We developed a mouse model to investigate the consequences of severe circadian dysfunction upon tumor growth. METHODS: The SCN of mice were destroyed by bilateral electrolytic lesions, and body activity and body temperature were recorded with a radio transmitter implanted into the peritoneal cavity. Plasma corticosterone levels and circulating lymphocyte counts were measured (n = 75 with SCN lesions, n = 64 sham-operated). Complete SCN destruction was ascertained postmortem. Mice were inoculated with implants of Glasgow osteosarcoma (n = 16 with SCN lesions, n = 12 sham-operated) or pancreatic adenocarcinoma (n = 13 with SCN lesions, n = 13 sham-operated) tumors to determine the effects of altered circadian rhythms on tumor progression. Time series for body temperature and rest-activity patterns were analyzed by spectral analysis and cosinor analysis. Parametric data were compared by the use of analysis of variance (ANOVA) and survival curves with the log-rank test. All statistical tests were two-sided. RESULTS: The 24-hour rest-activity cycle was ablated and the daily rhythms of serum corticosterone level and lymphocyte count were markedly altered in 75 mice with complete SCN destruction as compared with 64 sham-operated mice (two-way ANOVA for corticosterone: sampling time effect P<.001, lesion effect P =.001, and time x lesion interaction P<.001; for lymphocytes P =.001,.002, and.002 respectively). Body temperature rhythm was suppressed in 60 of the 75 mice with SCN lesions (P<.001). Both types of tumors grew two to three times faster in mice with SCN lesions than in sham-operated mice (two-way ANOVA: P<.001 for lesion and for tumor effects; P =.21 for lesion x tumor effect interaction). Survival of mice with SCN lesions was statistically significantly shorter compared with that of sham-operated mice (log-rank P =.0062). CONCLUSIONS: Disruption of circadian rhythms in mice was associated with accelerated growth of malignant tumors of two types, suggesting that the host circadian clock may play an important role in endogenous control of tumor progression.     

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.     

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.     

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.     

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.     

Circadian gene mPer2 overexpression induces cancer cell apoptosis

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-X(L) 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.     

Circadian properties of cancer stem cells in glioma cell cultures and tumorspheres

Increased cancer risk is linked to disruption of circadian rhythms. Cancer stem cells (CSCs) are a known cause of cancer aggressiveness, but their circadian properties have not been described. We discovered circadian rhythms in gene expression within C6 glioma tumorspheres enriched in CSCs and found that the circadian clock is particularly robust in medium lacking any growth factors. A method is introduced for identifying individual CSCs in culture for single-cell analysis. CSCs in monolayer cell culture failed to show a circadian rhythm in nuclear localization of mPER2 protein, suggesting that cell interactions or the tumor-like microenvironment within tumorspheres enable circadian timing.     

荷人鼻咽癌裸鼠骨髓细胞DNA合成及凋亡相关基因表达的生物节律的初步研究

背景与目的：有关鼻咽癌的生物节律特征尚未见文献报道,本文拟探索荷人鼻咽癌裸鼠骨髓细胞的DNA合成及几种凋亡相关基因表达的生物节律,为临床制定鼻咽癌时辰化疗方案提供必要的参数。方法：BALB/C裸小鼠69只,置于程控的独立光照系统（12h光照,12h黑暗）同步化至少3周。然后双侧腋窝皮下接种人鼻咽低分化鳞癌细胞（CNE-2）裸小鼠移植瘤,成瘤后按6个不同时间点分批处死小鼠,收集骨髓细胞,固定,染色后流式细胞仪测DNA含量,用ANOVA法检验各期细胞在6个时间点差异的显著性,Cosinor法考察G1、S、G2/M期细胞在24h的分布是否符合余弦函数,部分骨髓细胞经裂解,蛋白定量后用Western Blot法检测不同时间点p53,p21和Bcl-2三种基因蛋白表达的变化情况。结论：G1、S、G2/M期细胞在3、7、11、15、19、23HALO（Hours After Light Onset即灯亮后第3、7、11、19、23h)的分布随时间变化有显著性差异,G1、G2/M期细胞24h变化符合余弦节律,高峰值分别位于10.8HALO和1.8HALO,骨髓细胞p53和Bcl-2蛋白表达24h强度不同,p53在11HALO最强,在7HALO最弱,Bcl-2在15HALO最强,在19HALO最弱,p21蛋白未见表达。结论：荷人鼻咽癌裸小鼠骨髓细胞DNA合成随昼夜交替呈节律变化。p53和Bcl-2蛋白表达随昼夜时间变化而规律性波动。     

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.     

鼻咽癌患者外周血血浆可的松水平和全血还原型谷胱甘肽含量的昼夜节律

背景与目的:谷胱甘肽与细胞对抗癌药物的解毒作用以及对放射性损伤的保护机制密切相关。本文通过对鼻咽癌患者和健康志愿者外周血血浆可的松水平和全血还原型谷胱甘肽含量的昼夜节律进行研究,从而为鼻咽癌患者进行肿瘤的时间治疗提供参考资料。方法:13名鼻咽癌患者(实验组)和14名健康志愿者(正常对照组)参加本项研究。每位受试者均从中午12点开始抽取外周静脉血4.5mL,每隔4h抽血一次,在24h内共抽血6次。血浆可的松水平的检测采用放射免疫法,全血还原型谷胱甘肽含量则采用高效液相色谱法进行检测。结果:鼻咽癌患者组和正常对照组的血浆可的松水平均呈现出明显的昼夜节律的特征,且节律特性相似。两组的血浆可的松水平的峰值均出现在早晨,而谷值均出现在午夜。不同时间点时,两组的全血还原型谷胱甘肽含量不同,且均具有显著性差异(重复测量的方差分析,F=5.18,P=0.02)。余弦分析表明,鼻咽癌患者组呈现出昼夜节律变化趋势(P=0.06),峰值出现在早晨(05∶02),正常对照组也呈现出明显的昼夜节律的特征,峰值出现在早晨(07∶44±01∶56)(P<0.01)。鼻咽癌患者组外周全血中还原型谷胱甘肽的节律调整均值为(19.60±1.11)nmol/mgprotein,正常对照组为(8.95±0.46)nmol/mgprotein。结论:包括晚期患者在内的鼻咽癌患者仍然具有正常的生物节律。鼻咽癌患者全血中还原型谷胱甘肽水平呈现出昼夜节律变化的趋势,与正常对照组节律特性相似。这为临床上选择恰当的时间对患者进行化疗和放疗提供了有价值的参考资料。     

时钟基因PER 在肿瘤中的研究进展

昼夜节律钟控制着人类及大多数哺乳动物每天的体温、血压、激素、代谢等节律,这是由于昼夜节律基因调控的结果。PER（period）基因是核心昼夜节律基因之一,现已确认人类的PER 基因包括PER 1、PER 2 和PER 3,均是抑癌基因。PER 基因的表达异常不仅能促进肿瘤的发生,还能改变细胞对放射线和化疗药物的敏感性,为肿瘤患者的放疗和化疗提供新的治疗方向。本文在将对昼夜节律钟基因PER 在肿瘤中的研究进展进行综述。      

生物钟基因与肿瘤时辰治疗的研究进展

近年来,生物钟基因及昼夜节律在肿瘤发生、发展及转移中的研究已成为肿瘤研究领域内的热点.生物钟基因普遍存在于生物界,是生物昼夜节律运转的分子基础.其表达紊乱会增加肿瘤的发病率,通过对肿瘤时间节律性及时间生物学的研究,对指导肿瘤的治疗、预后及转归开辟了一条新思路.本文就生物钟基因及肿瘤的时辰治疗研究进展作一综述.     

Stage-dependent depression of melatonin in patients with primary breast cancer. Correlation with prolactin, thyroid stimulating hormone, and steroid receptors

Serum melatonin was determined over 24 hours in 35 patients with breast cancer with either a fresh primary tumor (n = 23) or a secondary tumor (n = 12) and in 28 patients with untreated benign breast disease (controls) having a fibroadenoma (n = 10), fibrocystic mastopathy (n = 14), or other breast diseases (n = 4). Circadian rhythms existed in all groups with acrophases at 2 a.m. A 50% depression of peak and amplitude occurred in the group of patients with primary breast cancer compared with age-matched controls (P less than 0.001, P less than 0.01). The peak declined with increasing tumor size: 27% at Stage T1, 53% at T2 (P less than 0.001), and 73% at T3 (P less than 0.05). In contrast, patients with secondary breast cancer, particularly those receiving antiestrogen therapy, had a melatonin peak similar to controls. These results demonstrated a transient depression of pineal melatonin secretion in primary breast cancer and indicated a dynamic role of the pineal gland in malignancy. To investigate some endocrine effects of a depressed melatonin peak, the 24-hour rhythms of prolactin (PRL) and thyroid stimulating hormone (TSH) were determined in patients with primary breast cancer and compared with patients with secondary breast cancer. The PRL had significant circadian rhythms in both groups; but acrophases occurred at midnight in patients with secondary breast cancer, and there were unusually high concentrations at noon in patients with primary breast cancer. Circadian rhythms were not seen for TSH, but the 24-hour average secretion was depressed by 45% (P less than 0.01) in patients with primary breast cancer. The abnormal concentrations of PRL and TSH in these patients could be due to a depressed melatonin peak normally serving as a central circadian synchronizer and modulator of the secretion of adenohypophysial hormones. Additionally, a positive correlation existed between the nocturnal melatonin peak and progesterone and androgen receptor concentrations in primary tumors indicating a direct involvement of melatonin in the growth control of breast cancer.     

移植于裸鼠的人鼻咽癌细胞DNA合成生物节律的初步研究

目的：探讨鼻咽癌细胞DNA合成的生物节律为临床制订鼻咽癌时辰化疗方案提供必要的参数。方法：BALB/C裸小鼠15只，置于积控的独立光照系统中（12h光照，12h黑暗）中同步化至少3周。然后双侧腋下接种人鼻咽低分化鳞癌细胞裸小鼠移植瘤CNE-2，成瘤后按灯亮后3、9、15、21h（即3、9、15、21 HALO）的时间点取瘤块，制成单个细胞悬液，固定，染色后流式细胞仪侧DNA含量。用SPSS软件系统ANOVA法检验各期细胞在4个时间点差异的显著性，用Cosinor法考察G1、S、G2/M期细胞在24h的分布是否符合余弦函数。结果：G1、S、G2/M期细胞在3、9、15、21 HALO的分布随时间变化有显著性差异，G1、G2/M期细胞变化符合余弦节律。结论：移植于裸鼠的人鼻咽癌细胞的DNA合成随昼夜交替呈节律性变化。     

Shift of circadian metabolic rhythm in BFO osteosarcoma-bearing mice

The diurnal rhythms of the contents of serum corticosterone and liver glycogen and the activity of liver tyrosine aminotransferase gradually shifted forward in C3H/He mice bearing BFO osteosarcoma. The production of alkaline phosphatase by the tumor showed a circadian rhythm. The eating behavior of tumor-bearing mice seemed to be responsible for their change in circadian rhythm.     

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.     

Epigenetic alterations in the suprachiasmatic nucleus and hippocampus contribute to age-related cognitive decline

Circadian rhythm dysfunction and cognitive decline, specifically memory loss, frequently accompany natural aging. Circadian rhythms and memory are intertwined, as circadian rhythms influence memory formation and recall in young and old rodents. Although, the precise relationship between circadian rhythms and memory is still largely unknown, it is hypothesized that circadian rhythm disruption, which occurs during aging, contributes to age-associated cognitive decline, specifically memory loss. While there are a variety of mechanisms that could mediate this effect, changes in the epigenome that occur during aging has been proposed as a potential candidate. Interestingly, epigenetic mechanisms, such as DNA methylation and sirtuin1 (SIRT1) are necessary for both circadian rhythms and memory. During aging, similar alterations of epigenetic mechanisms occur in the suprachiasmatic nucleus (SCN) and hippocampus, which are necessary for circadian rhythm generation and memory, respectively. Recently, circadian rhythms have been linked to epigenetic function in the hippocampus, as some of these epigenetic mechanisms oscillate in the hippocampus and are disrupted by clock gene deletion. The current paper will review how circadian rhythms and memory change with age, and will suggest how epigenetic changes in these processes might contribute to age-related cognitive decline.