Circadian clock,carcinogenesis, chronochemotherapy connections

The circadian clock controls the expression of nearly 50% of protein coding genes in mice and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate, or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.     

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ERA) gene also oscillates in a circadian fashion. In contrast, ERA-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ERA-positive breast cancer cells do not display circadian oscillation of ERA expression. Our findings suggest that estrogen signaling could be affected not only in ERA-negative breast cancer, but also in ERA-positive breast cancer due to lack of circadian availability of ERA. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.Key words: circadian rhythm, clock genes, estrogen receptor alpha (ERA), breast cancer cells, entrainment, serum shock     

Clock genes in cell clocks: roles, actions, and mysteries

Cellular events must be organized in the time dimension as well as in the space dimension for many proteins to perform their cellular functions effectively. The intracellular molecular oscillating loops that compose the cell's circadian clock coordinate the timing of the expression of a variety of genes with basic or specific cellular functions. In mammals, the temporal pattern of clock gene expression generated in each SCN neuron is coupled to those of other cells and, amplified, spreads its signals through the brain and then, via feeding behavior, glucocorticoids, and sympathetic nerves, to peripheral organs. These peripheral organs have their own circadian clocks. In some tissues, such as liver, there is also a clock-regulating cell cycle, which interacts strongly with the components and temporal organization of the circadian clock. Some tissues, however, such as testis, express clock genes whose function, if any, remains unclear. Furthermore, circadian clock function may be suspended in differentiating tissue. Thus, the prominence of circadian organization may not apply equally to all tissues under all conditions.     

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ER A) gene also oscillates in a circadian fashion. In contrast, ER A-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ER A-positive breast cancer cells do not display circadian oscillation of ER A expression. Our findings suggest that estrogen signaling could be affected not only in ER A-negative breast cancer, but also in ER A-positive breast cancer due to lack of circadian availability of ER A. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.     

The circadian conidiation rhythm inNeurospora crassa

The filamentous fungusNeurospora crassais one of the best organisms for analysing the molecular basis of the circadian rhythm observed in asexual spore formation, conidiation. Many clock mutants in which the circadian conidiation rhythm has different characteristics compared to those in the wild-type strain have been isolated since the early 1970s. With the cloning of one of these clock genes,frq, the molecular basis of the circadian clock inNeurosporahas become gradually clearer. Physiological and pharmacological studies have also contributed to our understanding of the physiological basis of the circadian clock inNeurospora. These studies strongly indicate that the circadian clock is based on or is closely related to a network of metabolic processes for cellular activities. Based on these studies, it may be possible to isolate new types of clock mutants which should contribute to a better understanding of the molecular basis of the circadian clock inNeurospora.     

周期节律与肿瘤关系的分子机理

周期节律是由内在时钟系统介导的多重生物过程的周期循环.周期节律系统是由位于大脑的视神经交叉上核的中央时钟系统和位于外周的几乎存在于所有细胞的外周时钟系统组成的.中央时钟与外周时钟都能够对生物体的生理过程进行调控,如激素的分泌、能量代谢、细胞增殖、DNA损伤修复等.而周期节律基因的表达失调,对其下游靶基因包括细胞周期相关基因的表达,以及细胞抗凋亡能力等产生重要的影响.而这一结果会导致细胞增殖加速及基因组不稳定,并可能促进肿瘤的发生.许多实验证据表明,肿瘤是一种节律相关的生理失调,在许多肿瘤中都发现周期节律遭到破坏,如乳腺癌、前列腺癌、子宫内膜癌等.本文将从周期节律对细胞周期进程及对细胞DNA损伤修复的影响来讨论分子水平上细胞的周期节律与肿瘤发生发展的关系.     

The intrinsic circadian clock within the cardiomyocyte

Circadian clocks are intracellular molecular mechanisms that allow the cell to anticipate the time of day. We have previously reported that the intact rat heart expresses the major components of the circadian clock, of which its rhythmic expression in vivo is consistent with the operation of a fully functional clock mechanism. The present study exposes oscillations of circadian clock genes [brain and arylhydrocarbon receptor nuclear translocator-like protein 1 (bmal1), reverse strand of the c-erbaalpha gene (rev-erbaalpha), period 2 (per2), albumin D-element binding protein (dbp)] for isolated adult rat cardiomyocytes in culture. Acute (2 h) and/or chronic (continuous) treatment of cardiomyocytes with FCS (50% and 2.5%, respectively) results in rhythmic expression of circadian clock genes with periodicities of 20-24 h. In contrast, cardiomyocytes cultured in the absence of serum exhibit dramatically dampened oscillations in bmal1 and dbp only. Zeitgebers (timekeepers) are factors that influence the timing of the circadian clock. Glucose, which has been previously shown to reactivate circadian clock gene oscillations in fibroblasts, has no effect on the expression of circadian clock genes in adult rat cardiomyocytes, either in the absence or presence of serum. Exposure of adult rat cardiomyocytes to the sympathetic neurotransmitter norephinephrine (10 microM) for 2 h reinitiates rhythmic expression of circadian clock genes in a serum-independent manner. Oscillations in circadian clock genes were associated with 24-h oscillations in the metabolic genes pyruvate dehydrogenase kinase 4 (pdk4) and uncoupling protein 3 (ucp3). In conclusion, these data suggest that the circadian clock operates within the myocytes of the heart and that this molecular mechanism persists under standard cell culture conditions (i.e., 2.5% serum). Furthermore, our data suggest that norepinephrine, unlike glucose, influences the timing of the circadian clock within the heart and that the circadian clock may be a novel mechanism regulating myocardial metabolism.     

Impaired light detection of the circadian clock in a zebrafish melanoma model

The circadian clock controls the timing of the cell cycle in healthy tissues and clock disruption is known to increase tumourigenesis. Melanoma is one of the most rapidly increasing forms of cancer and the precise molecular circadian changes that occur in a melanoma tumor are unknown. Using a melanoma zebrafish model, we have explored the molecular changes that occur to the circadian clock within tumors. We have found disruptions in melanoma clock gene expression due to a major impairment to the light input pathway, with a parallel loss of light-dependent activation of DNA repair genes. Furthermore, the timing of mitosis in tumors is perturbed, as well as the regulation of certain key cell cycle regulators, such that cells divide arhythmically. The inability to co-ordinate DNA damage repair and cell division is likely to promote further tumourigenesis and accelerate melanoma development.     

Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis

Altered estrogen receptor α (ERA) signaling and altered circadian rhythms are both features of breast cancer. By using a method to entrain circadian oscillations in human cultured cells, we recently reported that the expression of key clock genes oscillates in a circadian fashion in ERA-positive breast epithelial cells but not in breast cancer cells, regardless of their ERA status. Moreover, we reported that ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells. By using ERA-positive HME1 breast epithelial cells, which can be both entrained in vitro and can form mammary gland-like acinar structures in three-dimensional (3D) culture, first we identified a circuit encompassing ERA and an estrogen-regulated loop consisting of two circadian clock genes, PER2 and BMAL1. Further, we demonstrated that this estrogen-regulated circuit is necessary for breast epithelial acinar morphogenesis. Disruption of this circuit due to ERA-knockdown, negatively affects the estrogen-sustained circadian PER2-BMAL1 mechanism as well as the formation of 3D HME1 acini. Conversely, knockdown of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects ERA circadian oscillations and 3D breast acinar morphogenesis. To our knowledge, this study provides the first evidence of the implication of an ERA-circadian clock mechanism in the breast acinar morphogenetic process.     

Daily expression of clock genes in whole blood cells in healthy subjects and a patient with circadian rhythm sleep disorder

In recent years, circadian rhythm sleep disorders in humans have been increasing. Clinical features characteristic of this disorder are well known, but the specific causes remain unknown. However, various derangements of circadian expression of the clock gene are a probable cause of this disease. We have attempted to elucidate the relationship between the expression of the clock genes in whole blood cells and the clinical features characteristic of this disorder. In this study, we indicate the daily expression of clock genes period (Per) 1, 2, 3, Bmal1, and Clock in whole blood cells in 12 healthy male subjects. The peak phase of Per1, Per2, and Per3 appeared in the early morning, whereas that of Bmal1 and Clock appeared in the midnight hours. Furthermore, in one patient case with circadian rhythm sleep disorder, we observed variations of the peak phase in clock genes by treatments such as light therapy, exercise therapy, and medicinal therapy. This study suggested that the monitoring of human clock genes in whole blood cells, which may be functionally important for the molecular control of the circadian pacemaker as well as in suprachiasmatic nucleus, might be useful to evaluate internal synchronization.