Altered expression of circadian clock genes during peripheral blood stem cell mobilization induced by granulocyte colony-stimulating factor

Circulating hematopoietic stem cells exhibit robust circadian fluctuations, which influence the mobilized cell yield, even during enforced stem cell mobilization. However, alterations in the expression of circadian clock genes during granulocyte colony-stimulating factor (G-CSF)-induced peripheral blood stem cell (PBSC) mobilization are not fully elucidated. Therefore, we measured the expression of these genes in human peripheral blood leukocytes from 21 healthy donors. While CRY1 mRNA expression significantly increased by 3.9-fold (p?<?0.01), the expression of PER3, CRY2 and BMAL1 mRNAs significantly decreased (by 0.2-fold, 0.2-fold, and 0.6-fold, respectively; p?<?0.001) after G-CSF administration. Moreover, CRY1 mRNA expression was inversely correlated with the plasma level of noradrenaline (r?=??0.36, p?<?0.05), while PER3, CRY2, and BMAL1 mRNA expression directly correlated with the plasma level of noradrenaline (r?=?0.55, r?=?0.66, and r?=?0.57, respectively; p?<?0.001). Thus, significant correlations between the levels of circadian clock gene mRNAs and the plasma level of noradrenaline, a sympathetic nervous system neurotransmitter, were established. The modulation of sympathetic activation and of the circadian clock may be novel therapeutic targets for increasing stem cell yields in PBSC donors.     

Peripheral clock system circadian abnormalities in Cushing’s disease

ABSTRACT

In Cushing’s syndrome, the cortisol rhythm is impaired and can be associated with the disruption in the rhythmic expression of clock genes. In this study, we evaluated the expression of CLOCK, BMAL1, CRY1, CRY2, PER1, PER2, PER3 genes in peripheral blood leukocytes of healthy individuals (n = 13) and Cushing’s disease (CD) patients (n = 12). Participants underwent salivary cortisol measurement at 0900 h and 2300 h. Peripheral blood samples were obtained at 0900 h, 1300 h, 1700 h, and 2300 h for assessing clock gene expression by qPCR. Gene expression circadian variations were evaluated by the Cosinor method. In healthy controls, a circadian variation in the expression of CLOCK, BMAL1, CRY1, PER2, and PER3 was observed, whereas the expression of PER1 and CRY2 followed no specific pattern. The expression of PER2 and PER3 in healthy leukocytes presented a late afternoon acrophase, similarly to CLOCK, whereas CRY1 showed night acrophase, similarly to BMAL1. In CD patients, the circadian variation in the expression of clock genes was lost, along with the abolition of cortisol circadian rhythm. However, CRY2 exhibited a circadian variation with acrophase during the dark phase in patients. In conclusion, our data suggest that Cushing’s disease, which is characterized by hypercortisolism, is associated with abnormalities in the circadian pattern of clock genes. Higher expression of CRY2 at night outlines its putative role in the cortisol circadian rhythm disruption.     

Styrene Altered Clock Gene Expression in Serum-Shocked Cultured Human Fibroblasts

The circadian clock can regulate the metabolic process of xenobiotics, but little is known as to circadian rhythms can be perturbed by xenobiotics. Styrene is a organic chemical widely used in occupational settings. The effects of styrene on the circadian genes of HuDE cells were evaluated after serum-shocking synchronization. A subtoxic dose of 100 µM of styrene altered the expression of clock genes BMAL1, PER2, PER3, CRY1, CRY2, and REV-ERB-α.     

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.     

Haloperidol,but not olanzapine,may affect expression of PER1 and CRY1 genes in human glioblastoma cell line

Background: There is barely any evidence of antipsychotic drugs affecting the molecular clockwork in human, yet it is suggested that clock genes are associated with dopaminergic transmission, i.e. the main target of this therapeutics. We decided to verify if haloperidol and olanzapine affect expression of CLOCK, BMAL1, PER1 and CRY1 in a human central nervous system cell line model. Methods: U-87MG human glioblastoma cell line was used as an experimental model. The cells were incubated with or without haloperidol and olanzapine in the concentration of 5 and 20 μM for 24 h. Real-time quantitative polymerase chain reaction with the ΔC_T analysis was used to examine the effect of haloperidol and olanzapine on the mRNA expression of the genes. Results: At 5 μM, haloperidol decreased expression of CRY1 almost 20-fold. There was nearly a 1.5-fold increase in expression of PER1. Considering the 20 μM haloperidol concentration and both olanzapine concentrations, no other statistically significant effect was observed. Conclusions: At certain concentration, haloperidol seems to affect expression of particular clock genes in a human central nervous system cell line model, yet mechanism underlying this phenomenon remains elusive.     

Exploring the role of circadian clock gene and association with cancer pathophysiology

ABSTRACT

Most of the processes that occur in the mind and body follow natural rhythms. Those with a cycle length of about one day are called circadian rhythms. These rhythms are driven by a system of self-sustained clocks and are entrained by environmental cues such as light-dark cycles as well as food intake. In mammals, the circadian clock system is hierarchically organized such that the master clock in the suprachiasmatic nuclei of the hypothalamus integrates environmental information and synchronizes the phase of oscillators in peripheral tissues.

The circadian system is responsible for regulating a variety of physiological and behavioral processes, including feeding behavior and energy metabolism. Studies revealed that the circadian clock system consists primarily of a set of clock genes. Several genes control the biological clock, including BMAL1, CLOCK (positive regulators), CRY1, CRY2, PER1, PER2, and PER3 (negative regulators) as indicators of the peripheral clock.

Circadian has increasingly become an important area of medical research, with hundreds of studies pointing to the body’s internal clocks as a factor in both health and disease. Thousands of biochemical processes from sleep and wakefulness to DNA repair are scheduled and dictated by these internal clocks. Cancer is an example of health problems where chronotherapy can be used to improve outcomes and deliver a higher quality of care to patients.

In this article, we will discuss knowledge about molecular mechanisms of the circadian clock and the role of clocks in physiology and pathophysiology of concerns.     

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.     

Human Intestinal Circadian Clock: Expression of Clock Genes in Colonocytes Lining the Crypt

Biological clock components have been detected in many epithelial tissues of the digestive tract of mammals (oral mucosa, pancreas, and liver), suggesting the existence of peripheral circadian clocks that may be entrainable by food. Our aim was to investigate the expression of main peripheral clock genes in colonocytes of healthy humans and in human colon carcinoma cell lines. The presence of clock components was investigated in single intact colonic crypts isolated by chelation from the biopsies of 25 patients (free of any sign of colonic lesions) undergoing routine colonoscopy and in cell lines of human colon carcinoma (Caco2 and HT29 clone 19A). Per‐1, per‐2, and clock mRNA were detected by real‐time RT‐PCR. The three‐dimensional distributions of PER‐1, PER‐2, CLOCK, and BMAL1 proteins were recorded along colonic crypts by immunofluorescent confocal imaging. We demonstrate the presence of per‐1, per‐2, and clock mRNA in samples prepared from colonic crypts of 5 patients and in all cell lines. We also demonstrate the presence of two circadian clock proteins, PER‐1 and CLOCK, in human colonocytes on crypts isolated from 20 patients (15 patients for PER‐1 and 6 for CLOCK) and in colon carcinoma cells. Establishing the presence of clock proteins in human colonic crypts is the first step toward the study of the regulation of the intestinal circadian clock by nutrients and feeding rhythms.     

ASSOCIATIONS OF METABOLIC PARAMETERS AND ETHANOL CONSUMPTION WITH MESSENGER RNA EXPRESSION OF CLOCK GENES IN HEALTHY MEN

Recent studies suggest that the impairment of circadian clock function causes various pathological conditions, such as obesity, diabetes, and alcoholism, and an altered mRNA expression of clock genes was found under these conditions. However, it remains to be determined whether clock gene expression varies depending on metabolic conditions even in healthy people. To address this issue, we investigated the associations of metabolic parameters and alcohol consumption with mRNA expression of clock genes (CLOCK, BMAL1, PER1, PER2, and PER3) in peripheral blood cells obtained from 29 healthy non-obese elderly men (age 51–78 yrs) who adhered to a regular sleep-wake routine, through a single time-of-day venous blood sampling at ～09:00?h. There were significant correlations between (1) waist circumference and mRNA level of PER1 (r?=?0.43), (2) plasma glucose concentration and PER2 (r?=?0.50), (3) ethanol consumption and BMAL1 (r?=?0.43), and (4) serum γ-GTP concentration (a sensitive marker of alcohol consumption) and PER2 (r?=?0.40). These results suggest mRNA expression of clock genes is associated with obesity, glucose tolerance, and ethanol consumption even in healthy people. (Author correspondence: akiofuji@jichi.ac.jp)     

Loss of circadian clock gene expression is associated with tumor progression in breast cancer

Several studies suggest a link between circadian rhythm disturbances and tumorigenesis. However, the association between circadian clock genes and prognosis in breast cancer has not been systematically studied. Therefore, we examined the expression of 17 clock components in tumors from 766 node-negative breast cancer patients that were untreated in both neoadjuvant and adjuvant settings. In addition, their association with metastasis-free survival (MFS) and correlation to clinicopathological parameters were investigated. Aiming to estimate functionality of the clockwork, we studied clock gene expression relationships by correlation analysis. Higher expression of several clock genes (e.g., CLOCK, PER1, PER2, PER3, CRY2, NPAS2 and RORC) was found to be associated with longer MFS in univariate Cox regression analyses (HR<1 and FDR-adjusted P < 0.05). Stratification according to molecular subtype revealed prognostic relevance for PER1, PER3, CRY2 and NFIL3 in the ER+/HER2- subgroup, CLOCK and NPAS2 in the ER-/HER2- subtype, and ARNTL2 in HER2+ breast cancer. In the multivariate Cox model, only PER3 (HR = 0.66; P = 0.016) and RORC (HR = 0.42; P = 0.003) were found to be associated with survival outcome independent of established clinicopathological parameters. Pairwise correlations between functionally-related clock genes (e.g., PER2-PER3 and CRY2-PER3) were stronger in ER+, HER2- and low-grade carcinomas; whereas, weaker correlation coefficients were observed in ER- and HER2+ tumors, high-grade tumors and tumors that progressed to metastatic disease. In conclusion, loss of clock genes is associated with worse prognosis in breast cancer. Coordinated co-expression of clock genes, indicative of a functional circadian clock, is maintained in ER+, HER2-, low grade and non-metastasizing tumors but is compromised in more aggressive carcinomas.