Influence of circadian rhythm on drug metabolism in 3D hepatic spheroids.

Circadian rhythms are characterized as oscillations that fluctuate based on a 24 h cycle and are responsible for regulation of physiological functions. While the internal clock synchronizes gene expression using external cues like light, a similar synchronization can be induced in vitro by incubating the cells with an increased percentage of serum followed by its rapid removal. Previous studies have suggested that synchronization of HepG2 cell line induced the rhythmic expression of drug-metabolizing enzymes (DME) most specifically the cytochrome P450 enzymes. However, there is a lack of evidence demonstrating the influence of three-dimensional microenvironment on the rhythmicity of these genes. To understand this interplay, gene expression of the circadian machinery and CYP450s were compared using the model human hepatocarcinoma cell line, HepG2. Upon serum shock synchronization, gene and protein expression of core clock regulators was assessed and rhythmic expression of these genes was demonstrated. Further insight into the interrelations between various gene pairs was obtained using statistical analysis. Using RNA sequencing, an in-depth understanding of the widespread effects of circadian regulation on genes involved in metabolic processes in the liver was obtained. This study aids in the better understanding of chronopharmacokinetic events in humans using physiologically relevant 3D culture systems.

A Sextuple Knockout Cell Line System to Study the Differential Roles of CRY, PER, and NR1D in the Transcription-Translation Feedback Loop of the Circadian Clock.

The transcription-translation feedback loop (TTFL) is the core mechanism of the circadian rhythm. In mammalian cells, CLOCK-BMAL1 proteins activate the downstream genes by binding on the E-box sequence of the clock-controlled genes. Among these gene products, CRY1, CRY2, PER1, PER2, NR1D1, and NR1D2 can regulate the CLOCK-BMAL1-mediated transcription to form the feedback loop. However, the detailed mechanism of the TTFL is unclear because of the complicated inter-regulation of these proteins. Here, we generated a cell line lacking CRY1, CRY2, PER1, PER2, NR1D1, and NR1D2 (Cry/Per/Nr1d_KO) to study TTFL. We compared the Dbp transcription after serum-shock and dexamethasone-shock between Cry/Per/Nr1d_KO cells and cells expressing endogenous CRY (Per/Nr1d_KO) or NR1D (Cry/Per_KO). Furthermore, we found that CRY1-mediated repression of Dbp could persist more than 24 h in the absence of other proteins in the negative limb of the TTFL. Our Cry/Per/Nr1d_KO cells is a suitable system for the studying of differential roles of CRY, PER, and NR1D in the TTFL.

Identification of rhythmic human CYPs and their circadian regulators using synchronized hepatoma cells.

Cytochromes P450 (CYPs) catalyze a great number of metabolic reactions that have profound effects on the biological activities of xenobiotics and endobiotics. In this study, we aimed to characterize rhythmic expressions of drug-metabolizing CYPs using synchronized hepatoma cells, and to investigate the potential roles of cis-elements of circadian clock system (E-box, D-box and RevRE or RORE) in generating the rhythms.Serum was used to synchronize circadian cycles and to induce circadian gene expression in cultured hepatoma cells (HepRG and HepG2 cells). Regulation of CYP genes by circadian clock components was investigated by performing luciferase reporter, overexpression and knockdown experiments. mRNA and protein expression were determined by qPCR and Western blotting assays, respectively.Of ten major drug-metabolizing CYP genes, six are rhythmically expressed (CYP1A2, 2B6, 2C8, 2D6, 2E1 and 3A4), whereas other four are non-rhythmic (CYP1B1, 2A6, 2C9 and 2C19).The E-box binding protein BMAL1 directly controls the rhythmic expression of CYP1A2. Rhythmic expressions of CYP2E1 and CYP3A4 are generated via both E-box and D-box elements. The RevRE binding protein REV-ERBα contributes to rhythmic oscillations in CYP2B6 and CYP2C8.In conclusion, rhythmic expressions of five human CYPs (CYP1A2, 2B6, 2C8, 2E1 and 3A4) are generated and regulated by E-box-, D-box-, and/or RevRE-acting clock components. Our findings may have implications for understanding chronopharmacokinetic events in humans.

Identification of circadian-related gene expression profiles in entrained breast cancer cell lines.

Cancer cells have broken circadian clocks when compared to their normal tissue counterparts. Moreover, it has been shown in breast cancer that disruption of common circadian oscillations is associated with a more negative prognosis. Numerous studies, focused on canonical circadian genes in breast cancer cell lines, have suggested that there are no mRNA circadian-like oscillations. Nevertheless, cancer cell lines have not been extensively characterized and it is unknown to what extent the circadian oscillations are disrupted. We have chosen representative non-cancerous and cancerous breast cell lines (MCF-10A, MCF-7, ZR-75-30, MDA-MB-231 and HCC-1954) in order to determine the degree to which the circadian clock is damaged. We used serum shock to synchronize the circadian clocks in culture. Our aim was to initially observe the time course of gene expression using cDNA microarrays in the non-cancerous MCF-10A and the cancerous MCF-7 cells for screening and then to characterize specific genes in other cell lines. We used a cosine function to select highly correlated profiles. Some of the identified genes were validated by quantitative polymerase chain reaction (qPCR) and further evaluated in the other breast cancer cell lines. Interestingly, we observed that breast cancer and non-cancerous cultured cells are able to generate specific circadian expression profiles in response to the serum shock. The rhythmic genes, suggested via microarray and measured in each particular subtype, suggest that each breast cancer cell type responds differently to the circadian synchronization. Future results could identify circadian-like genes that are altered in breast cancer and non-cancerous cells, which can be used to propose novel treatments. Breast cell lines are potential models for in vitro studies of circadian clocks and clock-controlled pathways.

Understanding the Role of Cellular Molecular Clocks in Controlling the Innate Immune Response.

The importance of the 24-h daily cycle, termed circadian, on immune function has been highlighted by a number of recent studies. Immune parameters such as the response to bacterial challenge or immune cell trafficking change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. We are beginning to uncover that the key proteins that comprise the molecular clock, most notably BMAL1, CLOCK, and REV-ERBα, also control fundamental aspects of the immune response. Given the ubiquitous nature of the molecular clock in controlling many other types of physiologies such as metabolism and cardiovascular function, a more thorough understanding of the daily rhythm of the immune system may provide important insight into aspects of patient care such as vaccinations and how we manage infectious and inflammatory diseases. In this chapter, we describe a series of experiments to look at circadian expression and function in immune cells. The experiments described herein may provide an initial assessment of the role of the molecular clock on an immune response from any cell type of interest.

A role for 1α,25-dihydroxyvitamin d3 in the expression of circadian genes.

The active form of vitamin D, 1α,25-(OH)2D3, has been associated with metabolism control, cell growth, differentiation, antiproliferation, apoptosis, and adaptive/innate immune responses, besides its functions in the integrity of bone and calcium homeostasis. The circadian rhythm regulates a variety of biological processes, many of them related to the functions associated with 1α,25-(OH)2D3. In the present study, we determine whether 1α,25-(OH)2D3 alters the expression of circadian genes in adipose-derived stem cells (ADSCs). The effect of 1α,25-(OH)2D3 on the expression of circadian genes BMAL1 and PER2 was measured by qPCR, over a 60-h period every 4 h, in serum shocked ADSCs, serum shocked ADSCs supplemented with 1α,25-(OH)2D3, and ADSCs under the presence of only 1α,25-(OH)2D3. The results showed that 1α,25-(OH)2D3 was able to synchronize circadian clock gene expression in ADSCs. The expression of circadian genes BMAL1 and PER2 in ADSCs that contained only 1α,25-(OH)2D3 has a profile similar to that found in the ADSCs synchronized by a serum shock. The results suggest an important role of 1α,25-(OH)2D3 in the regulation of the molecular clock.

Daily rhythms of glycerophospholipid synthesis in fibroblast cultures involve differential enzyme contributions.

Circadian clocks regulate the temporal organization of several biochemical processes, including lipid metabolism, and their disruption leads to severe metabolic disorders. Immortalized cell lines acting as circadian clocks display daily variations in [(32)P]phospholipid labeling; however, the regulation of glycerophospholipid (GPL) synthesis by internal clocks remains unknown. Here we found that arrested NIH 3T3 cells synchronized with a 2 h-serum shock exhibited temporal oscillations in a) the labeling of total [(3)H] GPLs, with lowest levels around 28 and 56 h, and b) the activity of GPL-synthesizing and GPL-remodeling enzymes, such as phosphatidate phosphohydrolase 1 (PAP-1) and lysophospholipid acyltransferases (LPLAT), respectively, with antiphase profiles. In addition, we investigated the temporal regulation of phosphatidylcholine (PC) biosynthesis. PC is mainly synthesized through the Kennedy pathway with choline kinase (ChoK) and CTP:phosphocholine cytidylyltranferase (CCT) as key regulatory enzymes. We observed that the PC labeling exhibited daily changes, with the lowest levels every ~28 h, that were accompanied by brief increases in CCT activity and the oscillation in ChoK mRNA expression and activity. Results demonstrate that the metabolisms of GPLs and particularly of PC in synchronized fibroblasts are subject to a complex temporal control involving concerted changes in the expression and/or activities of specific synthesizing enzymes.

Oscillation of clock and clock controlled genes induced by serum shock in human breast epithelial and breast cancer cells: regulation by melatonin.

This study investigates differences in expression of clock and clock-controlled genes (CCGs) between human breast epithelial and breast cancer cells and breast tumor xenografts in circadian intact rats and examines if the pineal hormone melatonin influences clock gene and CCG expression. Oscillation of clock gene expression was not observed under standard growth conditions in vitro, however, serum shock (50% horse serum for 2 h) induced oscillation of clock gene and CCG expression in MCF-10A cells, which was repressed or disrupted in MCF-7 cells. Melatonin administration following serum shock differentially suppressed or induced clock gene (Bmal1 and Per2) and CCG expression in MCF10A and MCF-7 cells. These studies demonstrate the lack of rhythmic expression of clock genes and CCGs of cells in vitro and that transplantation of breast cancer cells as xenografts into circadian competent hosts re-establishes a circadian rhythm in the peripheral clock genes of tumor cells.