Recent studies in laboratory rodents have revealed that circadian oscillation in the physiologic functions affecting drug disposition underlies the dosing time-dependent change in pharmacokinetics. However, it is difficult to predict the circadian change in the drug pharmacokinetics in a diurnal human by using the data collected from nocturnal rodents. In this study, we used cynomolgus monkeys, diurnal active animals, to evaluate the relevance of intestinal expression of P-glycoprotein (P-gp) to the dosing time dependency of the pharmacokinetics of its substrates. The rhythmic phases of circadian gene expression in the suprachiasmatic nuclei (the mammalian circadian pacemaker) of cynomolgus monkeys were similar to those reported in nocturnal rodents. On the other hand, the expression of circadian clock genes in the intestinal epithelial cells of monkeys oscillated at opposite phases in rodents. The intestinal expression of P-gp in the small intestine of monkeys was also oscillated in a circadian time-dependent manner. Furthermore, the intestinal absorption of P-gp substrates (quinidine and etoposide) was substantially suppressed by administering the drugs at the times of day when P-gp levels were abundant. By contrast, there was no significant dosing time-dependent difference in the absorption of the non-P-gp substrate (acetaminophen). The oscillation in the intestinal expression of P-gp appears to affect the pharmacokinetics of its substrates. Identification of circadian factors affecting the drug disposition in laboratory monkeys may improve the predictive accuracy of pharmacokinetics in humans.

In mammals, circadian rhythms in physiological function are generated by a molecular oscillator driven by transcriptionaltranslational feedback loop consisting of negative and positive regulators. Disruption of this circadian clock machinery is thought to increase the risk of cancer development, but the potential contributions of each component of circadian clock to oncogenesis have been little explored. Here we reported that negative and positive transcriptional regulators of circadian feedback loop had different roles in oncogene-induced neoplastic transformation. Mouse embryonic fibroblasts prepared from animals deficient in negative circadian clock regulators, Period2 (Per2) or Cryptochrome1/2 (Cry1/2), were prone to transformation induced by co-expression of H-ras V12 and SV40 large T antigen (SV40LT). In contrast, mouse embryonic fibroblasts prepared from mice deficient in positive circadian clock regulators, Bmal1 or Clock, showed resistance to oncogene-induced transformation. In Per2 mutant and Cry1/2-null cells, the introduction of oncogenes induced expression of ATF4, a potent repressor of cell senescence-associated proteins p16INK4a and p19ARF. Elevated levels of ATF4 were sufficient to suppress expression of these proteins and drive oncogenic transformation. Conversely, in Bmal1-null and Clock mutant cells, the expression of ATF4 was not induced by oncogene introduction, which allowed constitutive expression of p16INK4a and p19ARF triggering cellular senescence. Although genetic ablation of either negative or positive transcriptional regulators of the circadian clock leads to disrupted rhythms in physiological functions, our findings define their different contributions to neoplastic cellular transformation.

Edited by Eric R. FearonDisruption of circadian rhythms has been implicated in an increased risk for cancer development. The Period2 (Per2) gene encodes one of the major components of the mammalian circadian clock, which plays a key role in controlling the circadian rhythms in physiology and behavior. PER2 has also been reported to suppress the malignant transformation of cells, but its role in the regulation of cancer susceptibility to chemotherapeutic drugs remains unclear. In this study, we found that oncogene-transformed embryonic fibroblasts prepared from Per2-mutant (Per2 m/m ) mice, which are susceptible to both spontaneous and radiation-induced tumorigenesis, were resistant against common chemotherapeutic drugs and that this resistance is associated with up-regulation of the aldehyde dehydrogenase 3a1 (Aldh3a1) gene. Co-expression of the oncogenes H-ras V12 and SV40 large T-antigen induced malignant transformation of both WT and Per2 m/m cells, but the cytotoxic effects of the chemotherapeutic agents methotrexate, gemcitabine, etoposide, vincristine, and oxaliplatin were significantly alleviated in the oncogene-transformed Per2 m/m cells. Although introduction of the two oncogenes increased the expression of Aldh3a1 in both WT and Per2 m/m cells, the ALDH3A1 protein levels in the Per2 m/m cells were ϳ7-fold higher than in WT cells. The elevated ALDH3A1 levels in the oncogenetransformed Per2 m/m cells were sufficient to prevent chemotherapeutic drug-induced accumulation of reactive oxygen species. Consequently, shRNA-mediated suppression of Aldh3a1 expression relieved the chemoresistance of the Per2 m/m cells. These results suggest a role for mutated PER2 in the development of multiple drug resistance and may inform therapeutic strategies for cancer management.

The chronic disruption of circadian rhythms has been implicated in the risk of cancer development in humans and laboratory animals. The gene product CLOCK is a core molecular component of the circadian oscillator, so that mice with a mutated Clock gene (Clk/Clk) exhibit abnormal rhythms in various physiological processes. However, we demonstrated here that Clk/Clk mice resisted chemical carcinogen-induced tumorigenesis by suppressing epidermal growth factor (EGF) receptor-mediated proliferation signals. The repetitive application of 7,12-dimethylbenz[α]anthracene (DMBA) to skin on the back resulted in the significant development of tumors in wild-type mice, whereas chemically-induced tumorigenesis was alleviated in Clk/Clk mice. Although the degree of DMBA-induced DNA damage was not significantly different between wild-type and Clk/Clk mice, EGF receptor-mediated Ras activation was not detected in DMBA-treated Clk/Clk mice. Genetic and biochemical experiments revealed that the suppression of EGF receptor-mediated signal transduction in DMBA-treated Clk/Clk mice was associated with the expression of the cellular senescence factor p16INK4a. These results suggest an uncovered role for CLOCK in the development of chemical carcinogen-induced primary tumors and offers new preventive strategies.

A number of diverse cell-surface proteins are anchored to the cytoskeleton via scaffold proteins. Na+/H+ exchanger regulatory factor-1 (NHERF1), encoded by the Slc9a3r1 gene, functions as a scaffold protein, which is implicated in the regulation of membrane expression of various cell-surface proteins. Here, we demonstrate that the circadian clock component PERIOD2 (PER2) modulates transcription of the mouse Slc9a3r1 gene, generating diurnal accumulation of NHERF1 in the mouse liver. Basal expression of Slc9a3r1 was dependent on transcriptional activation by p65/p50. PER2 bound to p65 protein and prevented p65/p50-mediated transactivation of Slc9a3r1. The time-dependent interaction between PER2 and p65 underlay diurnal oscillation in the hepatic expression of Slc9a3r1/NHERF1. The results of immunoprecipitation experiments and liquid chromatography-mass spectrometry analysis of mouse liver revealed that NHERF1 time-dependently interacted with fatty acid transport protein-5 (FATP5). Temporary accumulation of NHERF1 protein stabilized plasmalemmal localization of FATP5, thereby enhancing hepatic uptake of fatty acids at certain times of the day. Our results suggest an unacknowledged role for PER2 in regulating the diurnal expression of NHERF1 in mouse liver. This machinery also contributed to diurnal changes in the ability of hepatic cells to uptake fatty acids.