Sex-, feeding-, and circadian time-dependency of P-glycoprotein expression and activity - implications for mechanistic pharmacokinetics modeling.

P-glycoprotein (P-gp) largely influences the pharmacokinetics (PK) and toxicities of xenobiotics in a patient-specific manner so that personalized drug scheduling may lead to significant patient's benefit. This systems pharmacology study investigated P-gp activity in mice according to organ, sex, feeding status, and circadian time. Sex-specific circadian changes were found in P-gp ileum mRNA and protein levels, circadian amplitudes being larger in females as compared to males. Plasma, ileum and liver concentrations of talinolol, a pure P-gp substrate, significantly differed according to sex, feeding and circadian timing. A physiologically-based PK model was designed to recapitulate these datasets. Estimated mesors (rhythm-adjusted mean) of ileum and hepatic P-gp activity were higher in males as compared to females. Circadian amplitudes were consistently higher in females and circadian maxima varied by up to 10 h with respect to sex. Fasting increased P-gp activity mesor and dampened its rhythm. Ex-vivo bioluminescence recordings of ileum mucosae from transgenic mice revealed endogenous circadian rhythms of P-gp protein expression with a shorter period, larger amplitude, and phase delay in females as compared to males. Importantly, this study provided model structure and parameter estimates to refine PK models of any P-gp substrate to account for sex, feeding and circadian rhythms.

Critical cholangiocarcinogenesis control by cryptochrome clock genes.

A coordinated network of molecular circadian clocks in individual cells generates 24-hr rhythms in liver metabolism and proliferation. Circadian disruption through chronic jet lag or Per2 clock gene mutation was shown to accelerate hepatocarcinoma development in mice. As divergent effects were reported for clock genes Per and Cry regarding xenobiotic toxicity, we questioned the role of Cry1 and Cry2 in liver carcinogenesis. Male WT and Cry1-/- Cry2-/- mice (C57Bl/6 background) were chronically exposed to diethylnitrosamine (DEN) at ZT11. Rest-activity and body temperature rhythms were monitored using an implanted radiotransmitter. Serum aspartate and alanine aminotransferases (AST and ALT) were determined on four occasions during the progression stage. After 7 months, serum alkaline phosphatases (ALP) were determined, and livers were sampled for microscopic tumor nodule counting and histopathology. Five months after initiation of DEN treatment, we found that Cry1-/- Cry2-/- mice developed severe liver dysplasia, as evident from the increased AST, ALT and ALP levels, as compared to WT mice. DEN exposure induced primary liver cancers in nearly fivefold as many Cry1-/- Cry2-/- mice as compared to WT mice (p = 0.01). Microscopic study revealed no difference in the average number of hepatocarcinomas and a nearly eightfold increase in the average number of cholangiocarcinomas in Cry1-/- Cry2-/- mice, as compared to WT mice. This study validated the hypothesis that molecular circadian clock disruption dramatically increased chemically induced liver carcinogenesis. In addition, the pronounced shift toward cholangiocarcinoma in DEN exposed Cry1-/- Cry2-/- mice revealed a critical role of the Cry clock genes in bile duct carcinogenesis.

Clock gene Per2 as a controller of liver carcinogenesis.

Environmental disruption of molecular clocks promoted liver carcinogenesis and accelerated cancer progression in rodents. We investigated the specific role of clock gene Period 2 (Per2) for liver carcinogenesis and clock-controlled cellular proliferation, genomic instability and inflammation. We assessed liver histopathology, and determined molecular and physiology circadian patterns in mice on chronic diethylnitrosamine (DEN) exposure according to constitutive Per2 mutation. First, we found that Per2m/m liver displayed profound alterations in proliferation gene expression, including c-Myc derepression, phase-advanced Wee1, and arrhythmic Ccnb1 and K-ras mRNA expressions, as well as deregulated inflammation, through arrhythmic liver IL-6 protein concentration, in the absence of any DEN exposure. These changes could then make Per2m/m mice more prone to subsequently develop liver cancers on DEN. Indeed, primary liver cancers were nearly fourfold as frequent in Per2m/m mice as compared to wild-type (WT), 4 months after DEN exposure. The liver molecular clock was severely disrupted throughout the whole carcinogenesis process, including the initiation stage, i.e. within the initial 17 days on DEN. Per2m/m further exhibited increased c-Myc and Ccnb1 mean 24h expressions, lack of P53 response, and arrhythmic ATM, Wee1 and Ccnb1 expressions. DEN-induced tumor related inflammation was further promoted through increased protein concentrations of liver IL-6 and TNF-α as compared to WT during carcinogenesis initiation. Per2 mutation severely deregulated liver gene or protein expressions related to three cancer hallmarks, including uncontrolled proliferation, genomic instability, and tumor promoting inflammation, and accelerated liver carcinogenesis several-fold. Clock gene Per2 acted here as a liver tumor suppressor from initiation to progression.

The effect of circadian rhythm on pharmacokinetics and metabolism of the Cdk inhibitor, roscovitine, in tumor mice model.

Roscovitine is a selective Cdk-inhibitor that is under investigation in phase II clinical trials under several conditions, including chemotherapy. Tumor growth inhibition has been previously shown to be affected by the dosing time of roscovitine in a Glasgow osteosarcoma xenograft mouse model. In the current study, we examined the effect of dose timing on the pharmacokinetics, biodistribution and metabolism of this drug in different organs in B6D2F1 mice. The drug was orally administered at resting (ZT3) or activity time of the mice (ZT19) at a dose of 300 mg/kg. Plasma and organs were removed at serial time points (10, 20 and 30 min; 1, 2, 4, 6, 8, 12 and 24 h) after the administration. Roscovitine and its carboxylic metabolite concentrations were analyzed using HPLC-UV, and pharmacokinetic parameters were calculated in different organs. We found that systemic exposure to roscovitine was 38% higher when dosing at ZT3, and elimination half-life was double compared to when dosing at ZT19. Higher organ concentrations expressed as (organ/plasma) ratio were observed when dosing at ZT3 in the kidney (180%), adipose tissue (188%), testis (132%) and lungs (112%), while the liver exposure to roscovitine was 120% higher after dosing at ZT19. The metabolic ratio was approximately 23% higher at ZT19, while the intrinsic clearance (CLint) was approximately 67% higher at ZT19, indicating faster and more efficient metabolism. These differences may be caused by circadian differences in the absorption, distribution, metabolism and excretion processes governing roscovitine disposition in the mice. In this article, we describe for the first time the chronobiodistribution of roscovitine in the mouse and the contribution of the dosing time to the variability of its metabolism. Our results may help in designing better dosing schedules of roscovitine in clinical trials.

Functional genomics identify Birc5/survivin as a candidate gene involved in the chronotoxicity of cyclin-dependent kinase inhibitors.

The circadian timing system orchestrates most of mammalian physiology and behavior in synchrony with the external light/dark cycle. This regulation is achieved through endogenous clocks present in virtually all body cells, where they control key cellular processes, including metabolism, transport, and the cell cycle. Consistently, it has been observed in preclinical cancer models that both the efficacy and toxicity of most chemotherapeutic drugs depend on their time of administration. To further explore the molecular basis underlying the link between the circadian timing system and the cellular response to anticancer drugs, we investigated the circadian transcriptome and CDK inhibitor toxicity in colon mucosa cells. We first show here that among 181 circadian transcripts, approximately 30% of them drive the cell cycle in the healthy mouse colon mucosa, with a majority peaking during the early resting phase. The identification of 26 mitotic genes within this cluster further indicated that the transcriptional coordination of mitosis by the circadian clock participates in the gating of cell division in this tissue. Subsequent selective siRNA-mediated silencing of these 26 targets revealed that low expression levels of the mitotic and anti-apoptotic gene Birc5/survivin significantly and specifically increased the sensitivity of colon epithelial cells to CDK inhibitors. By identifying Birc5/survivin as a potential determinant for the circadian modulation of CDK inhibitor toxicity, these data provide a mechanistic basis for the preclinical development of future CDK inhibitor-based chronotherapeutic strategies.

Optimization of irinotecan chronotherapy with P-glycoprotein inhibition.

The relevance of P-glycoprotein (P-gp) for irinotecan chronopharmacology was investigated in female B6D2F1 mice. A three-fold 24h change in the mRNA expression of Abcb1b was demonstrated in ileum mucosa, with a maximum at Zeitgeber Time (ZT) 15 (p<0.001). No rhythm was found for abcb1a in ileum mucosa, or for Abcb1a/b in Glasgow osteosarcoma (GOS), a mouse tumor cell line moderately sensitive to irinotecan. Non-tumor-bearing mice received irinotecan (50mg/kg/day i.v.×4days) as a single agent or combined with P-gp inhibitor PSC833 (6.25mg/kg/day i.p.×4 days) at ZT3 or ZT15, respectively corresponding to the worst or the best irinotecan tolerability. Endpoints involved survival, body weight change and hematologic toxicity. Antitumor efficacy was studied in GOS-bearing mice receiving irinotecan (25, 30 or 40mg/kg/day×4days) and +/-PSC833 at ZT3 or ZT15, with survival, body weight change, and tumor growth inhibition as endpoints. Non-tumor bearing mice lost an average of 17% or 9% of their body weight according to irinotecan administration at ZT3 or ZT15 respectively (p<0.001). Dosing at ZT15 rather than ZT3 reduced mean leucopenia (9% vs 53%; p<0.001). PSC833 aggravated irinotecan lethal toxicity from 4 to ~60%. In tumor-bearing mice, body weight loss was ~halved in the mice on irinotecan or irinotecan-PSC833 combination at ZT15 as compared to ZT3 (p<0.001). PSC833-irinotecan at ZT15 increased tumor inhibition by ~40% as compared to irinotecan only at ZT15. In conclusion, P-gp was an important determinant of the circadian balance between toxicity and efficacy of irinotecan.

A circadian clock transcription model for the personalization of cancer chronotherapy.

Circadian timing of anticancer medications has improved treatment tolerability and efficacy several fold, yet with intersubject variability. Using three C57BL/6-based mouse strains of both sexes, we identified three chronotoxicity classes with distinct circadian toxicity patterns of irinotecan, a topoisomerase I inhibitor active against colorectal cancer. Liver and colon circadian 24-hour expression patterns of clock genes Rev-erbα and Bmal1 best discriminated these chronotoxicity classes, among 27 transcriptional 24-hour time series, according to sparse linear discriminant analysis. An 8-hour phase advance was found both for Rev-erbα and Bmal1 mRNA expressions and for irinotecan chronotoxicity in clock-altered Per2(m/m) mice. The application of a maximum-a-posteriori Bayesian inference method identified a linear model based on Rev-erbα and Bmal1 circadian expressions that accurately predicted for optimal irinotecan timing. The assessment of the Rev-erbα and Bmal1 regulatory transcription loop in the molecular clock could critically improve the tolerability of chemotherapy through a mathematical model-based determination of host-specific optimal timing.

Sex and dosing-time dependencies in irinotecan-induced circadian disruption.

Circadian disruption accelerates malignant growth; thus, it should be avoided in anticancer therapy. The circadian disruptive effects of irinotecan, a topoisomerase I inhibitor, was investigated according to dosing time and sex. In previous work, irinotecan achieved best tolerability following dosing at zeitgeber time (ZT) 11 in male and ZT15 in female mice, whereas worst toxicity corresponded to treatment at ZT23 and ZT3 in male and female mice, respectively. Here, irinotecan (50 mg/kg intravenous [i.v.]) was delivered at the sex-specific optimal or worst circadian timing in male and female B6D2F1 mice. Circadian disruption was assessed with rest-activity, body temperature, plasma corticosterone, and liver mRNA expressions of clock genes Rev-erbα, Per2, and Bmal1. Baseline circadian rhythms in rest-activity, body temperature, and plasma corticosterone were more prominent in females as compared to males. Severe circadian disruption was documented for all physiology and molecular clock endpoints in female mice treated at the ZT of worst tolerability. Conversely, irinotecan administration at the ZT of best tolerability induced slight alteration of circadian physiology and clock-gene expression patterns in female mice. In male mice, irinotecan produced moderate alterations of circadian physiology and clock-gene expression patterns, irrespective of treatment ZT. However, the average expression of Rev-erbα, Per2, and Bmal1 were down-regulated 2- to 10-fold with irinotecan at the worst ZT, while being minimally or unaffected at the best ZT, irrespective of sex. Corticosterone secretion increased acutely within 2 h with a sex-specific response pattern, resulting in a ZT-dependent phase-advance or -delay in both sex. The mRNA expressions of irinotecan clock-controlled metabolism genes Ce2, Ugt1a1, and Top1 were unchanged or down-regulated according to irinotecan timing and sex. This study shows that the circadian timing system represents an important toxicity target of irinotecan in female mice, where circadian disruption persists after wrongly timed treatment. As a result, the mechanisms underling cancer chronotherapeutics are expectedly more susceptible to disruption in females as compared to males. Thus, the optimal circadian timing of chemotherapy requires precise determination according to sex, and should involve the noninvasive monitoring of circadian biomarkers.

Strain- and sex-dependent circadian changes in abcc2 transporter expression: implications for irinotecan chronotolerance in mouse ileum.

BACKGROUND: ATP-binding cassette transporter abcc2 is involved in the cellular efflux of irinotecan. The drug is toxic for mouse ileum, where abcc2 is highly expressed. Here, we investigate whether circadian changes in local abcc2 expression participate in the circadian rhythm of irinotecan toxicity for ileum mucosa, and further assess whether genetic background or sex modify this relation. METHODOLOGY/PRINCIPAL FINDINGS: Ileum mucosa was obtained every 3-4 h for 24 h in male and female B6D2F(1) and B6CBAF(1) mice synchronized with light from Zeitgeber Time (ZT)0 to ZT12 alternating with 12 h of darkness. Irinotecan (50 mg/kg i.v. daily for 4 days) was administered at the sex- and strain-specific times corresponding to least (ZT11-15) or largest drug-induced body weight loss (ZT23-03-07). Abcc2 expression was determined with qRT-PCR for mRNA and with immunohistochemistry and confocal microscopy for protein. Histopathologic lesions were graded in ileum tissues obtained 2, 4 or 6 days after treatment. Two- to six-fold circadian changes were demonstrated for mRNA and protein mean expressions of abcc2 in mouse ileum (p<0.05). ZT12 corresponded to high mRNA and protein expressions, with circadian waveforms differing according to genetic background and sex. The proportion of mice spared from ileum lesions varied three-fold according to irinotecan timing, with best tolerability at ZT11-15 (p = 0.00003). Irinotecan was also best tolerated in males (p = 0.05) and in B6CBAF(1) (p = 0.0006). CONCLUSIONS/SIGNIFICANCE: Strain- and sex-dependent circadian patterns in abcc2 expressions displayed robust relations with the chronotolerance of ileum mucosa for irinotecan. This finding has strong potential implications for improving the intestinal tolerability of anticancer drugs through circadian delivery.

Circadian disruption accelerates liver carcinogenesis in mice.

BACKGROUND: The circadian timing system rhythmically controls behavior, physiology, cellular proliferation and xenobiotic metabolism over the 24-h period. The suprachiasmatic nuclei in the hypothalamus coordinate the molecular clocks in most mammalian cells through an array of circadian physiological rhythms including rest-activity, body temperature, feeding patterns and hormonal secretions. As a result, shift work that involves circadian disruption is probably carcinogenic in humans. In experimental models, chronic jet-lag (CJL) suppresses rest-activity and body temperature rhythms and accelerates growth of two transplantable tumors in mice. CJL also suppresses or significantly alters the expression rhythms of clock genes in liver and tumors. Circadian clock disruption from CJL downregulates p53 and upregulates c-Myc, thus favoring cellular proliferation. Here, we investigate the role of CJL as a tumor promoter in mice exposed to the hepatic carcinogen, diethylnitrosamine (DEN). METHODS: In experiment 1 (Exp 1), the dose-dependent carcinogenicity of chronic intraperitoneal (i.p.) administration of DEN was explored in mice. In Exp 2, mice received DEN at 10 mg/kg/day (cumulative dose: 243 mg/kg), then were randomized to remain in a photoperiodic regimen where 12 h of light alternates with 12 h of darkness (LD 12:12) or to be submitted to CJL (8-h advance of light onset every 2 days). Rest-activity and body temperature were monitored. Serum liver enzymes were determined repeatedly. Mice were sacrificed and examined for neoplastic lesions at 10 months. RESULTS: In Exp 1, DEN produced liver cancers in all the mice receiving 10 mg/kg/day. In Exp 2, mice on CJL had increased mean plasma levels of aspartate aminotransferase and more liver tumors as compared to LD mice at approximately 10 months (p = 0.005 and 0.028, respectively). The mean diameter of the largest liver tumor was twice as large in CJL vs LD mice (8.5 vs 4.4 mm, p = 0.027). In LD, a single histologic tumor type per liver was observed. In CJL, up to four different types were associated in the same liver (hepatocellular- or cholangio-carcinomas, sarcomas or mixed tumors). DEN itself markedly disrupted the circadian rhythms in rest-activity and body temperature in all the mice. DEN-induced disruption was prolonged for >or= 3 months by CJL exposure. CONCLUSIONS: The association of circadian disruption with chronic DEN exposure suggests that circadian clocks actively control the mechanisms of liver carcinogenesis in mice. Persistent circadian coordination may further be critical for slowing down and/or reverting cancer development after carcinogen exposure.

Liver circadian clock, a pharmacologic target of cyclin-dependent kinase inhibitor seliciclib.

Circadian disruption accelerates malignant growth and shortens survival, both in experimental tumor models and cancer patients. In previous experiments, tumor circadian disruption was rescued with seliciclib, an inhibitor of cyclin-dependent kinases (CDKs). This effect occurred at a selective dosing time and was associated with improved antitumor activity. In the current study, seliciclib altered robust circadian mRNA expression of the clock genes Rev-erb alpha, Per2, and Bmal1 in mouse liver following dosing at zeitgeber time (ZT) 3 (i.e., 3 h after the onset of the 12 h light span), when mice start to rest, but not at ZT19, near the middle of the 12 h dark span, when mice are most active. However, liver exposure to seliciclib, as estimated by the liver area under the concentration x time curve (AUC), was approximately 80% higher at ZT19 than at ZT3 (p = 0.049). Circadian clock disruption was associated with increased serum liver enzymes and modified glycogen distribution in hepatocytes, as revealed by biochemical determinations and optic and electronic microscopy. The extent of increase in liver enzymes was most pronounced following dosing at ZT3, as compared to ZT19 (p < 0.04). Seliciclib further up-regulated the transcriptional activity of c-Myc, a cell cycle gene that promotes cell cycle entry and G1-S transition (p < 0.001), and down-regulated that of Wee1, which gates cell cycle transition from G2 to M (p < 0.001). These effects did not depend upon drug dosing time. Overall, the results suggest the circadian time of seliciclib delivery is more critical than the amount of drug exposure in determining its effects on the circadian clock. Seliciclib-induced disruption of the liver molecular clock could account for liver toxicity through the resulting disruption of clock-controlled detoxification pathways. Modifications of cell cycle gene expression in the liver likely involve other mechanisms. Circadian clocks represent relevant targets to consider for optimization of therapeutic schedules of CDK inhibitors.

Circadian disruption in experimental cancer processes.

The circadian timing system (CTS) coordinated by the suprachiasmatic nuclei (SCN) of the hypothalamus regulates daily rhythms of behavior, physiology, as well as cellular metabolism and proliferation. Altered circadian rhythms predict for poor survival in cancer patients. An increased incidence of several cancers has been reported in flight attendants and in shift workers. To explore the contribution of the CTS to tumor growth, we developed experimental models of disrupted or enhanced circadian coordination through stereotaxic destruction of the SCN, modifications of photoperiodic or feeding synchronizers and/or the administration of pharmacologic agents. SCN ablation or exposure to experimental chronic jetlag (CJL, consisting of an 8-hour advance of the light-dark cycle every 2 days) caused alterations in circadian physiology and significantly accelerated tumor growth. CJL suppressed or altered the rhythms of clock gene and cell cycle gene expression in mouse liver. It increased p53 and decreased c-Myc expression, a result in line with the promotion of diethylnitrosamine -initiated hepatocarcinogenesis in jet-lagged mice. The accelerating effect of CJL on tumor growth was counterbalanced by the regular timing of food access over the 24-h. Meal timing prevented the circadian disruption produced by CJL and slowed down tumor growth. In synchronized mice, meal timing reinforced host circadian coordination, phase-shifted the transcriptional rhythms of clock genes in the liver of tumor-bearing mice and slowed down cancer progression. These results support the role of the CTS in cancer progression and call for the development of therapeutic strategies aimed at preventing or treating circadian clock dysfunctions.

Altered Stra13 and Dec2 circadian gene expression in hypoxic cells.

The circadian system regulates rhythmically most of the mammalian physiology in synchrony with the environmental light/dark cycle. Alteration of circadian clock gene expression has been associated with tumour progression but the molecular links between the two mechanisms remain poorly defined. Here we show that Stra13 and Dec2, two circadian transcriptional regulators which play a crucial role in cell proliferation and apoptosis are overexpressed and no longer rhythmic in serum shocked fibroblasts treated with CoCl(2,) a substitute of hypoxia. This effect is associated with a loss of circadian expression of the clock genes Rev-erbalpha and Bmal1, and the clock-controlled gene Dbp. Consistently, cotransfection assays demonstrate that STRA13 and DEC2 both antagonize CLOCK:BMAL1 dependent transactivation of the Rev-erbalpha and Dbp promoters. Using a transplantable osteosarcoma tumour model, we show that hypoxia is associated with altered circadian expression of Stra13, Dec2, Rev-erbalpha, Bmal1 and Dbp in vivo. These observations collectively support the notion that overexpression of Stra13 and Dec2 links hypoxia signalling to altered circadian clock gene expression.

Improved tumor control through circadian clock induction by Seliciclib, a cyclin-dependent kinase inhibitor.

The circadian timing system and the cell division cycle are frequently deregulated in cancer. The therapeutic relevance of the reciprocal interactions between both biological rhythms was investigated using Seliciclib, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Mice bearing Glasgow osteosarcoma received Seliciclib (300 mg/kg/d orally) or vehicle for 5 days at Zeitgeber time (ZT) 3, 11, or 19. On day 6, tumor mRNA 24-hour expression patterns were determined for clock genes (Per2, Rev-erbalpha, and Bmal1) and clock-controlled cell cycle genes (c-Myc, Wee1, cyclin B1, and CDK1) with quantitative reverse transcription-PCR. Affinity chromatography on immobilized Seliciclib identified CDK1/CDK2 and extracellular signal-regulated kinase (ERK) 1/ERK2, CDK7/CDK9, and casein kinase CK1epsilon as Seliciclib targets, which respectively regulate cell cycle, transcription, and circadian clock in Glasgow osteosarcoma. Seliciclib reduced tumor growth by 55% following dosing at ZT3 or ZT11 and by 35% at ZT19 compared with controls (P < 0.001). Tolerability was also best at ZT3. Mean transcriptional activity of Rev-erbalpha, Per2, and Bmal1 was arrhythmic in the tumors of untreated mice. Seliciclib induced rhythmic clock gene expression patterns with physiologic phase relations only after ZT3 dosing. c-Myc and Wee1 mRNAs displayed synchronous circadian rhythms in the tumors of control mice receiving vehicle only but not in those of mice given the drug. Seliciclib further enhanced Wee1 expression irrespective of dosing time, an effect that reinforced G(2)-M gating. Seliciclib also inhibited CK1epsilon, which determines circadian period length. The coordination of clock gene expression patterns in tumor cells was associated with best antitumor activity of Seliciclib. The circadian clock and its upstream regulators represent relevant targets for CDKIs.

Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow.

OBJECTIVE: Clock genes are known to mediate circadian rhythms in the central nervous system and peripheral organs. Although they are expressed in mouse hematopoietic progenitor and stem cells, it is unknown if they are related to circadian rhythms in these cells. We therefore investigated the 24-hour patterns in the activity of several clock genes in the bone marrow (BM) side population (SP) primitive stem cells, and compared these 24-hour patterns to clock gene variations in the whole BM and liver. METHODS: Cells were obtained from 84 B6D2F(1) mice in three replicate experiments on the second day after release into constant darkness from a standardizing light-dark schedule. mRNA expression of clock genes was measured with quantitative reverse transcriptase polymerase chain reaction. RESULTS: mPer2 displayed circadian rhythms in SP cells, whole BM, and liver cells. mPer1 and mRev-erb alpha showed a circadian rhythm in whole BM and liver, but not SP cells. mBmal1 was not expressed rhythmically in SP cells, nor in the whole BM, contrary to rhythms observed in the liver. CONCLUSIONS: With the exception of mPer2, most clock genes studied in primitive hematopoietic SP stem cells were not oscillating in a fully organized circadian manner, which is similar to immature cells in rapidly proliferating organs, such as the testis and thymus. These findings indicate that circadian clock gene expression variations in BM are developmentally regulated.

Effects of light and food schedules on liver and tumor molecular clocks in mice.

BACKGROUND: Disrupted circadian coordination accelerates malignant growth, but the molecular mechanism is unclear. METHODS: Healthy or Glasgow osteosarcoma-bearing mice (n = 162) were synchronized with light and darkness over 2-3 weeks, submitted to an 8-hour advance onset of light every 2 days (chronic jet lag) to disrupt circadian coordination, or submitted to chronic jet lag and meal timing to prevent molecular clock alteration. The expression of molecular clock genes and of the cell cycle genes c-Myc and p53 in liver and tumor was determined with quantitative reverse transcription-polymerase chain reaction at six circadian times over a 24-hour period of light and darkness and analyzed with analysis of variance and cosinor. Tumor weight was measured daily over the course of the experiment. All statistical tests were two-sided. RESULTS: In synchronized mice, mean mRNA levels of clock genes Rev-erbalpha, Per2, and Bmal1 varied by 206-, four-, and 26-fold, respectively, over the 24 hours in healthy mouse liver; by 36-, 35-, and 32-fold in the livers of tumor-bearing mice; and by 9.4-, 5.5-, and sixfold in tumor tissue (P = .046 to <.001). In mice subjected to chronic jet lag, the periodic changes were dampened and the clock gene rhythms were temporally shifted in liver and ablated in tumor, and tumor growth was accelerated. Meal timing reversed the chronic jet lag-induced alterations in Rev-erbalpha and Per2 expression in liver and of all three clock genes in tumor and slowed tumor growth. Tumor growth differed as a function of light and feeding schedules (P = .04). No obvious rhythm was detected for p53 or c-Myc in liver or tumor tissues of synchronized mice. In healthy mice subjected to chronic jet lag, the mean level of p53 expression was cut in half (P = .002), and a 12-fold circadian variation in c-Myc mRNA level (P = .03) was induced in the liver of healthy mice, whereas complex expression patterns were found in the liver and tumor of tumor-bearing mice. CONCLUSIONS: Altered light-dark or feeding schedules modified the expression of molecular clock genes and genes involved in carcinogenesis and tumor progression.

Circadian regulation of cell cycle and apoptosis proteins in mouse bone marrow and tumor.

Proapoptotic drugs such as docetaxel displayed least toxicity and highest antitumor efficacy following dosing during the circadian rest phase in mice, suggesting that cell cycle and apoptotic processes could be regulated by the circadian clock. In study 1, mouse bone marrow and/or tumor were obtained every 4 h for 24 h in C3H/HeN mice with or without MA13/C mammary adenocarcinoma in order to determine the circadian patterns in cell-cycle phase distribution and BCL-2 anti-apoptotic protein expression. In study 2, mouse bone marrow from B6D2F1 mice was sampled every 3 h for 24 h in order to confirm the BCL-2 rhythm and to study its relation with 24 h changes in the expression of proapoptotic BCL-2-associated X protein (BAX) protein and clock genes mPer2, mBmal1, mClock, and mTim mRNAs. The rhythms in G1-, S- or G2/M-phase cells were shifted in tumor compared with bone marrow. In the tumor, the mean proportion of G2/M-phase cells increased by 75% from late rest to late activity span (P from cosinor = 0.001). No 24 h rhythm was found for BCL-2 in tumors. In contrast to this, in the bone marrow, mean BCL-2 expression varied 2.8-fold in B6D2F1 mice (P=0.025) and 3- or 4.5-fold in tumor-bearing and nontumor-bearing C3H/HeN mice, with a peak during the early rest span (P=0.024 and P<0.001, respectively). BAX varied fivefold during the 24 h span with a major peak occurring near mid-activity (P=0.007). The mean mRNAs of mPer2, mClock, and mBmal1 varied twofold to threefold over the 24 h, with high values during the activity span (P<0.05). In the tumor, the circadian organization in cell-cycle phase distribution was shifted and BCL2 rhythm was ablated. Conversely, a molecular circadian clock likely regulated BCL-2 and BAX expression in the bone marrow, increasing cellular protection against apoptosis during the rest span.

Effects of chronic jet lag on tumor progression in mice.

Frequent transmeridian flights or predominant work at night can increase cancer risk. Altered circadian rhythms also predict for poor survival in cancer patients, whereas physical destruction of the suprachiasmatic nuclei (SCN), the hypothalamic circadian pacemaker, accelerates tumor growth in mice. Here we tested the effect of functional disruption of circadian system on tumor progression in a novel experimental model of chronic jet lag. B6D2F(1) mice were synchronized with 12 hours of light and 12 hours of darkness or underwent repeat 8-hour advances of the light/dark cycle every 2 days before inoculation of Glasgow osteosarcoma. The 24-hour changes were assessed for plasma corticosterone, clock protein mPER1 expression in the SCN, and mRNA expression of clock genes mPer2 and mRev-erbalpha in liver and tumor. Time series were analyzed by spectral analysis and/or Cosinor. Differences were compared with analysis of variance (ANOVA). The 24-hour rest/activity cycle was ablated, and the rhythms of body temperature, serum corticosterone, and mPER1 protein expression in the SCN were markedly altered in jet-lagged mice as compared with controls (ANOVA, P < 0.001 for corticosterone and P = 0.01 for mPER1). Tumor grew faster in the jet-lagged animals as compared with controls (ANOVA, P < 0.001), whereas exposure to constant light or darkness had no effect (ANOVA, P = 0.66 and P = 0.8, respectively). The expression of mPer2 and mRev-erbalpha mRNAs in controls showed significant circadian rhythms in the liver (P = 0.006 and P = 0.003, respectively, Cosinor) and in the tumor (P = 0.04 and P < 0.001). Both rhythms were suppressed in the liver (P = 0.2 and P = 0.1, respectively, Cosinor) and in the tumor (P = 0.5) of jet-lagged mice. Altered environmental conditions can disrupt circadian clock molecular coordination in peripheral organs including tumors and play a significant role in malignant progression.

Circadian rhythm of irinotecan tolerability in mice.

The toxicity of irinotecan (CPT-11), a topoisomerase-I inhibitor largely used in cancer patients, was investigated as a function of the circadian time of its administration in mice, with mortality, body weight loss, leukopenia, neutropenia, intestinal lesions, and bone marrow cell cycle phase distribution as end points. Four experiments were performed on a total of 773 male mice standardized with 12h light/12h darkness. Irinotecan was administered daily for 4 or 10 consecutive days (D1-4 and D1-10, respectively, in different experiments) at one of six circadian stages expressed in hours after light onset (HALO). The survival curves differed significantly as a function of the dosage and circadian time of drug administration by the D1-10 schedule, with 70% survival at 7 or 11 HALO and 51% at 19 or 23 HALO (p=0.039 from log rank test). CPT-11 administration at 19 or 23 HALO resulted in (1) greatest mean body weight loss at nadir; (2) most severe colic and bone marrow lesions and/or slowest recovery; and (3) deepest neutropenia nadir and/or slowest hematologic recovery. These circadian treatment time-related differences were statistically validated. The bone marrow cell cycle data revealed a four to eight-fold larger G2-M phase arrest following irinotecan administration at 19 or 23 HALO in comparison to the other times of drug administration, apparently representative of the repair of more extensive DNA damage (p < 0.001 from ANOVA) when the medication was given at these circadian times. Overall, CPT-11 was better tolerated by mice treated during the light (animals' rest) span. The results support the administration of CPT-11 to cancer patients in the second half of the night, during sleep, in order to improve drug tolerability.

Persistent twenty-four hour changes in liver and bone marrow despite suprachiasmatic nuclei ablation in mice.

Rest-activity or cortisol rhythms can be altered in cancer patients, a condition that may impair the benefits from a timed delivery of anticancer treatments. In rodents, the circadian pattern in rest-activity is suppressed by the destruction of the suprachiasmatic nuclei (SCN) in the hypothalamus. We sought whether such ablation would result in a similar alteration of cellular rhythms known to be relevant for anticancer drug chronopharmacology. The SCN of 77 B6D2F(1) mice synchronized with 12 h of light and 12 h of darkness were destroyed by electrocoagulation [SCN(-)], while 34 animals were sham operated. Activity and body temperature were recorded by telemetry. Blood and organs were sampled at one of six circadian times for determinations of serum corticosterone concentration, blood leukocyte count, reduced glutathione (GSH), and dihydropyrimidine dehydrogenase (DPD) mRNA expression in liver and cell cycle phase distribution of bone marrow cells. Sham-operated mice displayed significant 24-h rhythms in rest-activity and body temperature, whereas such rhythms were found in none and in 15% of the SCN(-) mice, respectively. SCN lesions markedly altered the rhythmic patterns in serum corticosterone and liver GSH, which became nonsinusoidal. Liver DPD mRNA expression and bone marrow cell cycle phase distribution displayed similar 24-h sinusoidal patterns in sham-operated and SCN(-) mice. These results support the existence of another light-dark entrainable pacemaker that can coordinate cellular functions in peripheral organs. They suggest that the delivery of anticancer treatments at an optimal time of day may still be beneficial, despite suppressed rest-activity or cortisol rhythms.