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 B6D2F1 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.
- circadian coordination
- cancer chronotherapeutics
- cell cycle
- dihydropyrimidine dehydrogenase
approximately 25% of cancer patients displayed profound alterations of rest-activity pattern and cortisol rhythm independently of tumor stage or general condition (32–34). Poorer tumor response and shorter survival were reported in these patients compared with those with near normal circadian function (36, 43). Such altered circadian function could also impair the therapeutic benefit that can result from chronotherapeutics, i.e., the delivery of anticancer treatments at selected times of day (12, 23, 26, 27).
This treatment optimization method is based on the regulation of cellular metabolism and proliferation by a molecular clock. This clock consists of interconnected molecular loops involving up to 12 specific clock genes. It has been uncovered in most mammalian tissues (21, 35, 41, 42) and rhythmically regulates the transcription of 5–10% of the genome (14, 21, 41, 42). Indeed, sustained rhythms in transcription or other cellular functions have been demonstrated in cultured mammalian cells, giving rise to the concept of “peripheral oscillators” (3, 14, 21, 41, 42). The autonomous cellular rhythms in liver, kidney, heart, or lung are presumably coordinated by the suprachiasmatic nuclei (SCN), a master pacemaker located in the hypothalamus. The SCN are ultimately responsible for the generation of the circadian rest-activity cycle and for the adjustment of the whole circadian time structure to the environmental light-dark cycle (21, 35, 41, 42).
In a prior study, we investigated the effect of SCN destruction on the rhythms in rest-activity, body temperature, plasma corticosterone concentration, and circulating lymphocyte count in 75 B6D2F1 mice with histologically confirmed SCN lesions and 64 sham-operated animals kept in a 12:12-h light-dark photoperiod (LD12:12) (16). Whereas all the mice with SCN destruction had an ablated circadian activity rhythm, an atypical body temperature rhythm was found in 15 animals. The plasma corticosterone rhythm was altered in mice with SCN lesions, with a peak advanced to midlight and a threefold reduction in the difference between peak and trough mean values, compared with sham-operated animals. Similarly, the rhythm in circulating lymphocytes was phase advanced by nearly 7 h and damped in mice with SCN lesions (16).
In the current study, we examined the effects of SCN ablation on rhythms in cellular detoxication processes and cell cycle-related events known as critical determinants for the dosing time dependency of anticancer drugs tolerability (5, 19, 26, 28, 35, 40, 46, 51). We selected cell cycle phase distribution in bone marrow and dihydropyrimidine dehydrogenase (DPD) mRNA expression in liver as determinants of 5-fluorouracil tolerability (17, 40, 45, 51) and reduced glutathione (GSH) in liver as a determinant of platinum complex tolerability (4, 6, 7, 28).
The study was conducted in accordance with the guidelines approved for animal experimental procedures by the French Ethical Committee, decree 87–848.
Seven-week-old B6D2F1 male mice were purchased from Charles Rivers (L'Arbresle, France). The experiment was performed in 111 mice synchronized to LD12:12 (with lights on from 0600 to 1800) to keep the possibility of an SCN-independent photoperiodic synchronization, which may be the case in clinically relevant situations. Animals had free access to food and water. All mice had a radiotransmitter (Physio Tel, TA 10 TA-F20, Data Sciences, St. Paul, MN) implanted into the peritoneal cavity to record locomotor activity and body temperature every 10 min throughout the experiment. Mice were randomly allocated to receive SCN lesions (77 mice) or sham operation (34 animals). Three or eight weeks after SCN destruction, blood, liver, femoral bone marrow, and brain were sampled at one of six different circadian times, 4 h apart over a single 24-h span, i.e., at 3, 7, 11, 15, 19, or 23 h after light onset (HALO), and processed as described below.
The SCN were destroyed by bilateral electrolytic lesion (25) [1 mA for 4 s; stereotaxic coordinates: anterior-posterior on bregma, mediolateral (±0.2 mm of midline), dorsoventral (0.55 mm below dural surface), incisor bar 0 mm below ear bars]. Sham-operated animals underwent the same stereotaxic procedure without electrolytic lesions. Effective SCN lesions were identified by the loss of any dominant periodicity (τ) of locomotor activity in the circadian domain (20 h < τ < 28 h) with Fourier transform spectral analysis after visual inspection (13). Complete SCN destruction was ascertained postmortem in all mice by both Nissl staining of the hypothalamus tissue and immunostaining for peptide histidine-isoleucine (PHI) (20).
Mice were anesthetized with a single intraperitoneal injection of 0.5 ml solution of 10 g of 2,2,2-tribromoethanol (Fluka, Saint-Quentin-Fallavier, France) in 10 ml of 2-methyl-2-butanol (Fluka) diluted 1:39 in 0.9% NaCl.
Plasma Corticosterone and Circulating Blood Cell Count Determination
Corticosterone concentration was measured by RIA [5 μl plasma were extracted with 3 ml diethyl ether; the residue was dissolved in 100 μl assay buffer before incubation with 1,2,6,7-[3H]corticosterone (Amersham, Orsay, France) and rabbit anti-corticosterone antibody (Valbiotech, Paris)]. Leukocyte count was determined with Cell Dyne 3500R (Abbott Diagnostics, Rungis, France). Within- and interassay coefficients of variation were 6 and 10% at a 5 ng/ml concentration and 5 and 9% at a 35 ng/ml concentration; all the samples were assayed in the same run.
Liver GSH Determination
Frozen tissues were suspended in 500 μl of 50 μm dithioerythritol (DTE) and homogenized with an Ultraturrax (Staufen, Germany) for 30 s at 4°C. The mixture was centrifuged at 13,000 g for 2 min; then 30 μl supernatant were mixed with 150 μl of 5% sulfosalicylic acid containing 50 μl DTE, and the precipitated proteins were removed by centrifugation. This protein precipitate was used for subsequent protein determination, dissolved in 200 μl of 20% sodium dodecyl sulfate, and measured with folin phenol reagent at 750 nm (Ultrospec II, LKB Biochrom, Cambridge, UK) (29). Free reduced glutathione (GSH) was determined in the acid extract according to GSH procedure 5 (45). The sensitivity of this method for GSH determination allows detection of quantities >2 pmol.
DPD Expression Measurement
DPD expression in liver was quantified according to a RT-PCR ELISA method previously published (40). Total RNA was isolated using the QIAGEN mini RNA/DNA kit (QIAGEN, Courtaboeuf, France). RNA quality was checked by agarose gel electrophoresis. RT was performed on 1 μg of total RNA. Because mouse DPYD gene was not cloned at the time of analysis, primer's oligonucleotides were selected from homology sequences obtained by alignment of cDNAs from the different species cloned so far (human, Rattus norvegicus, Bos taurus, Sus scrofa). The reference gene was β-actin. The oligonucleotides used for DPD amplification were AAT AGG TTT GCC AGA ACC CA [nucleotide (nt) 904–923 on R. norvegicus mRNA] for DPD sense strand and ACA TCA CCA CCT GCA AAT AC (nt 1484–1503 on R. norvegicus mRNA) for DPD antisense strand (600 bp product). Two specific capture probes, 5′-biotinylated and purified by HPLC (EUROBIO, les Ulis, France), corresponding to each amplification product, were used for ELISA detection. DPD capture probe was TCC CCA CGG AAG GTT ATA GT (nt 1232–1251 on R. norvegicus mRNA). PCR was conducted on 150-ng RNA equivalents in a 100-μl final volume containing 10 mM Tris·HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each deoxyribonucleotide triphosphate (dATP, dCTP, dGTP), 190 μM of dTTP, 10 μM of dUTP labeled with digoxigenin, 5 U of Taq polymerase, and 125 nM for the primer pair for actin and 500 nM for the primer pair for DPD. The multiplex amplification consisted of an initial 5-min incubation at 94°C followed by 23 amplification cycles (94°C for 30 s, 53°C for 30 s, and 72°C for 1 min) and 10-min incubation at 72°C. DPD and β-actin amplifications were performed using the PCR-ELISA DIG labeling and the PCR-ELISA DIG detection kits (Roche Diagnostics, Meylan, France) as previously described (11). The digoxigenin labeling reaction of the PCR products was carried out during coamplification of DPD and β-actin in the presence of digoxigenin-labeled dUTP. The bound hybrid was detected by an anti-digoxigenin antibody-peroxidase conjugate (measurement of peroxidase substrate absorption). Results were arbitrarily expressed as 1,000-fold the absorbance ratio (relative abundance, DPD/β actin). Intra- and interassay reproducibility evaluated on an RNA pool extracted from a rat liver gave coefficient of variation of 7.3% for intra-assay reproducibility (N = 6) and 19.4% for interassay reproducibility (N = 6).
Bone Marrow Nucleated Cells and Cell Cycle Phase Distribution
Each femur was flushed with 1 ml 0.9% NaCl. The cell suspension was centrifuged 5 min at 1,500 rpm at 4°C and washed in 0.9% NaCl. Nucleated cell count was determined with Cell Dyne 3500R (Abbott Diagnostics). One milliliter of ice-cold 75% ethanol was added to fix the cells. The cells were stored at 4°C until further processing.
A staining solution (250 μl) containing 50 μl/ml propidium iodide and 0.2 mg/ml RNase A (Sigma) was added to each sample (5 μl). They were incubated on ice for 24 h. Cell cycle was analyzed with a flow cytometer (FACScan, Becton Dickinson, Mountain View, CA) that measured the proportion of cells in G1, S, and G2-M phases (46).
Means and SEs were computed for each set of parameters. Intergroup differences were evaluated statistically using two-way ANOVA. Time series were analyzed by spectral analysis (Fourier transform analysis) using Mathcad 6.0. Statistical significance of circadian rhythmicity was further documented by cosinor analysis (13). This method characterized a rhythm by the parameters of the fitted cosine function best approximating all data. A period τ = 24 h was considered a priori. The rhythm characteristics estimated by this linear least squares method include the mesor (rhythm-adjusted mean), the double amplitude (2A, difference between minimum and maximum of fitted cosine function), and the acrophase (φ, time of maximum in fitted cosine function, with light onset as φ reference, so that units were in HALO). A rhythm was detected if the null hypothesis was rejected with P < 0.05; however, A and φ could be approximated if 0.05 < P < 0.10. All standard statistical tests were performed using SPSS for Windows software.
SCN Destruction, Rest-Activity, and Body Temperature Rhythms
Histological study using both Nissl stain and PHI immunostaining of the hypothalamus ascertained complete SCN destruction in 52 of 77 SCN-lesioned mice (68%), and only these animals were included in statistical analyses.
The rest-activity and the temperature patterns in sham-operated animals displayed a circadian rhythm validated by Fourier transform analysis, which showed a dominant 24-h period and was further confirmed with cosinor analysis (P < 0.001). All 52 lesioned mice lost circadian activity rhythm, with body temperature rhythm being suppressed in 44 of them (Fig. 1). In the eight remaining animals, Fourier transform analysis showed a dominant 24-h period for body temperature. Cosinor analysis further validated a statistically significant 24-h rhythm with markedly reduced amplitude and an acrophase shifted from middark to midlight (Fig. 2A). No apparent difference was found with regard to the extent of SCN destruction in these mice compared with those lacking any temperature rhythm (Fig. 2B)
The mean plasma corticosterone concentration displayed a large 24-h rhythm with a peak located at the light-dark transition in sham-operated mice. The 24-h variation was damped, its pattern was altered, and its peak advanced by 4 h in the lesioned mice. Furthermore, the 24-h mean concentration decreased by ∼30% in the lesioned mice compared with sham-operated animals (Fig. 3). These results were statistically validated with two-way ANOVA, which documented significant differences as a function of sampling time (P < 0.001), SCN lesioning (P = 0.026), and lesion × time interactions (P = 0.002). Cosinor analysis validated a sinusoidal 24-h rhythm in sham-operated mice (P < 0.001) but not in SCN lesioned ones (P = 0.14) (Table 1).
Circulating Leukocyte and Lymphocyte Counts
Mean levels of circulating white blood cells ranged from 3,154 cells/mm3 at 11 HALO to 5,760 cells/mm3 at 23 HALO in sham-operated mice. The circadian variation was also observed in lesioned mice, but it was damped, and its peak occurred 5 h earlier compared with sham-operated animals. SCN lesioning did not significantly affect the 24-h mean (ANOVA, P = 0.7). The circadian variation in leukocyte count was validated with two-way ANOVA (time effect, P = 0.04; time × lesion effect, P = 0.02) as well as cosinor analysis (sham operated, P = 0.02; lesioned, P = 0.04), with an amplitude that was halved in the SCN-lesioned group (Table 1).
The 24-h mean count in circulating granulocytes was nearly doubled in SCN-lesioned mice compared with sham-operated mice (ANOVA, P = 0.001), without any significant rhythm in either group (Table 1). Conversely, SCN lesions reduced the 24-h mean count in circulating lymphocytes by nearly 50% and severely damped its rhythm compared with sham operation (ANOVA, lesion, P < 0.0001; lesion × time, P = 0.025) (Table 1).
The average GSH concentration ranged from 16.3 nmol/mg protein at 11 HALO to 31.3 nmol/mg at 23 HALO in sham-operated mice. In lesioned animals, the rhythm was clearly flattened (Fig. 4). Significant effects of sampling time (P < 0.001) and lesion × time interactions (P = 0.003) were found by two-way ANOVA, indicating a profound modification of the circadian pattern by SCN destruction, although the 24-h means remained similar in both groups (P = 0.39). A 24-h sinusoidal rhythm was demonstrated with cosinor analysis in sham-operated mice (P < 0.001) but not in SCN-lesioned animals (P = 0.15) (Table 1).
The mRNA expression of DPD (ratio DPD/β actin) varied from 3,904 arbitrary units (AU) at 15 HALO to 2,635 AU at 3 HALO in the liver of sham-operated animals. A synchronous, circadian variation was observed in the mice with SCN destruction, with an 8% increase in 24-h mean level (P = 0.07) (Fig. 5). Thus, in both groups, high values in DPD mRNA expression plateaued during darkness and reached a nadir at 3 or 7 HALO. A circadian rhythm was validated with two-way ANOVA (sampling time effect, P < 0.001) without any significant interaction between the effects of sampling time and that of SCN lesioning (P = 0.82). Cosinor analysis further documented the similarity in the 24-h parameters in sham-operated mice and in SCN-lesioned animals (Table 1).
Bone Marrow Cell Count and Cell Cycle Phase Distribution
Femoral nucleated cell count.
The average count in nucleated bone marrow cells of sham-operated mice increased by ∼25% from a low point at 15 HALO to a high point at 3 HALO. Similar respective hours for peak and trough were found for the group of mice with SCN lesions, yet with a higher 24-h mean and a less regular 24-h pattern (Fig. 6). SCN destruction resulted in a modest yet significant increase of the average count in nucleated cells per femur from 16.4 × 106 to 18.4 × 106 (P from 2-way ANOVA = 0.006). The differences related to sampling time and SCN lesioning were statistically validated with two-way ANOVA (P = 0.012), without any significant time × lesion interactions (P = 0.64).
The adjustment of a 24-h cosine function to the data was close to statistical significance, indicating that the pattern was not adequately modeled with a single 24-h cosine curve in either group. Yet the similarity in the respective amplitude and acrophase estimates of both groups further supported the synchrony of the 24-h variations (Table 2).
Cell cycle phase distribution.
The proportions of cells in G1, S, and G2-M varied along the 24-h time scale, with a similar pattern in sham-operated mice and in mice with SCN lesions. For both groups, the mean maximum occurred in the second half of the light span for G1-phase cells, during the dark span for S-phase cells, and at the very end of the dark span for G2-M-phase cells (Fig. 7). SCN lesions decreased the proportion of S-phase cells compared with sham operation (ANOVA, F = 16.3, df = 1, P < 0.0001), but it displayed no significant effect on the proportion of G1- or G2-M-phase cells.
The circadian time-related differences were statistically validated with both ANOVA and cosinor. No significant interaction was found between sampling time and type of operation (sham operated vs. SCN destruction) with two-way ANOVA. This finding was further supported by the fact that the respective acrophases of each cell cycle phase differed by 1.5 h at the most between both groups (Table 2).
In the current study, the sham-operated and the SCN-lesioned mice were kept purposely under the same regular light-dark 24-h cycle throughout the study, as this condition is closer to normal life than constant darkness, a measure that would avoid masking by light (37). Despite this photoperiodic schedule, the rest-activity cycle was suppressed in all the mice with SCN ablation, as it has been widely reported. Indeed it has long been known that rodents with SCN lesions display no circadian pattern in locomotor activity, whether they are kept in constant darkness or in LD12:12 (24). However, despite the suppression of the circadian pattern in rest-activity, markedly altered yet persistent 24-h changes were documented for plasma corticosterone, circulating leukocyte count, and liver GSH in the SCN-lesioned mice in our study. Moreover, in these animals, DPD transcription in liver and cell cycle phase distribution in bone marrow remained rhythmic along the 24-h time scale, with temporal patterns similar to those found in sham-operated mice or reported in healthy controls (4, 19, 28, 40, 46). The rhythms were statistically validated with both ANOVA and cosinor analysis.
The adrenal corticosterone content of rats with SCN lesions was reported not to vary significantly along the 24-h time scale, yet only four time points separated by 6 h were studied (31). This design left the possibility of a peak occurring in between two time points. Indeed, our study involved the sampling of separate subgroups of eight to nine animals each, every 4 h for 24 h. Such scheme may well be a minimum requirement for the documentation of the damped and phase-shifted corticosterone rhythm in SCN-lesioned mice, which we already observed in a separate experiment (16).
In a two-time-point experiment [zeitgeber time (ZT) 3 and ZT15] involving 12 rats with SCN lesions and 9 sham-operated rats, we found significant time-related differences both in the liver concentration of GSH and in the proportion of S-phase bone marrow cells (44). These results prompted us to perform the current study in mice to obtain a sufficient number of animals at six time points. The data show for the first time that transcriptional and cell cycle-related rhythms can display near normal 24-h patterns in the absence of SCN and of circadian rhythms in rest-activity and body temperature. These findings indicate the ability of peripheral circadian oscillators to remain synchronized in the absence of the established central pacemaker. In a separate investigation, we found that lymphocyte subset rhythms persisted in rats with suppressed rest-activity and body temperature rhythms as a result of prolonged exposure to constant light (15).
The ability of peripheral oscillators to maintain circadian rhythms in the absence of a central pacemaker has been further demonstrated as cultured fibroblasts displayed sustained rhythms in gene expression (3, 17) and cultured murine bone marrow progenitors displayed sustained rhythms in response to hematopoietic growth factors (8). Nevertheless, these rhythms tended to fade away unless a stimulation (glucocorticoid, serum shock) or an environmental 24-h cycle (temperature, light) was introduced after a few days in culture, suggesting the need for a regular resetting of free-running peripheral oscillators in order for their coordination to be maintained (2, 3, 9, 21, 49). In rodents with lesioned SCN, a few transcriptional rhythms have been documented in vivo in peripheral tissues using DNA microarrays and very stringent amplitude criteria for rhythm detection (1, 39). A persistent circadian pattern in the feeding behavior of SCN-lesioned mice kept in LD12:12 may be questioned, as imposed feeding schedules entrained circadian rhythms in the peripheral oscillators of mice with destroyed SCN (18). Nevertheless, the circadian feeding pattern was clearly shown to be coupled with SCN function in rodents kept under constant darkness or exposed to LD12:12. Microlesions in the caudal part of the SCN were sufficient to jointly abolish circadian rhythms in locomotor activity as well as drinking and gnawing behaviors in Syrian hamsters (25). Furthermore, rats with SCN destruction kept in LD12:12 lost their circadian patterns in both rest-activity and feeding and ate 50% of their food during the light period and the remaining 50% during the dark period, compared with sham-operated rats, which ate 80% of their food during the dark period (38).
According to a recent report, a regular resetting of the molecular oscillator was not even mandatory for the circadian rhythm in clock gene mPer2 expression to persist in cultured cells from peripheral organs (50). The in vitro circadian rhythm in mPer2 transcriptional activity was also observed in liver, lung, or kidney cells from mice with prior SCN destruction kept in constant darkness before tissue sampling. Thus the main effect of SCN lesions consisted in an asynchrony of phase both within and among animals (50). Therefore, molecular clock components from cells in peripheral organs can continue to cycle in vitro with an ∼24-h period for a prolonged time span in the absence of any central coordination.
A circadian cycling in behavioral variables or peripheral oscillator functions has also been shown in vivo in rodents with SCN lesions. In rats or mice with destroyed SCN, the suppressed rest-activity circadian cycle was restored with metamphetamine treatment (22). In SCN-lesioned mice, daily epinephrine administration exerted no effect on the suppressed rest-activity cycle, but it restored the 24-h rhythm in the transcription of clock genes mPer2 and mBmal1 in liver. Interestingly, the circadian phase of the transcriptional rhythms was set by the time of daily epinephrine injection (47). In line with such results, forskolin induced the rhythmic expression of clock gene Per1 and Per2 and clock-controlled gene dbp in cultured rat fibroblasts (48). Both epinephrine and forskolin are known to stimulate adenylate cyclase and to increase cell content in cAMP, although through different mechanisms. Taken together, these results support a major role for the sympathetic system in the resetting of peripheral oscillators. In physiological conditions, this system is regulated by the SCN (10), yet in rodents with destroyed SCN, one (or several?) slave oscillator(s) in the brain could substitute for a defective SCN to ensure some aspects of circadian coordination (30). Indeed, mice with clock gene mutations displayed a complete loss of circadian rhythmicity after a few weeks in constant darkness, yet the exposure of these animals to LD12:12 photoperiodic regimens maintained or restored circadian function (21, 41, 42).
In our mice with SCN lesions, the rhythm parameters for DPD mRNA and for cell cycle phase distribution were similar to those found in sham-operated mice or reported in control animals of the same strain (40, 46). Based on these results and the literature review, we assume that light-dark periodic signals can be conveyed to peripheral oscillators via structures other than the SCN, and at least partly synchronize these molecular or cellular rhythms.
In conclusion, we here establish that cellular rhythms can be maintained, despite suppressed rest-activity and altered corticosterone rhythms in mice. These findings suggest that the delivery of anticancer treatments at an optimal time of day may still be beneficial, despite suppressed rest-activity or cortisol rhythms.
This research was supported by Grant 5853 from Association pour la Recherche sur le Cancer, Association pour la Recherche sur le Temps Biologique et la Chronothérapeutique, Institut de Cancer et d'Immunogénétique, Villejuif (France), and by a Research Training Fellowship from Medical Research Council to V. M. King.
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