Affiliations of authors: E. Filipski, X. Li, T. G. Granda, M.-C. Mormont, X. Liu, F. Lévi, Institut National de la Santé et de la Recherche Médicale (INSERM), Equipe Propre INSERM 0118 "Cancer chronotherapeutics" (Université Paris XI), Paul Brousse Hospital, Villejuif, France; V. M. King, Department of Anatomy, University of Cambridge, U.K.; B. Claustrat, Service Radiopharmacie et Radioanalyse, Hôpital Neurocardiologique, Lyon, France; M. H. Hastings, Laboratory of Molecular Biology, Medical Research Council, Cambridge, U.K.
Correspondence to: Francis Lévi, M.D., Ph.D., Institut National de la Santé et de la Recherche Médicale (INSERM) Equipe Propre INSERM 0118 "Cancer chronotherapeutics" (Université Paris XI), Paul Brousse Hospital, 94800 Villejuif, France (e-mail: chronbio{at}club-internet.fr).
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ABSTRACT |
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INTRODUCTION |
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Mammalian circadian rhythms are generated by interconnected molecular loops involving specific genes controlling the circadian clock [reviewed in (12)]. A central pacemaker consisting of suprachiasmatic nuclei (SCN), located in the hypothalamus, coordinates these rhythms and directly controls the restactivity circadian cycle (13). The ablation of SCN results in the suppression of these rhythms in several rodent species, whereas transplantation of SCN restores these rhythms in animals with prior SCN lesions (14). The SCN are responsible for the adjustment of the whole circadian time structure to the photoperiodic environment, the main synchronizer of bodily rhythms (14). As a result, most biochemical or molecular cell processes display predictable changes along the 24-hour time scale, which can modulate anticancer drug pharmacology (15).
We hypothesized that severe circadian dysfunction could accelerate tumor progression per se and tested this possibility in mice with ablation of SCN before tumor inoculation.
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METHODS |
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In a first series of experiments, we studied the effect of SCN destruction on the rhythms in restactivity body temperature, plasma corticosterone concentration, and circulating lymphocyte count in 188 B6D2F1 mice. Mice were randomly allocated to receive sham operation (64 animals) or SCN lesions (124 animals). Fourteen mice from the latter group were eliminated from the study as a consequence of postoperative death or obesity resulting from the destruction of other hypothalamic nuclei. In a second study, B6D2F1 mice were randomly assigned to receive either sham operation (25 animals) or introduction of SCN lesions [45 animals before receiving a fragment of either Glasgow osteosarcoma (GOS) (16) or the more slowly growing pancreatic adenocarcinoma (P03) (17)]. Two mice with SCN lesions were monitored as non-tumor-bearing controls, and 14 mice were eliminated from the study because of postoperative death or obesity. Both tumor cell lines are known to display a circadian rhythm-modulated susceptibility to chemotherapy (18,19). Male B6D2F1 mice, 56 weeks old, were purchased from Charles River (L'Arbresle, France). All the experiments were performed in mice synchronized to 12 hours of light and 12 hours of darkness (LD 12 : 12, with lights on from 6:00 AM to 6:00 PM) to allow for an SCN-independent photoperiodic synchronization, which may be the case in clinically relevant situations. All procedures were performed in accordance with French guidelines for experimental animal care.
Monitoring Rhythms
Locomotor activity and body temperature were measured every 10 minutes throughout each experiment with the use of a radio transmitter (Physio Tel, TA 10 TA-F20; Data Sciences, St. Paul, MN) implanted into the peritoneal cavity of each mouse.
SCN Destruction and Fourier Analysis
The SCN were destroyed by bilateral electrolytic lesion (20) (1 mA for 4 seconds; stereotaxic coordinates: approximately [anterior/posterior] on the Bregma line, mediolateral [± 0.2 mm of midline], dorsoventral [0.55 mm below dural surface], incisor bar at the same level as the 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 () of locomotor activity in the circadian domain (20 h <
< 28 h) with Fourier transform spectral analysis following visual inspection (21). Complete SCN destruction was ascertained postmortem in all mice for pathologic changes by investigators who had no knowledge of any data on these mice with respect to restactivity cycles, temperature patterns, or blood variables. Histologic methods involved both Nissl stain of the hypothalamus tissue and immunostaining for peptide histidineisoleucine (PHI) (20,22,23).
Anesthesia
Mice were anesthetized with a single intraperitoneal injection of a 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.
Assessment of Corticosterone Levels and Lymphocyte Counts
Blood samples were taken from separate groups of either animals with SCN lesions or sham-operated animals at one of six different times, 4 hours apart, over a 24-hour span (3, 7, 11, 15, 19, or 23 hours after light onset [HALO]), 37 weeks after introducing SCN lesions. Lymphocyte count was determined with a Cell DyneTM 3500R (Abbott Diagnostics, Rungis, France), and corticosterone concentration was measured by radioimmunoassay (RIA) (5 µL of plasma was extracted with 3 mL of diethyl ether; the residue was dissolved in 100 µL of assay buffer before incubation with 1,2,6,7 [3H]corticosterone [Amersham, Orsay, France] and rabbit anti-corticosterone antibody [Valbiotech, Paris, France]).
Tumor Models
GOS and P03 tumor cells were provided by the Research Center of Aventis Pharma (Vitry sur Seine, France). Both tumors were maintained in C57BL/6 female mice and passaged every 2 weeks (for GOS) or every 4 weeks (for P03) as bilateral subcutaneous implants. Three passages were required before tumor transplantation in the experimental mice. Tumor fragments of size 4 x 4 mm2 were prepared from dissected tumors grown in the donor mice and kept in Hanks' medium for approximately 1 hour. They were freshly implanted subcutaneously in each flank of male B6D2F1 mice with a 12-gauge trocar. The experiment followed a 2 x 2 factorial design to test the role of SCN lesions, that of tumor type, and an interaction term, with regard to tumor growth and survival.
Tumor size was measured three times a week using a caliper. Tumor weight (mg) was estimated from two perpendicular measurements (mm): tumor weight = (length x width2)/2.
Mice with tumor weight reaching approximately 2 g were sacrificed for ethical reasons and considered as dead from tumor progression on this date. Four mice (two with SCN lesions and two sham-operated) were not inoculated with tumor and served as healthy controls.
Statistical Analysis
Means and 95% confidence intervals were computed for each set of parameters. Intergroup differences were evaluated statistically using multiple-way analyses of variance (ANOVA). The effect of SCN lesions on tumor growth was assessed with 2-way ANOVA for repeated measures and followed by Student's t test. 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 (24). This method characterized a rhythm by the parameters of the fitted cosine function best approximating all data. A period = 24 hours was determined a priori. The rhythm characteristics estimated by this linear least squares method include the mesor (M, rhythm-adjusted mean), the double amplitude (2A, difference between minimum and maximum of fitted cosine function), and the acrophase (Ø, time of maximum in fitted cosine function, with light onset as Ø reference, so that units were in HALO). A rhythm was detected if the null hypothesis was rejected with P<.05; however, A and Ø were considered as valid imputations if .05<P<.10, as the statistical power could possibly be affected by the number of mice studied. Survival was computed with the KaplanMeier method, and differences in survival were validated with the log-rank test. All statistical tests were two-sided. All standard statistical tests were performed using SPSS for Windows software (Statistical Package for Social Sciences, Chicago, IL).
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RESULTS |
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The restactivity cycle and the temperature rhythms of all sham-operated mice were marked and were found to be regular from one day to the next (a representative example is given in Fig. 1, A, left panel, top and bottom, respectively). Spectral analysis adjusted best-fitting sinusoidal functions with decreasing periods to the data. The initial period (fundamental T) corresponded to the whole length of the time series (168 h in the examples shown in Fig. 1
). The subsequent harmonic periods tested were T/2, T/3, T/4.....T/50. The 7th harmonic (T/7) corresponded to a period of 24 h. Thus, this method identified the 24-h period as the most prominent one among the initial 50 harmonics tested with corresponding periods ranging from 168 to 3.36 hours (Fig. 1, A
, right panel, top and bottom). The restactivity cycle was suppressed in 79 of 110 mice with SCN lesions (a representative example is given in Fig. 1, B
, top left panel). Spectral analysis did not reveal any 24-hour periodicity that stood out among the initial 50 harmonics (Fig. 1, B
, top right panel). A postmortem histologic study ascertained complete SCN destruction in 75 animals. Only the animals with histologically verified SCN destruction were included in the SCN lesion group for comparison. None of the sham-operated mice were excluded from the analysis. Body temperature rhythm was suppressed in 60 of the 75 mice with histologically verified SCN lesions (Fig. 1, B
, lower left and right panels). An atypical body temperature rhythm was found in 15 of these 75 mice. It was confirmed by a dominant 24-hour periodicity with Fourier transform analysis and a statistically significant 24-hour rhythm with cosinor analysis (P<.001). The amplitude of temperature, however, was greatly decreased as compared with that of sham-operated mice; the maximum (acrophase) occurred between mid-light and early dark as compared with mid-dark in sham-operated animals (data not shown).
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Relevance of Circadian Coordination for Tumor Growth
Twenty-nine mice with histologically confirmed SCN lesions had received either GOS (n = 16) or P03 (n = 13) tumors. Sham-operated animals had been transplanted with GOS (n = 12) or P03 (n = 13) tumors. The results indicated a highly statistically significant effect of SCN lesions and tumor type on tumor size (2-way ANOVA, P<.001 for lesion and for tumor effects), without any statistically significant interaction (lesion x tumor) (P = .21). Accelerated growth of each tumor in mice with SCN lesions was further statistically validated with one-way ANOVA (GOS, P = .004; P03, P<.001). In GOS tumor-bearing mice, tumor growth rate was faster in mice with SCN lesions than in sham-operated mice. On day 12, i.e., before the death of the first animal, mean tumor weight was 1443 mg (95% confidence interval [CI] = 1009 mg to 1844 mg) versus 490 mg (95% CI = 273 mg to 707 mg) in mice with SCN lesions and in sham-operated mice, respectively (for t test, P = .002) (Fig. 3, A). In mice bearing the slower growing P03 tumors, tumor growth rate was faster in mice with SCN lesions as compared with that in the sham-operated mice. On day 22, i.e., before the death of the first animal, mean tumor weights were 1447 mg (95% CI = 695 mg to 2199 mg) in mice with SCN lesions and 749 mg (95% CI = 458 mg to 1040 mg) in sham-operated mice (for t test, P = .05) (Fig. 3, B
). Similarly, the survival of sham-operated mice was statistically significantly longer than that of mice with SCN lesions (median survival time in days: sham-operated mice = 26 (95% CI = 23 to 29); mice with SCN lesions = 22 (95% CI = 19 to 25); P for log-rank test = .0062). As we have observed in our previous studies, the mice with histologically verified partial SCN lesions (n = 4) displayed a normal circadian pattern in restactivity and body temperature. Tumor growth rate in these mice was similar with that in control mice (data not shown).
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DISCUSSION |
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SCN ablation accelerated malignant growth by twofold to threefold for two established tumors with different proliferation rates. In addition, tumor growth in the mice with partial SCN destruction was similar to that in controls, illustrating the need for complete SCN destruction to accelerate tumor proliferation. This remote antitumor effect of SCN may be mediated by either endocrine or neuroanatomic communications between the SCN and peripheral targets. For example, the existence of paracrine secretions from the SCN has been demonstrated by neural grafting experiments in which encapsulated SCN tissue can restore circadian patterning to locomotor activity in hosts with lesions (31). Additionally, the SCN have widespread anatomic associations with the autonomic nervous system (32), which may influence host responses and/or factors in the host environment that regulate tumor growth. Both tumor models have displayed sensitivity to immunotherapeutic agents (16,33), which suggests that SCN destruction could favor tumor progression through immune suppression. The mean lymphopenia that we observed in mice with SCN lesions was associated with a reduced mean serum corticosterone level, a finding divergent from what was expected, because lymphopenia usually results from glucocorticoid supplementation (34). This and the differential phase-shifting and amplitude-reduction effects of SCN destruction on serum corticosterone and lymphocyte count rhythms support distinct SCN control pathways of immunologic and adrenal rhythmic functions (35,36). Recent studies (30) highlight a role for corticosteroids in coordinating peripheral circadian oscillators located in a variety of tissues. The loss of temporal organization across tissues arising from SCN lesion-induced corticosteroid dysrhythmia may contribute to enhanced tumor growth. It is necessary to investigate further whether the endocrine or immune effects triggered by SCN lesions could differently affect transplanted or endogenous tumors. Yet, chemically induced carcinomas in rats were promoted by nonspecific methods of circadian alterations, such as continuous light exposure (37).
The current findings show that release from circadian regulation causes a dramatic acceleration of malignant growth, a result in line with recent clinical reports. In a study involving 200 patients with metastatic colorectal cancer, survival at 4 years was 22% in the patients with a marked restactivity cycle as compared with 11% in those with damped or altered rhythm. As indicated by multivariate analysis, the prognostic value of circadian rhythmicity in restactivity was independent of well-known prognostic factors such as performance status, number of organs with metastasis, or degree of liver tissue replacement by tumor (10). The relationship between salivary cortisol rhythm and survival was explored in 104 patients with previously treated metastatic breast cancer. A normal cortisol pattern, with peak concentration at 8:00 AM and decline thereafter, was found in 37% of the patients. This group had a 4-year survival rate of 55% as compared with 26% in the patients with an altered cortisol secretion pattern. Cortisol slope was an indepen-dent predictor of survival, as determined by the multivariate analysis (11). Two large epidemiologic studies (3841) further showed that disrupted circadian coordination induced by light at night increased the risk of developing breast cancer in women. Although the clinical data support the idea that circadian clock function partly controls several stages of tumor development, our experimental model clearly demonstrates a specific role for the hypothalamic clock with regard to cancer proliferation.
We expect that improved understanding of the biologic dynamics of neoplasia will stem from an examination of the temporal interplay between the central SCN clock, peripheral tissue-based oscillators, and malignant processes and will lead to novel therapeutic approaches to cancer.
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NOTES |
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E. Filipski, V. M. King, M. H. Hastings, and F. Lévi were involved in the conception of the study, study design, data acquisition, article drafting, revising, and final approval of the submitted version. XM. Li, T. G. Granda, M.-C. Mormont, XH. Liu, and B. Claustrat contributed to the study design, data acquisition, critical article revising, and approval of the final version.
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Manuscript received July 9, 2001; revised February 5, 2002; accepted February 28, 2002.
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