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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2004 as doi:10.1096/fj.04-2665fje. |
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* Cancer Chronotherapeutics, INSERM E 0354 and Université Paris XI, Hôpital Paul Brousse, Villejuif, France;
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS-INSERM, Université Louis Pasteur, Strasbourg, France; and
Department of Oncology and Medical Physics, Haukeland University Hospital, Bergen, Norway
1 Correspondence: INSERM E 0354, Chronothérapeutique des cancers, Hôpital Paul Brousse, 14 Ave. P.V. Couturier, Villejuif 94800, France. E-mail: levi-f{at}vjf.inserm.fr
SPECIFIC AIMS
We investigated the respective relevance of rhythms in cell cycle phase distribution and BCL-2 expression in bone marrow and in an experimental mammary cancer in C3H/He mice with nocturnal melatonin secretion. We also investigated BCL2 rhythmicity in bone marrow of B6D2F1 mice, whose melatonin secretion predominates during daytime. A comparison of both mouse strains would help us assess the physiological role of rhythmic melatonin secretion on BCL-2 expression. We further studied the relation between BCL-2 and BAX time courses and 24 h changes in mRNA expression of mouse Per1 (mPer1), mPer2, mTim, mClock, and mBmal1 clock genes in bone marrow of these mice.
PRINCIPAL FINDINGS
1. Cell cycle phase distribution
The average cell cycle phase distribution was similar in bone marrow of controls and in that of tumor-bearing mice. However, the mean proportion of G1 cells decreased and that of G2/M cells increased significantly in tumor compared with bone marrow. Conversely, the proportion of S-phase cells did not differ significantly in both tissues.
In bone marrow, the proportion of G1-phase cells peaked 15 h after light onset (HALO) in controls and at 19 HALO in tumor-bearing mice; the nadir of the circadian variation was reached at 3 HALO in both groups. The proportion of S-phase cells peaked at 3 HALO and decreased to consistently low values during the dark span in both groups. The proportion of G2/M phase cells peaked at 7 HALO in controls and at 23 HALO in tumor-bearing mice, while nadirs respectively occurred at 15 HALO and at 11 HALO (Fig. 1
); ANOVA: P = 0.045 and 0.014 for G1- and S-phase cells, respectively, but not significant for G2/M phase cells (P=0.79). According to cosinor analysis, significant 24 h rhythms were found for G1- and S-phase cells, with no difference in acrophases (time of maximum in fitted cosine function in 24 h) between healthy and tumor-bearing mice. Conversely, the rhythm in G2/M phase was statistically validated in healthy but not in tumor-bearing mice.
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In MA13/C adenocarcinoma, a significant circadian variation with a low amplitude, was found for the proportion of G1-phase cells but no rhythm was validated for S-phase cells. The proportion of G2/M phase cells displayed a prominent 24 h rhythm, with a mean value (expressed as a percentage of the 24 h mean) ranging from 84% at 15 HALO to 134% at 23 HALO (Fig. 1)
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2. BCL-2 expression in bone marrow and tumor
In the first study BCL-2 expression was largely increased in tumor tissue compared with bone marrow, where circadian changes were apparent. In bone marrow of controls, mean BCL-2 expression varied by 4.5-fold, from a nadir value of 35% at 15 HALO to a peak value of 159% at 3 HALO. A circadian variation with a similar range was seen in bone marrow of tumor-bearing mice, yet the nadir occurred at 23 HALO and the peak at 7 HALO. This alteration in timing was statistically validated as shown by a significant (time) x (tumor presence) interaction term (2-way ANOVA, P=0.04). Cosinor analysis further showed a circadian rhythm with similar amplitude in controls and in tumor-bearing mice, yet acrophase occurred 6.5 h later in mammary cancer-bearing animals than healthy controls. In tumor a high value was observed at 7 HALO and a nadir at 19 HALO, but no statistically significant difference was found as a function of sampling time.
3. BCL-2 and BAX expression in bone marrow
In the second study, relative abundance of BCL-2 and BAX proteins in bone marrow protein extracts varied as a function of sampling time whereas ß-actin (internal control) remained invariant. Magnitude of mean variation over 24 h (peak/trough value) was 2.8-fold for BCL-2 and 5-fold for BAX. Time-related differences were statistically validated by ANOVA for each variable (P=0.036 and 0.013, respectively). Mean relative abundance of BCL-2 and of BAX was highest at 4 HALO and at 16 HALO, respectively (i.e., in anti-phase). Circadian patterns were asymmetric, as the minima in BCL-2 and BAX occurred at 1 and 22 HALO, respectively. For BCL-2, cosinor further validated a 24 h rhythm (P=0.025), but no 12 h periodicity. For BAX, a statistically significant 12 h rhythm (P=0.007) was found; detection of a 24 h rhythm was close to statistical significance (P=0.07).
4. Clock gene expression in bone marrow
Almost undetectable levels of mPer1 mRNA were found in bone marrow of these mice, but relative abundance of mRNA could be quantified for mTim, mClock, mPer2, and mBmal1. No significant changes in mTim expression were found. Conversely, the average relative abundance of mClock mRNA varied 3.3-fold between late light (trough) and late darkness (peak) in the first experiment. The relative abundance of the mRNA of mPer2 and mBmal1, respectively, varied 2- and 2.6-fold between light (trough) and mid-dark (peak). A circadian rhythm was demonstrated with cosinor analysis for mPer2 (P=0.014) and mBmal1 (P=0.003).
5. Relation between clock genes and BCL-2 and BAX
A nearly coincidental temporal variation was confirmed for mPer2 and mBmal1 as the highest correlation between the time series obtained with no time shift (r=0.65, P=0.016) (Fig. 2
). The time series of BCL-2 was further correlated with that of mPer2 and that of mBmal1 with a –9 h time lag (r=0.54, P=0.05). A correlation was found between the time series of BAX and that of mPer2, with a time lag of –9 h (r=0.58, P=0.026) but not with mBmal1 time series.
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CONCLUSIONS AND SIGNIFICANCE
In the current study, average proportions of total bone marrow cells in G1-, S-, or G2/M-phases were not modified by MA13/C mammary adenocarcinoma or for circadian changes in G1- or S-phase cells. Rhythm in G2/M-phase cells, however, was suppressed in bone marrow of tumor-bearing animals, suggesting that tumor-associated factors could selectively alter circadian regulation in host tissues at the G2/M level. In healthy mice, the maxima of cell cycle phase distribution rhythms in total bone marrow occurred nearly 8 h later in C3H/HeN (current study) than in B6D2F1, a time lag similar to that found for the respective melatonin patterns in both strains. This supports a role for melatonin rhythm in regulating cell cycle phase distribution in total bone marrow.
A circadian regulation of cell cycle organization was retained in MA13/C mammary adenocarcinoma. The rhythm in G2/M phase cells was the most prominent: their proportion increased by 
75% from trough to peak. This finding was consistent with documentation of a rhythm in M-phase with a peak in the late dark phase in most experimental tumor models. Persistent circadian control of G2-M gating in malignant tumors likely contributed to the large dosing time dependency in the efficacy of docetaxel and other antimitotic drugs.
Circadian regulation of cell cycle phase distribution was markedly different in bone marrow and tumor, with maxima occurring several hours earlier in tumor than in bone marrow, similar to what is reported in humans.
BCL-2 expression varied 4.5- and 2.8-fold in bone marrow of C3H/He and B6D2F1 mice, respectively, with a peak at 3 or 4 HALO (in early rest phase) and a trough near 15 HALO (i.e., during the activity phase of the mice). BCL-2 rhythms were quite similar in bone marrow of both mouse strains, which suggests that circadian regulation of BCL-2 can be uncoupled from circadian regulation of cell cycles.
Together, these results emphasize the role of increased BCL-2 expression during the early stage of the rest span as a major determinant of improved tolerability of proapoptotic drugs at this circadian stage in mice.
The presence of the MA13/C tumor slightly but significantly shifted BCL-2 peak time by 4 h, which further suggested that tumor-associated factors may influence circadian regulation of cell cycle events and their related proteins in healthy tissues. No statistically significant rhythm was found for BCL-2 expression in the MA13/C adenocarcinoma itself.
The marked differences in circadian patterns of cell cycle phase distribution and BCL-2 expression in tumors compared with bone marrow may help explain why lowest hematologic toxicity and highest antitumor efficacy resulted from docetaxel dosing in the first half of the rest span of mice.
Whereas BCL-2 exerts antiapoptotic properties, BAX is proapoptotic. The finding that BAX expression displayed 5-fold changes over the 24 h span in mouse bone marrow, with a major peak at 16 HALO, indicated a well-coordinated circadian organization of apoptotic processes in this tissue. Nevertheless, BAX pattern was bimodal, with a lower peak occurring at 4 HALO and a statistically validated 12 h periodicity.
A single daily peak of mPer2 mRNA during darkness was observed, nearly coincidental with that of mBmal1 rhythm, in total mouse bone marrow. Rhythmic expression of mPer2 in bone marrow displayed a peak occurring at a time similar to that found in other peripheral tissues. The circadian transcription pattern of mBmal1 gene was similar to that found in mouse suprachiasmatic nucleus, the hypothalamic clock that coordinates the rhythms, as well as in liver or heart, in that mBmal1 oscillated with a broad peak during the night.
Through expression of clock genes, the circadian system generates circadian changes in cell functions via identified transcriptional and posttranscriptional regulatory processes (Fig. 3
). These mechanisms could account for circadian changes observed in BCL-2 and BAX in mouse bone marrow. Regulation of BCL-2 by clock genes and BAX by mPer2 was supported by correlation studies.
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Our data suggest a regulation of programmed cell death pathways by circadian clock genes in bone marrow. As a result, protection against BCL-2/BAX-driven apoptosis in this host tissue is favored during the early light span and apoptosis is facilitated during darkness. Altered circadian organization of cell cycle events and BCL-2-expression in tumor may account for optimal anticancer efficacy of docetaxel at a time close to that of best tolerability by the host.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2665fje;
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24 h for BCL-2 and P=0.007 with 