Disconnected circadian and cell cycles in a tumor-driven cell line

Abstract

Cell division occurs at a specific time of day in numerous species, suggesting that the circadian and cell cycles are coupled in vivo. By measuring the cell cycle rhythm in real-time, we recently showed that the circadian and cell cycles are not coupled in immortalized fibroblasts, resulting in a rapid rate of cell division even though the circadian rhythm is normal in these cells. Here we report that tumor-driven Lewis Lung Carcinoma (LLC) cells have perfectly temperature compensated circadian clocks, but the periods of their cell cycle gene expression rhythms are temperature-dependent, suggesting that their circadian and cell cycles are not connected. These data support our hypothesis that decoupling of the circadian and cell cycles may underlie aberrant cell division in tumor cells.

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Acknowledgements

We thank Dr. Muschel (University of Oxford) for the hCCNB1 promoter and Jia-Lin Liu for generating the LLC cells stably expressing Per2-luc. This work was supported by the National Institutes of Health (R01 NS051278 to S.Y.), the Research Foundation for Opto-Science & Technology (to S.Y.), the NEDO Project & Takeda Science Foundation (to Y.O.) and the Vanderbilt University Summer Research Program (to B.A.R.).

Figures and Tables

Figure 1 A new model: disconnection of the circadian and cell cycles in cancer cells. In normal (non-cancerous) cells, the circadian rhythm and cell division cycle are tightly linked resulting in a 24 hour rhythm of cell division. According to the current model, since the circadian clock and cell division cycle are linked, dysregulation of the biological clock results in uncontrolled cell division which causes cancer. According to our new model, the circadian and cell cycles are disconnected from each other in tumor cells.

Figure 2 The circadian rhythm is temperature compensated in Lewis lung carcinoma (LLC) cells. (A and B) LLC cells stably transfected with Per2-luc were treated with forskolin for 30 minutes and then the growth media was replaced with recording media. (A) Detrended data (counts/sec) was obtained by subtracting the 24-hour moving average from the raw data. (B) The period (mean ± SEM) of Per2-luc expression in LLC cells was determined from bioluminescence recorded by the LumiCycle at 31.5°, 32.5°, 33.5°, 34.5°, 35.5° and 36.5°C (Q₁₀= 1.02; n = at least four at each temperature).

Figure 3 The period of the cell cycle gene expression rhythm is not temperature compensated in Lewis lung carcinoma (LLC) cells. (A) Representative example of the optical density of CCNB1-dGluc bioluminescence in a single LLC cell imaged at 36.5°C. The timing of cell mitosis is indicated by red arrows. Seven cells from each of two independent dishes were analyzed. (B and C) LLC cells stably expressing CCNB1-dGluc were synchronized by cold shock (22°C for 24 hrs) and then bioluminescence was recorded. Representative traces of detrended CCNB1-dGluc expression (counts/sec) at 36.5°C (top part; 12-h moving average; n = 3) or 32.5°C (bottom part; 30-h moving average; n = 3) are shown. (C) The period (mean ± Sem) of CCNB1-dGluc expression in LLC cells was determined by measuring the peak-to-peak time of two cycles at 32.5°C or the average peak-to-peak time of three cycles at 36.5°C (Q₁₀ = 8.46).

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