Daily timed meals dissociate circadian rhythms in hepatoma and healthy host liver

Abstract

Dividing cells, including human cancers, organize processes necessary for their duplication according to circadian time. Recent evidence has shown that disruption of central regulation of circadian rhythms can increase the rate at which a variety of cancers develop in rodents. To study circadian rhythms in liver tumors, we have chemically induced hepatocellular carcinoma in transgenic rats bearing a luciferase reporter gene attached to the promoter of a core circadian clock gene (Period 1). We explanted normal liver cells and hepatomas, placed them into short-term culture, and precisely measured their molecular clock function by recording light output. Results show that isolated hepatocellular carcinoma is capable of generating circadian rhythms in vitro. Temporally restricting food availability to either day or night altered the phase of the rhythms in both healthy and malignant tissue. However, the hepatomas were much less sensitive to this signal resulting in markedly different phase relationships between host and tumor tissue as a function of mealtime. These data support the conclusion that hepatoma is differentially sensitive to circadian timing signals, although it maintains the circadian organization of the nonmalignant cells from which it arose. Because circadian clocks are known to modulate the sensitivity of many therapeutic cytotoxic targets, controlling meal-timing might be used to increase the efficacy of treatment. Specifically, meal and treatment schedules could be designed that take advantage of coincident times of greatest tumor sensitivity and lowest sensitivity of host tissue to damage.

Fig. 1

Bioluminescence measured in vitro from healthy liver and hepatocellular carcinoma tissue explants isolated from a PER1-LUC transgenic rat. A. Detrended bioluminescence counts smoothed with a 2h running average. The beginning of the record is the time of lights-on on the day of sacrifice, and the lighting cycle for that day is indicated by the white and shaded bars. The rat was killed about one hour before lights-off (ZT 11:00). B. Variance-weighted mean values for initial amplitude, circadian period and damping rate of healthy liver and hepatocellular carcinoma explants measured in vitro. Amplitude, in bioluminescence counts, is plotted on the left axis, while period and damping rate, in hours, are plotted on the right axis. * p<0.0001; Two-tailed Welch's t-test. Healthy n=14; Tumor n=21.

Fig. 2

A. Examples of detrended bioluminescence records made from an HCC and healthy liver explant both taken from a rat fed at ZT 1600 (night-fed). The arrows indicate the peak that was used for phase analysis. B. Mean (± SEM) peak phase in vitro for HCC (Open triangle) and healthy liver (closed circle) explants from rats fed ad-libitum, at ZT 0400 (RF Day), or at ZT 1600 (RF Night). The arrows indicate the mealtime for the restricted fed groups. White and dark bars on the bottom of the chart indicate the alternating light-dark cycle. Each datapoint indicates results from a minimum of 9 (RF Night) and a maximum of 21 (Ad-libitum) cultures. * p< 0.025.

Fig. 3

Photic resetting of healthy liver (closed circles) and HCC (open triangles). Mean ± SEM peak phase is shown for rats that have undergone a 6h phase delay (A), or a 6h phase advance (B) and then been killed for assessment of the circadian phase on either the first, third or sixth day in the new lighting conditions. The number of cultures that contribute to each datapoint are shown in the right margin (healthy, tumor). The hatching indicates the dark phase of the lighting cycle. The target, shown by a star, represents the theoretical phase of the tissue after the shift has been completed. It is calculated by adding or subtracting 6h from the baseline phase.