Several cytokinetic methods for showing circadian variation in normal murine tissue and in a tumor

Circadian rhythmicity in cell proliferation was studied by several cytokinetic techniques in 8-week-old CD2F1 and Swiss male mice. In separate experiments on four different dates, subgroups of six or seven mice (fed ad libitum and standardized to 8 hours light alternating with 16 hours of darkness) were killed at 3-hour intervals over a period of 24-48 hours. Cosinor analyses were used to determine the parameters of circadian periodicity in the data. Flow cytometric (FCM) analyses of DNA synthesis in tongue and stomach were compared with estimates of tritiated thymidine (3HTdR) incorporation into the DNA in these tissues. Circadian variation in tongue epithelium was reproducible in phasing and in range-of-change (100-140%) in seven 24-hour studies. The proportion of cells in G1 and G2 in tongue epithelium also demonstrated a circadian periodicity. When DNA synthesis in stomach was analyzed by the two methods, the results were entirely different and thus not comparable. The circadian rhythms in 3HTdR uptake agreed with those reported previously, but results of analyses by FCM were less certain, since epithelium of this organ could not be analyzed separately. Circadian rhythms were detected by FCM in G2, S, and G2M in Ehrlich ascites carcinoma (EAC). The mitotic index of this tumor varied with a circadian periodicity similar to that of the FCM-derived rhythm in G2M. This study validates the reliability of older techniques, such as 3HTdR uptake and mitotic indices, and suggests that circadian rhythms can be important in studies of cellular properties using FCM.