Circadian variation in the expression of cell-cycle proteins in human oral epithelium

Abstract

At the tissue level, there is experimental and clinical data to suggest a cytokinetic coordination of the cell cycle with a greater proportion of cycling cells entering S-phase and mitosis at specific times of the day. The association of certain cell-cycle proteins with defined events in the cell cycle is well established and may be used to study the timing of cell-cycle phases over 24 hours. In this study oral mucosal biopsies were obtained from six normal human volunteers at 4-hour intervals, six times over 24 hours. Using immunohistochemistry, the number of positive cells expressing the proteins p53, cyclin-E, cyclin-A, cyclin-B1, and Ki-67 was determined for each biopsy and expressed as the number of positive cells per mm of basement membrane. We found a statistically significant circadian variation in the nuclear expression of all of these proteins with the high point of expression for p53 at 10:56 hours, cyclin-E at 14:59 hours, cyclin-A at 16:09 hours, cyclin-B1 at 21:13 hours, and Ki-67 at 02:50 hours. The circadian variation in the nuclear expression of cyclins-E (G1/S phase), -A (G2-phase), and -B1 (M-phase) with a normal physiological progression over time suggests a statistically significant circadian variation in oral epithelial cell proliferation. The finding of a circadian variation in the nuclear expression of p53 protein corresponding to late G1 is novel. This information has clinical implications regarding the timing of chemotherapy and radiotherapy.

Figure 1.

Immunohistochemical detection of cell-cycle proteins in the basal and parabasal layers of normal human oral epithelium. A: Ki-67; B: cyclin A; C: cyclin B1; D: p53.

Figure 2.

Circadian variation in oral mucosa p53 in six subjects, showing both original data (left panel) and normalized data (right panel). Time point means (original value and normalized value) and SE of protein expression is depicted along the 24-hour time scale. The best fitting cosine curve is shown in both panels. The time for the high point of expression, overall mean, and P value for rhythm detection is shown. h, hours.

Figure 3.

Circadian variation in oral mucosa cyclin-E, cyclin-A, cyclin-B1, as well as Ki-67 in six subjects, using normalized data. Time point means (expressed as percentage of mean) and SE of protein expression is depicted along the 24-hour time scale. The best fitting cosine curve is shown. The time for the high point of expression, overall mean, and P value for rhythm detection is shown. h, hours.

Figure 4.

The high point for the best-fitting cosine for p53, cyclin-E, cyclin-A, cyclin-B1, and Ki-67 is depicted along the 24-hour time scale. The 95% confidence limit for each variable is shown. The cell-cycle phase that each protein is a marker for is shown in parenthesis. The normal physiological progression from one cell-cycle phase to the next is evident in this figure.