E. Thorud

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G2 phase DNA content were measured by flow cytometry of isolated basal cells, and the [3H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined.

EVIDENCE OF RAPID AND SLOW PROGRESSION OF CELLS THROUGH G2 PHASE IN MOUSE EPIDERMIS: A COMPARISON BETWEEN PHASE DURATIONS MEASURED BY DIFFERENT METHODS

Percentage labelled mitosis (PLM) measurements were initiated at four different times during a 24‐hr period and continued for 24 hr in hairless mouse epidermis. Estimates of G2 and S phase durations (mean TG2 and mean TS) were calculated. A significant number of labelled mitoses (10–20%) was seen after 30 min in all four PLM measurements and the estimated mean TG2 varied from 1.4 to 2.5 hr and was in agreement with values from PLM measurements in other epithelial tissues. These mean TG2 values were much shorter than expected from [3H]TdR double labelling experiments and from a multiparameter cell kinetic study in hairless mouse epidermis and did not reflect the circadian variations seen in these studies. the differences in estimates of phase durations can be explained by postulating two G2 cell populations; one with a rapid and another with a slow rate of cell cycle progression. the cells with the higher rate are mainly registered by the PLM method, whereas those with the lower rate largely escape detection by this method. TG2 estimates from PLM measurements in mouse epidermis therefore do not reflect the phase duration of the entire G2 population. It is also concluded that circadian variations in TS can not be accurately registered by the PLM method.

Subpopulations of Slowly Cycling Cells In S and G2 Phase In Mouse Epidermis

Evidence has been presented supporting the existence of heterogeneity in cell‐cycle progression in mouse epidermis, the present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell‐cycle analysis was combined with sorting of cells from windows in G1, S and G2, phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of sorted fractions were performed.

DNA synthesis in mouse epidermis: Labelled and unlabelled basal cells in S phase after administration of tritiated thymidine

SummaryEpidermal basal cells were isolated from hairless mouse skin 30 min after i.p. administration of various activities of tritiated thymidine (3H-TdR). Smears were prepared for autoradiography and the proportion of labelled cells (LI) was determined after one, three and five weeks of exposure. Flow cytometric DNA measurements were made from the same cell suspensions and the proportion of cells with S phase DNA content was estimated. In some experiments the LI in three fractions sorted from the S phase was determined.The lowest activities of3H-TdR resulted in a significant discrepancy between the LI and the proportion of cells with S phase DNA content, and confirmed previous results. As the3H-TdR activity increased, so did both the number of heavily labelled cells and the LI, which approached the estimated proportion of cells with S phase DNA content. Autoradiography of sorted cells with S phase DNA content from normal epidermis showed that about 50%–60% of the cells in the middle of the S phase were labelled at an activity of about 1 μ Ci3H-TdR/g animal, and that about 80% were labelled at an activity of 4 μ Ci/g animal (the activity giving the maximal labelling index). In contrast, about 93% of the cells in the middle of the S phase were labelled in regenerating epidermis. Thus, there is in normal non-stimulated mouse epidermis a significant fraction of cells with S phase DNA content that escapes detection by3H-TdR autoradiography, but this fraction is decreased to a minimum during rapid regenerative proliferation.

THE STATHMOKINETIC METHOD IN VIVO TIME‐RESPONSE WITH SPECIAL REFERENCE TO CIRCADIAN VARIATIONS IN EPIDERMAL CELL PROLIFERATION IN THE HAIRLESS MOUSE

Groups of hairless mice were injected i.p. with a stathmokinetic dose of 0·15 mg colcemid at seven different times of the day and animals killed 0, 15 and 30 min, 1, 2, 3 and 4 hr after the injection. The proportion of cells in metaphase and ana/telophase was determined in histological sections.

Model analysis of circadian rhythms in mouse epidermal basal cell proliferation

Abstract Computer simulations were made of circadian variations in six observed cell kinetic variables in the basal cell layer of hairless mouse epidermis. Different mathematical models were used and simultaneously fitted to all observed variables by an automatic method. The analysis shows that the origin of the circadian variations in cell kinetics in hairless mouse epidermis is not in the G1 phase or at the G 1 S transition alone as concluded from other studies, but must result from circadian variations in the duration of S and G2 phases. The results show that the data are compatible with the existence of circadian variations in the G1- and M-phase durations, although such variations, unlike the S and G2 variations, was not necessary to obtain a good fit of the model to the data. The results also indicate that the data are compatible with the existence of transient resting states of limited duration in S- and G2-phases.

Epidermis Extracts (Chalone) Inhibit Cell Flux At the G1‐S, S‐G2 and G2‐M Transitions In Mouse Epidermis

Epidermal cell flux at the G1‐S, S‐G2 and G2‐M transition was examined during the first 4 hr after injection of epidermis extract. the flux parameters were estimated by a combination of several methods. the G1‐S and S‐G2 transit rates were calculated on the basis of a double labelling technique with [3H]TdR, the G2‐M flux by means of colcemid and the relative proportion of cells in the S or G2 phase by means of flow cytometry. All experiments were performed both in early morning and late evening, corresponding to maximum and minimum rates of epidermal cell proliferation in the hairless mouse. the epidermis extract inhibited the S‐G and G2‐M transit rates to the same degree, while the inhibition of cell flux at the G1‐S transit was consistently stronger. In general, the inhibition of cell flux at the different transitions was most pronounced when the rate of cell proliferation was low and vice versa.

Circadian variation in the susceptibility of mouse epidermis to tumour induction by methylnitrosourea

SummaryTo study possible circadian differences in the sensitivity of hairless mouse epidermis to a small dose of a short-acting alkylating carcinogen, groups of animals were painted once with 0.2 mg methylnitrosourea (MNU) at 08.00, 12.00, 20.00 and 24.00 h. Other animals were painted three times at weakly intervals at 08.00 and at 20.00 h, respectively. Significantly higher tumour yields were found in animals painted at 20.00 h (when the cell cycle progression and DNA synthesis rate are lowest, and when relatively large numbers of late G1 cells may accumulate) than at any other time point investigated.Hence a circadian variation in sensitivity to MNU in mouse epidermis is confirmed. This may be due to the variations in flux of cells through the S phase. The formation of DNA/carcinogen adducts may be facilitated at times of low flux with many cells in late G1, and fixation of these errors in DNA may take place by the subsequent increased flux through S, before repair is possible.

Epidermal proliferation characteristics are similar in the pilary canal of mouse hair follicles and in interfollicular epidermis

SummaryProliferation characteristics of basal cells in the pilary canal of resting hair follicles were investigated and compared with corresponding parameters in interfollicular epidermis of hairless mice. The mitotic rates had similar 24-h means at both locations. Distinct circadian rhythms which showed phasing and amplitudes similar to that in interfollicular epidermis, were demonstrated by the3H-TdR labelling index, the mitotic rate and the mitotic index. Influx of cells to and efflux of cells from the S phase were measured in the early morning and in the evening by a3H-TdR double labelling method. The influx values were similar at both times of both locations. The efflux values recorded in the morning were more than twice the values seen in the evening in both the pilary canal and in interfollicular epidermis. The epidermal mitotic rate in the pilary canal was depressed by epidermal extracts, and increased after adhesive tape stripping in the same way as in interfollicular epidermis. The results indicate no heterogeneity in cell proliferation characteristics between the two locations, and suggest that similar mechanisms are responsible for maintainance of growth equilibrium at both sites.

Inhibition of methylcholanthrene‐induced skin carcinogenesis in hairless mice by the membranelabilizing agent DMSO

The effects of dimethyl sulphoxide (DMSO) are in some respects similar to those of retinoids. DMSO has the ability to penetrate cellular membranes and to enhance the penetration of other molecules. It may be reasonable to assume that DMSO treatment results in differentiation of cells, possibly through membrane‐mediated events. This may be of importance for the study of the carcinogenic process.