Cell population kinetics and the cell population mechanisms of the circadian rhythm of proliferative activity in mouse esophageal epithelium]

The dynamics of 3H-thymidine labeled mitosis and diurnal rhythm of proliferative activity was studied. The isotope was injected to BALB/C mice at the peak of diurnal rhythm of DNA synthesis activity of basal layer cells of oesophageus epithelium. It has been established that the increase in the mitotic index during 24 hours depends on the increase in number of cells being in S-period. The data show that the increase of mitotic index at diurnal rhythm occurs at the expense of 75% of new G0-cells which entered into the mitotic cycle, and of 25% of re-entering cells that had divided during the maximal mitotic activity a day before. It is found that the duration of mitotic cycle of cell population which entered into the mitotic cycle synchronously is almost equal to the period of diurnal rhythm of mitotic activity, i.e. 24 hours.     

STUDIES ON THE MECHANISM OF DIURNAL VARIATION OF PROLIFERATIVE INDICES IN THE SMALL BOWEL MUCOSA OF THE RAT

In the rat small bowel mucosa significant variation was found in both the labelling and the mitotic indices with time of day. The zenith and the nadir of labelling and mitotic activity coincided at 15.00 and 02.00 hours respectively. Small changes were found in the ‘cut-off’ position, but this variation in proliferative compartment size was insufficient to account for the comparatively wider fluctuations in proliferative indices. Measurements of the rate of entry into mitosis, using metaphase arrest with vincristine at three widely separated times during the day, showed no significant change. Changes in the growth fraction or in the birth rate as measured cannot account for diurnal variation in the proliferative activity of the small bowel mucosa. We propose a hypothesis which involves diurnal fluctuations in the transit times through G₁ and through G₂.     

The circadian variations in the epithelial growth of the hamster cheek pouch: quantitative analysis of DNA distributions

The pronounced diurnal rhythm in DNA distributions of the hamster cheek pouch epithelium both in the S fraction and in the (G2 + M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. The DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. The DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. The S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. The DNA distributions showed a diurnal release of G1 cells into S phase proceeding through (G2 + M) phase and returning to G1 phase within a 24 hr period.     

CIRCADIAN RHYTHMS OF PRESUMPTIVE STEM CELLS IN THREE DIFFERENT EPITHELIA OF THE MOUSE

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with ³H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.     

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques

Abstract. A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([³H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase-anti-alkaline-phosphatase technique, the peroxidase-anti-perox-idase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a GdG, peak nor a G₂+ M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.     

The Circadian Variations In the Eithelial Growth of the Hamster Cheek Pouch: Quantitative Analysis of Dna Distributions*

The pronounced diurnal rhythm in DNA distribution of the hamster check pouch epithelium both in the S fraction and in the (G₂+ M) fraction was compared with previous studies of the changes in tritiated thymidine labelling index and mitotic activity. the DNA distributions were obtained by flow cytometry after ultrasonic disaggregation of the isolated epithelium into a suspension of single nuclei. the DNA distributions were analysed with the computer program of J. Fried (1976) and by planimetry. the S fraction was higher than the autoradiographic labelling index during the whole 24 hr period. Only the computer fitted S fraction and the labelling index had the same difference between maximal and minimal values, and maxima at the same time of day. the DNA distributions showed a diurnal release of G₁ cells into S phase proceeding through (G₂+ M) phase and returning to G₁ phase within a 24 hr period.     

Epithelial cell kinetics in the descending colon of the rat

Epithelial cell loss was induced in the descending colon of the rat by temporary ischaemia to investigate whether this would lead to an increase in crypt cell proliferation. Shortly after the temporary ischaemia the number of cells per crypt was markedly reduced, and it was shown that the cell loss occurred mainly from the non-proliferating upper half of the crypt. The number of cells per crypt reached control values again after 24-48 h. There was a marked increase in proliferative activity, as reflected by the labelling index after 3HTdR and by the mitotic index, with peak values at 16 and 24 h after ischaemia. After 48 h the proliferative indices were normal again. The increase in crypt cell proliferation was characterized by an increase in the labelling index as well as in the mitotic index per crypt cell position. No enlargement of the proliferative cell compartment in the crypt was observed. It is most likely then that the increase in crypt cell proliferation was brought about by a shortening of the cell cycle, since the growth fraction in the lower half of the crypt approaches 1.0. The possible implications of the present data for the control of colonic cell proliferation and colonic carcinogenesis are discussed.     

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones

The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours. A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase). Labelled mitoses curves and metaphase accumulation after colchicine injection show that the duration of the S, G2 and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days. Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G2 duration. The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G1-S transition when it is higher in the evening.     

Cytostatic effect of cyclophosphane on esophageal tumors and epithelium of mice

Investigation of cytostatic activity of cyclophosphamide in sarcoma 37 and esophagus epithelium in albino mice with respect to the diurnal rhythm of mitotic activity and the number of labeled nuclei was performed. Apparently the tumour cells in the G1-phase and at the beginning of the S-phase of the mitotic cycle were the most sensitive to the inhibitory effect of this drug. During the completion of the DNA-synthesis period the cell resistance to the action of the cytostatic increased. Cells at the G1-phase of the mitotic cycle were sensitive to the inhibitory action of cyclophosphane in the esophageal epithelium.     

EPIDERMAL REGENERATION AFTER CELLOPHANE TAPE STRIPPING OF HAIRLESS MOUSE SKIN

After repeated applications of cellophane tape to the dorsal skin of hairless mice, the proliferative response in the treated epidermis was estimated by three different methods. The mitotic rate was determined in the interfollicular epidermis using the Colcemid technique, and the DNA synthetic activity was estimated after ³H-thymidine injection by counting labelled interfollicular cells in autoradiographs and by determining the specific activity of epidermal DNA. An initial 40–50% inhibition of DNA synthesis and mitosis was followed by an increase in the labelling index and the mitotic rate 8–10 hr after tape stripping. By 24 hr, peak values 5–6 times the controls were attained for both parameters. The labelling index and the mitotic rate were nearly normal at 3–4 days, but a second small peak was seen on day 5. Normal values were found on days 6 and 8. A similar pattern of response was found biochemically, but the peak of DNA specific activity was much broader and the extent of the increase was only about half as great as the increase in the labelling index. Possible reasons for these differences are discussed.     

Circadian synchronization of hepatocyte proliferation in young rats: the role played by adrenal hormones

Abstract. From the 20th day to the 30th day of life, the mitotic rhythm is progressively induced by a reduction in nocturnal values, while diurnal rhythms remain unchanged. Mitotic peaks emerge at 10.00 hours.
A labelling index wave occurs 8 hr before the corresponding mitotic wave, with a peak at 02.00 hours and a minimum in the evening, coincidental with the acrophase of plasma corticosterone level (activity phase).
Labelled mitoses curves and metaphase accumulation after colchicin injection show that the duration of the S, G₂ and M phases remain approximately constant and that the circadian variation is due to a variation in the rate of cells that enter these successive phases. During the synchronization period (from day 20 to 30), the growth fraction decreases progressively. Adrenalectomy at this time is followed by a higher cell proliferation and all rhythms disappear after 2 days.
Corticosterone injected before the triggering of the rhythmic activity in 17-day-old rats immediately reduces the labelling index, while the mitotic index is decreased 10 hr later; this delay is equal to the S + G₂ duration.
The results are discussed. They favour the hypothesis that the circadian variation of corticosterone is responsible for the induction of a circadian variation in developmental cell proliferation by inhibition of the G₁-S transition when it is higher in the evening.
The circadian rhythm of hepatic cell proliferation in rats appears on the 20th day of life, when the hypothalamo-adrenal axis is mature enough for circadian activity to occur.     

Proliferative activity of the epithelium of the rat large intestine during carcinogenesis]

Labelling index and mitotic regimen in the epithelium of the rat descending colon and the ileum was studied during the tumour induction with 1,2-dimethylhydrazine. One month after the beginning of the experiments there was a marked increase of abnormal mitoses (up to 51%) and a change in the proportion of the mitotic phases with the metaphase prevalence (up to 73%). Later, these parameters were unchanged. Beginning from the 3rd month of the experiment there was found an increase in the labelling index (especially, in the carcinoma in situ) and of the mitotic index. In the mucosa of the ileum (where the tumours never developed) no changes of the proliferative activity and of mitotic regimen were found.     

Radioprotection by pretreatment with deuterated water: cytokinetic changes in the small intestine of the mouse

All mice partially deuterated by ingestion of 29% heavy water for 12 days survived whole body gamma irradiation (8.5 Gy) from a 60Co source, whereas 42% of nondeuterated control animals died from bone marrow failure. The incorporation of 3HTdR into enterocytic DNA, as measured by autoradiography and liquid scintillation spectrometry, was used to assess the proliferative activity of small intestinal epithelium. The sequence and the magnitude of changes in tritium activity were in good agreement. Deuteration alone resulted in a reduced proliferative activity of small intestinal crypt epithelium, particularly in the basal cell positions and the first positions of the proliferation compartment. The number of positions occupied by the proliferative compartment and the crypt length were, however, barely affected by deuteration. The radiation-induced depression of DNA synthesis in the proliferative compartment was of similar magnitude in both groups. Crypt epithelium in deuterated mice, however, displayed signs of an accelerated and/or enhanced regeneration. The cytokinetic changes in deuterated animals are consistent with a protective effect for clonogenic intestinal epithelium at the time of irradiation.     

Epidermal cell kinetics of the pig: a review

Age-related changes in cell kinetic parameters for the epidermis of pigs have been shown to be small, indicating that young pigs may be used for experimental studies. It was not possible to draw any firm conclusions about any strain-related differences in the cell kinetics of the epidermis of the pig. Lower LI values have been quoted for the miniature pig and the Yorkshire pig than for the Large White pig. However, these differences may be related to variations in experimental technique. The cell kinetic data for the Yorkshire pig are not consistent. Very high values for the mitotic index suggested a high rate of cell turnover, whilst data from single pulse labelling and grain count halving studies indicate a relatively low rate of cell turnover. The results from continuous labelling studies on the epidermis of the Yorkshire pig suggest that the basal cell turnover time (TT) is a factor of two or more shorter (136 h) than the estimates obtained using other methods. In the Large White pig estimates of TT were similar using a variety of techniques and were comparable with the TT estimate for the Yorkshire pig obtained using the continuous labelling method. There is some degree of inconsistency in the literature with regard to possible diurnal variations in the cell kinetic parameters for the epidermis. In the study of Archambeau & Bennet (1984) distinct diurnal variations were found in the LI, although the reliability of this finding is questionable due to the small number of animals used. Later studies by Morris et al. (1987) have suggested that diurnal variations are negligible in the epidermis of the pig. The majority of labelled cells (80%) in pig epidermis are located in the basal layer, although a significant proportion (20%) occurs suprabasally, in the cell layer immediately above the basal layer. Therefore, the epidermis can be regarded as having a bilayered proliferative cell compartment. The results from studies on irradiated pig skin (Morris & Hopewell, 1986, 1988, 1989) are not consistent with the presence of a homogeneous proliferative compartment in the epidermis, and are best explained by the occurrence of an heterogeneous proliferative compartment consisting of a stem cell subpopulation and a much larger population of transit proliferative cells.     

Diurnal rhythm of changes in the DNA-synthesizing cell count and the mitotic activity of the cells of jejunal epithelium under conditions of subdiaphragmatic vagotomy

Bilateral subdiaphragmatic vagotomy in rats 7 days after surgery results in increasing proliferative activity of the jejunum epithelium not changing circadian rhythm of the mitotic process. Vagotomy induces changes in the diurnal rhythm structure of DNA-synthesis in the jejunum epithelial cells, i.e. monoapical rhythm in the norm becomes biapical.     

Annual mitotic activity in the intestinal epithelium of the musselCrenomytilus grayanus

The seasonal dynamics of cell reproduction in the intestinal epithelium of the musselCrenomytilus grayanus are described in detail. Mitotic indices in the intestinal epithelium varied throughout the year from 0.005 to 0.26% (averaged data) and from 0.003 to 0.37% (individual data). Cyclic seasonal changes were found in the mussel’s intestinal epithelium. In general, the average values of mitotic activity in the intestinal epithelium were low (the mitotic index was 0.13%); there was a rise in activity in late April–June and September and a decline in July–August and especially in January–March. The winter-early spring period was characterized by a profound inhibition of cell reproduction and the transition of cells to the resting state. An outburst of proliferation occurred in the spring, due to a manifold increase in the number of cells in the mitotic cycle. The musselC. grayanus may be a good model for the study of the two extreme states of proliferation and their alternation in marine animals in nature. The diel dynamics of mitotic activity in the intestinal epithelium were followed during the most active growth period (May). The mitotic index (MI) varied during the day within a narrow range, deviating from the daily average value by no more than one third; no pronounced diel rhythm was found. Optimum water temperatures for cell reproduction ranged from 5 to 18°C.     

Proliferation patterns in the canine endometrium during the estrous cycle

The proliferative activity in the endometrium of 58 bitches in different stages of the estrous cycle was assessed by immunohistochemical detection of the Ki-67 proliferation associated nuclear antigen and by counting mitotic figures. The Ki-67 labelling index and the mitotic index were determined in the surface epithelium, the stroma, the crypts and the basal glands by calculating the percentage of Ki-67 positive cells and mitotic figures, respectively, on a total of 500 cells of each category. Endometrial vascular proliferation was also verified by counting the number of Ki-67 positive cells on a total of 100 endothelial cells. The present study showed two proliferation peaks involving different cell groups. In the surface epithelium, the stroma, the blood vessels and the crypts, the highest labelling and mitotic indexes were noticed during proestrus, whereas for the basal glands these indexes significantly increased (P < 0.05) during estrus compared to late metestrus and anestrus. Furthermore, a slightly positive correlation (P < 0.05) was found between the labelling index in the basal glands and the serum progesterone levels, whereas the labelling indexes in the other cell groups were positively correlated with the estradiol-17 beta levels, although not always significantly. These findings suggest that regulation of the proliferation in the surface epithelium, the stroma, the blood vessels and the crypts is different from the proliferation in the basal glands.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9–13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13–14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Proliferation kinetics of mouse tongue epithelium under normal conditions and following single dose irradiation.

Epithelial proliferation in the ventral surface of mouse tongue follows a pronounced circadian rhythm with a peak in mitotic activity at 10.00 a.m., preceded by a wave of DNA synthesis 8 h earlier. Nearly all cells (85%) pass through G2 and mitosis immediately after the S-phase; they subsequently divide again, usually after 2 or 3 days, indicating cohorts of cells with different G1-duration. The fraction of all nucleated cells comprised in one daily proliferation wave is about 20%, indicating a turnover time of the nucleated cell compartment of about 5 days. Cytotoxic injury by a single radiation dose of 20 Gy causes a steep decrease in cell counts, leading to complete denudation after 9-13 days. The difference between the latent period before ulceration and the tissue turnover time is explained by a marked proliferative activity of the doomed cells. The mitotic index increases steeply after day 1 to three times the control level, but most mitotic figures display gross abnormalities such as multipolar spindles or chromosome clumping. As a consequence cells with abnormal or multiple nuclei appear in the basal layers 3 days post irradiation and subsequently migrate to the upper layers. After denudation the epithelium rapidly becomes restored, with a phase of transient hyperplasia on days 13-14. Normal architecture is regained by day 15. Over the whole healing period the mitotic index remains at a high level, with most of the mitoses appearing histologically normal.     

Mitotic and Labelling Activity In Normal Human Epidermis In Vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours > 09.00 hours by a factor of 2.6, P < 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.