Calculation of Steel's cell loss factor and of the parameters of cellular kinetics for a population with an exponential growth of the cell number and cell death at G0 phase with a probability equal to 1

The method of calculation of three cell kinetics parameters (the Steel's cell loss factor phi, the proliferative pool Pc, and the mean number m of the proliferating cells after mitotic division of one cell) was shown to be the same for the exponential growth state of cell number with cell death at the G0-phase, and for the exponential growth state with cell death occurring immediately after mitosis. The value of the mean number delta of non-proliferating cells that appeared after mitotic division of one cell is different for these two models of the exponential growth state with the equal values of the other three parameters (phi, Pc, and m). A method is proposed for calculating the parameter delta on the data of the percentage of labeled cells obtained in the experiments with continuous cultivation of cells in the nutrient medium containing 3H-thymidine. The kinetics of cell line HL-60 (the experimental data of Foa et al., 1982) can be described at the first approximation, by a model of the exponential growth state with the cell death at the G0-phase, with Pc = 0.80, phi = 0.24, m = 1.61, delta = 0.39, and the life time of the non-proliferating cells tQ = 24 hours.     

Effect of hydrocortisone on the mitotic cycle of epithelial cells in the mouse pylorus]

It was shown with the aid of thymidine-H3 that the mitotic cycle of mucous-forming cells (superficial epithelial mucosal cells of the neck) of the stomach pyloric glands of mice lasted 13.5 hrs (G1+1/2M = 7.6 hrs, S = 5.3 hrs; G2+1/2M = 0.6 hrs). With the administration of a physiological dose of hydrocortisone (0.1 mg) the duration of the mitotic cycle of mucous-forming cells of the stomach pyloric glands increased by 6.7 hrs (G1+1/2M = 11.6 hrs, S = 7.8 hrs; G2+1/2M = 0.8 hrs). A high dose of the hormone had a similar effect and increased the presynthetic period to 12.9 hours and the postsynthetic one--to 2.3 hours.     

Topographic distribution of dividing hepatocytes in the regenerating liver lobule in the period of maximum mitotic activity

Topographic distribution of dividing hepatocytes was studied in the liver lobule after hepatectomy performed at 10-11.30 a. m. The studies were conducted 20-32 h after surgery during the first increase in mitotic activity. It has been established that dividing hepatocytes appear simultaneously in all lobule areas, i. e. that all the hepatocytes are capable of responding to regenerating stimuli. However, further behaviour of dividing hepatocytes becomes different. It is concluded that hepatocytes from outer and intermediate lobule areas possess a more pronounced proliferative potential during peak mitotic activity after partial hepatectomy.     

Action of the chalone from Ehrlich's ascites, tumor in mice on the mitotic activity in this tumor after single and double administrations

Chalone from Ehrlich's ascites tumour exerts a short-lived and reversible inhibitory effect on cell proliferation in the tumour both after a single and two-fold administration. 10 hours following single and two-fold injection of chalone (second injection was given at 6 p.m.), the mitotic index in tumour cells rises as compared to controls an evidence of chalone action on G(2) cell population of the mitotic cycle and synchronization of cell division. Repeated injection of chalone at 9 p.m. results in a more prolonged effect on the cells and in a more pronounced synchronization wave of G(2) cell population comparatively to its injection at 6 p.m. Thus the duration of cell inhibition in G(2) phase of the mitotic cycle depends with repeated administration of chalone, on the condition of cell population affected by chalone.     

The nature of the lengthening of the G2 phase in the mitotic cycle of parenchymal cells of the mouse liver

The phenomenon of G2 phase prolongation was found in the population of mouse hepatocytes. In normal postnatal liver growth, G2 phase prolongation in not pronounced and occurs in a small fraction of proliferating hepatocytes. In case of liver regeneration after removal of 2/3 of the organ, G2 phase prolongation is observed in a population of hepatocytes, which response to the proliferative stimulus first. Estimation of individual variation in expression of prolonged G2 phase along with the detailed analysis of the structure of the process of proliferation in the main population of hepatocytes (cells with normal G2 phase) allows to define the biological meaning of the "G2-population" observed. The prolongation of G2 phase may result from non-specific cell damage in mitotic cycles, caused by destruction of trophic relations in liver during its growth and regeneration.     

Effect of thyroxine on the duration of the phases of the hepatocyte mitotic cycle at different times of day]

The lengths of the synthetic phase (S) and postsynthetic gap plus a half of the mitotic time (G2+1/2 M) has been investigated in hepatocytes of control and thyroxine-treated male white rats using percent labeled mitosis curves after injection of isotope at 10, 16, 22 and 4 o'clock. In the control, the minimum lengths of G2 lasted 3.0 hours without being changed during 24 hours. On the contrary, G2+1/2 M and S varied from 3.2 to 4.4 and from 8.0 to 9.5 hours, accordingly. A prolonged administration or hormone induced changes in duration of all the above phases whose alterations in thyroxine-treated group of animals showed 2.0--3.0, 2.9--3.4 and 6.4--11.3 hours, respectively. During 24 hours, there was observed a characteristic pattern of changes in the labeling index (LI) of both groups of animals. It has been established for both the groups that the increased in LI coincides with the shortening of S-phase. The data allow to conclude that some intracycle mechanisms may exist controlling the cell division and exerting their effects on the cells at the end of G1-phase and during G2-phase. Thyroxine is a regulator of cell proliferation, and its effect was found to occur due to the intracycle mechanisms of cell cycle kinetics.     

Regulation of mitotic activity in rat vaginal epithelium: relationships between the level of its inhibitor (G2-chalone) and estrogens.

The level of a tissue-specific inhibitor of mitotic activity (G2-chalone) and mitotic activity in the vaginal mucosa of cycling rats of varying age and castrated rats were studied. A direct correlation between the level of the inhibitor and mitotic index is found in cycling animals. Both parameters are maximal during estrus and minimal in proestrus, when estrogen level in blood circulation is the highest. The undulating variations in G2 inhibitor level during estrous cycle are less pronounced and the concentrations of the inhibitor in relevant phases are significantly lower in aged females than in adult rats. Administration of estradiol benzoate (1 microgram/100 g) to castrated female rats was followed by a significant decrease in mitotic inhibitor level in vaginal mucosa within 12 hrs. This, in turn, was followed by a rise in mitotic activity 18 hr after estrogen administration. Therefore, the estrogen exerts its effect on mitotic activity in target tissue after it has induced a decrease in the level of the antimitotic factor (G2-chalone).     

Synchronisation of ovarian follicular waves in the dromedary camel (Camelus dromedarius)

This study was designed to compare the efficacy of various treatments intended to synchronise follicular wave cycles in dromedary camels by removing the existing follicle of unknown size and replacing it with a follicle capable of ovulating at a known time. Camels were randomly assigned to one of five groups and treated with either (1) 5mg oestradiol benzoate (i.m.) and 100mg progesterone (i.m.; E/P, n=15), (2) 20 icrog GnRH analogue, buserelin (i.m.; GnRH, n=15), (3) 20 microg buserelin (i.m.) on Day 0 (T=0) and 500 microg prostaglandin on Day T+7 (GnRH/PG n=15), (4) transvaginal ultrasound-guided follicle ablation of all follicles > or =0.5 cm (ABL, n=15) or (5) 5 ml saline (i.m; Controls n=15). All camels were subsequently injected with 20 microg buserelin 14 days after the first treatment was given. The ovarian response was monitored daily by transrectal ultrasonography and the intervals from treatment to follicular wave emergence and also the day on which the new dominant follicle reached 1.3 cm was recorded. Amongst the treatment groups the mean interval from treatment to new follicle wave emergence and treatment to time taken for the new dominant follicle to reach 1.3 cm in diameter was shortest in the ABL group (2.3+/-0.5 days and 8.8+/-1.1 days respectively, P=0.044) and longest in the E/P group (6.4+/-0.8 days and 12.2+/-1.0 days respectively, P<0.001) whereas the GnRH and GnRH/PG groups were intermediate (3.0+/-0.5 days and 11.1+/-0.8 days GnRH; and 4.5+/-0.5 days and 10.7+/-0.7 days GnRH/PG). A total of 11/15 camels in both the GnRH and GnRH/PG groups had dominant follicles between 1.3 and 1.9 cm 14 days post treatment, of which 21 of the 22 follicles ovulated after GnRH injection on T+14. The ABL, E/P and control groups however, showed greater variability in follicle size with less camels having dominant follicles between 1.3 and 1.9 cm than the GnRH and GnRH/PG groups and more in the > or =2.0 cm or follicle regressing groups, therefore fewer of these camels ovulated (ABL n=7; E/P n=9; Control n=6) after GnRH injection on Day T+14. In conclusion, two GnRH injections 14 days apart or two GnRH injections 14 days apart and PG on Day 7 after the first GnRH were the most effective methods to synchronise ovulation rate in dromedary camels at a fixed time interval of 14 days after treatment.     

Changes in the mitotic activity of the corneal epithelium of white rats following coolong of the animals at different times of the day]

A study was made of the influence of moderate hypothermia on the mitotic activity of albino rat corneal epithelium. The animals were cooled by the contact method for one hour to 28 degrees C; such procedure was conducted at 6 a.m., at noon, and at 6 p.m.; the epithelial reaction to cooling proved to depend on the time, the greatest suppression of mitotic activity (14-fold) occurring at daytime 3 hours after the cooling. A tendency to normalization of cell division was observed 6 and 12 hours after the cooling. The number of mitoses decreased 3 hours after the evening cooling, no changes in the mitotic activity in 3 and 6 hours after the morning cooling; cell division was found to be suppressed in 12 hours.     

Renewal time, proliferative pool and duration of the mitotic cycle of the epithelial cells of the common bile duct]

Using cholchicine and H3-thymidine the following parameters of the mitotic cycle (in hours) were calculated: T=56.6; tm=0.9; tg2=1.2; ts=6; tg1=48.5 The proliferative pool was 7.5% and the time of epithelium renewal--754.5 hours. The common bile duct epithelium should be referred to the tissue systems with slow renewal.     

Splitting of phase G1 of the mitotic cycle of guinea pig large intestine crypt cells]

Changes in shape of the second wave of the labeled mitoses curve previously observed by Rowinski and Sawicki (1972) for three crypt zones of three different parts of guinea-pig ascending colon are explained by the complicated branching structure of the G1-phase. This structure is assumed to be the same for different crypt zones and for different sections of the intestine. Changes in shape of the second wave of the labeled mitoses curve are explained by the changes in distribution of proliferating cell stream between the alternative directions at the points of branching of the G1-phase, depending on the crypt zone, the intestine section, the cell state, and on the state of intestine.     

Circadian rhythm of the mitotic activity and of the number of nuclei synthesizing DNA in sarcoma 37 in white mice]

Circadian variations in the frequency of mitoses and the number of nuclei labed with thymidine-H3 in sarcoma-37 of mice were investigated. It was shown that the circadian rhythm of mitotic activity was composed of diurnal variations in the frequency of labeled and unlabeled mitoses. The G2-phase of mitotic cycle of the cells with labeled mitosis was approximately one hour. The G2-phase of the cells with unlabed mitosis lasted four hours and more. It is suggested that there are two cell populations in sarcoma-37.     

Prostaglandin E2-induced hyperplasia of the rat antral epithelium is followed by a secondary inhibition of the mitotic activity

The aim of the study was to examine the mitotic activity in the antral and duodenal epithelium of Sprague-Dawley rats given trophic doses of E2 prostaglandins during a prolonged period of time. Natural prostaglandin E2 (dose range: 0.2-5.0 mg.k-1) and 15 (R) 15 methyl prostaglandin E2 (dose range: 0.03-2.0 mg.kg-1) were administered for 11 days, and mitoses were arrested with vincristine for 4 h before estimation of the cumulative mitotic index. A dose-related hyperplasia of the antral glands was observed after treatment with prostaglandin E2 and the synthetic analogue (p less than 0.05). The proliferative zone was enlarged in rats treated with high doses of the analogue but natural prostaglandin E2 did not affect the limits of the proliferative zone. A dose-related reduction of the mitotic index was observed in animals treated with prostaglandin E2 despite the presence of hyperplastic changes. All doses of the analogue induced antral hyperplasia without affecting the mitotic index except in rats given the highest dose who had a significantly lower mitotic index than controls (p less than 0.05). Hyperplasia of both crypts and villi was observed in the duodenum of rats given high doses of E2 prostaglandins (p less than 0.05) whereas the mitotic index and the growth fraction were not affected by treatments. It is concluded that hyperplasia by prostaglandins is developed in absence of changes of the mitotic activity. The observed reduction of the mitotic index is interpreted as a secondary phenomenon, possibly mediated by a regulatory mechanism of cell proliferation which is triggered to reduce further epithelial growth. It is suggested that prostaglandin E2 might influence such regulatory mechanisms.     

Effect of thyroxine on the diurnal rhythm of the mitotic activity and parameters of the life cycle of cells of the liver and esophagus]

The duration of the cellular cycle and the diurnal rhythm of the amount of mitosis were studied in young rats in normality and under the influence of thyroxin. The parenchymal and connective-tissue cells of the liver and cells of the liver and the cells of the oesophagus epithelium basal layer were studied. It was found that under the influence of thyroxin there occured a shortening of the periods of the cellular cycle and a 3--6 h shift to the left of the diurnal rhythm curve of the amount of mitoses. In thyroxinized animals the 21--95% increase of the amount of mitoses in the period of maximum values of the mitotic index during a day was observed as compared with control animals. A conclusion is made about the diurnal rhythm of sensitivity of G0-phase cells to the synchronizing factor, suggesting the decisive significance of the state of the cell population in the interaction of the tissue and hormone cells. The data obatained in the work show that the thyroid hormones regulate the cellular reproduction in the organism by stimulating the cells in the division cycle, synchronization of greater amount of cells by the moment of beginning of the mitotic cycle at a definite time of day and by shortening the period of the cell mitotic cycle.     

Cyclic fluctuations of the mitotic Ca2+-ATPase during the cell cycle of the myxomycete Physarum polycephalum

The occurrence of the mitotic Ca2+-ATPase, resembling the enzyme described for higher organisms, is demonstrated in multinuclear plasmodia of the myxomycete Physarum polycephalum. The activity of this enzyme undergoes cyclic fluctuations during the synchronous nuclear cycle with a minimum in early G2-phase and a maximum around the time of mitosis.     

Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

Radiation-induced mitotic delay: duration, dose and cell position dependence in the crypts of the small intestine in the mouse

The cells of the proliferative compartment in the crypt of the small intestine undergo a step by step differentiation and/or maturation from stem cells to the functional cells on the villi. The consequent hierarchical organization of the proliferative cell population can be related to the actual position of cells within the crypt. The stem cells are found near the bottom of the crypt with the more mature cells occurring at increasingly higher positions. The sensitivity of proliferative cells in the crypt of small intestine to radiation-induced mitotic delay was investigated at each position within the crypt. Using the stathmokinetic method (vincristine accumulation), the following were noted. The yield of mitotic figures 3 h immediately after irradiation showed a strong cell position dependence with the cells at the base of the crypt being most inhibited and those at the top of the proliferative compartment least affected. The mitotic yields were largely unaffected for the first 15 min suggesting that there is a transition point (Tp) for radiosensitivity which is located about 15 min before metaphase for all crypt cells. Cells located less than 15 min from metaphase are unaffected while those more than 15 min from metaphase are inhibited from further cell cycle progression. After this initial delay all proliferative cells were inhibited in their progression through G2 but some recovered more quickly than others. The ratio of the time of division delay (Td) in stem cells to that in cells at the top of the proliferative compartment was about 3:1. In absolute values Td after 1.0 Gy was about 1 h and 2.8 h, for cells at the top of the crypt and at the base, respectively. After 2.5 Gy the corresponding values were less than 3 h and between 5 and 6 h for the mid-crypt and crypt base respectively. There is thus a dependence on dose for the duration of the mitotic inhibition which for the cells at the top of the crypt is similar to the widely quoted average value 1 h per Gy, but the duration depends strongly on cell position. Thus not all proliferative cells respond in the same way. The duration is shorter the closer the proliferative cells are to their last cell division in the proliferative hierarchy in the crypt and longest for cells situated where the stem cells are to be expected.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.