INTRAPOPULATION KINETICS OF THE MITOTIC CYCLE

Data obtained with time lapse cinemicrographic techniques showed that the distribution of generation times for exponentially proliferating human amnion cells in culture is skewed to the right and that reciprocals of generation times appear normally distributed. As shown for bacteria, the true age distribution is much broader than theoretical distributions which fail to take into account the dispersion of generation times. By means of the technique utilizing autoradiographic detection of tritiated thymidine in cells whose mitotic histories were recorded by time lapse cinemicrography, it was shown that the G1 distribution is similar to the generation time distribution but is more variable. In our experiments, the G2 + prophase distribution resembled the generation time and G1 distributions. The data suggested two possibilities for S: either it is relatively constant, or it is inversely related to the lengths of G1 and G2 + prophase. Since G1 is more variable than the total cycle, and G2 + prophase more variable than the computed sum of S + G2 + prophase + metaphase, it was concluded that the relationships between parts of the cycle are non-random and that compensating mechanisms apparently help regulate the lengths of successive parts of the mitotic cycle in individual cells.     

Some Characteristics of DNA Synthesis and the Mitotic Cycle in Ehrlich Ascites Tumor Cells

In vivo studies of Ehrlich ascites tumor cells during the first 5 days of growth in peritoneal cavities of mice consisted of the following: 1. Determination of growth curves by direct enumeration of cells. 2. Estimation of the duration of each phase of the mitotic cycle based on incidence of cells in different phases. 3. Radioautographic studies to determine the proportion of cells in different phases of the mitotic cycle that incorporate tritiated thymidine during a single brief exposure to this precursor of DNA. 4. Estimation of the rate of incorporation of tritiated thymidine at different times during the period of DNA synthesis by comparison of mean grain counts over nuclei in radioautographs at different times following exposure to tritiated thymidine. The assumptions underlying these experiments and our observations concerning the duration of the period of DNA synthesis and its relation to the mitotic cycle are discussed. It is concluded that DNA synthesis is continuous, occupying a period of 8.5 hours during the interphase and that the average rate of synthesis is approximately constant.     

MACROMOLECULAR SYNTHESES RELATED TO THE REPRODUCTIVE CYST OF TETRAHYMENA PATULA

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.     

The origin of variability in cell cycle durations of NHIK 3025 cells

The origin of cell cycle variability was investigated in NHIK 3025 cells synchronized by mitotic selection from an exponentially growing population. The variability in G1 durations was measured by flow cytometric analysis of the fraction of cells in G1 as a function of time after mitotic selection. Immediately before the first cells entered S, medium containing 2.0 mM thymidine was added to the cells, and removed when all the cells had reached S. Since the cells had approximately the same DNA content upon removal of the thymidine, the variability in the durations of S+G2+M was measured by counting the fraction of undivided cells as a function of time after removing the thymidine. Such a thymidine treatment did not affect the naturally occurring variability in cell cycle durations generated after the start of S. The results indicate that the cell cycle variability of NHIK 3025 cells can be adequately described by a cell cycle model consisting of at least two compartments, which the cells leave according to first order kinetics. The model accounts for the initial shoulder of the curve representing the fraction of undivided cells as a function of time after mitotic selection. Furthermore, it accounts for the reduction in the rate of entry into the subsequent cell cycle compared to the rate of entry into S. Both rate constants were equally reduced after serum stepdown.     

CELL POPULATION KINETICS OF EXCISED ROOTS OF PISUM SATIVUM

The cell population kinetics of excised, cultured pea roots was studied with the use of tritiated thymidine and colchicine to determine (1) the influence of excision, (2) the influence of sucrose concentration, (3) the average mitotic cycle duration, and (4) the duration of mitosis and the G₁, S, and G₂ periods of interphase.¹ The results indicate that the process of excision causes a drop in the frequency of mitotic figures when performed either at the beginning of the culture period or after 100 hours in culture. This initial decrease in frequency of cell division is independent of sucrose concentration, but the subsequent rise in frequency of division, after 12 hours in culture, is dependent upon sucrose concentration. Two per cent sucrose maintains the shortest mitotic cycle duration. The use of colchicine indicated an average cycle duration of 20 hours, whereas the use of tritiated thymidine produced an average cycle duration of 17 hours.     

A Stochastic Model For Cell Populations With Circadian Rhythms

A mathematical model for cell kinetics, based on a random walk, is developed. the model allows variations with time of the rates of passage of proliferating cells through the four phases of the mitotic cycle. Circadian variations in the mitotic and labelling indices of the Syrian hamster cheek pouch epithelium have previously been observed, and the random walk model has been used to simulate this phenomenon. Assuming that all basal cells are proliferative and that these cells leave the basal layer randomly throughout the mitotic cycle to become differentiated cells, it was found that the experimentally observed circadian rhythms of the mitotic and labelling indices could be reproduced in the model by postulating a circadian rhythm in the rate of passage of cells through the G₁ and S phases only. Moreover, the growth activity of cells in both the G₁ and S phases appears to reach a peak during the dark hours of the light-dark cycle, and to fall off rapidly in the early hours of daylight. the postulate of Møller, Larsen & Faber (1974) that injection of the animals with tritiated thymidine causes a shortening of the G₂ phase duration has been qualitatively confirmed by using the random walk model to simulate the FLM and MI curves after injection with tritiated thymidine.     

CELL PROLIFERATION KINETICS IN GOLDFISH ACCLIMATED TO VARIOUS TEMPERATURES

Cell proliferation has been studied in goldfish which have been acclimated to temperatures of 6, 10, 15, 20, 30 and 37°C. Hemopoietic tissue and intestinal epithelium show similar temperature effects, but they are seen more clearly in the latter. The fraction of intestinal epithelial cells labeled by a single injection of tritiated thymidine decreases as the temperature increases above 20°C. The total tissue incorporation of DNA precursor also decreases with increasing temperature. On the other hand, the grain counts per nucleus tend to increase slightly with increasing temperature. The mean generation time as indicated by the rate of DNA turnover (loss of ¹²⁵I-DNA) shows little if any change as the temperature increases above 20°C. These observations imply that in the rapidly proliferating cells of the goldfish the mitotic cycle is remarkably insensitive to temperature change, except for the DNA synthesis phase, which has a temperature dependence (Q₁₀= 2.6) characteristic of enzyme-mediated chemical reactions.     

An effect of exogenous thymidine on the cell cycle in Haplopappus gracilis

The study of an effect of exogenous thymidine on the mitotic cycle demonstrated that a 30 minute exposure to unlabeled and to tritiated thymidine at a concentration of 2.9 × 10^?6 M was sufficient to cause a significant increase in the mitotic index of root meristem cells of Haplopappus gracilis. An analysis of the data revealed that this was due to the prolongation of metaphase rather than to an increase in the actual number of cells entering division.     

A KINETIC ANALYSIS OF MYOGENESIS IN VITRO

Conditions which yielded reproducible growth kinetics with extensive, relatively synchronous differentiation are described for chick muscle cultures. The effects of cell density and medium changes on the timing of cell fusion were examined. Low-density cultures which received a change of medium at 24 hr after plating show the highest rate of cell fusion, increasing from 15 to 80% fused cells in a 10 hr period. These optimal culture conditions were employed to reexamine two questions from the earlier literature on muscle culture: (a) can cells which normally would fuse at the end of one cell cycle be forced to go through another cell cycle before fusion; and (b) how soon after its final S period can a cell complete fusion? In answer to the first question, it was found that if the medium is changed, many cells which would otherwise fuse can be made to undergo another cell cycle before fusion. In the second case, radioautographs were made from cultures incubated with tritiated thymidine for various times at the beginning of the fusion period. These show labeled nuclei in myotubes as early as 3 hr after the beginning of the incubation period. This indicates that cells can fuse as early as the beginning of the G₁ period, and suggests that there is not an obligatory exit from the cell cycle or a prolonged G₁ period before cell fusion and differentiation during myogenesis.     

Heterogeneous chromosomal radiosensitivity of phytohaemagglutinin-stimulated human blood lymphocytes in culture

Human blood lymphocytes were irradiated with hard X-rays, stimulated with phytohaemagglutinin (PHA), and grown in presence of amethopterin to accumulate the responding cells at the GI/S boundary of the first cell cycle in vitro. After reversal of the GI/S block with exogenous thymidine, the frequencies of asymmetric chromosome exchanges in relation to the position of metaphases within the first generation mitotic wave were compared. Significant differences of aberration yields within replicate culture series were found in several experiments. A gradual increase of aberration frequencies with increasing duration of S + G2 phases was the most constant feature encountered. In addition, in two parallel series of cultures from one donor, the highest frequency of aberrations was found in samples corresponding to the shortest S+G2 phase duration. A significant contribution of selective mitotic delay of aberration-carrying cells to the distribution of aberration frequencies was excluded. Therefore, it was inferred that the results reflect a true variability of radiosensitivity among the PHA-responsive cells, probably of a discontinuos character, ranging over the ratio of two.     

Proceedings: Heterochromatin and chromosome aberrations: a comparative study of normal and tumour cells of mouse with various distributions of heterochromatin.

Seedlings of Crepis capillaris were irradiated after pulse-labelling with tritiated thymidine ([³H]TdR), and both chromosomal aberrations and presence of silver grains were recorded in the same metaphase cells at various intervals throughout the whole mitotic cycle. The following results were obtained: (a) irradiated roots were homogeneous with respect to the number of aberrations, and heterogenous with respect to labelling index (LI); (b) time-effect curves for labelled (L) and unlabelled (U) cells showed no significant difference from one another; (c) no significant quantitative difference of aberration spectra produced in S and G₂ stages was found. These results support the view that the major factor which determines both quantitative and qualitative variation in the production of chromosomal aberrations by radiation is the time lapse between irradiation and fixation rather than relation of the time of irradiation to the time of DNA synthesis. In addition, it was found that labelling with [³H]TdR modifies the effect of radiation on chromosomes.     

Correlation between the high rate of protein synthesis during mitosis and the absence of G1 period in V79-8 cells

The object of this study was to investigate whether modification of culture conditions would induce G1 and G2 periods in the Chinese hamster cell line, V79-8, which has been reported to exhibit neither of these phases in its life cycle. The results of this study indicate that under optimum culture conditions this cell line multiplies rapidly, with a generation time of about 9.5 h, and exhibits no measurable G1 period. However, under conditions of confluent growth, deprivation of isoleucine or inhibition of polyamine biosynthesis, a significant fraction (44–85%) of the cell population is preferentially arrested in the G1 period. Transient G2 arrest can also be induced in these cells by replacing the amino acid phenylalanine by its analog p-fluorophenylalanine. We have observed that decreasing the concentration of serum in the medium from 16 to 1% resulted not only in the prolongation of generation time but also resulted in a significant increase in the length of G1 period. Culturing cells in medium with 1% serum had no measurable effect on the rate of protein synthesis in interphase cells but a 50% reduction was seen in that of mitotic cells. The ratio between the rates of protein synthesis in mitotic and interphase cells in the line V79-8 is considerably higher (0.373) than that of G1-1 (0.218), a variant of V79-8 that has a G1 period of 4.25 h. These data suggest that cell line V79-8 is unique in retaining a relatively high rate of protein synthesis during mitosis under most favorable conditions. Probably this feature allows the synthesis of the factors necessary for the initiation of DNA synthesis while the cells are still in mitosis. However, under subnormal conditions the protein synthesizing machinery in the mitotic cells becomes inefficient and the cells require a longer time to synthesize the inducers of DNA synthesis; hence a G1 period is expressed.     

X-ray Sensitivity of HeLa S3 Cells in the G2 Phase: Comparison of Two Methods of Synchronization

The sensitivity of HeLa S3 cells to 220 kv X-rays was measured in terms of cell survival (colony development) during the G2 phase of the cell generation cycle, employing two procedures designed to free G2 cultures from contaminating cells from other phases of the cycle. Treatment of synchronous cultures (obtained initially by mitotic selection) with high specific activity tritiated thymidine (HSA-³HTdR) selectively eliminated S phase cells, while addition of vinblastine permitted removal of cells as they entered mitosis. It was found that HeLa S3 cells become increasingly sensitive as they progress through G2. The pattern of sensitivity fluctuations observed in synchronous HeLa S3 populations selected by the foregoing method was compared with that found in synchronous cultures prepared by the HSA-³HTdR method of Whitmore. The latter method had been used previously with mouse L cells, which were found to undergo a different pattern of sensitivity fluctuations. The two methods yield similar results for HeLa cells in the S and G2 phases of the cycle. It may be concluded, therefore, that the discrepancies between HeLa and mouse L cells do not arise from methodological factors, but represent fundamental differences between the cell types.     

The cell cycle of epidermal cells during reprogramming in the pupa of Galleria

The epidermal cell cycle of the pupal mesonotum of Galleria was investigated by the determination of mitotic indices, [³H]thymidine incorporation and flow-cytophotometric analysis during the first 48 h after pupation.Immediately after the pupal ecdysis nearly all epidermal cells are arrested in G2. Thereafter only a few mitoses occur, leading to a slow increase in the number of G1 nuclei. With the onset of a mitotic wave at a pupal age of 21 h this increase becomes more rapid. On day 2, the cell population reaches a plateau in the number of G1 (resp. G2) cells, reflecting a steady state between mitotic activity and DNA synthesis.A comparison of these cell cycle changes with known data of the time course of reprogramming and ecdysteroid titre leads to the conclusion that there is no causal relationship between DNA synthesis and cellular determination in the sense of a quantal cell cycle, and that DNA synthesis can precede the definite rise in ecdysteroid titre.     

POPULATION DYNAMICS OF INTESTINAL EPITHELIA IN THE RAT TWO MONTHS AFTER PARTIAL RESECTION OF THE ILEUM

Sprague-Dawley rats that had been subjected 2 months previously to partial resection (10 per cent) of the small intestine and an equal number of control rats were injected with tritiated thymidine and sacrificed at intervals during the subsequent 16 hours. Segments of duodenum, jejunum and ileum were prestained by the Feulgen technique and radioautographed. The proportion of crypt cells bearing labeled nuclei, the percentage of labeled crypt cells in mitosis and the appearance of labeled crypt cells on the villi were determined. Comparison of control and resected rats showed that (a) the proportion of intestinal crypt cells incorporating thymidine was considerably greater and uniformly high throughout the shortened intestine, (b) the life cycle of crypt cells was slightly reduced, and was uniform throughout the shortened intestine, and (c) the time during which cells were retained in crypts was markedly reduced. On the basis of persistent, generalized increase in the production of crypt cells, and on prior evidence that the epithelial cells of shortened intestine continue to have a brief life span and evidence of metabolic immaturity, the existence of a humoral factor, tentatively called "intestinal epithelial growth hormone," is postulated.     

THE CELL GENERATION CYCLE OF THE ELEVEN-DAY MOUSE EMBRYO

The incorporation of tritiated thymidine into the DNA of erythroblasts, primitive ependymal cells, and mesenchymal cells of 11-day mouse embryos was studied by radioautography at different times between 25 minutes and 18 hours after injection intraperitoneally. There was no labeling of mitotic figures until 1 hour after injection. Following this, mitotic figures were labeled for about 5.5 hours in primitive ependymal cells and mesenchymal cells, and for a longer period in erythroblasts. The percentage of the labeled primitive ependymal cells at various times after injection indicate a periodic migration into and out of the mitotic zone. The cell generation cycle of primitive ependymal cells and mesenchymal cells is similar to some kinds of adult cells. The cycle of the erythroblasts is more like that of the cells of aging mice.     

Turnover and maturation kinetics in the hairless mouse epidermis. Continuous [3H]TdR labelling and mathematical model analyses

Hairless mice were continuously labelled with 10 microCi of tritiated thymidine ([3H]TdR) every 4 h for 8 d, and the proportions of labelled basal and differentiating cells were recorded separately. The mitotic rate was measured by the stathmokinetic method and the cell cycle distributions were measured by flow cytometry of isolated basal cells at intervals during the labelling period. The mitotic rate of the [3H]TdR-injected animals did not deviate from control values during the first 5 d. Computer simulations of the data based on various mathematical models were made, and three main conclusions were obtained: (1) a large spread in transit times through the G1 phase was found, together with a very narrow distribution in maturation time of differentiating cells; (2) about 20% of the differentiating cells were estimated to leave the basal cell layer directly after mitosis. This is consistent with results obtained from different sets of data; and (3) during continuous labelling more than 90% of the cells are labelled during each passage through the S phase.     

Duration of the Mitotic Cycle in Cell Cultures of Haplopappus gracilis

The duration of the cell cycle and its component phases in cell cultures of Haplopappus gracilis was estimated by means of pulse labelling with tritiated thymidine and subsequent autoradiographic techniques. The total duration of the mitotic cycle was found to be 22.0 hours. The average durations of the following component phases were: the synthetic period (S) 6.4 hours, the postsynthetic period (G₂) 4.86 hours, prophase (P) 0.64 hours, metaphase (M) 0.40 hours, anaphase + early telophase (AT) 0.36 hours, the presynthetic period (G₁) 9.34 hours. The results indicate that G₁ and G₂ are the phases, which are most prolonged in populations of cultivated cells when compared to the same phases in root lip cells from the same species.     

EVIDENCE FROM THYMIDINE-3H-LABELED MERISTEMS OF VICIA FABA OF TWO CELL POPULATIONS

Treatments with tritiated thymidine (TdR-³H) have revealed the existence of two populations of mitotically active cells in meristems of lateral roots of Vicia faba. A rapidly dividing population, with a cycle time of 14 hr, constitutes about half the cells in the meristem. A second population of cells, with a cycle time in excess of 30 hr, is also present. Estimates of the relative size of this slowly dividing population are more difficult to make, but we calculate that this population includes 27–43% of meristem cells. The remaining fraction of the meristem is made up of cells that divide rarely or not at all. Since, at all times, both populations contribute to the mitotic index, the curve of the percentage of labeled mitoses that can be determined after a pulse label with TdR-³H differs from the curve expected of an ideal population in an important way: the peak value of the curve of the percentage of labeled mitoses is always less than 100%, usually between 75 and 80%. This heterogeneity within a meristem must be borne in mind in terms of the response of meristems to disruptive treatments, the mechanisms controlling mitotic cycle duration, and the spatial organization of a heterogeneous population in an organ that shows polarized growth.     

CHANGES IN THE DNA SYNTHETIC PERIOD DURING THE PHASE OF RAPID GROWTH IN CULTURED DIPLOID FIBROBLASTIC CELLS

The DNA synthetic period in diploid fibroblastic cell populations was studied during the phase of rapid growth in vitro , using the labelled mitotic wave method and quantitative autoradiography.
It was found that the length of the DNA synthetic period increased progressively, from early passage levels, with the number of generations in vitro. the increase was mainly in the time taken to synthesize the first 50%, and especially the first 10%, of DNA.
The change was detectable despite the complication due to a low level of tritium incorporation after the removal of tritiated thymidine from the cultures.