Estimation of circadian variations in cell cycle phase durations in murine epidermal basal cells

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G2 and mitosis) have been disputed. The percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. The use of all three methods gives comparable and pronounced circadian variations in the duration of S and G2 phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.     

Analysis of the cell cycle in the root meristem of Allium cepa under the influence of ledakrin

The influence of a polish anticancer drug on the cell cycle using Allium test was studied. Methods of aceto-orcein squash slides, curve of labelled mitoses after 3H-thymidine incubation and cytophotometrics after Feulgen's reaction were employed. Ledakrin acts strongly antimitotically, but it does not block the cell cycle completely. The cytostatic activity of ledakrin results from its action on the interphase. The phases G1 and S are prolonged while M is unchanged after 6h incubation with ledakrin. During postincubation in water without ledakrin it was noted, at the beginning, that the mitotic activity decreases and it is brought about the lengthening of S and G2 phases. The duration of the cell cycle phases returns to the control level during further postincubation. The results of analysis of chromatin aberrations and the micronucleus test point to a mutagenic effect of ledakrin.     

A simple method for estimation of cell cycle parameters

A simple and convenient method of estimating cell cycle parameters was proposed. The means and the standard deviations of G1, S and G2 phase durations in the cell cycle of an ascites tumor, LY-1, a sub-line of Yoshida sarcoma, were estimated by plotting the fraction of the labelled mitoses on probit paper. The frequency distribution of the duration of each phase was assumed to follow a normal distribution. When this method was compared with the Monte Carlo program where each phase duration was assumed to follow a log-normal distribution, results by the two methods were in good agreement.     

Cell kinetics of a chondrosarcoma.

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G1, 10-5 hr; S, 9-5 hr; G2, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0-5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

CELL KINETICS OF A CHONDROSARCOMA

The cell kinetics of the transplantable DC-II mouse chondrosarcoma have been studied by the pulse labelled mitoses method. The analysis gave the following estimates for the phases of the cell cycle: G, 10-5 hr; S, 9-5 hr; G₂, 4 hr with an intermitotic time of 23-5 hr. Consideration of the overall growth of the tumour indicated that the growth fraction and cell loss factor both had values of about 0–5. The results are compared with cell kinetic data from sarcomas and other cartilage tissues.     

Duration of the cell cycle phases in mutants for the tumor suppressor Merlin in Drosophila melanogaster

The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulse-labeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.     

Cell kinetic studies in the epidermis of mouse. III. The percent labelled mitosis (PLM) technique

Full PLM curves have been obtained for four sites in the mouse. The first peaks have been analysed by computer and the duration of the G2 + M and S phases determined together with their standard deviations. The full curves showed a general similarity for all four sites with no clear second peak. The data are compared with the published data for mouse and human epidermis using the in vivo PLM technique. The timing and shape of the first peak can vary considerably even for one site in mice. Hence, both G2 + M and S can vary in their durations. Cells labelled at one time of day exhibit different kinetic properties to those labelled at another time of day. The duration of G2 + M is shortest in dorsum labelled at 03.00 hours (3 X 2 hr) and longest in tail (up to 7 X 5 hr). The S-phase is shortest in dorsum (6 X 3-7 X 2 hr) and longest in tail or ear (13 X 3-14 X 1 hr). There is also a very large standard deviation in tail and foot. There is little general variability when the psoriatic human data are considered, which is surprising. The general variability amongst the data from experimental mice might also be expected amongst humans which might make comparisons between the cell kinetics of normal and diseased skin difficult.     

Duration of the cell cycle phases in mutants for the tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The cell cycle duration was estimated in Drosophila melanogaster mutants for the tumor suppressor Merlin with the use of different approaches. Experiments on induction of mosaic clones in tissues of the larval wing imaginal disc showed that the cell cycle in mutant discs is shorter than that in control. Flow fluorescence cytometry revealed no differences between mutant and normal animals in the relative duration of the cell cycle phases, which suggests proportional shortening of the cell cycle phases. The study with pulselabeled mitoses confirmed these results and showed that the length of the cell cycle is 7 h (S phase duration 3 h) in control individuals and 5 h (S phase duration 2 h) in Merlin gene mutants.     

The kinetic significance of cell size : I. Variation of cell cycle parameters with size measured at mitosis

Using a cell line of human lymphoid cells, the kinetic significance of cell size measured at mitosis has been explored using fraction of labelled mitoses data. It was found that smaller cells tend to have progressively longer generation times. The principal mechanism for this generation time dilation is a progressively protracted G 1 duration as cell size decreases. There is a concomitant, but much slighter increase in S phase duration. G 2 duration remains essentially constant irrespective of cell size.     

Estimation of Circadian Variations In Cell Cycle Phase Durations In Murine Epidermal Basal Cells

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G₂ and mitosis) have been disputed. the percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. the use of all three methods gives comparable and pronounced circadian variations in the duration of S and G₂ phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.     

A modification of the grain count halving method for detailed analysis of cell kinetic parameters

A modification of the conventional grain count halving (GCH) method is presented. By determining the decrease of the mean grain number of all interphase cells in addition to that of all labelled interphase cells on the same autoradiographs, the potential doubling time Tpot (or the cell production rate kp) can be obtained in one and the same experiment. Thus the modified GCH method provides not only the cycle time of the cell population studied but also the growth fraction and, with additional cytofluorometric measurements, the duration of all cycle phases. By evaluating the cell production rate and the growth fraction this method leads to more reliable cell kinetic data of experimental tumours and human tumours growing in nude mice. In contrast to other cell kinetic methods, the modified GCH method can also be applied in special cases to human tumours in vivo, since only few points of measurement are needed. A comparison of the cell kinetic results obtained by the modified GCH method with those derived from the fraction of labelled mitoses method, both applied to allotransplants of the adenocarcinoma EO 771 in nude mice, shows good agreement.     

Interference of a synthetic C18 juvenile hormone with mammalian cells in vitro. II. Effects on cell cycle

Interference of a synthetic C18 juvenile (JH) with the cell cycle of mouse embryo cells (ME-cells) and mouse cells of established cell line (L-cells) was examined. After 3 hour in the medium with JH (20 mg/ml) the cells were transfered to the regular culture medium and labelled with H3-thymidine then incubated for 1 to 48 hours before processing them for autoradiography. The percentage of labelled mitosis was then calculated for all cells samples examined and the labelled mitosis curves were drown and analyzed. It was shown that in contrast to the solvent which had no effect on duration of any of the component phases of the cell cycle of ME-cells, the juvenile hormone under conditions of these experiments prolonged G1 and G2 intervals what resulted in prolongation of the total cell cycle of these cells. On the other hand it shortened G1 and prolonged G2 intervals of L-cells without changing duration of the total cell cycle. Thus, in the examined mouse cells, they were the G1 and G2 intervals which are affected by JH. This findings are considered as an argument for pleiotropic nature of the juvenile hormone interference with mouse cells, the more so as it interfered with both protein and DNA synthesis in these cells.     

A modification of the grain count halving method for detailed analysis of cell kinetic parameters

Abstract A modification of the conventional grain count halving (GCH) method is presented. By determining the decrease of the mean grain number of all interphase cells in addition to that of all labelled interphase cells on the same autoradiographs, the potential doubling time T _pot (or the cell production rate k _p) can be obtained in one and the same experiment. Thus the modified GCH method provides not only the cycle time of the cell population studied but also the growth fraction and, with additional cytofluorometric measurements, the duration of all cycle phases. By evaluating the cell production rate and the growth fraction this method leads to more reliable cell kinetic data of experimental tumours and human tumours growing in nude mice. In contrast to other cell kinetic methods, the modified GCH method can also be applied in special cases to human tumours in vivo , since only few points of measurement are needed. A comparison of the cell kinetic results obtained by the modified GCH method with those derived from the fraction of labelled mitoses method, both applied to allotransplants of the adenocarcinoma EO 771 in nude mice, shows good agreement.     

Transit times through the cycle phases of jejunal crypt cells of the mouse. Analysis in terms of the mean values and the variances.

Mean transit times as well as variances of the transit times through the individual phases of the cell cycle have been determined for the crypt epithelial cells of the jejunum of the mouse. To achieve this the fraction of labelled mitoses (FLM) technique has been modified by double labelling with [3H] and [14C]thymidine. Mice were given a first injection of [3H]thymidine, and 2 hr later a second injection of [14C]thymidine. This produces a narrow subpopulation of purely 3H-labelled cells at the beginning of G2-phase and a corresponding subpopulation of purely 14C-labelled cells at the beginning of the S-phase. When these two subpopulations progress through the cell cycle, one obtains FLM waves of purely 3H- and purely 14C-labelled mitoses. These waves have considerably better resolution than the conventional FLM-curves. From the temporal positions of the observed maxima the mean transit times of the cells through the individual phases of the cycle can be determined. Moreover one obtains from the width of the individual waves the variances of the transit times through the individual phases. It has been found, that the variances of the transit times through successive phases are additive. This indicates that the transit times of cells through successive phases are independently distributed. This statistical independence is an implicit assumption in most of the models applied to the analysis of FLM curves, however there had previously been no experimental support of this assumption. A further result is, that the variance of the transit time through any phase of the cycle is proportional to the mean transit time. This implies that the progress of the crypt epithelial cells is subject to an equal degree of randomness in the various phases of the cycle.     

Growth pattern of the human promyelocytic leukaemia cell line HL60

Abstract. In order to characterize the growth pattern of the human promyelocytic leukaemia cell line HL60, its kinetic parameters were studied. The doubling time was calculated from serial cell counts, the duration of the various cell cycle phases from the analysis of the labelled mitoses curve, and quiescent population from continuous labelling experiments. Proliferation in culture was exponential up to a saturation density of about 3.0 × 10⁶ cells/ml, with a doubling time of 34.0 hr. The cell cycle duration was 24.3 ± 4.1 hr (SD), and that of the cell cycle phases was: G₁, 3.8 ± 2.2 hr; S, 15.1 ± 3 hr; and G₂, 5.4 ± 1.2 hr. The growth fraction was 0.85, and cell loss was restricted to the quiescent cells. The HL60 cell line, with fully characterized kinetics, provides a useful tool for the in vitro study of substances which may affect human leukaemic myelopoietic proliferation.     

Tumour cell proliferation in relation to the vasculature.

The proliferation pattern of a transplantable mouse mammary carcinoma has been studied in relation to its macroscopic and microscopic structure. No significant differences were seen in the labelling or mitotic indices or in the percentage labelled mitoses curves for the peripheral 2.0 mm rim or for the central tumour core. When these parameters were scored for cells classified according to their position in relation to capillaries or to necrotic regions, marked differences were observed in all the parameters. Higher labelling and mitotic indices and higher grain counts were seen adjacent to the capillaries. These appear to result from a shorter cell cycle duration and a higher growth fraction. The variation in cell cycle is mainly due to a change in the duration of G1.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

The kinetics of isthmal cells in mouse antrum were examined in three ways: the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; the duration of interphase and mitotic phases was determined from how frequently they occurred; and mice were killed at various intervals after an intravenous injection of 3H-thymidine to time the acquisition of label by the various phases of mitosis. The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G1 and G2 stages were 6.8 and 1.0 hr, respectively. From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurrence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr. Ten minutes after an intravenous injection of 3H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase. We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G2. Also, nuclei are in telophase during the early half of G1 but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G1 into early and late periods is practical.     

Cell Kinetic Characteristics In Different Parts of Multicellular Spheroids of Human Origin

The growth fraction, the cell cycle time, and the duration of the individual cell cycle phases were determined as a function of distance from the surface of multicellular spheroids of the human cell line NHIK 3025. the techniques employed were percentage of labelled mitoses and labelling index measurements after autoradiography and flow cytometric measurements of DNA histograms. to separate cell populations from the different parts of the spheroid, fractionated trypsinization was employed. The results were compared with corresponding values in NHIK 3025 cell populations grown as monolayer cultures. While practically all cells in exponentially growing monolayer populations are proliferating, the growth fraction was between 0.6 and 0.7 in the outer parts of the spheroid. the inner region was mainly occupied by a necrotic mass. the proliferating fraction of the recognizable cells in the inner region was slightly below 0.5. the mean cell cycle time of NHIK 3025 cells in monolayer culture is 18 hr. the mean cell cycle time of proliferating cells in the periphery of the spheroid was 30 hr, compared to 41 hr in the inner region (150 μm from the spheroid surface). All phases of the cell cycle were prolonged compared to populations of exponentially growing monolayer cells. Within each part of the spheroid the distribution of cell cycle times was considerably broadened compared with monolayer populations.     

The dependence of the cytokinetic effects of alkylating antitumor preparations on the phase of the hepatocyte cell cycle in regenerating liver]

Effects of alkylating antitumor drugs on resting (G0 phase of cell cycle) and proliferating (G1, S, G2 and M phases) hepatocytes were studied in regenerating mouse liver. Cell cycle kinetics (fraction of labeled mitoses, labeling and mitotic indices) were determined by 3H-thymidine autoradiography. Dipin and fotrin as a DNA-damaging agents attack mainly resting (G0) and proliferating (G1) cells. Effect of the damage results in the inhibition of DNA synthesis and G2 phase arrest in the following mitotic cycle. An alkylating drug phopurin as well as ara-C both suppress the mitotic progression in proliferating hepatocytes and do not influence the resting cells.