Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G2 phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6-12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine [( 3H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [3H]TdR labelling was simulated. The presence of two distinct subpopulations in G2 phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G1 phase is suggested. The sizes of the slowly cycling fractions in G1 and G2 showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3-5%) was arrested in G2 phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G2 cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

Effects of Pulse Labelling With Tritiated Thymidine On the Circadian Rhythmicity In Epidermal Cell-Cycle Distribution In Mice

The influence of pulse labelling with 50 °Ci tritiated thymidine ([³H]TdR) (2 μCi/g) on epidermal cell-cycle distribution in mice was investigated. Animals were injected intraperitoneally with the radioactive tracer or with saline at 08.00 hours, and groups of animals were sacrificed at intervals during the following 32 hr. Epidermal basal cells were isolated from the back skin of the animals and prepared for DNA flow cytometry, and the proportions of cells in the S and G₂ phases of the cell cycle were estimated from the obtained DNA frequency distributions. the proportions of mitoses among basal cells were determined in histological sections from the same animals, as were the numbers of [³H]TdR-labelled cells per microscopic field by means of autoradiography. The results showed that the [³H]TdR activity did not affect the pattern of circadian rhythms in the proportions of cells in S, G₂ and M phase during the first 32 hr after the injection. the number of labelled cells per vision field was approximately doubled between 8 and 12 hr after tracer injection, indicating an unperturbed cell-cycle progression of the labelled cohort. In agreement with previous reports, an increase in the mitotic index was seen during the first 2 hr. These data are in agreement with the assumption that 50 °Ci [³H]TdR given as a pulse does not perturb cell-cycle progression in mouse epidermis in a way that invalidates percentage labelled mitosis (PLM) and double-labelling experiments.     

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

Evidence has been presented supporting the existence of heterogeneity in cell-cycle progression in mouse epidermis, the present study was undertaken to characterize this heterogeneity in more detail. Hairless mice were continuously labelled with tritiated thymidine every 4 hr for 4 days. Basal cell suspensions were prepared from slices of mouse skin at intervals during the experiment and subjected to DNA flow cytometry. Cell-cycle analysis was combined with sorting of cells from windows in G¹, S and G², phase, and the proportion of labelled cells within each window was determined in autoradiographs. Reanalysis and resorting to control the purity of sorted fractions were performed. Computer simulations of the data were made using a mathematical model assuming different S and G² phase characteristics. A good fit to the data was only obtained when heterogeneity in mouse epidermal cell-cycle progression was assumed, indicating the existence of slowly traversing, distinct subpopulations of cells in G² and S phase. These cells are assumed to contribute to about 40% of all cells in S phase and to about 70% of all in G² phase. the estimated residence times in the resting states were 38 and 32 hr in S and G² phase, respectively. Two-parameter sorting based on DNA and light scatter indicated that slowly cycling cells were larger than the average. There is no evidence of significant subpopulations of permanently non-proliferating keratinocytes in any of the cell-cycle phases.     

Kinetic Differences Between Fed and Starved Chinese Hamster Ovary Cells

When Chinese hamster (CHO-K1) cells are grown as monolayer cultures, they eventually reach a population-density plateau after which no net increase in cell numbers occurs. the kinetics of aged cells in nutritionally deprived (starved) or density-inhibited (fed) late plateau-phase cultures were studied by four methods: (i) Reproductive integrity and cell viability were monitored daily by clonogenic-cell assay and erythrosin-b dye-exclusion techniques. (ii) Mitotic frequencies of cells from 18 day old cultures were determined during regrowth by analysing time-lapse video microscope records of dividing cells. (iii) Tritiated-thymidine ([³H]TdR) auto-radiography was used to determine the fractions of DNA-synthesizing cells in cultures entering plateau phase and during regrowth after harvest. (iv) the rate of labelled nucleoside uptake and incorporation into DNA was measured using liquid scintillation or sodium iodide crystal counters after labelling with [³H]TdR or [¹²⁵]UdR. Non-cycling cells in starved cultures accumulate primarily as G₁, phase cells. Most cells not in G₁ phase had stopped in G₂, phase. Very few cells (< 2%) were found in S phase. In contrast, about half of the cells in periodically fed cultures were found to be in DNA-synthetic phase, and the percentage of these S phase cells fluctuated in a manner reflecting the frequency of medium replacement. Populations of both types of plateau-phase cultures demonstrate extremely coherent cyclic patterns of DNA synthesis upon harvest and reculturing. They retain this high degree of synchrony for more than three generations after the resumption of growth. From these data it is concluded that nutritionally deprived (starved) late plateau-phase cells generally stop in either G₁, or G₂, phase, whereas periodically fed late plateau-phase cultures contain a very large fraction of cycling cells. Populations of cells from these two types of non-expanding cultures are kinetically dissimilar, and should not be expected to respond to extracellular stimuli in the same manner.     

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING APPLICATION OF A SKIN IRRITANT (CANTHARIDIN)

The strong skin irritant cantharidin dissolved in benzene was applied to the back of hairless mice. Single cell suspensions of epidermal basal cells were obtained and flow microfluorometric measurements of cellular DNA content were made. Smears were made for autoradiography, and the [³H]TdR labelling index (LI) and mean grain count (MGC) were assessed up to 3 days after cantharidin application. Three successive peaks of cells with S phase DNA content accompanied by three LI peaks were observed. The first two peaks were follwed by peaks of cells in G₂ phase, indicating that after the acute cell injury caused by cantharidin the cells traversed the cell cycle in partial synchrony through two subsequent cell cycles, each of 10–12 hr duration. During this phase of rapid proliferation the LI reached the proportion of cells in S phase, contrary to what is observed in untreated mouse epidermis, where the labelled cells contribute to about half the proportion of cells with S phase DNA content. The first two peaks of cells in S phase and LI coincided with an increased MGC, whereas the third peak was accompanied by a MGC significantly below control values. This indicates that this latter peak is due to a longer DNA synthesis time rather than to a partially synchronized and increased cell proliferation. The duration of the G₁, S and G₂ phases seems to be reduced initially in rapidly proliferating epidermis.     

S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine

A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro*

Abstract. Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [³H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G₀/G₁, S, and G₂/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [³H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [³H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G₂ tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

Abstract. The present experiments with [¹⁴C]-thymidine (TdR) and [³H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight^-1 and that of BrdU is about 5·0 µg g body weight^-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [³H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [³H]-TdR.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

The present experiments with [14C]-thymidine (TdR) and [3H]-bromodeoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 microgram g body weight-1 and that of BrdU is about 5.0 micrograms g body weight-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [3H]-TdR doses commonly used in cell kinetic studies this proportion is only 0.1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [3H]-TdR.     

Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry.

Hairless mice were injected intraperitoneally with bromodeoxyuridine (Brd-Urd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate-buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cycle progression of a pulse-labelled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H-thymidine labelling studies. The percentage of cells in S phase was highest at night and lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase. Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation-keratin K10 was turned on only in G1 phase and after the last division.     

CIRCADIAN RHYTHMS IN MOUSE EPIDERMAL BASAL CELL PROLIFERATION

Several kinetic parameters of basal cell proliferation in hairless mouse epidermis were studied, and all parameters clearly showed circadian fluctuations during two successive 24 hr periods. Mitotic indices and the mitotic rate were studied in histological sections; the proportions of cells with S and G₂ phase DNA content were measured by flow cytometry of isolated basal cells, and the [³H]TdR labelling indices and grain densities were determined by autoradiography in smears from basal cell suspensions. The influx and efflux of cells from each cell cycle phase were calculated from sinusoidal curves adapted to the cell kinetic findings and the phase durations were determined. A peak of cells in S phase was observed around midnight, and a cohort of partially synchronized cells passed from the S phase to the G₂ phase and traversed the G₂ phase and mitosis in the early morning. The fluctuations in the influx of cells into the S phase were small compared with the variations in efflux from the S phase and the flux through the subsequent cell cycle phases. The resulting delay in cell cycle traverse through S phase before midnight could well account for the accumulation of cells in S phase and, therefore, also the subsequent partial synchrony of cell cycle traverse through the G₂ phase and mitosis. Circadian variations in the duration of the S phase, the G₂ phase and mitosis were clearly demonstrated.     

Unbalanced growth and cell death in HeLa S3 cultures treated with DNA synthesis inhibitors

Flow cytometry indicated that significant amounts of dsRNA were accumulated in HeLa S3 cells blocked at or near G₁/S boundary by hydroxyurea (HU) or excess thymidine (TdR). The dsRNA/DNA ratio increased in these cells in a manner characteristic of unbalanced cell growth. In HU-treated cells, dsRNA content was maximal 16 hours after addition of the drug and did not change significantly during the next 24 hours. The DNA content in blocked cells increased by 10%. Cell viability assessed by colony formation in soft agar decreased exponentially in HU-treated cultures after 16 hours of incubation. Correlation between loss of cell viability and rate of cell proliferation after removal of HU was observed, as determined by cell count and analysis of cell cycle progression. In TdR-treated cultures cells slowly progressed into mid S-phase during 40 hours and dsRNA accumulation continued during this period. Cell viability was not significantly affected by treatment with excess TdR, indicating that unbalanced growth per se, as measured by dsRNA accumulation, is not lethal for the cells. After reversal of DNA synthesis inhibition by removal of the drug, cells treated with HU for 16 hours or TdR for 16–24 hours promptly progressed through the cell cycle. This progression was accompanied by accumulation of significant amounts of dsRNA. As a result, cells in G₂ phase had a very high dsRNA content leading to retention of the unbalanced condition (increased dsRNA/DNA ratio) in the daughter cells. It is suggested that dsRNA accumulation in the cell is controlled to a certain degree by cell progression through the S phase. This type of control, evidently, was reflected in limited dsRNA accumulation in the cells blocked at or near G₁/S border, in continuous dsRNA accumulation in the cells slowly progressing through S phase, and in accumulation of large amounts of dsRNA after renewal of progression through the S phase.     

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

Percentage labelled mitosis (PLM) measurements were initiated at four different times during a 24-hr period and continued for 24 hr in hairless mouse epidermis. Estimates of G₂ and S phase durations (mean T_G2 and mean T_S) were calculated. A significant number of labelled mitoses (10–20%) was seen after 30 min in all four PLM measurements and the estimated mean T_G2 varied from 1.4 to 2.5 hr and was in agreement with values from PLM measurements in other epithelial tissues. These mean T_G2 values were much shorter than expected from [³H]TdR double labelling experiments and from a multiparameter cell kinetic study in hairless mouse epidermis and did not reflect the circadian variations seen in these studies. the differences in estimates of phase durations can be explained by postulating two G₂ cell populations; one with a rapid and another with a slow rate of cell cycle progression. the cells with the higher rate are mainly registered by the PLM method, whereas those with the lower rate largely escape detection by this method. T_G2 estimates from PLM measurements in mouse epidermis therefore do not reflect the phase duration of the entire G₂ population. It is also concluded that circadian variations in T_S can not be accurately registered by the PLM method.     

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

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

Comparative analysis of different approaches to investigate cell kinetics

The potential of different methods to investigate proliferative activity of cell populations was analysed for non-Hodgkin's lymphomas. Cells in S phase and all cycling cells were determined on cell suspensions obtained from fresh lymph node material by [3H]-thymidine autoradiography [( 3H]TdR LI), a monoclonal antibody to bromodeoxyuridine (BrdU LI), and the monoclonal antibody Ki67. A good correlation was observed between the values of [3H]TdR LI and BrdU LI (rs = 0.90; P less than 0.01), [3H]TdR LI and S phase (rs = 0.62; P less than 0.01) and [3H]TdR LI and Ki67 (rs = 0.64; P less than 0.01) in individual lymphomas. Using the median values obtained from the different approaches as cut-off points to define slowly and rapidly proliferating tumours, the best agreement was observed between [3H]TdR LI and BrdU LI (91%) and poorer agreements, even though statistically significant, were observed between [3H]TdR LI and S phase (73%) or Ki67 (76%). In conclusion, the kinetic information derived from different approaches was more or less concordant and newly proposed approaches should be directly and carefully verified for their prognostic relevance before using them as alternatives to conventional methods.     

Occurrence of two cell subpopulations with different cell-cycle durations in the central and peripheral zones of the vegetative shoot apex of Sinapis alba L.

The cell-cycle duration and the growth fraction were estimated in the vegetative shoot apical meristem of Sinapis alba L. The length of the cell cycle was about 86 h, i.e. 2.5 times shorter than the cell-doubling time (M. Bodson, 1975, Ann. Bot. 39, 547–554) and the growth fraction was between 32 to 41%. These data demonstrated that the cell population of this meristem was heterogeneous, including one subpopulation of rapidly cycling cells and one subpopulation of non-cycling cells, i.e. cells with a very long cell cycle compared with that of the rapidly cycling cells. Non-cycling cells had no particular localization within the meristem. Both the central and peripheral zones of the meristem were mosaics of rapidly cycling and non-cycling cells.Abbreviations G₁ pre-DNA-synthesis phase - G₂ post-DNA-synthesis phase - GF growth fraction - M mitosis phase - PLM pulse-labelled-mitoses method - S DNA-synthesis phase - T cell-cycle duration - TdR thymidine     

Retention of labelled DNA precursor by murine cells after a single administration of tritium labelled thymidine

The central zone of the rat lens epithelium, extending half way from the centre to the periphery of a whole mount preparation, normally has less than 1% of the cells in the cell cycle at any given time. Mechanical wounding initiates a burst of proliferation in the central zone. DNA synthesis begins 14 hr after wounding followed by mitosis 10 hr later. When [3H]TdR was applied at 2 hr prior to S phase, some moderately heavy and some light labelling was observed after the onset of S phase. When [3H]TdR was applied 5 hr before S phase (9 hr after wounding), all the cells were lightly labelled. Only small amounts of the label were available to these cells 5 hr after application. It is significant that there was labelling in this group because it indicates the persistence of relatively small intracellular pools of [3H]TdR for several hours after the initial 'pulse' labelling of cells. Determinations of the duration of S phase were based on the assumption that pulse labelling may be affected by the persistence of the pools of [3H]TdR and consequent light labelling of the cells.     

Sedimentation velocity characterisation of the cell cycle of granulocytic progenitor cells in monkey hemopoietic tissue

Sedimentation velocity separation of Rhesus monkey bone marrow cells has demonstrated a reproducible but heterogeneous size distribution of cells capable of forming granulocytic colonies in agar culture (CFC's). This heterogeneity is shown to be due to the cell cycle status of the progenitor cell population. In vitro exposure of bone marrow cells to lethal doses of tritiated thymidine (H³TdR) either before or after separation restricts the size distribution of CFC's, greatly reducing the proportion of rapidly sedimenting cells. The calculation of the volume distribution of such cells before and after H³TdR exposure indicates that 55% of total CFC's in adult marrow are in G₀ or G₁ with a volume of 410 μ³, 42% are in S phase and of volume 450–950 μ³, and the remainder are in G₂ and mitosis with a volume of between 600–950 μ³. CFC's in mid gestation fetal liver were larger than their adult counterparts and were of homogeneous volume indicative of a single non cycling population with no evidence of an S or G₂ component. H³TdR exposure confirmed the non-cycling status of these fetal progenitor cells.