DETERMINATION OF NON-PROLIFERATING CELLS IN CULTURE BY COMBINING FLOW CYTOMETRY WITH STATHMOKINETICS

Colcemid was added to the growth medium of L-cells in monolayer culture. The proliferating cells continued their progression through the cycle up to metaphase, where they were arrested. At different times after Colcemid addition the cells were trypsinized. suspended immediately in a solution of the DNA-specific fluorescent dye Hoechst 33258 and analysed with a flow cytometer. The histograms were evaluated to give the fraction of cells in the 2c peak as a function of time after Colcemid addition. The flux into the 2c compartment being interrupted, the peak content decreased until all proliferating (G₁) cells had entered S-phase. With increasing cell density or with increasing time after serum deprivation an increasing fraction of cells remained in the 2c peak at times greater than the normal G₁ duration. The possibility of applying this method to the determination of non-proliferating cells in a population is discussed.     

Flow cytometric BrdUrd-pulse-chase study of heat-induced cell-cycle progression delays

The flow cytometric, bromodeoxyuridine (BrdUrd)-pulse-chase method was extended by analysing five kinetic parameters to study perturbed cell progression through the cell cycle. The method was used to analyse the cell-cycle perturbations induced by heat shock. Exponentially growing, asynchronous Chinese hamster ovary (CHO) cells were pulse labelled with BrdUrd and simultaneously heated at 43°C for 5,10 or 15 min. The cells were then incubated in a BrdUrd-free medium and, at various times thereafter, were prepared for flow cytometry. Five compartments (BrdUrd-labelled divided and undivided, and unlabelled G₁, G₁S, and G₂) were defined in the resulting dual-parameter histograms. The fraction of cells and the mean DNA content, when appropriate, were calculated for each compartment. The rates of cell-cycle progression were assessed as time-dependent changes in the fraction of cells in a given compartment and/or the relative DNA content of cells within a given compartment. Linear regression analysis of the data revealed two distinct modes of alteration in cell progression: 1 a delay in cell transit (either out of or into a given compartment), and 2 a decrease in the rate of cell transit. Hyperthermia produced a delay in the exit of cells from the G₁ compartment of ≈ 16 min per minute of heat at 43°C with no threshold. In contrast, the delay in the exit of cells from all other compartments showed a threshold of from 3 to 5 min at 43°C. Above this threshold the delay in exit of cells from the BrdUrd-labelled, undivided compartment was 25 min per minute of heat at 43°C. The more complex dose-response function of this latter compartment may reflect the fact that it includes two cell-cycle phases, S and G₂+ M. The decrease in the rate of transit out of G₂ for cells heated in G₂ was significantly larger than that for any other compartment, consistent with previous studies, which showed a G₂ accumulation following hyperthermia. These results indicate that heat exposure induces very complex alterations in cell-cycle progression and that this flow cytometric method offers a straightforward approach for observing such alterations.     

FLOW CYTOMETRIC CELL CYCLE ANALYSIS USING THE QUENCHING OF 33258 HOECHST FLUORESCENCE BY BROMODEOXYURIDINE INCORPORATION

Cultures of L cells were grown in medium containing 2.0 mg/l bromodeoxyuridine (BUdR) and stained with the fluorescent dye 33258 Hoechst for flow cytometric analysis. During exposure to BUdR, the cells replace thymidine by BUdR in the newly synthesized DNA. The new DNA is not stainable with 33258 Hoechst, which is highly specific for thymidine. The temporal development of the fluorescence distributions after addition of BUdR to the growth medium has been investigated in the flow cytometer, and the data were used to calculate the mean durations of the phases G₁, S and G₂+ M in exponentially growing cultures as well as the cycle transit times in synchronized cultures. The percentage of non-cycling cells was determined in each experiment.     

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)-pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G₂, i.e. the fraction of BrdUrd-negative G₂ cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G₂ and in S are equally delayed. S phase transit of cells irradiated in S or in G₁ did not appear to be affected. There were apparent changes in flow of cells out of G₁, which could be explained by the delayed entry of G₂ cells into the compartment because of G₂ arrest. Thus, in asynchronous cells the method was able to detect G₂ delay in those cells irradiated in S and G₂ phases and demonstrate the absence of cell-cycle delays in other phases.     

A Rapid Automated Stathmokinetic Method For Determination of In Vitro Cell Cycle Transit Times

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G₁. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G₂+ M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.     

KINETIC ANALYSIS OF CELL SIZE AND DNA CONTENT DISTRIBUTIONS DURING TUMOR CELL PROLIFERATION: EHRLICH ASCITES TUMOR STUDY

In order to study the growth dynamics of proliferating and non-proliferating cells utilizing discrete-time state equations, the cell cycle was divided into a finite number of age compartments. In analysing tumor growth, the kinetic parameters associated with a retardation in the growth rate of tumors were characterized by computer simulation in which the simulated results of the growth curve, the growth fraction, and the mean generation time were adjusted to fit the experimental data. The cell age distribution during the period of growth was obtained and by a linear transformation of the state transition matrices, was employed to specify the cell size and DNA content distributions. In an application of the model, the time-course behavior of cell cycle parameters of Ehrlich ascites tumor is illustrated, and the parameters important for the transition of cells in the proliferating compartment to the non-proliferating compartment are discussed, particularly in relation to the G₁-G₀ and G₂-G₀ transitions of non-cycling cells as revealed by the variation of cell size distribution.     

ON THE EXISTENCE OF 'ARRESTED G2 CELLS' IN MOUSE EPIDERMIS

It has been postulated that mouse epidermis contains two populations of resting cells, one of which is blocked at the G₁-S boundary and the other between G₂ and mitosis. the ‘arrested G₂ cells’ were estimated, by the labelled mitosis method, to comprise 510% of the epidermal population and presumed to function as a ‘reserve pool’ which could be activated by wounding. A comprehensive search has now been carried out for arrested G₂ cells in mouse epidermis using the direct methods of single cell and flow through cytophotometry. No evidence was obtained which supports the existence of such a cell compartment. Suitable control experiments were carried out to ensure that G₂ cells were not lost during the isolation of epidermal nuclei.     

Cell cycle analysis of tumour-derived cultures ofCrepis capillaris: A kinetic analogy with proliferation of animal tumour cells

Summary Analysis of the cell cycle by three methods has revealed unusual kinetics of proliferation in tumour derived suspensions ofCrepis capillaris. The different methods of analysis yield different estimates of cycle phase durations, and such discrepancies have been explained in terms of low growth fractions with rapid total cycle traverse. Specifically, confidence in the estimation of G₂ duration by the fraction of labelled mitosis analysis, and comparison with shorter G₂ estimates obtained by the two other methods, suggests that cells drop out in G₁. However, cells which do not drop out of the proliferative compartment traverse G₁ extremely rapidly. Extremely short cell cycle durations in which the G₁ phase is virtually non-existent are uncharacteristic of plant cell suspension cultures, in which the G₁ phase has previously been shown to be extended as compared with meristematic root tip cells. A model has been proposed in which a central core of rapidly dividing cells continuously loses cells into a subpopulation of resting or G₀ cells with the G₁ DNA content. Similarities between plant and animal tumours with respect to cell growth and division are discussed.     

Cell Synchrony Techniques. I. A Comparison of Methods

Abstract Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G₁ S, or G₂+ M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G₁ phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G₁ peak yielded a relatively pure but heterogeneous G₁ population (i.e. early to late G₁). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

THE PROLIFERATION KINETICS OF NHIK 1922 CELLS IN VITRO AND IN SOLID TUMOURS IN ATHYMIC MICE

The proliferation kinetics of cells of the line NHIK 1922 grown in vitro and as solid tumours in the athymic mutant nude mouse has been studied. In vitro, growth curves were determined for exponentially growing populations and for populations synchronized by mitotic selection. The phase durations for these populations were determined by flow cytofluorometric measurements of DNA-histograms and pulsed incorporation of [³H]TdR respectively. The generation time and the phase durations for synchronized populations were found to be about equal to those for exponentially growing populations. The duration of the phases G₁, S and G₂+ M was found to be 8·5–9·5, 11·0–12·0 and 6·0–6·5 hr respectively, i.e. the generation time was 26·5–27·0 hr. The proliferation kinetics in vivo were studied by flow cytofluorometry and by the technique of percentage labelled mitoses. The median duration of S-phase and (G₂+ M)-phase in vivo was found to be approximately the same as that observed in vitro, while the median duration of G₁-phase was found to be approximately 5 hr longer in vivo than under the present in vitro growth conditions. The growth fraction in vivo was estimated to be approximately 50%. The non-proliferative compartment of the tumour cells was found to consist mainly of cells with the DNA-content of cells in G₁-phase. It is concluded that the reduced rate of proliferation of NHIK 1922 cells in vivo is correlated with alterations in the duration of G₁-phase and, hence, the proportion of cells in G₁-phase.     

Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0

The cell cycle is at the center of growth, productivity, and death of mammalian cell cultures. There exists a need to identify and quantify major landmarks in the cell cycle of industrially relevant mammalian cell lines and its association with productivity; central for designing productivity optimization strategies. Herein, we studied the expression of three cyclins, under both perturbed and unperturbed growth, by flow cytometry in batch cultures of GS-NS0. The perturbed systems involved two different DNA synthesis inhibitors, thymidine and dimethyl sulfoxide (DMSO). This approach enables the establishment of characteristic cyclin profiles, timings, and thresholds. In particular, two G₁ class cyclins (D1 and E1), and one G₂ cyclin (B1) were investigated. Cyclin B1 showed a clear cell cycle phase-specific expression increasing during G₂ phase where it was approximately 40% higher when compared to G₁ phase. Similarly, cyclin E1 showed a clear pattern being expressed approximately 10% higher in G₁ compared to G₂ phase and decreased through S phase. Cyclin D1 expression was fairly invariable throughout the cell cycle phases. The observed patterns provide a blueprint of the cell line's cell cycle, which can be used for the development of biologically accurate and experimentally validated distributed cell cycle models.     

A lymphocyte blastogenesis inhibitory factor (LBIF) reversibly arrests a human melanoma cell line, A375, at G1 and G2 phases of cell cycle

A lymphocyte blastogenesis inhibitory factor, LBIF, has been found in the culture supernatant of a human macrophage-like cell line, U937. The factor has been purified by fast protein liquid chromatography. Partial amino acid sequencing analysis showed that LBIF was a novel immunoregulatory factor. Recent study has demonstrated that LBIF possesses a remarkable tumor growth inhibitory activity. In this study, the cell growth inhibitory activity of LBIF was characterized on the proliferation of a human melanoma cell line A375 in vitro. LBIF strongly inhibits the proliferation of A375 cells. The inhibitory activity was cytostatic and reversible by Day 5 although the lethal effect became apparent at Day 7. Cell cycle analysis by flow cytometry showed that LBIF arrested A375 cells at both G₁ and G₂/M phases. Mitotic index analysis indicated that A375 cells were arrested in G₁ and G₂ phases. LBIF function was not attributed to the elevation of intracytoplasmic cyclic-AMP levels. Thus, these results suggest that LBIF plays an important role in controlling cell cycle and there is a similarity between the mechanisms of G₁ and G₂ arrests in eukaryotic cell proliferation. LBIF-induced reversible cell-cycle arrest of A375 cells can be a useful system to analyze the signal transduction for cell proliferation and cell-cycle arrest.     

An RXR-Selective Analog Attenuates the RARα-Selective Analog-Induced Differentiation and Non-G1-Restricted Growth Arrest of NB4 Cells

NB4, a human acute promyelocytic leukemia cell line expressing the promyelocyte–retinoic acid receptor α (PML–RARα) hybrid protein was treated with RAR- and retinoid X receptor (RXR)-selective analogs to determine their effects on cell proliferation, retinoblastoma (RB) tumor-suppressor protein phosphorylation, and differentiation. An RAR- or just RARα-selective analog alone induced similar cell population growth arrest, cell cycle arrest without restriction to G₁, hypophosphorylation of RB, and myelomonocytic cell surface differentiation marker expression (CD11b). In addition, an RARα antagonist could inhibit the effects of the RARα agonist completely. The RARα-selective analog-elicited response was attenuated by simultaneous addition of various RXR-selective analogs. In contrast, each of the RXR-selective analogs was unable to induce any of the cellular responses analyzed. The growth arrest of NB4 cells is not G₁-restricted and occurs at all points in the cell cycle. Cells growth arrested by treatment with an RARα-selective analog show primarily hypophosphorylated RB. When these cells are sorted into G₁or S + G₂/M subpopulations by flow cytometry, hypophosphorylated RB protein was in G₁as well as S + G₂/M cells. This suggests that the hypophosphorylated RB protein may be mediating the growth arrest of NB4 cells at all points in the cell cycle. These results are consistent with an involvement of PML–RARα and/or RARα in the transduction of the retinoid signal in NB4 cells.     

Human Cytomegalovirus UL69 Protein Induces Cells To Accumulate in G1 Phase of the Cell Cycle

Earlier studies have revealed that human cytomegalovirus rapidly inhibits the growth of fibroblasts, blocking cell cycle progression at multiple points, including the G₁-to-S-phase transition. The present study demonstrates that the UL69 protein, a virus-encoded constituent of the virion, is able to arrest cell cycle progression when introduced into uninfected cells. Expression of the UL69 protein causes U2 OS cells and primary human fibroblasts to accumulate within the G₁ compartment of the cell cycle, and serum fails to induce the progression of quiescent human fibroblasts into the S phase when the protein is present. Therefore, the UL69 protein is at least partially responsible for the cell cycle block that is instituted after infection of permissive cells with human cytomegalovirus.     

New distinct compartments in the G2 phase of the cell cycle defined by the levels of γH2AX.

Induction of DNA double strand breaks leads to phosphorylation and focus-formation of H2AX. However, foci of phosphorylated H2AX (γH2AX) appear during DNA replication also in the absence of exogenously applied injury. We measured the amount and the number of foci of γH2AX in different phases of the cell cycle by flow cytometry, sorting and microscopy in 4 malignant B-lymphocyte cell lines. There were no detectable γH2AX and no γH2AX-foci in G₁ cells in exponentially growing cells and cells treated with PARP inhibitor (PARPi) for 24 h to create damage and reduce DNA repair. The amount of γH2AX increased immediately upon S phase entry, and about 10 and 30 γH2AX foci were found in mid-S phase control and PARPi-treated cells, respectively. The γH2AX-labeled damage caused by DNA replication was not fully repaired before entry into G₂. Intriguingly, G₂ cells populated a continuous distribution of γH2AX levels, from cells with a high content of γH2AX and the same number of foci as S phase cells (termed “G₂H” compartment), to cells that there were almost negative and had about 2 foci (termed “G₂L” compartment). EdU-labeling of S phase cells revealed that G₂H was directly populated from S phase, while G₂L was populated from G₂H, but in control cells also directly from S phase. The length of G₂H in particular increased after PARPi treatment, compatible with longer DNA-repair times. Our results show that cells repair replication-induced damage in G₂H, and enter mitosis after a 2–3 h delay in G₂L.     

Sequential flow cytometric analysis of cell-cycle related changes in LFA-1 (CD18/CD11a) expression by Trisomy 21 (Down's syndrome) lymphoblastoid cells*

Abstract Lymphocyte function associated antigen 1 (LFA-1) is a heterodimeric leucocyte adhesion molecule comprising non-covalently associated 95 kD, CD18 and 180 kD, CD11a subunits. Lymphoblastoid cell-lines (LCL) derived from persons with Down's syndrome (Trisomy 21) exhibit increased expression of LFA-1 compared with normal LCL. Although this is probably due to a gene-dosage related increase in the synthesis of CD18, cell-cycle differences between Trisomy 21 (T21) and normal LCL could also influence LFA-1 expression. We have therefore analysed expression of CD18 on G₁ and G₂M cells using sequential flow cytometry. T21 and normal LCL were co-stained with the DNA-binding vital dye HO342 (Hoechst 33342), and with a CD18 monoclonal antibody. The LCL were first sorted on the basis of HO342 staining into G₁ and G₂M populations, and these fractions then analysed for CD18 expression. Irrespective of the stage of the cell-cycle, expression of CD18 was increased on T21 compared with normal LCL. Although more CD18 was detected on both T21 and normal G₂M compared with G₁ cells, the relative density of CD18 in G₂M was less than in G₁ because G₂M cells were larger.     

Cultured Human Tumour Cells May Be Arrested In All Stages of the Cycle During Stationary Phase: Demonstration of Quiescent Cells In G1, S and G2 Phase

Six human colon carcinoma cell lines were induced to enter stationary phase of growth by nutrient deprivation and cell crowding. Growth kinetics parameters (cell number, flow cytometric analysis of DNA distribution, and labelling and mitotic indices) were measured sequentially for all lines during the various stages of in vitro growth. Our results demonstrated that a substantial fraction of cells (9–18%) were located in G₂, phase when they changed from an exponential to a stationary mode of growth. Moreover, a large number of cells in stationary phase of growth had an S-phase DNA content, as determined by flow cytometry, but failed to incorporate radioactive DNA precursors (up to 15-fold difference). to substantiate these findings. cells in stationary phase of growth were induced to enter exponential growth by re-seeding in fresh medium at a lower density. Subsequently observed changes in DNA-compartment distribution, and in labelling and mitotic indices were those expected from cells that had been arrested at different stages of the cycle during their previous stationary phase. Thus, the non-proliferating quiescent state (Q), traditionally located ‘somewhere’ in G₁, phase, appears to be composed also of cells that can be arrested at other stages of the cycle (Q_s, and Q_G). Although the proportion of such cells is rather small, their contribution to the growth kinetics behaviour of human in vivo tumours will become apparent following ‘recruiting’ or ‘synchronizing’ clinical manoeuvres and will prevent the formation of a clear-cut wave of synchronized cells.     

Effects of environmental stresses on the cell cycle of two marine phytoplankton species

Cell cycle phase durations of cultures of Hymenomonas carterae Braarud and Fagerl, a coccolithophore, and Thalassiosira weissflogii Grun., a centric diatom, in temperature-, light- or nitrogen-limited balanced growth were determined using flow cytometry. Suboptimal temperature caused increases in the duration of all phases of the cell cycle (though not equally) in both species, and the increased generation time of nitrogen-limited cells of both species was due almost wholly to expansion of G₁ phase. In H. carterae light limitation caused only G₁ phase to expand, but in T. weissflogii both G₂ + M and G₁ were affected. These results are discussed in relation to cell division phasing patterns of these two species and to models of phytoplankton growth. Simultaneous measurements of protein and DNA on individual cells indicated that under all conditions, the protein content of cells in G₁ was a constant proportion of that of G₂ + M cells. Simultaneous measurements of RNA and protein on each cell indicated that the amounts of these two cell constituents were always tightly correlated. Under conditions of nitrogen limitation both protein and RNA per cell decreased to less than one-third of the levels found in nonlimited cells. This indicates, at least for nitrogen-replete cells, that neither protein nor RNA levels are likely to act as the trigger for cell cycle progression. Strict control by cell size is also unlikely since mean cell volume decreased as growth rates were limited by light and nitrogen supply, but increased with decreasing temperature.     

In vitro DNA synthesis as indicator of mammary epithelial cell division: [14C]thymidine uptake versus flow cytometry cell cycle analysis

Summary Mammary and adipose explants from eight mid-lactation Holstein cows were co-cultured for 24 h in the presence or absence of liver explants, 1 μg/ml pituitary bovine somatotrophin, or 100 ng/ml insulinlike growth factor-I. Liver explants in the media significantly depressed DNA and protein synthesis by mammary tissue as measured by [¹⁴C]-thymidine and amino acid incorporation. As measured by flow cytometry, the concentration of DNA in the G₀G₁ and G₂M cells and the percentage of cells in the G₀G₁ population of mammary tissue was also significantly depressed by liver tissue. Changes in the percentage of cells in the S and G₂M phases were not significant. Insulinlike growth factor-I in the presence of liver explants depressed protein synthesis, thymidine incorporation, and the concentration of DNA in the G₀G₁ and G₂M cells compared to control but did not affect the percentage of cells in the G₀G₁, S, or G₂M phases. Previously it was assumed that changes in [¹⁴C]thymidine incorporation indicated that changes in cell division were occurring. Flow cytometry revealed that changes in DNA content of mammary cells as a result of liver or hormonal stimulation were not due to changes in cell division. Indications are that differences in cellular DNA content result from changes in the rate of amplification of individual genes responsible for milk protein synthesis.