Non-random distribution of abnormal mitoses in heteroploid cell lines.

We have performed absorption-cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa-11 and HL-60 lines, as well as the WCHE-5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase-cell pairs of the heteroploid MCa-11 and HL-60 lines than in those of the chromosomally stable WCHE-5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non-random, unblanced divisions of heteroploid cells with non-modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.     

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.     

Method For Probing Cells In Radiation-Induced G2Arrest: Demonstration of Potentially Lethal Damage Repair

Abstract. Chinese hamster ovary cells were arrested in the G₂ phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G₂, for the investigation of G₂ arrest. Comparisons were made of the number of cells retrieved from G₂ arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. the retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. the repair curves (surviving fraction us time) were each described by a single exponential. G₂ cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.     

Expression of G1 and G2 cyclins measured in individual cells by multiparameter flow cytometry: a new tool in the analysis of the cell cycle

Abstract. Multivariate analysis of the expression of cyclin proteins and DNA content has opened new possibilities for the study of the cell cycle. By virtue of their cell cycle phase specificity, the expression of cyclins may serve, in addition to DNA content, as another marker of a cell's position in the cycle, and provide information about the proliferative potential of cell populations. Several applications of the methodology based on bivariate analysis of DNA content v . expression of B, E and D type cyclins are reviewed: 1 expression of cyclins by individual cells during their progression through the cycle can be studied, using exponentially growing cells without the necessity of cell synchronization or other perturbations of the cycle; 2 cells having the same DNA content but residing in different phases of the cycle (e.g. G₂ diploid v. G₁ tetraploid) can be distinguished; 3 cell transition from G₀ to G₁ and progression through G₁ (e.g. mitogen stimulated lymphocytes) can be assayed; 4 the population of proliferating cells can be distinguished from noncycling cells based on dual cell labelling with a G₁ and G₂ cyclin antibody; 5 cyclin restriction points can serve as additional cell cycle landmarks to map the point of action of antitumour drugs; 6 unscheduled expression of cyclins (e.g. the presence of cyclin B1 during G₁ and S) can be detected in several tumour transformed cell lines, possibly indicating disregulation of the machmery of cell cycle progression. The last finding 6 is of special importance, because such disregulation may be of prognostic consequence in human tumours.     

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells

Abstract. To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96 h exposures to 4 μM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G₁ compartments of the second and third cell cycles of serum stimulated cells. The exit from the G₀/G₁ compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2–3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G₂ phase arrests and delayed G₀/G₁ phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G₁ phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.     

New methods for calculating kinetic properties of cells in vitro using pulse labelling with bromodeoxyuridine

Abstract. The transit times of Chinese hamster ovary cells through the phases of their cell cycle were measured using dual parameter flow cytometry to measure DNA content and the presence of monoclonal antibodies to bromodeoxyuridine. Up to four separate populations can be accurately measured: unlabelled cells in G₂+ M; labelled cells that have not yet divided; labelled cells that have already divided; and the unlabelled cells that were originally in G₁ plus the cells that were originally in G₂+ M and have since divided. The fractions of cells in these populations can be easily followed in time and the usual kinetic properties can be estimated from these fractions, or combinations thereof, including the times through G₁, S, G₂+ M and the cycle time. We present equations for analysing this type of data and comment on which equations are most appropriate for measuring specific kinetic properties of the cells.     

Cell cycle progression in human cells following re-oxygenation after extreme hypoxia: consequences concerning initiation of DNA synthesis

Abstract. The initiation of DNA synthesis and further cell cycle progression in cells during and following exposure to extremely hypoxic conditions in either G₁ or G₂+M has been studied in human NHIK 3025 cells. Populations of cells, synchronized by mitotic selection, were rendered extremely hypoxic (< 4 p.p.m. O₂) for up to 24n h. Cell cycle progression was studied from flow cytometric DNA recordings. No accumulation of DNA was found to take place during extreme hypoxia. Cells initially in G₁ at the onset of treatment did not enter S during up to 24 h exposure to extreme hypoxia, but started DNA synthesis in a highly synchronous manner within 1.5 to 2.25 h after reoxygenation. The duration of S phase was only slightly affected (increased by ≅10%) by the hypoxic treatment. This suggests that the DNA synthesizing machinery either remains intact during hypoxia or is rapidly restored after reoxygenation. Cells initially in G₂ at the onset of hypoxia were able to complete mitosis, but further cell cycle progression was blocked in the subsequent G^ Following reoxygenation, these cells progressed into S phase, but the initiation of DNA synthesis was delayed for a period corresponding to at least the duration of normal G₁ and did not appear in a synchronous manner. In fact, cell cycle variability was found to be increased rather than decreased as a result of exposure to hypoxia starting in G₂. We interpret these findings as an indication that important steps in the preparation for initiation of DNA synthesis take place before mitosis. Furthermore, the change in cell cycle duration induced by hypoxia commencing in G₁ is of a nature other than that induced by hypoxia commencing in other parts of the cell cycle.     

The effects of interleukin 4 on the cell cycle of a human B-cell lymphoma line

Abstract. Exposure of Farage, a human B-cell lymphoma line, to IL-4 for 3–11 days led to inhibition of tritiated thymidine ([³H]dT) uptake by the cells. Study of the incorporation of 5-bromodeoxyuridine by Farage cells showed that IL-4 reduced significantly the number of cells in the S phase of the cell cycle and increased the proportion of cells in the G₁ phase. Limiting dilution analysis of proliferation demonstrated that IL-4 decreased the frequency of clone-forming cells by 40%. IL-4 did not reduce the viability of Farage cells. On the contrary, IL-4 diminished the spontaneous death of Farage cells in culture, as determined by pulse chase analysis of cells which were labelled with [³H]dT. Moreover, the pre-treatment of Farage cells with IL-4 prevented their death induced by exposure to a high dose of staurosporine. IL-4 abrogated the staurosporine-induced arrest of cells in the G₂+ M phase and replaced it by accumulation of cells in the G₁ phase. IL-4 protected Farage cells from the radioactive suicide caused by the uptake of [³H]dT by dividing cells. The cytokine failed to prevent the damage to Farage cells exerted by mitomycin C, which affected cellular DNA regardless of the phase of the cell cycle. The data obtained showed that IL-4 inhibited the division of B cells by arresting their progression through the early stages of the cell cycle. This inhibition of the cell efflux from G₁ phase plays an important role in the protection against cell death during further stages of the cell cycle.     

Unbudded G2 as well as Gj arrest in the stationary phase of the basidiomycetous yeast Cryptococcus neoformans

Abstract Stationary-phase cells of Cryptococcus neoformans displayed two morphological characteristics: virtually all the cells were unbudded even in the early stationary phase and even when grown in rich media, and average cell size increased from that of exponential-phase cells. DNA contents for small and large stationary-phase cells were determined by quantitative fluorescence microscopy after DNA staining with propidium iodide or DAPI. Small cells contained G, DNA, whereas large unbudded cells had either a G₂ or G₁ DNA content, indicating that Cr. neoformans can enter into the stationary phase from either the G₁ or G₂ period.     

ON THE RESTING STAGES OF THE JB-1 ASCITES TUMOUR

Cytophotometric determination of single-cell DNA after repeated ³H-thymidine labelling of the JB-1 ascites tumour in the plateau phase of growth showed a massive accumulation of unlabelled cells with both G₁ and G₂ content. Autoradiography combined with cytophotometry or colcemid block demonstrated that some of these unlabelled cells were rapidly triggered into the cell cycle when plateau tumours were transferred to new hosts. This indicated that tumour cells may be held up in non-cycling stages corresponding to both the G₁ and the G₂ phase of the cell cycle.     

Cytokinetic studies on the switch from glucose to uridine metabolism, and vice versa, of Ehrlich ascites tumour cells in vitro

Abstract. Glucose is normally required as the energy source and for the proliferation of neoplastic cells. For Ehrlich ascites tumour cells, kept under glucose-free culture conditions, this requirement was alleviated by uridine, indicating that the supply of ribose is obligatory for sustaining growth capacity.
In a 96-hr culture experiment with mouse-derived cells, the increase in cell number from cultures supplemented with 5 mM uridine was 50–70%, whilst lactate production was 5% that of controls. An increase in the number of multinucleate cells was observed from cell-smears; DNA histograms indicated the presence of cells with a DNA content higher than 4c and an increased portion of cells in G₂ phase. For precise determination of changes in cell cycle distribution on transfer of cells from glucose-supplemented to glucose-free conditions, the progression of phase-accumulated cells (by centrifugal elutriation) was monitored by DNA distribution analysis; G₂ cells continued the cycle at a rate comparable to controls but were delayed, in the following cycle, predominantly in S and G₂ phases. This was also observed with G₁ cells from a G₁-accumulated fraction in the first cycle.
The addition of glucose to cells kept for some hours in glucose-free, uridine-supplemented medium resulted in an immediate increase in mitotic index (amplification by the colcemid method).
The results are interpreted and support our concept that the delivery of compounds, necessary for normal growth, i.e. hexoses for glycoproteins and glycolipids, are limited as a consequence of the 'metabolic channelling' of pentose from uridine in Ehrlich ascites tumour cells. Therefore, the constantly lowered growth-rate in uridine-supplemented cells observed with long-term culture experiments could reflect an adaption of growth-cycle to these limitations.     

Laser scanning cytometry (LCS) allows detailed analysis of the cell cycle in PI stained human fibroblasts (TIG-7)

We have demonstrated a method for the in situ determination of the cell cycle phases of TIG-7 fibroblasts using a laser scanning cytometer (LSC) which has not only a function equivalent to flow cytometry (FCM) but also has a capability unique in itself. LSC allows a more detailed analysis of the cell cycle in cells stained with propidium iodide (PI) than FCM. With LSC it is possible to discriminate between mitotic cells and G₂ cells, between post-mitotic cells and G₁ cells, and between quiescent cells and cycling cells in a PI fluorescence peak (chromatin condensation) vs. fluorescence value (DNA content) cytogram for cells stained with PI. These were amply confirmed by experiments using colcemid and adriamycin. We were able to identify at least six cell subpopulations for PI stained cells using LSC; namely G₁, S, G₂, M, postmitotic and quiescent cell populations. LSC analysis facilitates the monitoring of effects of drugs on the cell cycle.     

Elucidation of the DNA Synthetic Cycle of Entamoeba Spp. Using Flow Cytometry and Mathematical Modeling

ABSTRACT. We developed a method to study the DNA synthetic cycles of Entamoeba histolytica and Entamoeba invadens by flow cytometry (FCM) based on a preparative procedure to reduce both high levels of natural fluorescence and non-specific adsorption of fluorochromes. We modeled G₁, S, and G₂ phases as a series of overlapping Gaussian curves. Both E. histolytica and E. invadens displayed G₁, S, and G₂ proportions that are consistent with eukaryotic cell populations in exponential or stationary growth phase. Exponential phase E. histolytica populations contained a hypodiploid subset with a mass of about 20% less than the diploid value which we estimate by FCM to be 24 × 10^-14 g DNA/cell. Exponential phase E. invadens populations contained a hypodiploid subset with a mass of about 6% less than the diploid value which we estimate by FCM to be 30 × 10^-14 g DNA/cell.     

Flow cytometric correlation of the c-myc oncoprotein and cell cycle kinetics of HL60 leukaemia during induced maturation with cytosine arabinoside and dimethylsulphoxide

Abstract The c-myc oncogene codes for a DNA binding protein that functions in a cell cycle-related manner. A useful model for studying the relationship of c-myc expression with cell cycle kinetics is the HL60 cell line. HL60 cells constitutively express high levels of c-myc mRNA; however, the level can be down-regulated as the cells are induced to differentiate. We have developed a flow cytometric assay for correlating c-myc oncoprotein levels with DNA content. C-myc oncoprotein levels were additionally correlated with c-myc mRNA levels as determined by slot blot hybridization. Dimethylsulphoxide (DMSO) and cytosine arabinoside were used to induce granulocytic and monocytic maturation respectively. Treatment of HL60 cells with DMSO leads to an increase in the per cent of cells in G₁/G₀ and a decrease in mean c-myc mRNA and oncoprotein levels. The cells with G₁ DNA content show the greatest decrease in c-myc protein. ARA-c treatment of HL60 cells leads to a slowing and an accumulation of cells in S phase with a moderate decrease in mean mRNA and only a slight decrease in mean c-myc protein levels. These data support the hypothesis that c-myc is involved in the switch from G₁ to G₀.     

Cultured human keratinocytes: discrimination of different cell cycle compartments based upon measurement of nuclear RNA or total cellular RNA content

Abstract Correlated measurements of total cellular RNA and DNA of cultured human keratinocytes by flow cytometry, followed by multivariate analysis, discriminate three distinct subpopulations of cells differing in RNA content. The first subpopulation is comprised of small cells resembling basal cells of epidermis, with low RNA content and long (100–300 h) generation times. The second subpopulation consists of keratinocytes resembling cells in the spinous layer of epidermis, characterized by increased RNA content and shorter (35–40 h) generation times. The third subpopulation consists of the largest, keratinohyalin-containing cells which remain in G₁ and undergo terminal differentiation. In contrast to total cellular RNA, correlated measurements of DNA and nuclear RNA reveal that: (1) entrance of all cultured cells from G₁ into S phase occurs only after accumulation of the same, threshold amount of nuclear RNA; hence there is only a single population of S + G₂+ M-phase cells; (2) there are two distinct subpopulations in G₁, one with minimal nuclear RNA content and another with increased RNA. Stathmokinetic experiments indicate that the G₁-phase cells with low nuclear RNA have distinctly longer residence times in G₁ compared to cells with high nuclear RNA content. Thus, measurements of the total cellular RNA versus nuclear RNA content reveal kinetically distinct cell subpopulations. Whereas total cellular RNA content correlates more with differentiation, nuclear RNA content reflects primarily the kinetic properties of the cell.     

CELL CYCLE COMPARTMENT ANALYSIS OF CHINESE HAMSTER CELLS IN STATIONARY PHASE CULTURES

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G₁ and G₂ portions of the cycle; the relative proportion of these cells in G₁ increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G₂ than in G₁. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.     

Monocytic differentiation of HL60 cells induced by potent analogs of vitamin D3 precedes the G1/G0 phase cell cycle block

Abstract. Differentiation of mammalian cells is accompanied by reduced rates of proliferation and an exit from the cell cycle. Human leukemic cells HL60 present a widely used model of neoplastic cell differentiation, and acquire the monocytic phenotype when exposed to analogs of vitamin D₃ (VD₃). The maturation process is accompanied by two blocks in the cell cycle: an arrest in the G₁/G₀ phase, and a recently described G₂+ M block. In this study we have analyzed the traverse of the cell cycle phases of the well-differentiating HL60-G cells exposed to one of ten analogs of VD₃, and compared the cell cycle effects of each compound with its potency as a differentiation-inducing agent. We found that in general there was a good correlation between the effects of these compounds on the cell cycle and on differentiation, but the best cell cycle predictor of differentiation potency was the extent of accumulation of the cells in the G₂ compartment. All analogs induced a marked decrease in the mitotic index, and polynucleation of HL60 cells was produced, especially by compounds which were effective as inducers of differentiation. Time course studies showed that induction of differentiation was accompanied by a transient increase of the proportion of cells in the G₂+ M compartment, but preceded the G₁ to S, and the G₂ compartment blocks. These studies indicate that complex changes in the cell cycle traverse accompany, but do not precede, the acquisition of the monocytic phenotype by HL60 cells.     

Synthesis of protein and mRNA is necessary for transition of suspension-cultured Catharanthus roseus cells from the G1 to the S phase of the cell cycle

The effects of inhibition of the synthesis of protein, mRNA or rRNA on the progression of the cell cycle have been analyzed in cultures of Catharanthus roseus in which cells were induced to divide in synchrony by the double phosphate starvation method. The partial inhibition of protein synthesis at the G₁ phase by anisoniycio or cycloheximide caused the arrest of cells in the G₁ phase or delayed the entry of cells into the S phase. When protein synthesis was partially inhibited at the S phase, cell division occurred to about the same extent as in the control. When asynchronously dividing cells were treated with cycloheximide, cells accumulated in the G₁ phase, as shown by flow-cytometric analysis. The partial inhibition of mRNA synthesis by α-amanitin at the G₁ phase caused the arrest of cells in the G₁ phase, although partial inhibition of mRNA synthesis at the S phase had little effect on cell division. In the case of inhibition of synthesis of rRNA by actinomycin D at the G₁ phase, initiation of DNA synthesis was observed, but no subsequent DNA synthesis or the division of cells occurred. However, the addition of actinomycin D during the S phase had no effect on cell division. These results suggest that specific protein(s), required for the progression of the cell cycle, are synthesized in the G₁ phase, and that the mRNA(s) that encode these proteins are also synthesized at the G₁ phase.     

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry

Abstract. Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming asynchronously growing cell populations and (i) measurement coefficients of variation ( CV ) from 2 to 16%; (ii) constant measurement CV or CV 's increasing from G₁ to G₂ phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time.
The fractions of cells in the G₁, S, and (G₂+ M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10–20%. However, most methods tended to overestimate G₁ fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental The errors were only slightly larger than for histograms from asynchronously growing cell populations.