Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90 degrees and forward scatter measurements

Mitotic cells could be well discriminated from the cells in the G1-, S- and G2-phases of the cell cycle using pulse labeling of S-phase cells with bromodeoxy-uridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2- and M-phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M-phase cells showed lower propidium iodide fluorescence emission compared to G2-phase cells. The fluorescence difference of M- and G2-phase cells was caused by the different thermal denaturation of their DNA. Best separation of M- and G2-phase cells was obtained after 30-50 min heat treatment at 95 degrees C. Mitotic index could be measured if no unlabeled S-phase cells were present in the cell culture. With additional measurements of 90 degree scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2- and S-phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulse-labeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrd-unlabeled S-phase cells.     

BrdUrd/DNA flow cytometry analysis demonstrates cis-diamminedichloroplatinum (II)-induced multiple cell-cycle modifications on human lung carcinoma cells.

The treatment of cultured human cells by cis-diamminedichloroplatinum (II) (cis-DDP) has been shown to induce complex modifications in the cell cycle. Using dual parameter DNA/BrdUrd flow cytometric analysis, we were able to monitor the cell cycle traverse of a pulse-labeled cohort of cells in an asynchronous culture of the A549 cell line (human lung adenocarcinoma). Two major modifications of the cell cycle following cis-DDP treatment were observed: 1) after 24 h of treatment, the labeling index was significantly increased and was linked with a prolonged S-phase; the S-phase delay occurred rapidly after cis-DDP and was dose dependent but not exposure time dependent; 2) an accumulation of cells at the S/G2 transition with an onset approximately 12 h after cis-DDP contact, which was found to be dependent on both dose and duration of exposure. The cytokinetic results also predict maximal sensitivity to cis-DDP for G1 cells and minimal for G2 cells. In our model the late S/G2 accumulation was always preceded by a slowing down of the S-phase. However, only the former should be the correct indicator of cytotoxicity since it was correlated with cell survival as evidenced by a colony formation assay, under all treatment conditions.     

Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions.

An immunofluorescent staining procedure has been developed to identify, with flow cytometry, replicating cells of Saccharomyces cerevisiae after incorporation of bromodeoxyuridine (BrdUrd) into the DNA. Incorporation of BrdUrd is made possible by using yeast strains with a cloned thymidine kinase gene from the herpes simplex virus. An exposure time of 4 min to BrdUrd results in detectable labeling of the DNA. The BrdUrd/DNA double staining procedure has been optimized and the flow cytometry measurements yield histograms comparable to data typically obtained for mammalian cells. On the basis of the accurate assessment of cell fractions in individual cell cycle phases of the asynchronously growing cell population, the average duration of the cell cycle phases has been evaluated. For a population doubling time of 100 min it was found that cells spend in average 41 min in the replicating phase and 24 min in the G2+M cell cycle period. Assuming that mother cells immediately reenter the S phase after cell division, daughter cells spend 65 min in the G1 cell cycle phase. Together with the single cell fluorescence parameters, the forward-angle light scattering intensity (FALS) has been determined as an indicator of cell size. Comparing different temporal positions within the cell cycle, the determined FALS distributions show the lowest variability at the beginning of the S phase. The developed procedure in combination with multiparameter flow cytometry should be useful for studying the kinetics and regulation of the budding yeast cell cycle.     

A rapid and simple estimation of cell cycle parameters by continuous labeling with bromodeoxyuridine

The DNA synthesis time (Ts) and other related cell cycle parameters were roughly estimated in HeLa cells labeled with bromodeoxyuridine (BrdUrd) for various durations by using the flow cytometrical technique. The labeling indices increased in proportion to time after addition of BrdUrd. The Ts can be calculated from the slope of the regression line obtained by plotting the serial labeling indices against the labeling time and was equivalent to the value determined by fraction labeled cells in mid S-phase (FLSm) method. These parameters would be determined by only two samples labeled for different times. This simple method using BrdUrd provides rough but rapid estimation of Ts and other cell cycle parameters without complicated mathematical procedures, in addition to cell cycle partition of cell populations.     

Subdivision of S-phase by analysis of nuclear 5-bromodeoxyuridine staining patterns

After pulse labeling of mammalian cells in vivo or in vitro with 5-bromodeoxyuridine (BrdUrd), followed by immunostaining with a monoclonal antibody to DNA-incorporated BrdUrd, various intranuclear staining patterns are observed. These results are obtained in various labeling, fixation, denaturation, and staining conditions. We defined five different patterns in immunoperoxidase-stained monolayers of human and rodent cancer cells and compared mean nuclear areas as measured by computerized planimetry. Furthermore, we evaluated frequency distributions of these patterns in partly synchronized cell populations and correlated these results with flow cytometric DNA histograms. The results indicate that the observed patterns reflect the spatial and temporal organization of DNA synthesis, which seems to be characterized by at least three successive stages of replication. Evaluation of these patterns may have various applications in studies on cell cycle kinetics.     

Flow cytometric analysis of the kinetic effects of cisplatin on lung cancer cells

The effects of cisplatin on the cell cycle and DNA synthesis of human lung adenocarcinoma cell line PC-9 were examined by flow cytometry. The cellular DNA content and the bromodeoxyuridine (BrdUrd) incorporation rate were measured simultaneously using a monoclonal anti-BrdUrd antibody. Following exposure to cisplatin (1.0 micrograms/ml) for 1 and 24 hr, the bivariate DNA/BrdUrd distributions revealed a delayed S-phase transit and an accumulation of cells in the G2M phase. The BrdUrd-linked green fluorescence intensity continued to decrease with the lapse of time. However, early- and mid-S-phase cells soon recovered DNA synthesis activity, and the former showed higher activity than the control cells. These findings suggested the vigorous DNA synthesis of cells in early S phase. However, for quantitative analysis of chemotherapeutic effects, some problems remained to be resolved regarding the condition for DNA denaturation and its alteration by the agents.     

Lymphoblasts already in the DNA synthesis phase of the cell cycle can be reversibly arrested at the R/G transition

Early and late S-phase of the cell cycle are separated by the R-band/G-band (R/G) transition. This corresponds to the time at which R-band synthesis has been completed while G-band synthesis has yet to begin. The aim of this work was to study cell cycle kinetics during S-phase using different blocking agents: mimosine, methotrexate, 5-fluorouracil, 5-fluoro-2'-deoxyuridine and an excess of thymidine. The stage at which these blocking agents arrest the cell cycle and their efficiency at blocking Epstein-Barr virus transformed lymphoblasts at the R/G transition were evaluated using flow cytometric techniques. Mimosine blocked 90% of the cells near the G1/S-phase boundary. Methotrexate, 5-fluoro-2'-deoxyuridine and 5-fluorouracil, and particularly thymidine, let a significant proportion of cells enter S-phase. The cells were released from the arrest state and their progression through early S-phase was monitored by flow cytometry. Before the cells reached the R/G transition, a second agent was added to inhibit cell cycle progression. For example, the use of mimosine followed by thymidine allowed up to 60% of the cells to be blocked at the R/G transition. The arrest of DNA replication at the R/G transition was confirmed by a marked decrease of 5-bromo-2'-deoxyuridine (BrdUrd) incorporation, revealed by using bivariate flow cytometric analysis. The blocking agent was then removed and the cell cohort was released in the presence of BrdUrd so that replication banding analysis could be performed on the harvested mitotic cells. This yielded a mitotic index of approximately 10% and chromosomes showing replication bands. Flow cytometric analysis combined with cytogenetic banding analysis suggested that the R/G transition is an arrest point within the S-phase of the cell cycle and allowed us to conclude that only cells that have already initiated S-phase are blocked at this point. It corresponds to a susceptible site where S-phase can be arrested easily. The R/G transition could also be a regulatory checkpoint within S-phase, a checkpoint that could respond to imbalance in deoxyribonucleotide pools.     

Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008-32 microM bromodeoxyuridine (Brd-Urd). After dual labeling with monoclonal anti-BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8-32 microM--a 40-fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady-state plasma concentrations in the 8.0 microM range can be expected to provide a reliable estimate of the S-phase fraction.     

Cell cycle analysis using numerical simulation of bivariate DNA/bromodeoxyuridine distributions

This report describes a mathematical model of cell proliferation for simulation of bivariate DNA/bromodeoxyuridine (BrdUrd) distributions. The model formulates the change with time in the frequency of cells with any DNA content and in the amount of incorporated BrdUrd, according to given cytokinetic parameters, i.e., durations and dispersions of cell cycle phases and DNA synthesis rate during S-phase. We have applied this model to sequential DNA/BrdUrd distributions measured for Chinese hamster ovary cells asynchronously grown in vitro, 1) for 30 min in 10 microM BrdUrd followed by growth in BrdUrd-free medium for 0 to 24 h, or 2) during continuous incubation in 3 microM BrdUrd plus 30 microM thymidine for 2 to 24 h. The matches between the experimental and simulated distributions give the G1, S, G2M, and total cell cycle durations (and coefficients of variation) of 5.6 h (0.08), 7.0 h (0.07), 1.4 h (0.16), and 14.0 h (0.05), respectively. The model is shown to be useful for quantitative interpretation of the bivariate distributions.     

Expression of cell cycle related proteins—proliferating cell nuclear antigen (PCNA) and statin—during adaptation and de-adaptation of EUE cells to a hypertonic medium

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.     

Cell kinetic studies in mouse tongue mucosa by autoradiographic, immunohistochemical and flow cytometric techniques

Abstract. A number of techniques, including autoradiography after in vivo administration of tritiated thymidine ([³H]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytometry (FCM) with and without BrdUrd detection were compared in the epithelium of ventral mouse tongue. Investigation of the diurnal proliferative rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phosphatase-anti-alkaline-phosphatase technique, the peroxidase-anti-perox-idase method, and an indirect method with a polyclonal peroxidase-conjugated secondary antibody yielded results similar to standard autoradiography. Preparation of single cell suspensions for flow cytometry was not successful. A maximum yield of about 8.5% of the original cell number was achieved by ultrasound disintegration in combination with trypsin and dithioerythrol treatment, but neither a GdG, peak nor a G₂+ M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensions of nuclei using citric acid and the detergent Tween 20 in combination with magnetic stirring. Both S-phase index and BrdUrd labelling index could be determined by FCM and showed the normal diurnal variations. However, the BrdUrd labelling index in suspensions of nuclei was significantly higher than the labelling index determined after immunohistochemistry. The FCM S-phase index at times of day with low DNA synthesizing activity was higher than the BrdUrd index, indicating a fraction of unlabelled S-phase cells. In conclusion, detection of incorporated BrdUrd in oral mucosa by immunohistochemical techniques or flow cytometry is feasible and provides a useful tool for fast measurements of proliferation.     

Cell cycle analysis using the monoclonal antibody Ki-67

We determined by flow cytometry the proportion of cells in cycle with the monoclonal antibody Ki-67 and also in S-phase after incorporation of bromodeoxyuridine (BrdUrd) with monoclonal antibody to BrdUrd. The monoclonal antibody Ki-67 was useful to detect a nuclear antigen present only in proliferating cells but not expressed in resting (Go) cells. Cell preparation to measure BrdUrd amount incorporated into cellular DNA was difficult but this anti-BrdUrd antibody was useful for measuring the rate of DNA synthesis and for the analysis of precious cell kinetics. These antibodies may provide useful information of cell kinetics.     

G(1)/S but not G(0)/G(1)cell fraction is related to 5-fluorouracil cytotoxicity

BACKGROUND: Bromodeoxyuridine (BrdU) cell cycle analysis using flow cytometry is of clinical interest for making treatment decisions or for predicting response and survival, through proliferation rate (labeling index or S-phase fraction) assessment or T(pot) calculation. Thymidylate synthase expression was tested in vitro, in vivo, and clinically as a prognostic factor for 5-fluorouracil (5FU) sensitivity. However, results were still controversial. Moreover, we had reported that 5FU sensitivity was related to the labeling index of untreated cell cultures. METHODS: We used six human cancer cell lines that exhibited a wide range of 5FU sensitivity. Cell cycle analysis was performed using flow cytometry monovariate propidium iodide (PI) analysis and bivariate distributions of BrdU incorporation versus DNA content. 5FU sensitivity was assayed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay. RESULTS: In all cell lines, 5FU exposure resulted in a statistically significant G(1)/S accumulation. No statistically significant relationship was seen between G(0)/G(1) delay determined by monovariate analysis and 5FU sensitivity. However, 5FU sensitivity was statistically correlated to the labeling index and G(1)/S subpopulation assessed with bivariate analysis using BrdU incorporation versus DNA content. CONCLUSIONS: Cellular proliferation parameters using BrdU incorporation are more informative than PI for in vitro 5FU sensitivity. Because BrdU incorporation could be assessed clinically, it could also be informative for 5FU clinical response prediction.     

Analytical methods for the study of liver cell proliferation.

Various cytometric methods for analysis of regenerating rat liver growth (DNA ploidy distributions, binucleation, and DNA synthesis by in vivo BrdUrd incorporation) were evaluated. The overall hepatocellular growth rate (labeling index), the binucleation rate, and separate indices for mononuclear and binuclear cells could be measured simply by microscope counting of collagenase-isolated hepatocytes immunostained for BrdUrd. Flow cytometry of cells stained for BrdUrd and DNA provided labeling indices for the various hepatocellular DNA ploidy classes as well as for nonparenchymal cells (identified by their size-dependent light scatter), but could not distinguish between mononuclear and binuclear hepatocytes. Image cytometry, using fluorescence or Feulgen staining, was inferior to flow cytometry in terms of speed and DNA resolution, but allowed a complete analysis of all hepatocellular DNA ploidy and nuclearity classes. It may therefore be the method of choice, particularly for analysis of liver cell cultures from which single cells are not easily obtained. Fluorescence staining would seem to be preferable to Feulgen staining, since the latter could not be used simultaneously with BrdUrd staining and therefore required a two-step analysis. A non-immunological method, based on the ability of incorporated BrdUrd to quench DNA staining by a Hoechst dye, could only be applied to isolated nuclei, thus giving no information about binucleation. The latter method may be useful for analysis of tumors which are difficult to dissociate to intact whole cells.     

Centrifugal elutriation of human lymphoid cells: synchronization and separation of aneuploid cells into diploid and tetraploid populations

Both normal and leukemic human lymphoid cell lines were separated into populations corresponding to different positions in the cell cycle by centrifugal elutriation. Each population was analyzed for cell concentration, cell volume, [3H]thymidine incorporation, percentage S phase by autoradiography, and percent G1, S, and G2/M phases by flow cytometry. The smallest cells, collected at the lowest flow rate, were in G1 phase. Cells collected at increasing flow rates progressively increased in volume and represented distinct positions in the cell cycle transition from G1 phase, through S phase, and into G2/M phase. The purity of the G1 population varied according to cell load. One hundred percent of cells were recovered and cells collected in G1- and S-phase populations proliferated in culture with patterns characteristic of synchronized cells. An aneuploidy leukemia cell line, CEM, was separated into near-diploid and near-tetraploid populations by centrifugal elutriation. This method of cell separation provides large numbers of human lymphoid cells at different positions in the cell cycle for investigating the relationship between the cell cycle and various surface membrane and metabolic properties of cells. Aneuploid leukemia and lymphoma cells can be separated by centrifugal elutriation into populations which contain different numbers of chromosomes for comparisons of their biologic properties.     

Gradient fractionation of cycling and resting cells monitored by BrdUrd incorporation

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium Iodide (PI), showed the S compartment highly concentrated in the 1.073/77 g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.     

Evaluation of four methods of DNA distribution data analysis based on bromodeoxyuridine/DNA bivariate data

Four published methods of DNA-content histogram analysis (those of Fried, Dean and Jett, simplified Dean, and Fox) were compared using a double labeling of different cell populations. Partially synchronized and asynchronous cell populations were incubated with bromodeoxyuridine (BrdUrd) and then stained with an anti-BrdUrd monoclonal antibody and propidium iodide (PI). The fractions of cells in the G1, S, and G2 + M phases were calculated by each method and compared with those derived from G1, S, and G2 + M areas plotted on BrdUrd/DNA bivariate histograms, taken as the "true" values. This procedure enabled an optimal choice of method for a given cell population.     

Requirement for phosphatidylinositol-3 kinase activity during progression through S-phase and entry into mitosis

Phosphatidylinositol-3 kinase (PI3K) proteins are important regulators of cell survival and proliferation. PI3K-dependent signalling regulates cell proliferation by promoting G1- to S-phase progression during the cell cycle. However, a definitive role for PI3K at other times during the cell cycle is less clear. In these studies, we provide evidence that PI3K activity is required during DNA synthesis (S-phase) and G2-phase of the cell cycle. Inhibition of PI3K with LY294002 at the onset of S-phase caused a 4- to 5-h delay in progression through G2/M. LY294002 treatment at the end of S-phase caused an approximate 2-h delay in progression through G2/M, indicating that PI3K activity functions for both S- and G2-phase progression. The expression of constitutively activated Akt partially reversed the inhibitory effects of LY294002 on mitotic entry, which demonstrated that Akt was one PI3K target that was required during G2/M transitions. Inhibition of PI3K resulted in enhanced susceptibility of G2/M synchronized cells to undergo apoptosis in response to DNA damage as compared to asynchronous cells. Thus, similar to its role in promoting cell survival and cell cycle transitions from G1 to S phase, PI3K activity appears to promote entry into mitosis and protect against cell death during S- and G2-phase progression.