Analysis of regenerating hepatic cell replication in vivo by differential chromatid staining

Abstract. The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.     

14C-Methylated beta-casein as a substrate for plasmin, and its application to the study of milk protein transformations

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about 1/6 the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G1 and early S phases had the lowest membrane fluidity.     

Interdependence of the cell cycles of mammalian sister cells in monolayer cultures detected through their desynchronization by UV microirradiation

The duration and variability of cell cycles in epithelial and fibroblast-like mammalian sister cells with different types of intercellular contacts were estimated using time-lapse cinemicrographic technique. To study a possible interrelation between cell cycles of the sister cells, one cell in each pair of sister cells was inactivated by selective UV microbeam irradiation at the beginning of its cell cycle. It is shown that this action may delay the cycle of the intact cell as well. Such an interrelation of sister cells was found only at the G1 phase of the cell cycle and only in epithelial cells.     

Electron spin resonance studies of changes in membrane fluidity of Chinese hamster ovary cells during the cell cycle

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about $\text{1}\text{6}$ the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G₁ and early S phases had the lowest membrane fluidity.     

Quantitative determination of the induction of apoptosis in a murine B cell line using flow cytometric bivariate cell cycle analysis.

WEHI-231 cells have been used extensively as a model of tolerance induction in B cells. Recent evidence has shown that anti-IgM treatment of WEHI-231 cells resulted in the induction of apoptosis. In this study, using acridine orange staining and flow cytometric analysis, we demonstrated that apoptotic cells are detected as a distinct population of cells separate from the cells in normal cell cycle progression. The validity of analysis gates was confirmed by cell sorting of the apoptotic population versus normal cells and subsequent gel analysis. Using this technique, we have demonstrated that F(ab')2 anti-mu, A23187, or PMA induced apoptosis in the WEHI-231 cells. The addition of LPS reversed apoptotic induction as seen previously with the WEHI-231 cell line. In contrast, however, PMA did not prevent the induction of apoptosis in anti-mu-treated cells. Additionally, we were interested in determining if the induction of apoptosis was restricted to a specific phase of cell cycle. Since growth inhibition results in most cells arresting in the G1 phase of cell cycle, we wanted to demonstrate apoptosis as a G1-dependent event. This was examined with WEHI-231 cells treated with known cell cycle inhibitors. Interestingly, inhibition of cells in each phase of cycle resulted in the induction of apoptosis. LPS was able to inhibit the induction of apoptosis with each of the cell cycle inhibitors except actinomycin D. Furthermore, we have demonstrated that the WEHI-231 cells contain a Ca(2+)-Mg(2+)-dependent preexisting endonuclease.     

Characterization of a CV-1 cell cycle. V. An assessment of velocity sedimentation for cell separation and synchrony.

Velocity sedimentation at unit gravity has been used to enrich populations of logarithmically growing cells in different cell cycle phases. In order to evaluate the degree of synchrony obtained by this method of cell separation, synchronous populations of CV-1 cells, initially obtained by the selective detachment of mitotic cells from roller cultures, were separated by velocity sedimentation. It was found that although the mean cell volume increased linearly, the cells remained heterogeneous with respect to size during all phases of the cell cycle. Since the velocity sedimentation technique depends upon discrimination of cell size, the size heterogeneity of cells throughout the cycle limits the degree of synchrony which can be obtained by this method.     

High resolution analysis of cell cycle-correlated vimentin expression in asynchronously grown,TPA-treated MPC-11 cells by the novel flow cytometric multiparameter BrdU-hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley-Liss, Inc.     

Roles of volume-activated Cl- currents and regulatory volume decrease in the cell cycle and proliferation in nasopharyngeal carcinoma cells

OBJECTIVES: Previously it has been shown, that the volume-activated plasma membrane chloride channel is associated with regulatory volume decrease (RVD) of cells and may play an important role in control of cell proliferation. We have demonstrated that both expression of the channel and RVD capacity are actively regulated in the cell cycle. In this study, we aimed to further study the role of the volume-activated chloride current and RVD in cell cycle progression and overall in cell proliferation. MATERIALS AND METHODS: Whole-cell currents, RVD, cell cycle distribution, cell proliferation and cell viability were measured or detected with the patch-clamp technique, the cell image analysis technique, flow cytometry, the MTT assay and the trypan blue assay respectively, in nasopharyngeal carcinoma cells (CNE-2Z cells). RESULTS: The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and tamoxifen, inhibit the volume-activated chloride current, RVD and proliferation of CNE-2Z cells in a dose-dependent manner. Analysis of relationships between the current, RVD and cell proliferation showed that both the current and RVD were positively correlated with cell proliferation. NPPB (100 microM) and tamoxifen (20 microM) did not significantly induce cell death, but inhibited cell proliferation, implying that the blockers may inhibit cell proliferation by affecting cell cycle progression. This was verified by the observation that tamoxifen (20 microM) and NPPB (100 microM) inhibited cell cycle progress and arrested cells at the G0/G1 phase boundary. CONCLUSIONS: Activity of the volume-activated chloride channel is one of the important factors that regulate the passage of cells through the G1 restriction point and that the Cl- current associated with RVD plays an important role in cell proliferation.     

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

Isolation by a replica-plating technique of chinese hamster temperature-sensitive cell cycle mutants

Isolation of a wide variety of temperature-sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild-type cells with agents that kill DNA-synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures, We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl-methane-sulfonate (EMS) and used a replicaplating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all ts mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.     

Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle

Flow cytometric multiparameter analysis of two proliferation-associated nuclear antigens (proliferating cell nuclear antigen (PCNA)/cyclin and Ki-67) was performed on seven human hematopoietic cell lines. PCNA/cyclin, an S phase-related antigen, was detected using an autoantibody and a fluorescein isothiocyanate-labeled anti-human antibody. The Ki-67 antigen, which in cycling cells is expressed with increasing levels during the S phase with a maximum in the M phase, was detected using a monoclonal antibody and a phycoerythrin-conjugated anti-mouse antibody. In some experiments the PCNA/Ki-67 staining was combined with a DNA stain, 7-amino actinomycin D, and simultaneous detection of the three stains was performed by a single laser flow cytometer. Using this technique four distinct cell populations, representing G1, S, G2, and M, respectively, could be demonstrated in cycling cells on the basis of their PCNA/cyclin and Ki-67 levels. The cell cycle phase specificity could be verified using metaphase (vinblastine, colcemide) and G2 phase (mitoxantrone) blocking agents, as well as by stainings with a mitosis-specific antibody (MPM-2). Also, G0 cells could be discriminated from G1 cells in analysis of a mixture of resting peripheral mononuclear blood cells and a proliferating cell line. This technique can be valuable in detailed cell cycle analysis, since all cell cycle phases can be visualized and calculated using a simple double staining procedure.     

Accelerated entry of aortic smooth muscle cells from spontaneously hypertensive rats into the S phase of the cell cycle.

The present study was designed to characterize the growth kinetics of the exaggerated proliferative response to mitogens of vascular smooth muscle cells from spontaneously hypertensive rats compared with cells from normotensive Wistar-Kyoto controls. Cellular DNA content, analyzed by flow cytometry, demonstrated a 4-h accelerated entry into the S phase of the cell cycle of vascular smooth muscle cells from spontaneously hypertensive rats; the significant (4.5-fold) increase in the percentage of cells in the S phase occurred between 8 and 12 h after calf serum stimulation. A 3.9-fold increase of cells in the S phase was seen in the normotensive controls only between 12 and 16 h. Transit through the cell cycle was quantitated by flow cytometry using the Hoechst 33,342--bromodeoxyuridine substitution technique. Vascular smooth muscle cells from spontaneously hypertensive rats went through the cell cycle 4 h ahead of cells from normotensive Wistar-Kyoto rats. This accelerated transit of spontaneously hypertensive rat cells was mostly due to an earlier entry into the S phase. Persistence of this new intermediate phenotype in cell culture suggests its primary pathogenetic role in spontaneous hypertension.     

Correlating cell cycle with metabolism in single cells: combination of image and metabolic cytometry.

BACKGROUND: We coin two terms: First, chemical cytometry describes the use of high-sensitivity chemical analysis techniques to study single cells. Second, metabolic cytometry is a form of chemical cytometry that monitors a cascade of biosynthetic and biodegradation products generated in a single cell. In this paper, we describe the combination of metabolic cytometry with image cytometry to correlate oligosaccharide metabolic activity with cell cycle. We use this technique to measure DNA ploidy, the uptake of a fluorescent disaccharide, and the amount of metabolic products in a single cell. METHODS: A colon adenocarcinoma cell line (HT29) was incubated with a fluorescent disaccharide, which was taken up by the cells and converted into a series of biosynthetic and biodegradation products. The cells were also treated with YOYO-3 and Hoechst 33342. The YOYO-3 signal was used as a live-dead assay, while the Hoechst 33342 signal was used to estimate the ploidy of live cells by fluorescence image cytometry. After ploidy analysis, a cell was injected into a fused-silica capillary, where the cell was lysed. Fluorescent metabolic products were then separated by capillary electrophoresis and detected by laser-induced fluorescence. RESULTS: Substrate uptake measured with metabolic cytometry gave rise to results similar to those measured by use of laser scanning confocal microscopy. The DNA ploidy histogram obtained with our simple image cytometry technique was similar to that obtained using flow cytometry. The cells in the G(1) phase did not show any biosynthetic activity in respect to the substrate. Several groups of cells with unique biosynthetic patterns were distinguished within G(2)/M cells. CONCLUSIONS: This is the first report that combined metabolic and image cytometry to correlate formation of metabolic products with cell cycle. A complete enzymatic cascade is monitored on a cell-by-cell basis and correlated with cell cycle.     

Analysis of cell cycle phases and progression in cultured mammalian cells

Fluorescence activated cell sorting (FACS) analysis has become a standard tool to analyze cell cycle distributions in populations of cells. These methods require relatively large numbers of cells, and do not provide optimal resolution of the transitions between cell cycle phases. In this report we describe in detail complementary methods that utilize the incorporation of nucleotide analogs combined with microscopic examination. While often more time consuming, these protocols typically require far fewer cells, and allow accurate kinetic assessment of cell cycle progression. We also describe the use of a technique for the synchronization of adherent cells in mitosis by simple mechanical agitation (mitotic shake-off) that eliminates physiological perturbation associated with drug treatments.     

Asynchronous replication of p53 and 21q22 loci in chronic lymphocytic leukemia

In this study, in order to evaluate the replication pattern and the cell cycle dynamics of normal and malignant cells from patients with chronic lymphocytic leukemia, we applied the FISH technique with the p53 gene. Asynchrony was determined by the presence of one single and one set of double dots in the same cell. The rate of asynchronous replication was significantly higher in malignant cells than in normal cells (a mean of 28 vs 13, respectively, P = 0.023). There were proportionately more cells with two single dots among the normal cells (P = 0.0047). These results probably reflect the changes in gene replication and cell cycle progression that occur in malignant cells. Received: 25 March 1997 / Accepted: 28 July 1997     

Significance of the cell cycle in commitment of murine erythroleukemia cells to erythroid differentiation.

The relationship between differentiation of murine erythroleukemia cells (MEL) induced by DMSO and the cell division cycle has been analyzed. We demonstrate that incubation in the presence of DMSO increases the length of the G1 phase of the cell cycle. A method of synchronization of MEL cells by unit gravity sedimentation has been developed and characterized. Using this method, a series of synchronized cell populations covering the entire cell division cycle can be generated simultaneously. Cells synchronized by this technique were challenged with DMSO and analyzed for kinetics of commitment to the differentiation program. Our results indicate that populations of cells in G1 or G2 at the time of addition of inducer give rise to a greater proportion of committed cells than an unfractionated population, while cells in S phase result in a lower percentage of committed cells than the unfractionated population when cultured in DMSO.     

New insights into the cell cycle profile of Paracoccidioides brasiliensis

The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus' multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus' unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature.     

Cutting edge: B7/CD28 interactions regulate cell cycle progression independent of the strength of TCR signaling

The role of B7/CD28 signals in Ag-induced cell cycle progression of CD4(+) T cells was examined using the technique of CFSE dye dilution and flow cytometry. In wild-type T cells, proliferation was directly related to the concentration of Ag available to the APC. Consistent with this, the rate of G(0)-->G(1) cell cycle progression varied with the concentration of Ag. However, cell division by T cell blasts occurred at a constant rate, independent of Ag concentration. G(0)-->G(1) phase progression by CD28-deficient CD4(+) T cells or wild-type T cells cultured in the presence of neutralizing anti-B7 mAbs was slowed, confirming that a synergy does exist between TCR and CD28 signaling in the initial activation of the T cells. However, unlike the TCR, the strength of CD28 stimulation was also shown to play a unique role in controlling the rate of cell division by T cell blasts.     

Regulatory volume decrease is actively modulated during the cell cycle

Nasopharyngeal carcinoma cells, CNE-2Z, when swollen by 47% hypotonic solution, exhibited a regulatory volume decrease (RVD). The RVD was inhibited by extracellular applications of the chloride channel blockers tamoxifen (30 microM; 61% inhibition), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM; 60% inhibition), and ATP (10 mM; 91% inhibition). The level and time constant of RVD varied greatly between cells. Most cells conducted an incomplete RVD, but a few had the ability to recover their volume completely. There was no obvious correlation between cell volume and RVD capacity. Flow cytometric analysis showed that highly synchronous cells were obtained by the mitotic shake-off technique and that the cells progressed through the cell cycle synchronously when incubated in culture medium. Combined application of DNA synthesis inhibitors, thymidine and hydroxyurea arrested cells at the G1/S boundary and 87% of the cells reached S phase 4 h after being released. RVD capacity changed significantly during the cell cycle progression in cells synchronized by shake-off technique. RVD capacity being at its highest in G1 phase and lowest in S phase. The RVD capacity in G1 (shake-off cells sampled after 4 h of incubation), S (obtained by chemical arrest), and M cells (selected under microscope) was 73, 33, and 58%, respectively, and the time constants were 435, 769, and 2,000 sec, respectively. We conclude that RVD capacity is actively modulated in the cell cycle and RVD may play an important role in cell cycle progress.     

Multiparametric cell cycle analysis by automated microscopy

Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.