Improved Flow Cytometric Analysis of the Budding Yeast Cell Cycle

The budding yeast, Saccharomyces cerevisiae has been a remarkably useful model system for the study of eukaryotic cell cycle regulation. Flow cytometric analysis of DNA content in budding yeast has become a standard tool for the analysis of cell cycle progression. However, popular protocols utilizing the DNA binding dye, propidium iodide, suffer from a number of drawbacks that confound accurate analysis by flow cytometry. Here we show the utility of the DNA binding dye, SYTOX Green, in the cell cycle analysis of yeast. Samples analyzed using SYTOX Green exhibited better coefficients of variation, improved linearity between DNA content and fluorescence, and decreased peak drift associated with changes in dye concentration, growth conditions or cell size.

Key Words:

Flow cytometry, Cell cycle, Saccharomyces cerevisiae, SYTOX Green, Propidium iodide     

Detection of S-Phase Cells in Smear Cytology Using in Vitro Bromodeoxyuridine Labeling

A technique Is described for rapid detection of S-pha?e cells of tumor tissues in smear specimens using bromodeoxyuridine (BrdU) immunostaining. Mouse NR-S1 tumors and human tumor specimens were prepared for smear cytology after incubation in RPMI 1640 culture medium containing 200 μM BrdU at 37 °C under 3 atm for 1 hr. Samples were fixed in 70% ethanol for 30 min and used immediately or air dried for 30 min. Samples were then denatured in either 4 N HC1 or 0.07 N NaOH to prepare partially single-stranded DMA. Fixation with air drying for 30 min followed by 30 min in 70% ethanol and 1 min denaturation with 0.07 N NaOH resulted in satisfactory staining quality. Cultured tumor specimens were processed for routine paraffin sections after smears were made for cytology. The labeling indices of the smear specimens and of the paraffin sections gave similar results. This technique should be useful in evaluating the cell proliferative potential of tumor tissue in smear cytology without processing paraffin sections.     

A Flow Cytometric In Vivo Chalone Assay Using Retransplanted Old Murine Jb-1 Ascites Tumour Cells

A flow cytometric in vivo chalone assay is described. Transplantation of old JB-1 ascites tumour cells to new hosts induced an influx of tumour cells, with G₁ DNA content, to the S phase. This induction could be reversibly and specifically blocked by injections of an ultrafiltrate of old JB-1 ascites fluid. The method described is superior to a previously published in vivo chalone assay using regenerating ascites tumours. Owing to a reduced variability in time of onset of DNA synthesis, a smaller scatter of observations is achieved and thus the number of mice per group may be reduced using the new method. In contrast to the older technique, the present one does not necessitate killing of mice during the observation period.     

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.     

Kinetics of Cell Volume Changes of Murine Lymphoma Cells Subjected to Different Agents In Vitro

Cell volume distributions obtained with an electronic particle analyzer were used to study the changes in volume of individual cells in the absence of cell division. Cultures of murine lymphoma (strain L5178-Y) cells in suspension were used in these studies. During a division delay following ionizing radiation, individual cells increased exponentially in volume with equal rate constants; these rate constants were indistinguishable from that describing the increase in cell number of an unirradiated population. When an originally log phase population of cells was prevented from increasing in number by inhibitors of DNA synthesis, individual cells increased exponentially in volume for about one generation time with the same rate constant as observed after exposure to ionizing radiation; thereafter, only the cells defining the upper half of the volume distribution continued to increase in volume, and they apparently did so with a first order rate constant proportional to their amount of DNA exceeding that present in one diploid complement of chromosomes in G(1). Cells arrested in mitosis with colchicine increased in volume for approximately 4 hr after which they remained constant in volume for almost one generation time; eventually these cells again increased in size. Inhibitors of protein and RNA synthesis inhibited the cell volume growth of irradiated cells.     

Analysis of Cell Flow and Cell Loss Following X-Irradiation Using Sequential Investigation of the Total Number of Cells In the Various Parts of the Cell Cycle

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. the generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G₂ blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. the experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.     

Flow Cytometric Quantification of All Phases of the Cell Cycle and Apoptosis in a Two-Color Fluorescence Plot

An optimal technology for cell cycle analysis would allow the concomitant measurement of apoptosis, G0, G1, S, G2 and M phases in combination with cell surface phenotyping. We have developed an easy method in flow cytometry allowing this discrimination in an only two-color fluorescent plot. It is based on the concomitant use of 7-amino-actinomycin D and the antibodies anti-Ki67 and anti-phospho(Ser10)-histone H3, both conjugated to Alexa Fluor®488 to discriminate G0 and M phases, respectively. The method is particularly valuable in a clinical setting as verified in our laboratory by analyzing human leukemic cells from marrow samples or after exposure to cell cycle modifiers.     

Nucleic Acid Profile of the Emt6 Cell Cycle In Vitro

Simultaneous RNA and DNA estimations were carried out during the cell cycle of EMT6/M/CC cells growing in vitro following synchronization by mitotic selection. the determinations were performed with a flow cytofluorimeter on individual cells stained with acridine orange. It was found that the RNA content increased during G₁ then remained virtually constant between early and mid S phase, but a second increase occurred during late S. the rate of uptake of tritiated uridine paralleled these changes in RNA levels, and it was also found that the rate of uptake in metaphase and anaphase was virtually zero, but a rapid increase occurred in telophase. the increase in DNA during S was approximately linear, and the intermitotic phase and cycle durations were very similar to previously reported results.     

G1 Cell Cycle Regulatory Proteins in Chemically-Induced Rat Mammary Adenocarcinomas In Vivo and Tumor Promotion-Sensitive, -Resistant and Transformed Mouse Epidermal Cells In Vitro

Tumor promotion is characterized by selective proliferation of initiated cells resulting in their clonal expansion. Cyclin D1 is frequently upregulated in this process, but its expression does not necessarily correlate positively with cyclin A. In the present article, expression of G₁ cell cycle regulatory proteins was systematically analyzed using two models of carcinogenesis: (a) N-methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas and normal rat mammary epithelial cells in vivo and (b) promotion- sensitive, -resistant, and transformed JB6 mouse epidermal cells in vitro. The results of this analysis revealed that p27Kip1 negatively correlated with cyclin D1. In addition, there were two types of correlations between p27Kip1 and cyclin A. First, p27Kip1 negatively correlated with cyclin A (type-I correlation). This scenario was observed in normal rat mammary epithelial cells in vivo and promotion-sensitive (P+) JB6 mouse epidermal cells, stimulated with phorbol ester (TPA) in vitro. Second, p27Kip1 positively correlated with cyclin A (type-II correlation). This correlation was observed in MNU-induced rat mammary adenocarcinomas in vivo and TPA-stimulated (P+) JB6 cells, treated with retinoic acid in vitro.     

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.     

Quantitative Determination of Gap Junction Intercellular Communication Using Flow Cytometric Measurement of Fluorescent Dye Transfer

Gap junction intercellular communication (GJIC) is involved in several aspects of normal cell behaviour, and disturbances in this type of communication have been associated with many pathological conditions. Reliable and accurate methods for the determination of GJIC are therefore important in studies of cell biology. (Tomasetto, C., Neveu, M.J., Daley, J., Horan, P.K. and Sager, R.(1993) Journal of Cell Biology, 122, 157–167) reported some years ago the use of flow cytometer to determine transfer between cells of a mobile dye, calcein, as a measure of cell communication through gap junctions. In spite of this being a method with potential for quantitative and reliable determination of GJIC, it has been modestly used, possibly due to technical difficulties. In the present work we have illustrated several ways to use flow cytometric data to express cell communication through gap junctions. The recipient cells were pre-stained with the permanent lipophilic dye PKH26, and the donor cell population were loaded with the gap junction permeable dye, calcein. We show that the method may be used to measure the effect of chemicals on GJIC, and that the information is reliable, objective and reproducible due to the large number of cells studied. The data may give additional information to that obtained with other methods, since the effect observed will be on the establishment of cell communication as compared to what is observed for microinjection or scrape loading, where the effect is on already established communication. This is probably the reason for the more potent effects of DMSO on GJIC measured by the present method than on already existing GJIC measured by microinjection or quantitative scrape loading. We also show that the problem related to the mobile dye calcein not being fixable with aldehydes will not affect the results as long as the cells are kept on ice in the dark and analysed by flow cytometer within the first hours after formalin cell fixation.     

Estimation of Circadian Variations In Cell Cycle Phase Durations In Murine Epidermal Basal Cells

Circadian variations in the proliferative activity of squamous epithelia are well known. However, circadian variations in the duration of the various cell cycle phases (S, G₂ and mitosis) have been disputed. the percent labelled mitoses method, which is traditionally used to obtain duration of cell cycle phases, is poorly suited for identification of circadian variations. Therefore methods combining changes in compartment size (cell cycle phase) and cellular flux through the compartments have been used. Three different methods using such data are presented. These incorporate various simplifying assumptions that cause methodological errors. Limits for use of the different methods are indicated. the use of all three methods gives comparable and pronounced circadian variations in the duration of S and G₂ phase. These results are also compatible with circadian variations in the mitotic duration, but they may also represent artefacts due to sensitivity to model errors.     

A Comparison Between Melanotic and Amelanotic Retinal Pigment Epithelial Cells In Vitro Concerning the Effects of L-Dopa and Oxygen on Cell Cycle

Melanin precursors and free radicals, cytotoxic substances, are produced during melanin synthesis by tyrosinase. We compared these cytotoxic effects of L-dopa and oxygen on the cell cycle of melanotic retinal pigment epithelial (RPE) cells with amelanotic RPE cells because of the differences of tyrosinase activities between melanotic and amelanotic RPE cells. Flow cytometric DNA analysis of RPE cells exposed to L-dopa (100 μM and 250 μM) were conducted at several oxygen concentrations (20%, 10%, and 5%). The dose-dependent effect of L-dopa to arrest the cell cycle (the S phase) was more pronounced in melanotic than in amelanotic RPE cells, and oxygen caused arrest in the G₁ phase.     

Ultrastructural Characteristics of the In Vitro Cell Cycle of the Protozoan Pathogen of Oysters, Perkinsus marinus

Ultrastructural characteristics of vegetative and zoosporangial stages of cultured Perkinsus marinus, a pathogen of the eastern oyster, Crassostrea virginica, were examined by transmission electron microscopy. An axenic cell culture was propagated from infected Chesapeake Bay oyster hemolymph. Different stages of the in vitro cell cycle, including schizonts and different size trophonts, were examined. Trophonts had spherical nuclei with wide perinuclear spaces, mitochondria with tubular cristae, and vacuoles with vacuoplasts. There were micropores on the inside of cell walls. A tubular network in the cytoplasm connected lomasomes to vacuoles, and contained vacuoplast precursor material. Vacuoplasts and precursor material diminished when cell cultures were not fed, suggesting a function in metabolite storage. Cells divided by schizogony or binary fission. Daughter cells in a schizont were not alike, and may specialize for different functions. Some of the daughter cells in a schizont died. Some hypnospores, directly isolated from infected oyster hemolymph enlarged in Ray's fluid thioglycollate medium, and were induced to zoosporulate. Zoosporangia contained varicose, hypha-like structures, whose apical tips gave rise to prezoospores. Ultrastructural characteristics of the vegetative and zoosporangial stages did not resemble any apicomplexan parasites other than members of the genus Perkinsus.     

Flow Cytometric Analysis of DNA Content of Living and Fixed Cells: a Comparative Study Using Various Fixatives

The majority of studies dealing with DNA analyses are made on fixed cells. In this context, the efficiency as fixatives of ethanol, methanol, acetone, Carnoy, Boehm-Sprenger and aldehydes was determined using two different DNA fluorescent probes, Hoechst 33342 and propIDium iodIDe. The purpose of our study was to find the fixative that would provIDe the best results with respect to the following parameters: aggregates, cell size and granularity, and DNA staining analysis. Using murine fibroblasts, we found that 68% ethanol, 85% methanol and aldehydes dID not increase aggregate formation, whereas Carnoy, acetone or Boehm-Sprenger fixatives dID. The results show that aldehydes seem to alter cell size least. All fixatives induce an increase in cell granularity, which is very pronounced with alcohols, but aldehydes alter morphology less than alcohols. We observed that the fixatives giving the best resolution with Hoechst 33342 staining lead to a lower measurement variabili ty than with propIDium iodIDe staining. This study leads us to conclude that 68% ethanol and 85% methanol can be consIDered as appropriate fixatives for flow cytometry studies of DNA content.     

Effects of Exogenous Zinc on the Cellular Zinc Distribution and Cell Cycle of A549 Cells

Carotenogenesis in Nocardioform actinomycete Rhodococcus rhodochrous was investigated using carotenogenesis mutants and inhibitors, and a postulated carotenogenesis pathway was proposed. At the end of the synthesis, fatty acid or mycolic acid was esterified by different esterases to the same C-6 hydroxyl group of β-D-glucoside.