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.     

Long-term cytogenetic effects in children prenatally-exposed to radiation as a result of the accident at the Chernobyl Atomic Energy Station

Forty-two children exposed to ionizing radiation in prenatal period and 15 children of control group were examined in the remote terms after the accident using the method of differential G-staining of chromosomes in lymphocytes of peripheral blood. It was found that the average group rate of aberrant cells and chromosome aberrations was reliably higher in the children exposed in utero compared to control. Long-term cytogenetic consequences of the pre-natal exposure were characterized by prevalence of aberrations of a chromosome type, mainly stable chromosome lesions. At chronic exposure to low doses of ionizing radiation the increase in the rate both stable and unstable chromosome aberrations.     

Restoration of pine plantations after effect of ionizing radiation in the region of the accident at the Chernobyl Atomic Energy Station

The main results of the 12-year radiation-genetic monitoring of radiobiological, cytogenetic, and genetic parameters in the Pinus sylvestris L. forest plantations from the zone of the accident at the Chernobyl nuclear power plant presented. Acute ionizing irradiation at doses > 1 Gy was shown to induce the formation of morphoses and depressed growth; at doses > 2 Gy, the reproductive ability of the trees declined. The radiobiological parameters showed a linear (or close to linear) dose-effect relationship. Acute irradiation at a dose of 0.5 Gy induced cytogenetic and genetic effects that were significantly higher than the corresponding control values. The relationship between the cytogenetic effects and the absorbed dose was exponential. The dependence of the mutation frequency at specific loci on the absorbed dose was described by a nonlinear curve. The results of cytogenetic analysis of sprouts obtained from seeds annually (1986-1998) collected in zones of slight, moderate, and strong damage of Pinus sylvestris L. are presented.     

The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 9. The morphofunctional indices of the immunocompetent organs in mice]

The permanent action of small doses of low-intensity radiation on the immune status of 2.5-3.5 month CC57W mice has been investigated. Total doses of internal and external irradiation were about few cGy. The permanent action of low-level radiation on the experimental animals of the first and fourth generations was shown to change spleen and lymph nodes weights and the count of lymphocytes isolated from these organs. Cellularity and DNA synthesis in the lymph-node lymphocytes and their proliferative response to polyclonal mitogens also changed. The alterations in the parameters that characterized T-lymphocyte population were statistically significant.     

The structural-functional disorders in the liver of wild rodents from areas of the accident at the Chernobyl Atomic Electric Power Station]

A study was made of the morphological status of hepatocytes, the antioxidant activity of lipids and composition of phospholipids, and dehydrogenase activity in the liver of field mice taken from seven regions of the Chernobyl A.P.S. zone with different levels of contamination in 1987. There observed multiple types of destructive damages to the organ; depletion of liver lipids by antioxidants; diminution of phospholipids within the total lipid level; considerable increase in the phosphatidyl choline/phosphatidyl ethanolamine ratio and in the relative content of phospholipid lysoforms; and inhibition of dehydration processes. In the absence of a strict correlation between the changes in the biophysical and biochemical parameters or between the severity of degenerative changes in hepatocytes and the level of external irradiation, certain relationship was followed up between liver lipid depletion by antioxidants, inhibition of dehydration processes and the number of wild rodents which developed dystrophic changes in the organ. These structural and functional changes were found in the liver of wild rodents taken from all the regions: this indicated a considerable sensitivity of the parameters of the regulatory cell systems and hepatocytes to the effect of technogenic contamination.     

The effect of the accident at the Chernobyl Atomic Electric Power Station in 1986 on the fish population of a cooling pond]

The cytogenetic analysis of Cyprinus carpio eyes, developing Blicca bjoerkna L. eggs, and eggs and larvae of Hypophtalmichthys molitrix Val. has demonstrated that the yield of cells with chromosome aberrations in the fish species under study is normal, while the level of variability of morphometric indices in offspring is considerably higher than that in parent fish.     

A rapid method for flow cytometric cell counting and cell cycle analysis from monolayer cultures of tumor cells.

A rapid, simple, and reliable flow cytometric method using the histochemical fluorescent stain Hoechst 33342 in presence of the non-ionic detergent Triton X-100 has been reported. The processing of melanoma cell cultures to get nuclei stained with the fluorescent dye was accomplished in one step and within an hour permitted concurrent flow cytometric measurement of cell density and cell cycle analysis. The preparation is stable for more than three weeks at room temperature for flow cytometry. The histograms are reproducible and exhibit a coefficient of variation of less than 2.5% (G1 peak). The cell density measurements varied within +/- 5% limits.     

Flow cytometric cell cycle analysis of cultured porcine fetal fibroblast cells

Normal development of nuclear transfer embryos is thought to be dependent on transferral of nuclei in G0 or G1 phases of the cell cycle. Therefore, we investigated the cell cycle characteristics of porcine fetal fibroblast cells cultured under a variety of cell cycle-arresting treatments. This was achieved by using flow cytometry to simultaneously measure cellular DNA and protein content, enabling the calculation of percentages of cells in G0, G1, S, and G2+M phases of the cell cycle. Cultures that were serum starved for 5 days contained higher (p < 0.05) percentages of G0+G1 (87.5 +/- 0. 7) and G0 cells alone (48.3 +/- 9.7) compared with rapidly cycling cultures (G0+G1: 74.1 +/- 3.0; G0: 2.8 +/- 1.2). Growth to confluency increased (p < 0.05) G0+G1 percentages (85.1 +/- 2.8) but did not increase G0 percentages (6.0 +/- 5.3) compared to those in cycling cultures. Separate assessment of small-, medium-, and large-sized cells showed that as the cell size decreased from large to small, percentages of cells in G0+G1 and G0 alone increased (p < 0.05). We found 95.2 +/- 0.3% and 72.2 +/- 12.0% of small serum-starved cells in G0+G1 and G0 alone, respectively. Cultures were also treated with cell cycle inhibitors. Treatment with dimethyl sulfoxide (1%) or colchicine (0.5 microM) increased percentages of cells in G0 (24.8 +/- 20.0) or G2+M (37.4 +/- 4.6), respectively. However, cells were only slightly responsive to mimosine treatment. A more complete understanding of the cell cycle of donor cells should lead to improvements in the efficiency of nuclear transfer procedures.     

Flow cytometric cell cycle analysis of cultured brown bear fibroblast cells

The aim of this study was to assess by flow cytometry the cell cycle of brown bear fibroblast cells cultured under different growth conditions. Skin biopsies were taken in Cantabria (Spain) from a live, anaesthetized brown bear. DNA analysis was performed by flow cytometry following cell DNA staining with propidium iodide. Serum starvation increased (P<0.01) the percentage of G0/G1 phase cells (92.7+/-0.86) as compared to cycling cells (39.7+/-0.86) or cells cultured to confluency (87.3+/-0.86). DMSO included for 48h in the culture significantly increased (P<0.01) the percentage of G0/G1 phase of the cell cycle at all concentrations used and decreased percentages of S phase in a dose-dependent fashion. Roscovitine increased the G0/G1 phase of the cell cycle (P<0.01) at 15microM concentration. Interestingly, the G2/M stage significantly increased at 30 and 50microM compared to the control and 15microM (P<0.02). The cell cycle of brown bear adult fibroblast cells can be successfully synchronized under a variety of culture conditions.     

Level of chromosome aberrations in peripheral blood lymphocytes in evacuees from the 30-kilometer zone of the Chernobyl Atomic Energy Station and residents of radioactively-polluted territories in the long term after the Chernobyl accident

Eighteen Ukrainian evacuees from the Chernobyl exclusive zone, twenty one inhabitants of radioactively contaminated areas of Belarus and twelve control donors age-matched to the exposed persons were investigated 14-15 years after the Chernobyl accident for chromosomal aberration yields detected in blood lymphocytes by fluorescence in situ hybridisation technique. Unstable aberration yields measured in both Chernobyl cohorts were close to the background frequencies. Positive age-dependence trends in control donors were determined for the all type stable aberration levels. In evacuees the tendency for diminishing the difference between them and controls for stable aberration levels with persons' age increasing was found. The total stable chromosome exchange yields in evacuees 46-55 years old and inhabitants of areas with low contamination level didn't exceed the control values, but for younger evacuees and inhabitants of sufficiently contaminated regions the statistical increase above the age relevant background meanings was detected for this end-point. The advantages of using the FISH-detectable stable aberrations and particularly the total level of stable chromosome exchanges as the end-points for retrospective biological indication of past radiation exposure in Chernobyl cohorts were discussed.     

Leukaemia and thyroid cancer in emergency workers of the Chernobyl accident:

This work focuses on the direct epidemiological assessment of the risks of radiation-induced leukaemia and thyroid cancer in emergency workers (EW) after the Chernobyl accident. The Russian National Medical Dosimetric Registry (RNMDR) contains data for 168 000 EW as of January 1, 1996. The analysis relates to 48 leukaemias and 47 thyroid cancers, diagnosed and verified. Radiation risks are estimated by comparing the EW data with national data for a male population of the same age distribution. For leukaemia, an excess relative risk per Gy (ERR/Gy) of 4.30 (95% CI: 0.83, 7.75) is obtained, while the excess absolute risk per 10⁴ person-years (PY) Gy (EAR/10⁴ PY Gy) is found to be 1.31 (95% CI: 0.23, 2.39); for thyroid cancer an ERR/Gy of 5.31 (95% CI: 0.04, 10.58) is obtained, and an EAR/10⁴ PY Gy of 1.15 (95% CI: 0.08, 2.22).     

Simultaneous cell surface phenotype and cell cycle analysis of lymphocytes by flow cytometry

A method for the simultaneous measurement of cell surface components and nucleic acids (DNA and RNA) of human lymphocytes by flow cytometry has been developed, thereby providing a means of analyzing cell surface changes during the various phases of the cell cycle. Unfixed cells were coated with fluorescein-conjugated concanavalin A (F Con A) or surface antigen-specific antibody, fixed sequentially with paraformaldehyde and methanol, treated with specific nucleases, and then stained with propidium iodide. Neither portion of the procedure (cell surface staining, nucleic acid staining) interfered significantly with the other. Cell cycle phases of phytohemagglutinin-stimulated human lymphocytes as determined by this method were comparable with those identified by acridine orange staining. Cell cycle-specific blocking agents were used to additionally demonstrate the specificity of the staining procedure. Simultaneous measurement of cell cycle phase and detection of surface receptors for Con A and T lymphocyte surface determinants was performed with this method.     

Genetic effects in mice exposed to the 10-km area around the Chernobyl Atomic Energy Station

Mice (CBAxC57BL) F of both sexes were exposed within the 10 km zone of Chernobyl nuclear power station. Genetic damage of phone chronic effect of increased radiation in exposed adult mice and in the course of embryogenesis was studied. The total absorbed radiation doses in testes varied from 1.85 to 0.42 Gy in embryos and from 3.4 to 2.7 Gy in adult males. Increase of dominant lethal mutations (DLM) and abnormal sperm heads (ASH) was only observed right after the end of exposure of adult males. The yield of reciprocal translocations (RT) in these males was relatively low. Among the males exposed at the stage of early embryogenesis, 4 heterozygotes for RT were revealed. In other males of this group the RT yield was low.     

The biological effects in animals in relation to the accident at the Chernobyl Atomic Electric Power Station. 5. Longevity and the carcinogenic effects]

Remote effects in laboratory animals living in conditions of exposure to a mixture of external and internal radiation resulted from the Chernobyl A.P.S. disaster have been investigated. It has been found that the rate of deaths from non-tumor illnesses grows, the incidence of neoplasms increases and their latency time decreases. The redistribution within the spectrum of benign and malignant tumors and the increase in the multiplicity coefficient have been revealed. It is concluded that chronic exposure of animals to low-level radiation from the radionuclides, resulted from the accident, brings about a much larger number of negative stochastic and nonstochastic remote effects as compared to those expected from the extrapolation of the effects produced by high radiation doses.     

Preparation and stability of sixteen murine tissues and organs for flow cytometric cell cycle analysis

Three different technical protocols were used to prepare samples for flow cytometric (FCM) analysis. Each protocol developed worked best for only certain organs. Protocol I involved mincing small pieces of fresh tissue in the propidium iodide (PI) staining solution and filtering through packed glass wool. The organs that were prepared by protocol I were: submandibular gland, urinary bladder, liver, thymus, bone marrow, spleen, lung, kidney and testis. Protocol II involved exposure of the organ to 0.5% acetic acid for 48 h prior to mincing in the PI. The organs that were prepared by protocol II were: uterus, rectum, colon, ileum, and heart. Protocol III utilized an exposure to 0.5% acetic acid, pepsinization, and then staining with PI. The tissues that were prepared by protocol III were the epithelium of the anterior surface of the cornea and the epithelium of the surface of the tongue. A total of 16 different organs and tissues were successfully prepared. For each organ, averaged DNA histograms were analyzed by nonparametric and parametric programs and the results (phase fractions) are presented in tabular form. Several of the organs used came from animals exposed to 1.0 mg/kg vincristine (VC) for 5-6 h to test the capability of the different protocols to detect the enlargement of the G2 + M compartment by the accumulation of VC-arrested mitotic figures. The stability of the many different sample preparations was tested by comparing averaged DNA histograms obtained on the day of sample preparation to averaged DNA histograms of the same set of samples after storage at 4 degrees C, with or without fixation in 10% phosphate-buffered formalin, for days to weeks. After staining with propidium iodide, fixation of the sample with a final concentration of 2-3% phosphate-buffered formalin, was the procedure adopted to assure sample stability. The demonstration of sample stability permits sample preparation to occur at one site followed by transport of the samples to the FCM laboratory at another geographical location. The major findings of this work were a) technical protocols were developed which resulted in acceptable nuclear suspensions for FCM from 16 different murine organs or tissues, b) the stability of these samples can be assured by fixing the PI stained nuclear suspension with formalin, and c) each different protocol was capable of detecting and preserving at least some of the mitotic figures arrested and collected by vincristine.     

Automated identification of subpopulations in flow cytometric list mode data using cluster analysis

The application of K-means (ISODATA) cluster analysis to flow cytometric data is described. The results of analyses of flow cytometric data for mixtures of fluorescent microspheres and samples of peripheral blood mononuclear cells are presented. A method for simultaneously displaying list mode data for any number of parameters, which had previously been applied to a continuous set of parameters such as multi-angle light scattering data, is used to present the results of cluster analysis on physically unrelated parameters; this method allows rapid evaluation of the success of subpopulation identification. The factors that influence automated identification of subpopulations are examined, and methods for determining optimal values for these factors are described.     

Sensitivity of flow cytometric data to variations in cell cycle parameters

Abstract We investigated to what extent flow cytometric DNA histograms are informative of cell cycle parameters. We created a computer program to simulate cell cycle progression in a generic and flexible way. Various scenarios, characterized by different models and distributions of cell cycle phase transit times, have been analysed in order to obtain the percentages of cells in the different cell cycle phases during exponential growth and their time course after mitotic block.
Cell percentages during exponential growth were insensitive to intercell variability in phase transit times and thus can be employed to estimate the relative mean phase transit times, even in the presence of non-cycling cells. However, this information is ambiguous if re-entry of such cells into the cycling status is permitted. The stathmokinetic outline gives the mean phase transit times, but also provides information about the spread, but not the form, of the phase transit time distributions, being particularly sensitive to the spread of G1 phase duration. The stathmokinetic outline also helps distinguish between scenarios considering only cycling cells, those forecasting a fraction of definitively non-cycling cells and those admitting a Go status with first-order output kinetics.