Peculiarities of cytometrical methods of DNA content determination in the nucleus

This work has proposed a new theoretical approach to analysis of histograms of DNA content, which are obtained by the method of flow cytometry, in cells of Drosophila melanogaster imaginal discs. The precision of measurements of the DNA amount in G₁ and G₂(M) phases has been shown to be limited by precision of instrument tuning of zero of the flow cytometer. Use of the calculative zero of the flow cytometer and of dividing cells as standards of the DNA content is able to increase severalfold the precision of the DNA measurements in nuclei of the species. Comparative analysis of errors of various methods of measurement of the DNA content in cell nuclei is also performed. For methods of flow fluorescent cytometry, confocal scanning, and cytophotometry of the Feulgen-stained nuclei, it has been shown that, at present, the mean square errors of the DNA content measurements are within the interval of values considered acceptable for biological studies (0.02 < CV < 0.06).     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

Cell cycle distributions, growth characteristics, and variation in prolactin and growth hormone production in cultured rat pituitary cells.

Clonal strains of rat pituitary tumour cells (GH3 cells) spontaneously produce and secrete prolactin and growth hormone. Chromosome analysis and DNA ploidy measurements revealed that the GH3 cells in the present study were triploid and had a decreased chromosome number compared to the parent strain. Monolayer cultures of these cells grow exponentially for 6-7 days with a mean doubling time of 54 h. Cell cycle distributions and phase durations were determined by micro-flow fluorometric measurements of cellular DNA content combined with computer calculations. During exponential growth the cell cycle distribution did not change (65.4% cells with a G1 phase DNA content, 24.9% with an S phase DNA content, and 9.7% with a (G2 + M) phase DNA content). Counting of mitoses gave 1.4% cells in M phase. The 3H-Tdr labeling indices were determined by autoradiography, and the results were in good agreement with the number of cells in S phase as calculated by micro-flow fluorometry. The phase durations were: Ts=15.9 h, TG2=6.2 h, TM=1.1 h, and TG1=30.9 h. TS and TM calculated from 3H-Tdr labeled and Colcemid treated cultures gave corresponding results. In plateau phase cultures the number of cells with a G1 DNA content increased to 80% and the number of cells with an S phase DNA content decreased to between 5% and 10%. The specific production of prolactin and growth hormone determined by radioimmunoassay showed two and four-fold increases respectively, during exponential growth. The hormone values decreased to initial or subinitial values (day 2 values) when approaching plateau phase. We conclude: that changes in the cell cycle distribution of the cell population cannot be responsible for the spontaneous alterations in hormone production during growth and that most of the hormone-producing cells must be in the G1 phase.     

Increased accuracy of absorption cytophotometric DNA values by control of stain intensity

A method of improving absorption cytophotometric cellular DNA values by making measurements on Feulgenstained cells at optimal stain absorbances has been developed. Stain intensity can be controlled either by alteration of the Feulgen staining reaction or by selection of "off-peak" wavelengths of light for cytometry. The use of chicken red blood cells as an internal standard, and of a computerized cytometer for the measurements, allows selection of the appropriate off-peak light wavelengths, correction for staining variability at different sites on the same slide, and rapid calculation of cellular DNA values. Cytometry can also be performed at controlled absorbance levels on autoradiographs of 3H-thymidine-labeled cells to allow direct study of the DNA content of nonlabeled G1/G0 and G2/M cells. Use of this technique on mixtures of mouse thymocytes, spleen cells, bone marrow cells, and liver cells gave essentially identical values for G1/G0 cellular DNA content, with coefficients of variation of less than 3%.     

Estimation of kinetic cell-cycle-related gene expression in G1 and G2 phases from immunofluorescence flow cytometry data

BACKGROUND: Flow cytometry of immunofluorescence and DNA content provides measures of cell-cycle-related gene expression (protein and/or epitope levels) for asynchronously growing cells. From these data, time-related expression through S phase can be directly measured. However, for G1, G2, and M phases, this information is unavailable. We present an objective method to model G1 and G2 kinetic expression from an estimate of a minimum biological unit of positive immunofluorescence derived from the distribution of specific immunofluorescence of mitotic cells. METHODS: DU 145 cells were stained for DNA, cyclin B1, and a mitotic marker (p105) and analyzed by flow cytometry. The cyclin B1 immunofluorescence (B1) distribution of p105-positive cells was used to model the B1 distribution of G2 and G1 cells. The G1/S and S/G2 interface measurements were used to calculate expression in S phase and test the validity of the approach. RESULTS: B1 at S/G2 closely matched the earliest modeled estimate of B1 in G2. B1 increased linearly through G1 and S but exponentially through G2; mitotic levels were equivalent to the highest G2 levels. G1 modeling of B1 was less certain than that of G2 due to low levels of expression but demonstrated general feasibility. CONCLUSIONS: By this method, the upper and lower bounds of cyclin B1 expression could be estimated and kinetic expression through G1, G2, and M modeled. Together with direct measurements in S phase, expression of B1 throughout the entire cell cycle of DU 145 cells could be modeled. The method should be generally applicable given model-specific assumptions.     

Cell cycle perturbation of cultured C6 glioma cells following short-term contact with a low dose of ACNU

The purpose of this study was to investigate the cell cycle perturbation of cultured C6 rat glioma cells induced by 1-(4-amino-2-methyl-5-pyrimidyl)methyl-3-(2-chloroethyl)3-nitrosourea hydrochloride (ACNU) using simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdU) content. A new graphic computer program permitted the quantification of cell density in hexagonal subareas and allowed the fraction of BrdU-labeled cells with mid-S phase DNA content (FLS) to be defined in a narrow window. The cell kinetic parameters such as cell cycle time (Tc) and S phase time (Ts) were estimated from a manually plotted FLS curve at 18 and 6 hr, respectively. The major effect of ACNU on the cell cycle was an accumulation of the cells in the G2M phase 12 to 24 hr posttreatment when compared to G2M traverse of untreated cells. For the two-dimensional analysis, cells were labeled with BrdU and then treated with ACNU, or treated with ACNU and then labeled with BrdU. It was concluded that the cells in the S and G2M phases at the time of ACNU administration progressed to mitosis but that the G1 phase cells accumulated in the subsequent G2M phase. Two-dimensional FCM analysis using BrdU provided a useful tool in studying cell cycle perturbation.     

A new method to discriminate G1, S, G2, M, and G1 postmitotic cells

A new flow cytometric method combining light scattering measurements, detection of bromodeoxyuridine (BrdU) incorporation via fluorescent antibody, and quantitation of cellular DNA content by propidium iodide (PI) allows identification of additional compartments in the cell cycle. Thus, while cell staining with BrdU-antibodies and PI reveals the G1, S, and G2 + M phases of the cell cycle, differences in light scattering allow separation of G2 phase cells from M phase cells and subdivision of G1 phase into two compartments, i.e., G1A representing postmitotic cells which mature to G1B cells ready to initiate DNA synthesis. The method involves fixation of cells in 70% ethanol, extraction of histones with HC1, and thermal denaturation of DNA. This treatment appears to enhance the differences in chromatin structure of cells in the various phases of the cell cycle to the extent that cells could be separated on the basis of the 90 degrees scatter. Mitotic cells show much lower scatter than G2 phase cells, and G1 postmitotic cells (G1A) show lower scatter than G1 cells about to enter the S phase (G1B). Light scattering is correlated with chromatin condensation, as judged by microscopic evaluation of cells sorted on the basis of light scatter. The method has the advantage over the parental BrdU/DNA bivariate analysis in allowing the G2 and M phases of the cell cycle to be separated and the G1 phase to be analyzed in more detail. The method may also allow separation of unlabeled S phase cells from mitotic cells and distinguish between labeled and unlabeled mitotic cells.     

Cytometry of chromatin bound Mcm6 and PCNA identifies two states in G1 that are separated functionally by the G1 restriction point<Superscript>1</Superscript>

Background  

Cytometric measurements of DNA content and chromatin-bound Mcm2 have demonstrated bimodal patterns of expression in G1. These patterns, the replication licensing function of Mcm proteins, and a correlation between Mcm loading and cell cycle commitment for cells re-entering the cell cycle, led us to test the idea that cells expressing a defined high level of chromatin-bound Mcm6 in G1 are committed - i.e., past the G1 restriction point. We developed a cell-based assay for tightly-bound PCNA (PCNA*) and Mcm6 (Mcm6*), DNA content, and a mitotic marker to clearly define G1, S, G2, and M phases of the cell cycle. hTERT-BJ1, hTERT-RPE-1, and Molt4 cells were extracted with Triton X-100 followed by methanol fixation, stained with antibodies and DAPI, then measured by cytometry.     

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.     

Flow cytometric investigation of heterogeneous copper-sensitivity in asynchronously grown Saccharomyces cerevisiae

The variable stress-sensitivity of individual cells within pure cultures is widely noted but generally unexplained. Here, factors determining the heterogeneous susceptibility to copper toxicity in Saccharomyces cerevisiae were examined with a rapid non-perturbing approach based on flow cytometry. By determination of the DNA content (with propidium iodide) in cell fractions gated by forward angle light scatter (an indicator of the cell volume), it was shown that forward angle light scatter measurements gave an approximation of the cell cycle stage. Thus, our observation that cells in different forward angle light scatter fractions displayed differing Cu-sensitivities indicated that heterogeneous Cu-sensitivity is a function of the cell cycle stage. Furthermore, cells sorted by their Cu-sensitivity and-resistance and subsequently analyzed for DNA content were found predominantly to occupy G1/S and G2/M cell cycle stages, respectively. The oxidant-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate was used to show that the Cu-sensitivity of G2/M phase S. cerevisiae was correlated with greater levels of pre-existing reactive oxygen species in these cells. The results indicate that differential Cu-sensitivity in a S. cerevisiae culture is linked to the cell cycle stage and this link may be determined partly by cell cycle-dependent fluctuations in basal reactive oxygen species generation.     

Combined flow and absorption DNA measurements of [3H]TdR-labelled tumour cells. I. Studies of MCa-11 cells grown as tumours in vivo and as exponential cultures in vitro

Flow cytometry of cellular DNA content provides rapid estimates of DNA distributions, i.e. the proportions of cells in the different phases of the cell cycle. Measurements of DNA alone, however, yield no kinetic information and can make it difficult to resolve the cell cycle distributions of normal and transformed cells present in tumour biopsy specimens. The use of absorption cytophotometry of the Feulgen DNA content and [3H]TdR labelling of the same nuclei provides objective criteria to distinguish the ranges of DNA content for G0/G1, S, and G2/M cells. We now report on a study in which we combined flow and absorption cytometry to resolve the cell cycle distributions of host and tumour cells present in biopsy specimens of MCa-11 mouse mammary tumours labelled in vivo for 0.5 hr with [3H]TdR. A similar analysis of exponential monolayer cultures, labelled for 5 min with [3H]TdR under pulse-chase conditions, revealed a highly synchronous traversal of almost all cells through the different phases of the cell cycle. Combination of the flow and absorption methods also allowed us to detect G2 tumour cells in vivo and a minor tumour stem-line in vitro, to show that these two techniques are complementary and yield new information when they are combined.     

Flow cytometric analysis of the cell cycle: Mathematical modeling and biological interpretation

Estimation of the repartition of asynchronous cells in the cell cycle can be explained by two hypotheses: the cells are supposed to be distributed into three groups: cells with a 2c DNA content (G0/1 phase), cells with a 4c DNA content (G2 + M phase) and cells with a DNA content ranging from 2c to 4c (S phase); there is a linear relationship between the amount of fluorescence emitted by the fluorescent probe which reveals the DNA and the DNA content. According to these hypotheses, the cell cycle can be represented by the following equation: [formula: see text] All the solutions for this equation are approximations. Non parametric methods (or graphical methods: rectangle, peak reflect) only use one or two phase(s) of the cell cycle, the remaining phase(s) being estimated by exclusion. In parametric methods (Dean & Jett, Baisch II, Fried), the DNAT(x) distribution is supposed to be known and is composed of two gaussians (representative of G0/1 and G2 + M) and a P(x,y) function representative of S phase. Despite the generality, these models are not applicable to all sample types, particularly heterogeneous cell populations with various DNA content. In addition, the cell cycle is dependent on several regulation points (transition from quiescence to proliferation, DNA synthesis initiation, mitosis induction) and biological perturbations can also lead to cytokinesis perturbations. Before the emergence of flow cytometry, the current view of cell cycle resided in the assessment of cell proliferation (increase in cell number) or the kinetic of molecules incorporation (DNA precursors).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of cytoskeletal proteins throughout the cell cycle of the MRC-5 fibroblastic cell line

Fluorescent dyes were used to stain actin, vimentin, tubulin and DNA in the same MRC-5 fibroblastic cells. Cytofluorometry and image analysis were then used to quantitatively evaluate the F actin, vimentin and tubulin content throughout the cell cycle. The results showed that different cells can have the same DNA content while their cytoskeletal protein content is variable. The data also showed that cytoskeletal protein content variations exist throughout the cell cycle of the fibroblastic cell line. The F actin content increased during the cell cycle from G1 to G2 phases and decreased in M phase. The amount of tubulin in the G2 was about twice as much as that in the G1 phase, before decreasing in the M phase; there was a threshold of tubulin content for G2 cells entering S phase.     

Simultaneous analysis of DNA content and surface antigens in human bone marrow

In order to identify when cellular expansion occurs during hematopoietic maturation, a method was developed for the simultaneous analysis of one or two cell-surface antigens and DNA content on bone marrow cells while preserving their light-scatter properties. Proliferation in a population defined by light-scatter and surface-antigenic characteristics was assessed by measuring the percentage of cells in this population having more than 2C amount of DNA ("proliferation index"). Viable, low-density (1.077 g/cm3), bone marrow cells, stained with monoclonal antibodies conjugated with fluorescein or phycoerythrin, were fixed with paraformaldehyde and subsequently treated with the detergent, Tween 20. The UV-excitable DNA stain Hoechst 33342 was used to quantify DNA content in the cells without interference with immunofluorescence. A FACS IV flow cytometer was used, equipped with the first laser at 488 nm emitting for light scattering and immunofluorescence measurements and the second laser emitting at 360 nm for the Hoechst excitation. The Hoechst uptake was the same for all bone marrow populations, yielding a tight coefficient of variation (CV) (average 5.0%) for the G0/G1 DNA peak. This permitted high sensitivity of cell detection in S, G2, and M phases of the cell cycle, while preserving light-scattering properties of the cells and maintaining cell surface immunofluorescence. The lowest "proliferation index" detected using this technique was 0.08% in a sample obtained from a patient with chronic lymphocytic leukemia. Normal helper T lymphocytes in marrow had approximately 0.5% of the cells in S, G2, or M phase. We show that the erythroid lineage, in the adult normal bone marrow, is the most active in proliferation among all hematopoietic lineages.     

Cell cycle regulator geminin is dispensable for the proliferation of vascular smooth muscle cells

The proliferation of vascular smooth muscle cells (VSMCs) plays a major role in the pathogenesis of many cardiovascular diseases. Geminin regulates DNA replication and cell cycle progression and plays a key role in the proliferation of cancer cells. We therefore hypothesized that geminin regulates the proliferation of VSMCs. The present study demonstrates that the level of geminin expression was low in quiescent VSMCs (approximately 90% and 10% of cells in the G1 and in S/G2/M phases of the cell cycle, respectively), increased as more cells entered in S/G2/M, and then decreased as cells exited S/G2/M. Further, angiotensin II and norepinephrine stimulated expression of geminin in VSMCs. However, the DNA content, nuclear morphology, percentage of cells at different stages of the cell cycle, and rate of proliferation of VSMCs from which geminin was either depleted or overexpressed were all similar. These findings indicate geminin functions differently in VSMCs than it does in cancer cell lines and that it may provide a target for treating cancers without affecting normal cells.     

Improved method for computing potential doubling time from flow cytometric data

Relative movement methods use the timed progression of the mean fluorescence of cells which have been labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, TS. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity v is introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to ln(2)Ts/Tpot so that Tpot (equal to ln(2)Ts/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.     

Estimating cell death in G(2)M using bivariate BrdUrd/DNA flow cytometry.

BACKGROUND: In an accompanying paper (Asmuth et al.) it was found necessary to include cell death explicitly to estimate parameters of cell proliferation. The use of bivariate flow cytometry to estimate the phase durations and the doubling times of cells labeled with thymidine analogues is well established. However, these methods of analysis do not consider the possibility of cell death. This report demonstrates that estimating cell death in G(2)/M is possible. METHODS: Mathematical models for the experimental quantities, the fraction of labeled undivided cells, the fraction of labeled divided cells, and the relative movement were developed. These models include the possibility that, of the cells with G(2)/M DNA content, only a certain fraction will divide, with the remainder dying after some time T(R). Simulation studies were conducted to test the possibility of using simple methods to estimate phase durations and cell death rates. RESULTS: Cell death alters the estimates of phase transit times in a rather complex manner that depends on the lifetime of the doomed cells. However, it is still possible to obtain estimates of the phase durations of cells in S and G(2)/M and the death rates of cells in G(2)/M. CONCLUSIONS: The methods presented herein provide a new way to characterize cell populations that includes cell death rates and common measurements of cell proliferation.     

Alterations of growth fraction and DNA content in K562 cells by differentiating agents

The growth fraction, estimated by the monoclonal antibody Ki-67 labeling, and DNA content, assessed by ethidium bromide staining, were determined simultaneously in K562 leukemic cells by flow cytometry. A multiparametric analysis enabled the fraction of the cell population with G1, S, and G2 + M contents in Ki-67-positive and Ki-67-negative cells to be evaluated. Butyric acid (BUT) was used as positive control. The fraction of Ki-positive cells decreased with the BUT concentration, while the proportion of cells with G1 DNA content increased only in the Ki-negative cells. Adriamycin, aclacinomycin A, and fagaronine induced differentiation, as assessed by benzidine staining and glycophorin A expression. These drugs decreased the fraction of Ki-positive cells by more than 50% for both anthracyclines and by 25% for fagaronine. Following treatment, Ki-negative cells displayed a G1, but also a G2 and a S DNA content in different proportions, indicating that induction of quiescent cells by differentiating agents is not a uniform process and is worthy of interest.     

Analysis of origin recognition complex in saccharomyces cerevisiae by use of Degron mutants

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.     

不同倍性鱼的血细胞和精子DNA含量比较

我们以前的研究表明, 以红鲫 (2n=100) 为母本及湘江野鲤 (2n=100) 为父本的杂交后代的F1-F2 为二倍体 (2n= 100)。在二倍体 F2 个体中, 存在能分别产生二倍体卵子和二倍体精子的雌、雄个体, 二倍体卵子和二倍体精子结合, 形成了两性可育的四倍体鱼 (F3)。目前四倍体鲫鲤已连续繁殖了 12 代 (F3-F14), 形成了一个遗传性状稳定的四倍体鱼群体 (4n= 200) (Liu et al.,2001; 孙远东等, 2003)。雌性四倍体鲫鲤产生的二倍体卵子经紫外线照射的散鳞镜鲤精子激活后,无须染色体加倍处理, 可发育为全雌性二倍体雌核发育后代 (G1) (2n=10…