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.     

Recovery of Pisum root meristems after mitotic-inhibitory treatments with 3H-thymidine. Inhibition of cell-cycle progression by unincorporated 3H-thymidine.

Primary root meristems of Pisum sativum recover form a 3H-thymidine-induced reduction in mitotic activity once the roots are no longer exposed to exogenous 3H-thymidine. Cells arrested in G2 during 3H-thymidine treatment apparently do not divide for at least 16 hours after treatment, whereas cells remaining in G1 and S do divide and thereby account for recovery. Recovery occurs only when meristems are no longer exposed to exogenous (i.e. unincorporated) 3H-thymidine, suggesting that cytoplasmic irradiation from unincorporated 3H-thymidine prevents cellular recovery from 3H-thymidine-induced inhibition of cell progression through the mitotic cycle. Concentrations of 14C-thymidine which result in cytoplasmic irradiation nearly equivalent to that achieved with 3H-thymidine, but much lower levels of nuclear irradiation, also prevent recovery from 3H-thymidine-induced inhibition of mitotic activity, but do not alone produced such inhibition. These results support the contention that cytoplasmic irradiation prevents recovery from the effects of nuclear irradiation. Unincorporated 3H-thymidine also prevents recovery from sucrose deprivation in stationary phase G2 cells which have not incorporated 3H-thymidine into nuclear DNA.     

The blocking of the proliferation of the human endothelial cells in culture by beta-irradiation from internal and external sources of the radiation. S-block is induced by the 3H2O beta-particles

Recently we shown that low doses (0.12-0.46 Gy) of (methyl-3H)-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. The temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the induction of the G2-block estimated by flow cytometry analysis of DNA content and in the induction of the chromosome aberrations (bridges and fragments in anaphase). The aim of this study was the comparative investigation of efficiency of beta-rays emitted 3H from 3H-thymidine and 3H2O by several of the cellular parameters. Here we shown that at the equal conditions of the incubation of the cells in medium with 3H2O induced the accumulation cells in S-phase without decreasing of the mitotic activity and without increasing of the chromosome aberrations level. Unlike from 3H2O the incubation of the cells with 3H-thymidine induced the accumulation cells in G2-phase with decrease of the mitotic activity and with increase of the chromosome aberrations level. Concurrent treatment cells with 3H-thymidine and thymidine abrogate these cellular effects of the 3H-thymidine. Inhibitor ATM-kinase caffeine abrogate as G2-block as S-phase block. These results suggest that the low-dose beta-radiation activates S-phase and G2-phase checkpoints requiring ATM-mediated signal transduction pathway. The factors, which impact on the efficiency of the internal and of the external sources of the irradiation, depend on theirs disposition in relation to radiosensitive target--DNA was discussed.     

Nuclear protein content and cell progression kinetics following X irradiation

After irradiation with 4 Gy of X rays the nuclear protein and DNA contents (to determine cell-cycle position) of HeLa cells were determined by isolating nuclei and staining them with the fluorescent dyes fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for DNA. Immediately following irradiation there was no change in the shape of the bivariate (FITC-PI) histogram. At 3 and 4 h after irradiation the region of the histogram which corresponds to mitotic cells had disappeared. At 6 h nuclei reappeared in this region. The maximum rearrangement of the histogram (i.e., maximum accumulation of cells in G2 with minimum cells in G1) occurred at 10.5 h after irradiation, which is later than the time required for mitotic recovery. No change in nuclear protein content of cells in G1 and S was observed. However, beginning at 4 h after irradiation and continuing throughout the period of observation, a small (10-20%) but significant increase in nuclear protein content was observed for nuclei isolated from cells in G2. The increase in nuclear protein content may be part of the mechanism of G2 arrest and/or may reflect unbalanced growth.     

The effect of herbicide "Chwastox p?ynny 30" on the cell cycle parameters in root meristem of Vicia faba L. subsp. minor

The duration of the cell cycle and its phases after the treatment with herbicide "Chwastox p?ynny 30" was calculated using 3H-thymidine labelling method. Inhibition of DNA synthesis and marked prolongation of G2 + 1/2 M phase were observed. Tested herbicide caused a significant lowering in the mitotic activity and accumulation of metaphase cells.     

Subpopulation analysis of drug-induced cell-cycle delay in human tumor cells using 90 degrees light scatter

A mitotic cell subset has been identified with nuclear light scatter. Colcemid-treated T-47D human breast cancer cells were permeabilised, stained with ethidium bromide, and analysed by flow cytometry. Cells with G2M DNA content exhibited a unimodal distribution for DNA fluorescence and forward scatter, but two peaks were discernible with 90 degrees light scatter. A discrete low-scattering cell cluster could be distinguished from the G2 cell subset on two-dimensional contour plots of 90 degrees light scatter vs. DNA fluorescence; this cluster was reproduced by mitotic shake-off experiments and varied quantitatively with mitotic indices determined either by microscopy or by stathmokinetic cell-cycle analysis of DNA fluorescence. Cell sorting confirmed that the low-scattering cell cluster comprised predominantly metaphase and anaphase cells. Identification of mitotic cells with this one-step technique enables rapid analysis of drug-induced cell-cycle delay in cell populations with different rates of cell-cycle traverse. Hence, vincristine-induced cytostasis is shown to arise in part because of premitotic G2 arrest, whereas etoposide is shown to affect cycling cells with equal sensitivity in quiescent and activated cell populations. The use of light scatter to discriminate mitotic cells in this way facilitates analysis of drug-induced cell-cycle delay and supplements the information obtainable by conventional cell-cycle analysis.     

Human Parvovirus B19 Induces Cell Cycle Arrest at G2 Phase with Accumulation of Mitotic Cyclins

Human parvovirus B19 infects specifically erythroid progenitor cells, which causes transient aplastic crises and hemolytic anemias. Here, we demonstrate that erythroblastoid UT7/Epo cells infected with B19 virus fall into growth arrest with 4N DNA, indicating G(2)/M arrest. These B19 virus-infected cells displayed accumulation of cyclin A, cyclin B1, and phosphorylated cdc2 and were accompanied by an up-regulation in the kinase activity of the cdc2-cyclin B1 complex, similar to that in cells treated with the mitotic inhibitor. However, degradation of nuclear lamina and phosphorylation of histone H3 and H1 were not seen in B19 virus-infected cells, indicating that the infected cells do not enter the M phase. Accumulation of cyclin B1 was persistently localized in the cytoplasm, but not in the nucleus, suggesting that B19 virus infection of erythroid cells raises suppression of nuclear import of cyclin B1, resulting in cell cycle arrest at the G(2) phase. The B19 virus-induced G(2)/M arrest may be the critical event in the damage of erythroid progenitor cells seen in patients with B19 virus infection.     

Mitotic Entry: A Matter of Oscillating Destruction

Entry into mitosis is essentially driven by cyclin B1 and its associated catalytically active partner Cdk1. While cyclin B1 is kept cytoplasmic throughout interphase, nuclear accumulation occurs just prior to mitosis. This restriction is thought to be part of an oscillating mechanism to properly time mitotic entry. A novel nuclear SCF-type mammalian E3 ligase defined by the F-box containing protein NIPA (nuclear interaction partner of ALK), SCFNIPA, targets nuclear cyclin B1 in interphase while it allows for accumulation at G2/M. Thus, oscillating ubiquitination of nuclear cyclin B1 driven by the SCFNIPA complex contributes to the timing of mitotic entry in the mammalian cell cycle.     

Expression of p60v-src in Saccharomyces cerevisiae results in elevation of p34CDC28 kinase activity and release of the dependence of DNA replication on mitosis.

Expression of the oncogenic protein tyrosine kinase p60v-src in the yeast Saccharomyces cerevisiae has been shown to result in rapid cell death (J. S. Brugge, G. Jarosik, J. Andersen, A. Queral-Lustig, M. Fedor-Chaiken, and J. R. Broach, Mol. Cell. Biol. 7:2180-2187, 1987). Work described here demonstrates that v-Src expression results in accumulation of large-budded cells and a nuclear division block without blocking cytokinesis. Flow-cytometric analysis indicates that the DNA content of these cells is elevated beyond the G2 DNA content, and genetic studies indicate that v-Src expression causes aneuploidy. The activity of Cdc28 kinase, which controls the G1/S and G2/M transitions in S. cerevisiae, increases during galactose induction in a Src+ strain but not in an isogenic Src- strain. These observations indicate that v-Src expression disrupts p34CDC28 kinase regulation, allowing DNA replication to proceed in the absence of a prior mitotic event.     

The dependence of the cytokinetic effects of alkylating antitumor preparations on the phase of the hepatocyte cell cycle in regenerating liver]

Effects of alkylating antitumor drugs on resting (G0 phase of cell cycle) and proliferating (G1, S, G2 and M phases) hepatocytes were studied in regenerating mouse liver. Cell cycle kinetics (fraction of labeled mitoses, labeling and mitotic indices) were determined by 3H-thymidine autoradiography. Dipin and fotrin as a DNA-damaging agents attack mainly resting (G0) and proliferating (G1) cells. Effect of the damage results in the inhibition of DNA synthesis and G2 phase arrest in the following mitotic cycle. An alkylating drug phopurin as well as ara-C both suppress the mitotic progression in proliferating hepatocytes and do not influence the resting cells.     

Changes in a nuclear matrix antigen during the cell cycle: interphase and mitotic cells

We studied the behaviour in interphase and mitotic human cells of a 125 kDa (pI 6.5) antigen, associated with the nuclear matrix and detected in proliferating cells. Indirect immunofluorescence with a specific monoclonal antibody reveals that during interphase in WISH and Namalwa cells, as well as phytohaemagglutinin-stimulated lymphocytes, the antigen displays a speckled distribution in the nucleoplasm of all cells. At early prophase the fluorescence intensity of the coalesced speckles increases markedly. During metaphase and anaphase the antigen gives maximal fluorescence distributed diffusely in the nucleoplasm, while chromosomes remain negative. At anaphase and cytokinesis the antigen is still cytoplasmic, but fluorescence intensity decreases. Two-dimensional gel electrophoresis and immunoblotting reveal that the p125/6.5 antigen displays a net increase in isolated mitotic cells as compared to interphase cells. These results suggest that the p125/6.5 protein participates in late G2 phase and G2/M transition events preparing the cell for mitosis.     

Mechanism of action of interferon is linked to its ability to protect the cell from interstrand cross-linking of DNA, induced by 8-methoxypsoralen, activated by long-wave UV-light

Treatment of human lymphocytes in the G1 phase of mitotic cycle with human lymphoblastoid interferon (Ly-IFN) decreased the frequencies of chromosome aberrations induced by 8-methoxy-psoralen-induced interstrand cross-links. Anticlastogenic effect of Ly-IFN was accompanied by stimulation of unscheduled synthesis of DNA in the G2 phase of mitotic cycle, as shown by increased percent of labeled cells registered by 3H-thymidine autoradiography. The data obtained seem to indicate that the mechanism of Ly-IFN protection is connected with stimulation of postreplicative repair.     

A DELAY IN THE G2 PERIOD OF CULTURED HUMAN LEUCOCYTES AFTER TREATMENT WITH RUBIDOMYCIN (DAUNOMYCIN)

Human leucocytes were cultured for 3 days at 37°C, and during this time treated with rubidomycin (also known as daunomycin) for periods up to 48 hr. The effects of this treatment were studied by examining mitotic indices, uptake of ³H-thymidine, and patterns of DNA content (measured by microdensitometry on Feulgen-stained cells). A low concentration of rubidomycin (0.1 μg/ml) caused accumulation of cells in the G₂ period, which in turn resulted in a decrease in the mitotic index. A secondary effect was a slight drop in ³H-thymidine uptake after 12 hr. Higher doses (up to 10 μg rubidomycin per ml) caused an inhibition of DNA synthesis, with accumulation of unlabelled cells between G₂ and G₂. The probable mode of action of rubidomycin, as presented by earlier authors, is the intrusion of the drug molecule between DNA strands, forming a complex with DNA, and hindering its normal folding. This is discussed with respect to the present findings.     

Transient G2M arrest and subsequent release of apoptotic and mitotic cells in vanadyl(4)-prepulsed human Chang liver cells

The relationship between cell cycling and apoptosis/programmed cell death has been perceived as either checkpoint arrests or mitotic aberration where common pathways between mitosis and apoptosis seem suggested. We show here evidence implicating both perceptions of cell cycle involvement. The process was initiated by hydroxyl free radicals (OH*) generated intracellularly from internalized vanadyl(4). Intranuclear sequestration of vanadyl(4) was verified by nuclear microscopy. Resultant high oxidative reactivity in the nucleus was shown by the redox indicator methylene blue, suggesting direct oxidative damage to genomic DNA. Oxidative stress was further enhanced by depletion of glutathione which is the main cellular reducing agent. Genomic degradation and fragmentation was confirmed by flow cytometric evaluation of terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH end-labelling (TUNEL) of DNA nicks, and cell cycle DNA profiling demonstrating sub-G1 (sub-2N) accumulation. With DNA degradation, there was a G2M transient with hyperdiploid right-shifting, consistent with G2 arrest. G2 arrest was subsequently 'released' with abolition of G2M and all other cell cycle phases except for a solitary sub-G1 (apoptotic) peak. The cytological profile of this 'release' phenomenon was initially marked by the appearance of clusters of mitotic and apoptotic cells. At later stages, the cell population was composed exclusively of nuclear ghosts, apoptotic cells, mitotic cells, and mitotic cells with both chromosomes and apoptotic condensations. Concurrent and conjoint expression of cell death and cell division as the exclusive process of an entire cell population refuted the notion of mutual exclusivity between life and death. Zn2+, an endonuclease inhibitor, abolished all observed cytological and DNA profile changes.     

Targeted inactivation of nuclear interaction partner of ALK disrupts meiotic prophase

NIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa-/- mouse embryonic fibroblasts show no severe phenotype, Nipa-/- spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G2/M kinase activity in Nipa-/- spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.     

Regulation of the cell cycle of B-lymphocytes in mice by substances elevating the levels of intracellular cAMP and cGMP

Spontaneous velocity sedimentation of B lymphocytes activated by intraperitoneal injection of ovalbumin into mice was used to obtain cell cycle synchronized cells, evidenced by differences in the incorporation of labeled precursors of protein and nucleic synthesis (14C-methionine and 3H-thymidine). The effects of acetylcholine and adrenaline, cAMP and cGMP on the intensity of 3H-thymidine incorporation into mouse B lymphocytes and on the amount of the cells entering mitosis were examined. It was shown that acetylcholine is capable of stimulating whereas adrenaline of inhibitin B lymphocyte entry into the stage of DNA synthesis and egress of these cells from the stage of DNA synthesis to the stage of mitosis. Adrenaline was found to have a reciprocal action. The acetylcholine effect could be mimetized by exogenous cGMP, that of adrenaline by cAMP. Stimulation of the G1/S transition was mediated by intracellular calcium ions but did not depend on exocellular calcium.     

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.     

Flow cytometric discrimination of mitotic nuclei by right-angle light scatter

Flow cytometry has been used to demonstrate alterations in protein, RNA, and DNA content of cells as they traverse the cell cycle. Employing fluorescein isothiocyanate (FITC) to stain protein and propidium iodide (PI) to stain nucleic acids, multiple regions within the G1 and G2 phases of the cell cycle, in addition to the M phase, can be distinguished. In this study, cytograms of the 90 degree light scatter signal vs. PI fluorescence were remarkably similar to those of FITC fluorescence vs. PI fluorescence, suggesting a relationship between 90 degree light scatter and protein content. M-phase nuclei can be distinguished from G2-phase nuclei on cytograms of 90 degree light scatter vs. PI fluorescence. However, the percentage of mitotic nuclei obtained by this technique is less than that found by light microscopic analysis. Flow cytometric parameters of nuclei prepared by nonionic detergent (NP40) lysis in Dulbecco's PBS, Vindelov's buffer, or Pollack's hypotonic EDTA/Tris buffer were compared. The best resolution of mitotic nuclei was obtained in Pollack's buffer. However, the stainability of the M-phase nuclei is reduced, and the nuclei are located in the late S/G2 region of the single-parameter histogram.     

Internal and external sources of the radiation induce the blocking of the proliferation of the human endothelial cells in culture. G2-block is induced by beta-particle 3H-thymidine and gamma-rays 137Cs

We found that low doses (0.12-0.46Gy) of (methyl-) 3H-thymidine incorporated into human endothelial cells induce the accumulation cells in G2-phase of the cell cycle. Temperate doses of (1-6 Gy) gamma-rays 137Cs were less effective in the G2-block estimated by flow cytometry analysis of DNA content. Furthermore, the induced the high level of the chromosome aberrations (bridges and fragments in anaphases). 1Gy of gamma-ray 137Cs and 0.005 Gy of beta-rays induced the same per cent of the aberrant anaphases. Apparently, that the damages of the cellular hereditary structures are responsible for the blocking of the cellular proliferation in G2-phase. We suggest, that the disposition 3H-thymidine into radiosensitive target (DNA) defines the high cytotoxic of the beta-rays.