Cell cycle kinetics by BrdU-Hoechst flow cytometry: an alternative to the differential metaphase labelling technique

Human lymphocyte cell cycle kinetics was studied in parallel in whole blood and in isolated lymphocyte cultures by differential metaphase labelling and by flow cytometry, employing the principle of quenching of Hoechst fluorescence by BrdU substituted DNA. The BrdU-Hoechst flow technique yields information on the kinetics of cell recruitment and cell cycle progression superior to the differential metaphase staining, since it provides data from interphase cells, including cycle compartment durations, non-cycling cell fractions and transition probabilities. The Smith and Martin model, modified to include a fraction of non-cycling cells, yields excellent correspondence to the experimental data. We show that lymphocytes isolated from Ficoll gradients respond to PHA stimulation with a 4-6 hr delay compared to whole blood cultures or to cultures with autologous serum supplementation. A detailed study of the effects of such culture supplements on lag phase duration, cell cycle compartment length, non-cycling cell fractions and transition probabilities illustrates the application and reproducibility of the flow assay. The potential of the method is further documented with two examples showing the dependence of lymphocyte proliferation on donor age and donor genotype.     

BrdU-Hoechst flow cytometry reveals regulation of human lymphocyte growth by donor-age-related growth fraction and transition rate

An improved BrdU-Hoechst flow assay was applied to cell kinetic studies of human lymphocyte cultures during a 24-96 hr interval after PHA stimulation. The assay shows that the duration of the initial lag phase and the proportions of noncycling cells increase as a function of donor age, whereas the rates of transition from each cell cycle compartment to the next decrease. Cell cycle arrest occurs in the first S and G2 phase after stimulation of lymphocytes from a 75-year-old donor but not from younger donors. The data are consistent with several models of cell cycle kinetics, so long as these models are modified to include a fraction of noncycling cells in each cell cycle compartment.     

Chromosomal radiosensitivity of human leucocytes in relation to sampling time

Frequencies of chromosomal aberrations after irradiation with X-rays of peripheral blood lymphocytes in vitro were determined at different times after initiation of cultures. In each culture, the kinetics of cell multiplication was followed by using BrdU labelling and differential staining of chromosomes.The results indicate that the mixing up of first and second cell cycle cells at later sampling times cannot explain the observed variation in the frequencies of chromosomal aberrations but that donor-to-donor variation is a predominant factor influencing yields of aberrations. The condition of a donor seems to be most important because repeats on the same donor also showed marked variability.     

Alteration of cell cycle kinetics by reducing agents in human peripheral blood lymphocytes from adult and senescent donors

For improving cell proliferation reducing agents are routinely used as medium supplements in murine cell cultures, however, they are rarely used for human peripheral blood lymphocytes (PBLs). Data on changes in cell kinetics induced by reducing agents are not available. Here cell kinetic alterations induced by reducing agents in human lymphocytes are revealed by applying flow cytometric BrdUrd/Hoechst cell cycle analysis and by using the exit kinetic model of Smith and Martin. Applying alpha-thioglycerol (a-TG) as a model compound it was shown that the major cell kinetic effect is a shortening of the mean duration of the G0/G1 phase. The minimum G0/G1 phase duration and the percentage of the non-cycling G0/G1 cell fraction decrease only slightly. Moreover, a lower number of PBL's are arrested in the G2/M phase of the 1st cell cycle. The durations of the S and G2/M phase in the 1st and G1 phase in the 2nd cycle are not affected. These cell kinetic effects are identical for lymphocytes from both adult and senescent donors. The supplementation of the cell cultures with recombinant IL-2 did not induce similar cell kinetic alterations compared with a-TG. This indicates that the variation of the cell cycle progression factor IL-2 is not solely responsible for improvement of the cell activation process in the G0/G1 phase.     

Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non-stoichiometric Hoechst fluorochrome binding.

Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X-rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non-cycling cell fraction; and a distinctive, non-cycling G-/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst 33258-ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non-cycling cell fraction in the control culture to 29% (X-rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3-aminobenzamide, this aberrant cell population increased significantly after X-ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non-cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non-cycling and cycling cell fractions, this subpopulation with non-stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of non-cycling cells represents damaged cells with altered dye binding properties.     

Age-related alterations in cell division and cell cycle kinetics in control and trimethyltin-treated lymphocytes of human individuals

Trimethyltin chloride induced age-related suppression of cell division and cell cycle kinetics in human peripheral blood lymphocytes cultured in RPMI 1640 culture medium supplemented with human AB serum, phytohemagglutinin and bromodeoxyuridine. A high frequency of M₁ (first metaphase) cells was seen in cultures treated with a high dose (C ₁ = 1.0 g per culture) and in lymphocytes from donors in the age range 40–70 years. The delay in cell division and cell cycle kinetics may indicate a longer duration in DNA synthesis induced by trimethyltin chloride in aged lymphocytes.     

Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo

Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin's lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12-15%) was found in two independent sets of experiments (P=0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P<0.01), in accordance with the in vitro finding of increased RI at low doses.     

Chromosomal response of human lymphocytes to streptozotocin

The induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) by the methylating agent streptozotocin (STZ) and the effect of this compound on mitotic index (MI) and cell cycle progression in human lymphocytes were investigated. Unstimulated (G(0)) or cycling lymphocytes derived from whole blood or purified lymphocyte cultures were pulse-treated with increasing doses of STZ for 0.5-24h. Induction of CAs by STZ was only observed in cycling lymphocytes derived from whole blood cultures (WBC) (P<0.05). On the contrary, STZ produced a significant and dose-response increase in the yield of SCEs in unstimulated as well as cycling lymphocytes (P<0.05). In addition, STZ induced a dose-dependent decrease in the MI but had a slight effect on cell cycle progression. These results suggest that SCEs are the most sensitive endpoint for evaluating the chromosomal effects of STZ on these cells.     

Cell cycle analysis of human peripheral blood T lymphocytes in long-term culture

We have studied the cell cycle of resting T lymphocytes from long-term (LT) cultures following stimulation with phytohemagglutinin (PHA) and recombinant Interleukin 2 (IL-2). We examined the kinetics of entry into S phase by autoradiography, the accumulation of cellular RNA by microfluorometric techniques, and ultrastructural morphology by electron microscopy. In addition, we examined the expression at the mRNA level of six cell cycle-dependent growth-regulated genes (c-fos, c-myc, KC-1, JE-3, vimentin, and histone H3). We show that T lymphocytes of LT cultures respond differently to mitogenic stimulation than the T lymphocytes of freshly isolated peripheral blood mononuclear cell cultures. At the ultrastructural, biochemical, and molecular levels, resting T lymphocytes of LT cultures can be distinguished from physiological (G0) lymphocytes of peripheral blood.     

Co-cultivation of whole blood from male and female muntjacs and the cell proliferation kinetics in vitro

A mixed blood culture (MBC) of heparinized whole blood from male and female Indian muntjac has been done using the BrdU-Hoechst-sunlight-Giemsa method to study the cell-cycle kinetics in vitro. Blood lymphocytes of both male and female muntjacs show a much shorter cell cycle time, roughly, 10-12 h for the initial but only 8 h for the subsequent cycles. There is a significant difference in the rate of cell proliferation between male and female cells. The male blood cells constitute a majority of the 'slow'-dividing cells which reach a peak at the first cycle of mitosis at 40 h, whereas a similar peak of first cycle mitosis is reached by female cells at 32 h, indicating the occurrence of a high frequency of 'fast' dividing female cells as compared to those of males. This novel sex-based differential cell proliferation kinetics is observed both in mixed and separate cultures. This type of MBC method which is free of interculture variations can be reliably used for comparative studies where two genomes can be distinguished.     

Micronucleus frequency and proliferation in human lymphocytes after exposure to herbicide 2,4-dichlorophenoxyacetic acid in vitro and in vivo

Widespread use of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) and its association with non-Hodgkin’s lymphoma (NHL) and other cancers has raised public concern. Here, micronucleus (MN) formation has been used as a biomarker of genotoxicity, and replicative and mitotic indices (MIs) as biomarkers of cell cycle kinetics in human lymphocytes. Cells were cultured either as whole blood or isolated lymphocytes and treated with pure or commercial forms of 2,4-D at doses between 0.001 and 1 mM for 48 h. Exposure to 2,4-D produced a minimal increase in MN in whole blood and even smaller one in isolated lymphocyte cultures. This induction took place only at levels approaching cytotoxicity and was accompanied by a significant inhibition of replicative index (RI). At a low (0.005 mM) dose of commercial 2,4-D, a small, marginally significant increase in RI (12–15%) was found in two independent sets of experiments (P=0.052). Additionally, we found that lymphocyte RI was more affected by commercial 2,4-D containing 9.4% of the chemically pure 2,4-D, than with an equal concentration of the latter suggesting that other ingredients present in the commercial pesticide may be responsible or may enhance the effect of 2,4-D. Mitotic index, however, did not show any significant change with either commercial or pure 2,4-D. The lymphocytes of 12 male applicators exposed solely to 2,4-D during a 3-month period had a significantly higher RI than the same group prior to exposure and than a control group (P<0.01), in accordance with the in vitro finding of increased RI at low doses.     

Glucocorticoid receptors in subpopulations of human lymphocytes defined by monoclonal antibodies

Glucocorticoid receptors (GR) were investigated in subpopulations of lymphocytes identified by monoclonal antibodies. Purified T (OKT3+) and non-T lymphocyte subpopulations were isolated from human peripheral blood using Degalan bead columns coated with rabbit anti-human IgG. Purified subpopulations of OKT4+ and OKT8+ lymphocytes were obtained by coating the nonadherent population (T cells) from the first column with OKT4+ or OKT8+ and pouring it into a second Degalan column, coated with goat anti-mouse IgG. GR content and affinity were analyzed by a whole cell assay with [3H]dexamethasone as tracer. The numbers of GR in lymphocyte subpopulations (OKT3+ cells, non-T cells, OKT4+, and OKT8+ cells) were nearly equal. It is concluded that the differential effects of glucocorticoids on the circulatory kinetics of OKT4+ and OKT8+ cells probably are not related to differences in glucocorticoid receptors of these T-cell subpopulations.     

A compartmental model for the study of diurnal rhythms in cell proliferation

A mathematical model taking into account the observed diurnal variations in cell kinetics is presented. Its principle is to divide each phase of the cell cycle into a definite number of compartments and to assume time-dependent probabilities of transition from one compartment to the following; general properties of the model are derived.The particular case where the only time-dependent transition probabilities are those corresponding to the G₁ phase is studied. A characterization of the joint percentages of S and M cells variations is given. The application of the model to interpretation of published experimental data obtained in hamster cheek pouch epithelium is given.     

Proliferation kinetics of human B- and T-lymphocytes

Human peripheral blood B- and T-lymphocytes, highly purified by immunological methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohaemagglutinin, respectively. Proliferation kinetics of the cell populations were investigated using 5-bromodeoxyuridine (BrdU) labelling of the cell cultures and chromosome preparation at different times after stimulation. The percentages of metaphase cells having replicated for one, two or three generations in the presence of BrdU were determined following sister chromatid differential staining. In all donors, the changes in these percentages were faster in B- than in T-lymphocytes, indicating a longer cell cycle time in the latter population.     

Quantification of entry into and exit from the cell cycle in human lymphocyte cultures

A mathematical model is presented for the analysis of transition between cycling and non-cycling compartments by cells responding to a growth stimulus. The cellular age distribution as a function of time is derived from sequential [3H]thymidine pulse labeling indices. Rates of entry into and exit from the cycling compartment are determined on the basis of labeling indices obtained after instantaneous and long duration [3H]thymidine pulses. Analysis of an experiment involving sequential measurements over the whole lifespan of a human lymphocyte culture stimulated by phytohemagglutinin is presented as an example of the application of this method.     

Growth of human T lymphocyte colonies from whole blood: culture requirements and applications

Growth of human lymphocyte colonies from whole blood following stimulation with PHA, Con A, or PPD is described. Individual colony cells were identified as T lymphocytes on the basis of surface marker and enzyme cytochemical characterizations. Colony formation increased as a power function over a wide range of cell concentrations above a critical minimal concentration. The whole blood culture system eliminates possible selective effects of lymphocyte colony techniques utilizing gradient-enriched lymphocyte fractions and more closely approximates the in vivo milieu. The whole blood colony method is more sensitive for the detection of low-level radiation effects on lymphocytes than widely used tests that measure 3H-thymidine incorporation. In preliminary studies, we used the whole blood method to determine the relative radiosensitivity of lymphocytes from humans with various hematopoietic disorders, and observed abnormalities in mitogen responsiveness and colony formation in some of the patient groups. This method has wide application for studies in cellular and clinical immunology.     

Prolongation of cell cycle transit time and the presence of non-cycling cells in human lymphoblastoid cells cultured under adverse conditions

The growth characteristics of B lymphocytes infected with Epstein-Barr virus (lymphoblastoid cells) have been investigated by flow cytometric analysis of DNA content and by estimation of cell culture doubling times. It was found that the manipulative procedures involved in the cell cycle analysis resulted in a slowing of the growth rate. This slowing of growth was brought about by the prolongation of cell cycle transit times and by the entry of cells into a short-lived non-cycling pool. The entry of a proportion of the cells into the non-cycling pool may be the normal response of lymphoblastoid cells to non-optimal conditions. The non-cycling cells survived in culture with a T 1/2 of approximately 30-60 hr and continued to secrete immunoglobulin. Their surface transferrin receptors were considerably reduced, which suggests that the failure to divide may have resulted from a failure of growth factor receptors to reach a threshold value following mitosis.     

Accuracy of routine flow-cytometric bitmap selection for three leukocyte populations

A double-blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS-C flow cytometer using the two-parameter 90 degrees light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.     

Large granular lymphocytes inhibit the in vitro growth of autologous Epstein-Barr virus-infected B cells

The effect of lymphocyte subsets, separated on the basis of cell density, on Epstein-Barr virus (EBV)-induced B-cell proliferation was studied. The experiments were performed with lymphocytes of seropositive individuals. After 2 weeks of culture, the growth of B cells was inhibited by the T subset, which is also active in natural killer assays, i.e., the low-buoyant density lymphocyte fractions. However, if the cultures were observed for a longer time, the initial growth regressed even in cultures containing the subsets which did not have natural killing (NK) function, i.e., those with high cell density. The initial cell concentration at which the cultures were seeded determined the outcome of the experiments and the demonstration of inhibitory effects. An important difference was seen between the subsets with regard to radiosensitivity. The prompt inhibitory effect of the NK-positive subset remained after irradiation, while the function of the NK-negative one was abrogated. In the presence of the irradiated T-enriched total population, infected B cells (BEBV) grew. Consequently, the radiation-resistant effector compartment, represented by the low-density cells, was not sufficient to counteract the establishment of BEBV lines. They contributed, nevertheless, to the regression because the kinetics of B-cell growth were different in cultures containing separated high-density cells or the total population. In the former, growth continued for a longer time and complete regression occurred only in the cultures initiated with high cell concentrations. The experiments showed that two types of cells contribute to the regression of BEBV growth in cultures initiated with lymphocytes of seropositive donors. One acts promptly and is independent of cell proliferation; another is activated for proliferation by encounter with B blasts.     

Induction of sister-chromatid exchanges by 2-aminofluorene in cultured human lymphocytes with and without erythrocytes.

2-Aminofluorene (2-AF), an indirect mutagen reported to be metabolically activated by erythrocytes in the Salmonella mutagenicity test, was studied for the induction of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro with (whole-blood cultures) and without erythrocytes (isolated lymphocyte cultures). 2-AF (0.025-0.8 mM) was present in the cultures for the last 48 h of 72-h cultures. In both types of culture, SCEs increased in a dose-dependent manner, with a statistically significant elevation already at the lowest concentration of 2-AF tested and maximum responses of 2.4-fold (whole blood) and 2.1-fold (isolated lymphocytes), in comparison with mean SCEs/cell in control cultures, at 0.4 and 0.2 mM concentrations (respectively). Thus, the induction of SCEs by 2-AF was not dependent on the presence of erythrocytes. Styrene (2 mM), a positive control chemical known to require erythrocytes for efficient SCE induction in vitro, was shown to produce a 4.9-fold increase in SCEs in whole-blood cultures, but only a slight (1.3-fold) effect in isolated lymphocyte cultures. The results suggest that leukocytes, but not erythrocytes, are important in the metabolic activation of 2-AF in the human lymphocyte SCE assay.