Cell Cycle Compartments Research Articles

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.

Analysis of cell flow and cell loss following X-irradiation using sequential investigation of the total number of cells in the various parts of the cell cycle.

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

Expression of a mouse long terminal repeat is cell cycle-linked.

The expression of the long terminal repeat (LTR) of intracisternal A particle retroviral sequences which are endogenous to the mouse genome has been shown to be linked to the early G1 phase of the cell cycle in Friend erythroleukemia cells synchronized by density arrest and also in logarithmically growing cells fractionated into cell-cycle compartments by centrifugal elutriation. Regions of homology were found in comparing the LTR sequence to a repetitive Syrian hamster sequence specifically expressed in early G1 in hamster cells.

Transferrin receptor expression during exponential and plateau phase growth of human tumour cells in culture.

Transferrin receptor expression by the human tumour cell lines CCRF-CEM leukaemia and PMC-22B melanoma was studied, measuring the specific binding of fluorescein isothiocyanate (FITC)-labelled transferrin using a fluorescence-activated cell sorter. By measuring the fluorescence of cells stained at subsaturating concentrations of conjugate it was possible to calculate the average numbers of receptors per cell and the binding affinity by Scatchard analysis. These values (1.9 X 10(5) binding sites/cell, KA 1.2 X 10(9) M-1 for CCRF-CEM during exponential growth and 6.9 X 10(4) binding sites/cell, KA 1.4 X 10(-9) M-1 for PMC-22B) are in close agreement with previously published data obtained using radiolabelled transferrin. The present method, however, allowed the transferrin receptor expression of individual cells within a population to be measured and thus it has been possible to test the hypothesis that transferrin receptor is a marker for cycling cells. Frequency-distribution histograms of transferrin receptor showed a wide range of values for both cell lines during exponential growth. When the extreme ranges were sorted and the cells examined for cellular DNA content it was found that those with the highest transferrin receptor expression were enriched with cells in S, G2, and M phases of the cell cycle, whereas those with low transferrin receptor expression were mainly in G1. However, two-parameter-correlated dot plots of transferrin receptor expression versus DNA content showed there was considerable overlap between the ranges of receptor expression for the different cell cycle compartments. Using a stathmokinetic method we have measured the proportion of quiescent cells in fed plateau phase cultures. Transferrin receptor expression was downgraded under these growth conditions but, contrary to expectation, the decline affected the population uniformly, without the emergence of a distinct, transferrin receptor-negative subpopulation corresponding to the increasing proportion of quiescent cells. Thus, although transferrin receptor expression bears some relation to cell cycle phase and reflects the proliferative activity of populations of cells, it is incapable of identifying individual cells which are out of cycle.

Interferon-induced growth modulation: low dose maintenance of the antiproliferative response.

Treatment of the Burkitt's lymphoma-derived Daudi cell line with human beta interferon (HuIFN-beta) results in a dose-dependent antiproliferative response. We have defined three phases including: (1) initiation, (2) maintenance, and (3) termination of the antiproliferative state. Each phase is characterized by specific growth modulatory properties. Initiation of the antiproliferative state with a single interferon dose requires 200 International Reference Units (IRU) per ml, depending on such factors including cell density, serum content in the medium and the length of IFN exposure. Initiation of the IFN response occurred within a 4 h incubation period. Even following this short exposure, the vast majority of cells would be sequestered in the G0/G1 cell cycle compartment. As measured by cytofluorimetry there was a 16-20 h delay in the initiation of the antiproliferative response. The antiproliferative response, as measured by changes in the distribution of cells in the cell cycle, was maximal at 24 h after treatment. This delay was equal to the doubling time of the Daudi cell. The antiproliferative response initiated by 200 IRU/ml of IFN was reversible if the IFN was removed and not replenished. Cells showed renewed growth approximately 40 h after treatment. The antiproliferative state could be maintained by additional interferon, but, even after such treatment, the antiproliferative state decayed in a dose-dependent fashion. This decay was proportional to the decrease in the biological activity of the interferon molecule itself. Finally, we showed that the antiproliferative state could be maintained by using only 40 IRU/ml. These cells were maintained in the antiproliferative state for an extended period of time.

Flow cytometric analysis of factors which influence the BrdUrd-Hoechst quenching effect in cultivated human fibroblasts and lymphocytes.

Human fibroblasts and lymphocytes were grown in bromodeoxyuridine (BrdUrd) containing medium, harvested after 27-71 hours and stained with the fluorochrome Hoechst 33258. Parameters that influence the BrdUrd quenching effect of Hoechst fluorescence at a given pH and ionic strength of the staining solution were determined. The parameters were determined in order to obtain high resolution histograms with differentiation of each cell cycle compartment for noncycling cells and for cycling cells in the first and second cycle. The BrdUrd concentration was found to be the most significant determinant, but cell density, cell type, dye concentration, type of Hoechst stain and incubation temperature during staining also contribute to the modulation of fluorescence of BrdUrd substituted nuclei. Optimization of these factors is required in order to achieve complete and well resolved cell cycle histograms by the BrdUrd-Hoechst flow cytometric technique.

Prognostic implications of ploidy and proliferative activity in human solid tumors

Ploidy and cell cycle compartment distribution were measured by DNA flow cytometry in 261 patients with a variety of different tumors. Eighty-one percent of all tumors were aneuploid, and 72% were hyperdiploid. Ploidy levels spanned a wide range from hypodiploid (maximum 30% < diploid controls) to hyperoctaploid (440% in excess of diploid controls) with mean and median values coinciding at a near-triploid DNA content. The proportion of cells with G 1 DNA content decreased with increasing hyperdiploid abnormality. While unrelated to biopsy site and to a number of host factors such as age, sex and race, both ploidy and cytokinetic parameters were markedly affected by histopathologic diagnosis. Patients with metastatic lung, breast, and GI cancer had higher ploidy levels than individuals with the corresponding primary tumors. Ploidy (except for one patient) remained constant, and G 1/0 proportions showed only minor variation by disease site and over a median observation time of 6 months. Prognostic factor analysis was performed in the subgroup of patients studied within 6 months from diagnosis. The adverse impact of low tumor G 1/0 proportion on survival was lost as the proportional hazard analysis was extended to include diagnostic subgroups. Accounting for histopathologic diagnosis, stage of disease, ploidy, and the proportion of tumor G 1/0 cells, the following sequence of adverse prognostic factors in order of their relative ranks was established: (1) absence of breast cancer (p = 0.001), (2) hypertriploid DNA index (p = 0.049), and (3) presence of metastatic disease (p = 0.079). Our study demonstrates that DNA content-derived information on instrinsic tumor cell features pertaining to cytogenetics and cytokinetics may provide an objective means of biologically relevant cancer classification.

Analysis of cell cycle in root meristems

We have analyzed the cell proliferation in a meristem assuming a single file model for root architecture. The meristem file appears to be built up by two clearly separated zones: the first going from the initial cell to the middle of the meristem and the second from the middle to the meristem boundary. The first half of the meristem shows an exponential age distribution for the cell population. In contrast, in the second half of the meristem, the cell kinetics of cycling cells strongly disagree with exponential kinetics and due to the compensation between the observed deviations in both halves, cell supply in the file meristem is in line with linear kinetics. However, we proposed that exponential kinetic equations offer a suitable approach to problems of cell cycle compartments and population age distributions in real meristems, where non-cycling cells cannot be identified inside the meristem, whether we consider the meristem as a whole or study a “window” inside it. Nevertheless, for more exact kinetic analysis, when estimating the proliferative fraction, the width of the “window” and its location along the axis must carefully be taken into account.

The ratio of RNA to total nucleic acid content as a quantitative measure of unbalanced cell growth.

Use of the metachromatic dye, acridine orange, to stain cells in suspension for flow cytometry allows for the simultaneous measurement of DNA and RNA content in individual cells. The relative RNA content as a function of total cellular nucleic acid content [alpha r = RNA/(RNA + DNA)] is a constant value, characteristic for particular cell lines during their exponential growth under optimal conditions. This ratio can be estimated for the G1A, G1B, S, and G2 + M cell cycle compartments. Changes in growth rate or the addition of antitumor drugs induces characteristic changes in the ratio either evenly throughout or at a particular phase of the cell cycle. Under such conditions, measurement of cellular DNA and RNA content provides a sensitive assay of any deviation from balanced cell growth. Unbalanced growth caused by suboptimal culture conditions or as a result of incubation with various antitumor agents is illustrated. Examples of unbalanced growth which are not correlated with cell viability as measured by cell clonogenicity are discussed.

Studies on the control of the cell cycle in cultured plant cells

When suspension-cultured sycamore (Acer pseudoplatanus L.) cells are deprived of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) cell division activity ceases after a brief period of exponential growth. Feulgen microdensitometry analyses showed that sycamore cultures starved of 2,4-D do not exhibit exclusive arrest in any particular cell cycle compartment. Regrowth, following readdition of 2,4-D to 2,4-D-starved cells, produced periodic oscillations in mitotic index and discontinuous increases in cell number. Detailed characteristics of the regrowth kinetics indicated involvement of periodic, rather than synchronous, growth. The results of this study suggest that the stimulation of cell division by 2,4-D is indirect and not due to the activation of a cell cycle control point to commit cells to division.

A rapid automated stathmokinetic method for determination of in vitro cell cycle transit times.

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G1. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G2 + M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.

New cell cycle compartments identified by multiparameter flow cytometry.

AbstractSimultaneous measurements of cellular DNA and RNA as well as estimates of the sensitivity of DNA in situ to denaturation by acid (which correlates with the degree of chromatin condensation) and cell ability to incorporate 5‐bromodeoxyuridine (BUdR), performed on over 40 different cell systems enabled us to subclassify cells into 12 functionally distinct cell cycle compartments. Quiescent cells had low RNA values, DNA very sensitive to denaturation, and they did not incorporate BUdR. Although in most cell systems they had 2C DNA content (G1Q), quiescent cells with higher DNA values (SQ and G2Q) were also seen. The G1 phase of exponentially growing cells had two distinct compartments, A and B. G1 cells entered S phase only from the B compartment. An increase in RNA and a decrease in the sensitivity of DNA to acid denaturation beyond a specific level characterized the transition of cells from the A to B compartments. Since the G1A to G1B transition was not linear but exponential, it may be assumed that a non‐deterministic event triggering cell progression into the cycle resides within G1A. Under adverse growth conditions the probability of a G1A to G1B transition decreased. Cells In G2 had DNA more sensitive to acid denaturation than S or G1 phase cells. Mitotic cells had the most condensed chromatin and their RNA content was twice that of G1A cells. Differentiated cells were characterized by 2C DNA (G1D) but, depending on the cell type, had varying RNA content and different degrees of chromatin condensation. A hitherto undescribed category of cells undergoing transition from quiescence to the cycle (or vice versa) was distinguished based on their intermediate values of RNA, sensitivity of DNA to acid denaturation and inability to incorporate BUdR at the rate characteristic for S cells. Depending on their DNA content (C) at the time of transition these cells could be classified as G1T (2C), ST (4&gt;C&gt;2) or G2T (4C).

A New Analytical Method For Determining Duration of Phases, Rate of Dna Synthesis and Degree of Synchronization From Flow‐Cytometric Data On Synchronized Cell Populations

L-cells synchronized by mitotic selection were investigated by flow-cytometry and the fractions of cells in the various cell cycle compartments were determined as a function of time. A new analytical evaluation procedure was developed, by which the mean transit-times of cells through various cell cycle phases can be calculated from these data. Three examples for application of the method are presented: (1) determination of the duration of G1, S, G2 + M and of the whole cell cycle; (2) calculation of the rate of DNA synthesis in several subcompartments of the S-phase; and (3) evaluation of the degree of synchronization at different stages of the cell cycle.

Analysis of cell cycle compartments of hepatocytes after partial hepatecomy.

An analysis of cell kinetic parameters as a function of intralobular localization of hepatocytes was performed using autoradiographic methods to obtain a basis for a quantitative description of cell cycle compartments after partial hepatectomy. The influx into the S compartment revealed a maximum in a lobular zone not immediately adjacent to the portal tract, with a decrease towards the perivenous and periportal area of the lobule. The maximum influx was found in the intermediate zone at 34 hr and a lower one in the perivenous parts of the lobule at 40 hr. The influx pattern at 56 hr was similar to the situation at 18 hr. The fraction of labelled mitoses in the whole liver and in subunits of the lobule disclosed a constant duration of S whereas the duration of G2 + M was prolonged from the periportal to the perivenous zone. A graphical model of kinetic events after partial hepatectomy is proposed, which describes the sizes of the various cell cycle compartments; the model successfully predicted the results of a continuous labelling experiment.

Cell cycle compartment analysis of Chinese hamster cells in stationary phase cultures.

The distribution of Chinese hamster cells with respect to the compartments of the cell generation cycle was studied in cultures in the stationary phase of growth in two different media. A measure of the state of depletion of the nutrient medium was formulated by defining a quantity termed the nutritive capacity of the medium. This quantity was used to verify that the cessation of cell proliferation is due to nutrient deficiencies and not to density dependent growth inhibition. Cell cultures in stationary phase were diluted into fresh medium and as growth resumed, mitotic index, cumulative mitotic index, label index and viability were measured as a function of time. The distribution of cells with respect to compartments of the cell generation cycle in stationary phase populations was reconstructed from these data. Stationary phase populations of Chinese hamster cells that retained the capacity for renewed growth when diluted into fresh medium were found to be arrested in the G1 and G2 portions of the cycle; the relative proportion of these cells in G1 increased with time in the stationary phase, but the sequence differs in the two media. In early stationary phase, in the less rich medium, more cells are in G2 than in G1. Also in this medium a fraction of the population was observed to be synthesizing DNA during stationary phase, but this fraction was not stimulated to renewed growth by dilution into fresh medium.

Dynamics of cell populations in the rat lens epithelium

Some statistics and kinetic parameters of the cell populations in the different regions of the normal 3·5–4·0-month-old rat lens epithelium have been determined. In all regions where cell proliferation occurred, the average duration of DNA synthesis was 14·5 hr, the average duration of mitosis was 1·42 hr, and a period of 6–7 hr intervened between DNA synthesis and mitosis. The 147,000 cells of the entire (aggregate) population were divided into 3–4 subpopulations based on location (regions or zones) in the epithelium, histological appearance, and differences in proliferative activity. The subpopulations were composed of cells in different functional states or compartments. These compartments, which imply no spatial grouping, were (1) metabolically active cells able to divide but not in the cell cycle, (2) proliferating cells, and (3) cells committed to differentiation into lens fibers. Most of the cells in the central region were in the first compartment; no more than 1–2% of the cells were in the second (cell cycle) compartment. This subpopulation, 27% of the aggregate population, had 4758 cells/mm2 with 0·05% in mitosis and 0·36% in DNA synthesis. All three compartments were represented in the peripheral region. This subpopulation had 11,015 cells/mm2 with 0·26% in mitosis and 2·63% in DNA synthesis. All of the cells in the equatorial subpopulation (12% of the aggregate population) were committed to differentiation. Cell turnover times in the central and peripheral regions were estimated to be 198 and 23 days, respectively.