Mammalian Cell Populations Research Articles

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.

Mitotic anomalies as the source of mutation of the genome: polyploidization cycles—segregation in mammalian cell population

Mitotic anomalies as the source of mutation of the genome: polyploidization cycles—segregation in mammalian cell population

Mitotic anomalies as the source of mutation of the genome: polyploidization cycles—segregation in mammalian cell population

Mitotic anomalies as the source of mutation of the genome: polyploidization cycles—segregation in mammalian cell population

Different drugs arrest cells at a number of distinct stages in G2

SEVERAL physical and chemical agents can induce susceptible mammalian cell populations to accumulate in the G2 phase of the cell cycle1–8. The question arises whether cells treated with different agents all accumulate at a single ‘restriction’ stage within G2 or, alternatively, stop at a variety of stages.

Aging in mammalian cell populations: A review

Research on in vitro aging and development is reviewed within a framework provided by three aspects of population growth — birth, canalization and death. Although there are periodic changes in the birth rate coupled to periodic changes in the in vitro environment, there do not appear to be any age-related changes in the birth rate of dividing cells. There appear to be distinct subpopulations of dividing and nondividing cells, each with its own characteristic morphological and functional properties; thus, Weiss-Kavanaugh theory may be applicable to the growth, maturation and senescence of mammalian cell populations. Substantial cell losses occur during subcultivation and transfer; correction for these losses requires substantial reinterpretation of existing experimental findings. The various measures of age are poorly correlated with one another; there is wide variation from population to population. A uniform measure of age has been found in the cell generation, the number of ancestors of a given cell. Studies of intrinsic variation in development can provide additional insight into the developmental process. The framework of birth, canalization and death permits reduction in the number of competing hypotheses of aging in mammalian cell populations. Additional experimental evidence is needed to discriminate among the remaining hypotheses.

Chapter 5 The Accumulation and Selective Detachment of Mitotic Cells

This chapter discusses the accumulation and selective detachment of mitotic cells. The sequence of events required for mitosis in a synchronous population must be comparable to the sequence observed in randomly dividing cells. Thus, the need for normalcy in the resulting population limits the synchrony technique employed. Synchronized populations of mammalian cells are frequently established by the selective harvest of mitotic figures from monolayer cultures. This approach avoids the problem of unbalanced growth reported for other procedures involving inhibitors of DNA synthesis. However, its application is restricted in two ways. First, the yield of mitotic cells is low since a small percentage of most asynchronous populations are dividing at any one time. Second, cells which demonstrate great cell-to-cell adhesion or enhanced attachment to culture vessels may have little advantage for selection at mitosis. Increased agitation of the culture fluids to displace mitotic cells may also remove loosely attached cells in interphase. Thus, only a few of the established lines of cells are routinely utilized in cell cycle studies and rarely are reports found using diploid cell strains. Preliminary attempts to obtain synchronous populations of a simian virus 40 transformed human cell line by selective harvest following incubation in Colcemid were not consistent owing to the detachment of interphase cells.

Kinetic Cell-Cycle Analysis of a Cultured Mammalian Cell Population

The parameters of the cell cycle are analyzed in terms of the stochastic theory of cell proliferation for a murine mastocytoma line. The cells were grown in suspension culture under steady-state conditions in a chemostat. Initial estimates of the parameters from synchronous growth indicate that agreement of the data with the model is obtained only if the model is modified to include an initial proliferating fraction of less than 100%, and a cell loss continuing throughout the course of the experiment. The analysis verifies that the modified theory adequately describes the data, and that similar parameters are obtained from both desynchronization and percent labeled mitosis experiments. The average cycle time from 10 desynchronization experiments was 8.24 +/- 0.52 h with a cellular standard deviation of 1.28 +/- 0.18. The combined parameter obtained by dividing the cellular standard deviation by the cycle time is shown to be a useful measure of biological variability well defined over many different experiments. The rate constant for cell loss is about 0.009 which gives an 8% cell loss per cycle. The cell loss is sufficient to account for the apparent deficit in initially proliferating cells. The initial distribution of the synchronous cells is qualitatively examined and is found to be peaked late in G(1) or early in S.

Determination of rates of DNA synthesis in cultured mammalian cell populations.

In cultures of a murine mastocytoma, endogenous synthesis of thymidine phosphates, as determined by the incorporation of [(3)H]deoxyuridine into DNA, was reduced within 15 min to less than 3% of control values by the addition of amethopterin (10 microM) in combination with hypoxanthine and glycine. If [(3)H]thymidine and unlabeled thymidine were added simultaneously with amethopterin, the increase with time of radioactivity in cellular DNA was linear at least between 30 and 90 min, while radioactivity in the acid-soluble nucleotide fraction remained constant during this time interval, indicating that intracellular thymidine nucleotides had the same specific activity as exogenously supplied [(3)H]thymidine. This permitted calculation of the amount of thymidine incorporated per hour into DNA of 10(6) cells. In conjunction with the base composition of mouse DNA, these results were used to calculate rates of DNA synthesis. Cell proliferation rate, cell cycle time, and the duration of the S period were not affected to any appreciable extent by the addition of amethopterin and thymidine. Rates of DNA synthesis, as derived from thymidine incorporation rates, were in good agreement with those derived from the measured mean DNA content of exponentially multiplying cells and rates of cell proliferation.

Editorial: The gene for the poliovirus receptor.

In cultures containing mixed populations of mammalian somatic cells, two cells of diverse origin may fuse and produce mono-nucleated hybrid descendants that can be grown indefinitely. Even cells of different species can be hybridized in this way. The nature of the human-mouse hybrid cell lines that Mary Weiss and I prepared in 19671 suggested that these hybrids could be used to determine in which chromosome a particular human gene is located. The property that distinguished the human-mouse lines from other interspecies hybrids made earlier in the laboratory of Ephrussi2 was that during growth, they eliminated human chromosomes preferentially. Since conditions . . .

A cytokinetic model for heterogeneous mammalian cell populations: II. Tritiated thymidine studies: The per cent labeled mitosis (PLM) curve

A model is described for the simulation of tritiated thymidine radiotracer studies in mammalian cell populations, which takes into account intracycle age-specific variations in the rate of DNA synthesis and tritiated thymidine incorporation, and their relation to tritium detection characteristics by radioautographic methods. The per cent labeled mitosis curve is considered in detail. The model suggests that the per cent labeled mitosis curve alone does not contain sufficient information for the precise characterization of age structure and growth behavior in heterogeneous cell populations; however, in many instances certain features can be identified which permit the classification of population growth behavior in accordance with broad, but nonetheless useful criteria.

Alternative mechanisms for homeostatic regulation of mammalian cell populations

A model for homeostatic regulation in mammalian tissues is analyzed. The model treats resting and active dividing cells, immature and mature non-dividing cells as separate populations. In the model, regulation is accomplished through control of the proportion of newly-formed cells that will become non-dividers. Four possible regulating substances, produced by dividing cells, non-dividing cells, mature non-dividing cells, and newly-formed cells respectively, are considered. Stability theorems are provided. System behavior in each instance depends on the relative values of the rate at which cells divide and the rate at which non-dividers die.

A cytokinetic model for heterogeneous mammalian cell populations: I. Cell growth and cell death

A general model is proposed for describing the growth behavior of mammalian cell populations, which features:(a) a cell cycle time distribution function with properties such that mean and variance increase with increasing population size; (b) maturation age and maturation rate functions which constrain the maturational pathways of individual cells; and (c) a death rate function, where cell death is construed as irreparable damage to a cell's reproductive apparatus. The biological implications of the model are discussed, and methods for relating the model to real cell systems by means of commonly used experimental techniques are described. The model is compared with earlier models.

Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells.

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [(3)H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [(3)H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis.

The effects of x-irradiation on histone acetylation and methylation in cultured mammalian cells

Random populations of mammalian cells in suspension culture with generation times ranging from 16–17 hr were subjected to 1600 rads of x-irradiation. Irradiated cells displayed a postirradiation division delay period of approximately 16 hr, during which period protein accumulation continued. DNA and histone biosynthesis ceased between 10 and 12 hr postirradiation. Approximately 15% of the irradiated population completed their first postirradiation division 16 hr after irradiation and very few were able to complete a second division. The DNA, protein and histone contents of the cell population rose during the division delay period, approximating levels attained by normal cells in the G 2 portion of their life cycle. Accumulation of labeled acetate in histone fractions F1, F2b and F3 of irradiated cells paralleled control values, while accumulation of incorporated acetate into histone fraction F2a of irradiated cells was depressed below control values between 16 and 24 hr after irradiation. Accumulation of labeled methyl groups as methyl-lysine derivatives in histone fractions F2a, F2b and F3 of irradiated cells was depressed below control values 24 hr postirradiation. No methylation of the lysine residues of histone fraction F1 was observed in either control or irradiated cultures during this period. Histone acetyltransferase and methyltransferase activity levels increased during the division delay period to follow the accumulation of cellular protein in irradiated cells. However, enzyme activity increased more rapidly than total protein, reached G 2 levels at 16 hr and decreased with resumption of cell division. These observations provide confirmatory evidence that mammalian cells irradiated under these conditions are arrested within the cell cycle in G 2 or a G 2-like state.

Synchronisation in vivo: Temporary inhibition of DNA synthesis in the rat embryo with hydroxyurea

As part of experimental studies on the synchronisation of mammalian cell populations in vivo, the proliferative response of rat embryo cells (18th day of gestation) was investigated following transplacental administration of hydroxyurea (HU). DNA synthesis was strongly inhibited by i.p. injection of a single dose of 0.25 or 0.5 mg HU/g body weight, for periods of 2.5 and 4.5 h, respectively. Reversal of blocks occurred rapidly due to the short half-life (45 min) of the inhibitor in the embryo. The resulting synchronizing effects were analysed in terms of mitotic indices and rate of 3H-TdR incorporation for a period of 18 h after administration of HU.

Natural and induced synchronous cultures.

It is probable that the initial impetus to develop synchronized populations of mammalian cells came from the demonstration, first in plant (1) and later in animal cells (2-4), that deoxyribonucleic acid (DNA) synthesis was a discontinuous process separated from the preceding and succeeding mitotic events by the G1 and G, phases respectively. The existence of discrete events within the lifetime of a cell means that most populations are in fact heterogeneous and contain subpopulations, each of which is undergoing certain events at any given time. From almost any biological point of view, each of these subpopulations would be expected to have different properties. Consequently, studies on such mixed populations yield some sort of average phenotype and in many instances might be uninformative or perhaps misleading. Recognition of this situation has led to attempts to obtain synchronized cultures where the entire population of cells is confined to a small segment of the cell cycle. The success of these attempts has made possible many important investigations. It may well be that as the technology becomes more sophisticated and the advantages of synchronous populations become even clearer the majority of experiments with mammalian cells will be carried out with such populations. Therefore, consideration of the methods of inducing synchrony, their advantages and disadvantages, and some of the more recent findings resulting from these methods seem appropriate. My purpose is to discuss some of the terminology and concepts involved in cell cycles and synchrony and the general uses and requirements of synchronous populations, and finally to attempt to analyse the advantages and disadvantages of various methods with respect to these requirements. Recent findings obtained with these and other techniques are not discussed in this paper. Since the nomenclature in this field is somewhat varied I first define my meaning for some of the terms to be used. "Natural synchronization" refers to the selection of specific populations from a growing population and "induced synchronization" describes situations where a population has been exposed to external perturbatio s to induce synchrony. In both cases the resulting populations are referred to as "synchronous." For reasons which will become cle rer later it is difficult to formulate a more precise general definition of a synchronous population. In some cell lines the cycle still consists of only four recognized events, the beginning and the end of mitosis, and the beginning and end of DNA synthesis. Considering how long the existence of th se events has been known, it is perhaps surprising how little information we now possess on the timing of other events which must occur. In a recent review (5) Mitchison was able to list only eight enzymes in four cell systems for which the cyclic pattern had been determined and even in this short list there appeared to be inconsistencies. It is likely, however, that other markers will be added in the near future.

Synchronization of Mouse L-Cells by a Velocity Sedimentation Technique

Differential velocity sedimentation at unit gravity has been used to separate an asynchronous population of mammalian cells into fractions synchronized in all phases of the cell cycle. Better enrichment was obtained for G(1) and S phases than for G(2)-M phase. Electronic cell volume measurements of the fractions indicated that the separation was primarily dependent on cell size, and an experimentally determined sedimentation coefficient agreed very well with its predicted value. Sources of dispersion in the separation (including the contribution of cell density heterogeneity) were quantitated and found to be insufficient to explain all of the observed dispersion. Both the limitations and the applications of the technique are discussed.

A formalism describing the kinetics of some mammalian cell populations

Mammalian cell populations are represented mathematically in terms of occupancy numbers of subdivisions of the mitotic cycle. The occupancy numbers that determine the fraction of cells in these subdivisions form components of a finite-dimensional vector (the state vector). Events in the life of a cell population are described as transformations of the state vector. A sampled data system formulation permits the operators effecting these transformations to be expressed as real matrices. Specific matrices are presented which describe normal growth, exposure to X-irradiation, and exposure to chemotherapeutic agents. The resulting equations are applied to calculating decay of synchrony, percentage of labeled mitoses curves, and labeling index, as well as to the problem of control of murine leukemia.

The action of arabinosylcytosine on synchronously growing populations of mammalian cells

The action of arabinosylcytosine on synchronously growing populations of mammalian cells

Cytogenetical Polymorphism and Evolution in Mammalian Somatic Cell Populations in vivo and in vitro

Co-operation as well as competition between cells affects the evolution of cell populations. Thus studies on chromosomal variation in relation to age, environment and mechanisms of cytogenetical instability have led to the discovery of a new kind of balanced chromosomal polymorphism.