D. Killander

A quantitative cytochemical investigation of the relationship between cell mass and initiation of DNA synthesis in mouse fibroblasts in vitro

The mass of cells entering interphase after cell division, initial mass (mo), and the cell mass at the stage of initiation of DNA synthesis (mG1) were determined in 21 populations of mouse fibroblasts in vitro (L-cells) which exhibited different mo values. Cells in populations with low initial masses had to synthesize more mass before the onset of DNA synthesis than cells in populations with high initial masses. In addition, these smaller cells required more time for this mass synthesis than the bigger cells. The generation times were found to be almost identical for the different L-cell populations. Cells in populations with low initial masses therefore spent a relatively long time in the G1 and a relatively short time in the S + G2 phases of the cell cycle. The opposite applied when the cell populations had high initial masses. These findings support the hypothesis that initiation of DNA synthesis is dependent on the cell mass.

Quantitative cytochemical studies on interphase growth: I. Determination of DNA, RNA and mass content of age determined mouse fibroblasts in vitro and of intercellular variation in generation time☆

A combination of quantitative cytochemistry and time-lapse cinematography has been used for studying the DNA, RNA and protein (dry mass) synthesis during interphase of mouse fibroblasts in vitro. This method is mainly associated with two error factors, i.e., variation in generation time and variation in the amount of DNA, RNA and protein of cells in the same stage of interphase. The magnitude of these factors were compared and were discussed in connection with the relation between growth and division. The cellular DNA, RNA and mass content doubled during interphase; there was no evidence for loss of material during mitosis. There was an error in RNA and, to greater extent, in mass distribution to the daughter cells at mitosis, while the error of DNA distribution was of the same order as the error of measurements. The G 1 (preceding DNA synthesis), the S (DNA synthesis) and the G 2 (following DNA synthesis) periods were 8, 6 and 5 hr, respectively, and the rate of DNA synthesis was constant. RNA and mass increased throughout the entire interphase. The rate of mass synthesis was higher in the latter part of interphase. It was not possible to judge whether the rate of RNA synthesis was constant or changed during interphase. DNA synthesis seemed to be initiated at a critical cellular mass. The importance of such a critical mass was discussed with regard to maintenance of the size homogeneity of the cell population, intercellular variations in generation times and the “trigger” mechanism for mitosis.

Cytochemical characterization of deoxyribonucleoprotein in individual cell nuclei: Techniques for obtaining heat denaturation curves with the aid of acridine orange microfluorimetry and ultraviolet microspectrophotometry

Abstract Simple methods for obtaining heat denaturation curves on the deoxyribonucleoprotein DNP of individual cell nuclei or intact cells are described. Cells or nuclei adhering to microscopic slides were heated in a salt solution (SSC) containing formaldehyde. After heating the slides were rapidly cooled. Both the formaldehyde and the rapid cooling served to prevent renaturation of denatured DNA. The heated preparations were analyzed for changes in single- and double-strandedness by UV microspectrophotometry and by microfluorimetry after acridine orange staining. These techniques were used to obtain melting profiles of DNP from non-stimulated and PHA stimulated lymphocytes, erythrocyte nuclei activated by cell fusion with HeLa cells, normal erythrocyte nuclei and isolated HeLa nuclei. The results obtained show that the activation of a large number of genes in both human lymphocytes and hen erythrocyte nuclei is paralleled by striking changes in the melting profiles of nuclear DNP.

Quantitative cytochemical studies on interphase growth: II. Derivation of synthesis curves from the distribution of DNA, RNA and mass values of individual mouse fibroblasts in vitro

Abstract An investigation was made of the kinetics of nucleic acid and protein (dry mass) synthesis during interphase of mouse fibroblasts in vitro. Quantitative microspectrophotometric and microinterferometric methods were employed for the determination of the cellular DNA, RNA and mass content. The time course of DNA, RNA and mass synthesis during interphase was obtained by analyzing the distribution of the DNA, RNA and mass content of individual cells from large samples in fixed populations. The main source of error in this type of analysis lies in momentary variation, i.e. variation in the amount of DNA, RNA and mass among cells in the same physiological stage of the cell cycle. The influence of momentary variation on this method of analysis was examined in order to be able to make corrections for it. The accuracy of the results obtained was discussed in relation to sample size and momentary variation. The G1, S and G2 periods were found to be 9, 6 and 4 hr respectively. During the S period DNA was synthesized at a constant rate. Contrary to DNA, RNA and mass were synthesized throughout the whole interphase. The rate of synthesis of both RNA and mass was 2–2.5 times faster at the end of interphase than it was at the beginning. The increase in the rate of RNA synthesis seemed to occur predominantly during the second half of interphase at the same time as the doubling of the amount of DNA in the cell. The rate of mass synthesis, however, seemed to increase over the whole of the interphase period in proportion to the amount of RNA.

Activation of deoxyribonucleoprotein in human leucocytes stimulated by phytohemagglutinin: I. Kinetics of the binding of acridine orange to deoxyribonucleoprotein☆

Abstract In accordance with previous findings a pronounced and rapid increase in the fluorescence intensity of fixed human leucocytes stained with acridine orange (AO) was observed only a few minutes after stimulation with phytohemagglutinin (PHA). This increase in fluorescence intensity was ascribed to an increased accessibility of AO binding sites in the deoxyribonucleoprotein (DNP) complex. The measured AO fluorescence was not caused by staining artifacts due to altered autofluorescence of leucocytes, nonspecific AO binding or changes in cell membrane permeability. On the average, the number of AO binding sites increased by a factor of 2 after PHA stimulation. In activated cells, about half of all binding sites in DNP were accessible to AO. PHA-P and PHA-M induced the same final increase in AO binding sites in DNP although the rate of increase in AO binding sites was lower when PHA-M was used. Since the same decrease in rate could be obtained by lowering the PHA-P doses, it was concluded that the difference in the activity between the two PHA fractions might be due to different contents of the stimulating factor. Leucocytes sampled from infected or recently vaccinated donors exhibited a spontaneous increase in AO binding sites in DNP, which, in most cases, could not be further increased by PHA. The increase in AO binding sites in DNP, which seems to be one prerequisite for later cell growth, is apparently due to weakened interaction between DNA and concomitant proteins. This hypothesis was supported by the finding that acetylation of amino groups in the protein moiety of the DNP complex resulted in a marked increase in AO binding sites in nonstimulated cells while no such increase was found in PHA stimulated cells having a large number of accessible groups for AO binding even without acetylation.

Quantitative cytophotometric and autoradiographic studies on the rate of protein synthesis during interphase in mouse fibroblasts in vitro.

An investigation was made of the rate of the overall protein synthesis during interphase growth in mouse fibroblasts in vitro (L-cells). Quantitative microspectrophotometric and microinterferometric methods were used for the determination of the cellular content of DNA, RNA and dry mass. The stage which the cell had reached in its interphase development was determined in two different ways. Firstly, the cell age, expressed in hr after the last cell division, was determined in a direct way by means of time-lapse cinematography. Secondly, the cell age was determined in an indirect way from the relationship between cell mass and relative cell age (the fraction of the total interphase time). This relation was derived from the mass distribution of individual cells in the growing fibroblast population. The amount of 14C-leucine, 3H-leucine and 35S-methionine incorporated into the cell protein after pulse incubation was quantitated by means of autoradiographic methods and used as a measure of the overall rate of protein synthesis in the cell. The absorption of tritium radiation in the cell was taken into account. The rate of incorporation of protein precursors into the growing cell increased over the whole of interphase by a factor of approximately 2. This result is in very strong agreement with previously published interferometric data on L-cells [17], which showed that the cellular dry mass also grew at an increasing rate over the whole of interphase by a factor of 2. The correlations between the number of grains over the cells and their DNA, RNA and mass content were investigated. The grain counts were found to have the best correlation with the cellular content of RNA and mass. These results support the idea that the total capacity for protein synthesis during interphase growth is controlled by the existing number of ribosomes in the cell.

Cytochemical observations on the nucleolus-ribosome system: Effects of actinomycin D and nitrogen mustard☆

Abstract Cytochemical techniques have been developed for the quantitative determination of the DNA, RNA and proteins (dry mass) in individual cells in large cell populations. These methods have been employed for the study of the effects of nitrogen mustard and actinomycin D on the nucleolar system of Ehrlich ascites cells in vivo and a line of normal mouse fibroblasts maintained in cell culture. Parallel studies on the nucleoli of individual cells have been made with ultraviolet microscopy and spectrophotometry. Nitrogen mustard, in certain concentrations, blocked mitosis but did not block DNA synthesis, as adjudged by the accumulation of DNA to pre-mitotic levels in non-dividing cells. Concurrently, the total cellular RNA and the total cellular mass of such cells increased far beyond normal values, with an overdevelopment of the nucleolar apparatus. The experiments with actinomycin D support the conclusion that this antibiotic inhibits the total synthesis of DNA-dependent RNA, and consequently, the synthesis of cellular proteins. Exposure to effective concentrations of actinomycin D results in marked inhibition of the synthesis of nucleolar RNA and proteins, as well as cytoplasmic RNA and proteins. Cytochemical analyses of cell populations exposed to actinomycin D and nitrogen mustard, either alone or in combination, together with studies of the nucleolar apparatus in individual cells in such populations, support the concept that the nucleolus-associated chromatin is concerned primarily with the synthesis of ribosomal RNA—either directly, or by the mediation of a “messenger” fraction of RNA. Such a function is considered to be consistent with the polygenic character of the nucleolus-associated chromatin, suggesting the presence of many repeating DNA cistrons complementary to ribosomal RNA, as described recently in E. coli .

Activation of deoxyribonucleoprotein in human leucocytes stimulated by phytohemagglutinin. II. Structural changes of deoxyribonucleoprotein and synthesis of RNA.

Abstract The increase in acridine orange (AO) binding sites in the DNP complex occurring in human leucocytes during the first 60 min after PHA stimulation was found to be paralleled by a decreased heat stability as well as a more regular arrangement and stretching of the DNP complex. These findings support the theory that the binding between DNA and surrounding proteins in the DNP complex is altered by the action of PHA and that thereby increasing numbers of DNA phosphate groups become uncovered. Increasing amounts of low ordered ribonucleic acids were found in mononuclear leucocytes if more than half of all AO binding sites became accessible to AO. Above this value a correlation was found between the number of AO binding sites in DNP uncovered during the initial phase of PHA stimulation, and the amount of low ordered RNA found 1 and 48 h after PHA administration. The data suggest that this activation of the DNP complex acts as one necessary trigger for RNA synthesis and that the role of basic proteins in gene regulation might be confined to this trigger process preceding a more specific and refined mechanism for gene regulation.

CYTOCHEMICAL EVALUATION OF METABOLIC INHIBITORS IN CELL CULTURE.

Abstract A method for the cytochemical evaluation of the mechanism of action of potential metabolic inhibitors in cell culture, utilizing the rapid-scanning, high-resolution, automatically integrating biophysical instrumentation developed for cell population analyses is described. This method permits the in situ determination of the amount of DNA, RNA, and protein in individual cells in populations of heterogeneous cells, and the effects of inhibitory agents on these biosynthetic processes. The utility of the method has been demonstrated by the cytochemical evaluation of a series of inhibitory agents selected on the basis of what is known of their mechanisms of action with respect to inhibition of mitosis, synthesis of nucleic acids, or protein. The results of cytochemical analyses of populations of cells exposed to these agents agreed in general with what is known of their mechanisms of action from other studies. In addition to the advantages provided by a method based upon the evaluation of the effects of an inhibitory agent on single cells in heterogeneous populations, cytochemical analyses allow determination of the phase of the cell growth cycle in which inhibition occurs; information which is not readily obtained from other methods of assay. The advantages of such a method for the selection of candidate inhibitory agents for sequential or combination therapy of neoplastic disease, as well as detailed studies in cell physiology, are evident.

Cytochemical differences between mammalian cell lines of normal and neoplastic origins: Correlation with heterotransplantability in Syrian hamsters

Abstract The cytochemical characteristics of uncloned cell lines derived from normal and neoplastic sources have been determined by means of recently developed biophysical “population study techniques”. Cell lines derived from neoplastic sources exhibited a greater variability than cell lines similarly derived from non-neoplastic sources with respect to the amount of cytoplasmic proteins and cytoplasmic RNA per cell, an observation which is in accord with the results of earlier studies on populations of neoplastic cells derived from in vivo sources. It is of interest that certain cytochemical attributes of populations of neoplastic cells in vivo are retained by such populations when isolated and maintained in vitro, and that these cytochemical attributes correlate with the heterotransplantability of such cell lines to the cheek pouch of the Syrian hamster. The variability of the per cell amounts of cytoplasmic proteins and cytoplasmic RNA in these populations of neoplastic cells is of such magnitude that it must represent quantitative variations in the ribosomal RNA content of neoplastic cells. In view of the evidence for the participation of the nucleolar system in the synthesis of ribosomal RNA, the cytochemical variability of neoplastic cells may reflect disturbances in the nucleolus-nucleolus-associated chromatin biosynthetic system.