G.E. Foley

Chemical differentiation with fluorescent alkylating agents in Vicia faba metaphase chromosomes

With the purpose of searching for chemical bases for the transfer of differentiation information through metaphase, efforts have been made cytochemically to identify chemical and physiochemical differences between different parts of metaphase chromatin in Vicia. n nEquipment has been developed for very high resolution quantitative spectrophotometric determinations on chromosomes and for fluorimetric work with low irradiation load on the object. n nA bifunctional highly fluorescent quinacrine mustard was shown to give chromosome breaks preferentially localized to heterochromatic chromosome regions (identifiable by cold treatment) in the M-chromosome. This and other fluorescent mustards of different types accumulated preferentially in the same regions, which then appear as strongly fluorescent bands. In these bands the fluorescence per unit DNA is higher than in the rest of the chromosome. Possible mechanisms for the fluorochrome-binding are discussed.

DNA-binding fluorochromes for the study of the organization of the metaphase nucleus.

Fluorescent DNA-reagents with different modes of binding have been used for the purpose of identifying chemical differences along metaphase chromosomes. The DNA-distribution pattern along the chromosomes, determined with very high resolution procedures, has been used as reference when studying the differential uptake of different chromosome regions of the compounds. For quantitative measurements of the fluorescence of very small objects a special system has been developed. Several types of plant material and also Chinese hamster have been investigated. Great and reproducible differences in the uptake of the fluorescent reagents by different chromosome regions were observed. In Trillium a good correlation was found between regions reacting with several different fluorescent compounds and heterochromatin as defined by cold treatment. The looseness of the several presently used definitions of heterochromatin is stressed.

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 .

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.

Inhibition of DNA synthesis and chromosome aberrations in cultured Ehrlich ascites tumor cells following treatment with luteoskyrin

Abstract The effect of the hepatotoxic and tumorigenic bis-polyhydroxydihydroanthraquinone luteoskyrin on nucleic acid and protein metabolism was studied in asynchronous cultures of Ehrlich ascites tumor cells. Exposure to luteoskyrin (1.0 μg/ml; 1.74 × 10−6 M) resulted in a gradual inhibition of DNA synthesis and cell multiplication, although RNA and protein synthesis continued for some time after DNA synthesis was almost completely arrested. Pulse-labeling with 14C-2-thymidine, 14C-2-uridine and 14C-2-leucine in the presence of luteoskyrin revealed an exponential decline of the per cell rate (cpm/10 μg DNA) of DNA synthesis, while the per cell rates of RNA and protein synthesis were only partially inhibited over a period of 126 h; for example, after 73 h in the presence of luteoskyrin the per cell rate of DNA synthesis was inhibited by 93% whereas RNA and protein synthesis were inhibited only 48% and 40%, respectively. Luteoskyrin-treated cultures contain increased numbers of multinucleate cells, and cells which accumulate Sudan Black positive material. Luteoskyrin-resistant cells (growing in the presence of 1.0 μg/ml and at higher concentrations) were isolated by gradual adaptation to increasing concentrations of luteoskyrin. Abnormally long and partially “stretched” metaphase chromosomes were observed in these resistant cells after cultivation for 3–5 months in the presence of luteoskyrin (1.0 μg/ml). It is suggested that luteoskyrin exerts the described effects primarily by interference with the mechanisms of DNA replication.

Cytochemical population analyses of continuous cultures of human lymphoblasts derived from acute lymphoblastic leukemia

Abstract The cytochemical characteristics of populations of human lymphoblasts (CCRF-CEM cells) in continuous log-phase suspension cultures derived from the peripheral blood buffy coat of a child with acute lymphoblastic leukemia have been determined by the use of high-resolution, rapid-scanning biophysical population analysis techniques. The pattern of distribution of the DNA content in individual cells was consistent with that of a diploid or near-diploid population in asynchronous log-phase growth, and there was no suggestion of two distinct populations, despite the presence of an extra “minute” chromosome in ca 50 per cent of the cells. The mean RNA : DNA ratio of these cells was 0.7:1.0. The distribution patterns of total nucleotide content and dry mass per cell indicated a somewhat greater degree of variability than would be expected in populations of normal cells, although this variability was not as great as that observed with populations of neoplastic cells in which individual cells were characterized by a large cytoplasmic mass. These continuously cultured human leukemic lymphoblasts resemble those examined directly in the peripheral blood buffy coat of patients with acute lymphoblastic leukemia with respect to the pattern of variability of total nucleotide and dry mass per cell.

Effects of 4-aminopyrazolo (3, 4-d) pyrimidine in combination with guanine on nucleic acid and protein synthesis by Erlich ascites cells in culture.

Abstract Quantitative microspectrophotometric and microinterferometric analyses in combination with biochemical methods were used to examine the effects of 4-aminopyrazolo(3,4-d)pyrimidine (APP, 5 × 10−6 M) in combination with guanine (2 × 10−4 M) on the synthesis of DNA, RNA, and protein in asynchronously growing Ehrlich ascites cells in culture. Exposure to such concentrations of APP + guanine resulted in a nonlethal inhibition of cell multiplication. Pulse-labeling with 14C-thymidine, 14C-uridine and 14C-1-leucine in the presence of APP + guanine revealed an exponential decline in the per cell rate of DNA synthesis, while the per cell rate of RNA synthesis, as adjudged by 14C-uridine incorporation, was not significantly inhibited, and the per cell rate protein synthesis was inhibited only ca 50 per cent. Determination of the cellular content of DNA, RNA, and protein by cytochemical population analyses revealed an accumulation of cells in G1 or early S-phase in APP + guanine-treated cultures. These viable, but non-dividing cells continued to incorporate 14C-uridine and 14C-leucine, but there was no net increase in either RNA or protein per cell; thus continued synthesis must be limited to RNA and protein which is constantly in the process of degradation and resynthesis. It suggested that APP + guanine exerts its inhibitory effect by interference with the synthesis and pool sizes of intracellular purine nucleotides.