REGULATION OF INITIATION OF DNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Induction of DNA Synthesis and Cell Division by Isoleucine and Glutamine in G1-Arrested Cells in Suspension Culture
TL;DR: Suspension cultures of Chinese hamster cells, which had stopped dividing and were arrested in G1 following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine.
Abstract: Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G1 following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G1, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 105 cells/ml. Under similar conditions, about 4 x 10-6 M isoleucine was required for all G1-arrested cells to progress through cell division. In contrast, 1 x 10-4 M glutamine was necessary for maximum initiation of DNA synthesis in G1 cells, along with sufficient isoleucine. A technique for rapid production of G1-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G1 after 30 hr.
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TL;DR: It is concluded that the factors that control growth are probably polypeptide hormones, or hormone-like materials, as well as a variety of low molecular weight nutrients.
Abstract: Recent studies of the control of growth of mammalian cells are considered and it is concluded that the factors that control growth are probably polypeptide hormones, or hormone-like materials, as well as a variety of low molecular weight nutrients. An understanding of the control of growth of mammalian cells will probably be found in the complex interactions of cells with these common types of materials.
523 citations
TL;DR: This chapter reviews the current state of knowledge of autonomous parvovirus structure and replication and explores the strategies employed by these viruses—at the molecular and cellular levels—to parasitize their various hosts.
Abstract: Publisher Summary Various aspects of the natural history of autonomous parvoviruses are beginning to be understood in some detail, mostly through the analysis of tissue culture analogs of the pathogenic processes observed in the whole animal This chapter reviews the current state of knowledge of autonomous parvovirus structure and replication The chapter explores the strategies employed by these viruses—at the molecular and cellular levels—to parasitize their various hosts Members of the autonomous parvovirus group are capable of productive replication without the aid of a helper virus in the majority of host cells Cell cycling—although necessary—is not sufficient for the lytic, productive replication of individual parvovirus strains The differentiated state of the host cell is of paramount importance It has also been reported that the surface structure of the viral particle—as monitored by the expression or absence of certain antigenic configurations—may have a dramatic influence on the ability of the virus to replicate in a particular host cell type, and that this capsid-mediated specificity may well involve intracellular interactions with host cell factors, as well as, or rather than, differences in binding to a specific cell surface receptor
494 citations
Journal Article•
TL;DR: Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G1 phase of the cell cycle.
Abstract: Synchronization of mammalian cells is essential for investigations involving cell proliferation. A simple method for obtaining synchrony in all types of cells, through several cycles and with minimal overall metabolic perturbations, has not yet been available. We describe a procedure for synchronizing normal as well as tumor cells reversibly in the G 1 phase of the cell cycle using Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. This method of synchronization was successful with all cell lines tested, including normal and tumor cells of mouse, hamster, and human origins. For example, when MCF-7 human breast cancer cells were synchronized with Lovastatin and released by the addition of mevalonic acid (the product of the reaction catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase), 3 phases of accelerated thymidine incorporation into DNA corresponding to 3 S phases of the cell cycle occurred during a 90-h period of cell replication. Thymidine incorporation was decreased to ≤4% during the initial lag of 18 h before the first S phase, and maximum incorporation was then achieved after only 6 h. The antibody Ki-67, which detects a nuclear antigen associated with proliferation, was present in cells arrested with Lovastatin. This fact, together with the lack of thymidine incorporation during the initial lag time, indicates that the cells were arrested in the G 1 and not in the G 0 phase of the cell cycle. Furthermore, in synchronized tumor-derived human breast epithelial cells, histone H4 RNA was low after Lovastatin release and increased with the onset of DNA synthesis. Concomitant synthesis of DNA and histone H4 RNA expression could be observed for 2 cycles. Minimal perturbations of general metabolic functions occurred since the rate of RNA, protein, and initial DNA synthesis were unaffected by Lovastatin, as evidenced by [ 3 H]uridine, [ 3 H]leucine, and initial [ 3 H]thymidine incorporation. Finally, while the Lovastatin-induced synchronization was overcome by mevalonic acid, addition of squalene or cholesterol-ethanol had no such effect. Thus, Lovastatin appears to prevent formation of an early intermediate in the cholesterol pathway that is essential for progression of cells through early G 1 phase.
467 citations
TL;DR: Results are strongly suggestive of the operation of a cell size requirement for entry into nuclear division, confirmed by a nutritional shift-down, where nuclear division and cell division are stimulated after the shift.
422 citations
TL;DR: Especially significant in view of the importance of glutamine metabolism are an insensitivity of the new system to stimulation by either insulin or glucagon, and its distinct enhancement on starvation of the cells with respect to amino acids, suggesting a second system has been found to show adaptive regulation.
395 citations
References
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TL;DR: A new nutrient mixture, F10, has been prepared in which each component has been added at the experimentally determined optimum concentration and revealed improved growth rate and reliability in a fetuin and albumin supplemented medium and excellent short term growth of human white blood cells for chromosome preparations.
865 citations
TL;DR: Evidence is provided which firmly associates the agent first recovered by Eaton with lower respiratory tract illness of man and the demonstration that naturally acquired antibody offered protection against such illness supports the contention that the agent is a respiratory tract pathogen.
Abstract: Recent volunteer and controlled epidemiologic field studies have provided evidence which firmly associates the agent first recovered by Eaton in 1944 with lower respiratory tract illness of man.1-3 A serologic response to the Eaton agent occurs in approximately 90 per cent of pneumonia illnesses in which cold agglutinins develop during convalescence as well as in a significant but variable proportion of cold agglutinin-negative pneumonias.2 4 The development of pneumonia and other forms of respiratory disease following the administration of tissue culture-grown Eaton agent to volunteers and the demonstration that naturally acquired antibody offered protection against such illness supports the contention that the agent is a respiratory tract pathogen.5 For many years, the agent was tentatively classified as a virus. The large size of the agent (180-250 m,i) and its sensitivity to streptomycin and various tetracycline derivatives, however, posed some difficulty with such a classification.6-8 Recently, Marmion and Goodburn were able to visualize small cocco-bacillary bodies on the mucous layer covering the bronchial epithelium of the Eaton agent infected chick embryo.9 The distribution of these bodies corresponded with the localization of Eaton agent as visualized by the fluorescent antibody technique. These workers also demonstrated that the Eaton agent was inhibited by an organic gold salt. Clyde visualized extracellular "colony-like" structures in stained preparations of infected tissue culture; these structures corresponded with the areas of specific immunofluorescence.U? Both groups of workers suggested the possibility that the Eaton agent may be a "pleuropneumonia-like organism" (PPLO) rather than a virus. Cultivation of the organism in cell-free media, however, was not achieved. T
683 citations
TL;DR: A high-speed flow system for quantitative determination of fluoresence of cells containing fluorochrome has been developed and results compare well with results of other independent methods.
Abstract: A high-speed flow system for quantitative determination of fluoresence of cells containing fluorochrome has been developed Feulgen-DNA distributions in populations of tissue culture cells and human leukocytes havebeen measured at a rate of 104 to 105 cells per minute and compare well with results of other independent methods
485 citations
TL;DR: A convenient, reliable method for chromosome delineation of animal cells grown as monolayers on glass has been applied to human, opossum, and Chinese hamster cells, finding that cells of malignant, aneuploid constitution have been maintained in active growth for 3 years and hundreds of generations, with stable chromosomal and metabolic characteristics.
Abstract: A convenient, reliable method for chromosome delineation of animal cells grown as monolayers on glass has been applied to human, opossum, and Chinese hamster cells. Tissue cultured cells from 5 different, normal organs of 7 different human subjects uniformly displayed the expected chromosome number of 46 and showed no variations in morphology or number other than the expected sex differences and a small incidence of polyploidy. The chromosomes of normal cells from the American opossum were as uniform as those of human cells. Cells of the inbred Chinese hamster demonstrated appreciable karyotype variability, the cause of which is under investigation. The chromosome number and morphology of cells from normal human tissues have remained constant after more than 5 months of continuous, rapid growth in tissue culture involving scores of vessel transfers and a number of generations equivalent to many billions of progeny. By the use of routine recloning, even cells of malignant, aneuploid constitution have been maintained in active growth for 3 years and hundreds of generations, with stable chromosomal and metabolic characteristics. The cells of the American opossum and Chinese hamster which possess only 22 chromosomes have been established in vitro and are especially suitable for genetic studies. The readily recognizeable Y and X chromosomes of the male opossum are particularly favorable as cytological markers. Photomicrographs of the chromosomes of the various cells employed are presented.
370 citations
TL;DR: Equations are presented describing the accumulation of cells at any part of the life cycle as a result of addition of specific blocking agents, which makes possible analysis with relatively high resolution of the distribution of cells throughout thelife cycle in normal cultures or those treated with various agents.
332 citations