G<sub>2</sub> + M arrest of cultured mammalian cells after incorporation of tritium‐labeled nucleosides

März, Richard; Zylka, J M; Plagemann, Peter G.W.; Erbe, John; Howard, Roger B.; Sheppard, J. R.

doi:10.1002/jcp.1040900102

Cited by 32 publications

(14 citation statements)

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“…The cell density of attached cells did not increase with time in culture as it did in cultures not continuously exposed to [3H]thymidine (Table 2, 7th column). This observation was partly due to detachment of cells which could be seen floating in the medium, possibly because of radiation damage resulting from the continuous exposure to [3H]thymidine (Marz et al, 1977).…”

Section: Ld V Replication In Long-term Primary Maerophage Culturesmentioning

confidence: 99%

Replication of Lactate Dehydrogenase-elevating Virus in Macrophages: 1. Evidence for Cytocidal Replication

Stueckemann

Ritzi

Holth

et al. 1982

Journal of General Virology

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SUMMARYCultures of starch-elicited peritoneal mouse macrophages in medium containing macrophage growth factor (MGF) were infected with lactate dehydrogenaseelevating virus (LDV) and, after various times in culture, LDV production was monitored as a function of time by infectivity titrations in mice, by measuring [3H]uridine incorporation into LDV RNA and extracellular LDV, by autoradiographic analysis of the proportion of productively infected cells and by electron microscopy. Regardless of the age of the cultures when infected with LDV, only a small proportion of the macrophages (generally between 3 and 20% of the total) became productively infected after a primary infection; maximum virus RNA synthesis and virus production occurred during the first 24 h after infection and then decreased precipitously. Productively infected macrophages could be readily recognized in electron micrographs of 24-h infected macrophage cultures and in sections of spleens from 24-h infected mice by characteristic morphological alterations. These consisted of formation of clusters of double-membrane vesicles with a diameter of 100 to 300 ~m, budding of nucleocapsids into vesicles with single membranes and accumulation of mature virions in these vesicles. One to 4 days later, however, such cells were no longer found in infected cultures or spleens of infected mice and superinfection did not restimulate LDV replication. Cultures established with macrophages from 1-day LDV-infected mice also did not support LDV replication. We conclude that LDV replication in cultures or mice is limited to a single cycle in a subpopulation of macrophages and that infection leads to cell death and rapid phagocytosis of the dead cells by the resistant, uninfected macrophages.

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Section: Ld V Replication In Long-term Primary Maerophage Culturesmentioning

confidence: 99%

Replication of Lactate Dehydrogenase-elevating Virus in Macrophages: 1. Evidence for Cytocidal Replication

Stueckemann

Ritzi

Holth

et al. 1982

Journal of General Virology

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“…Amplification of the sensitivity of those methods through the use of radioactive precursors is restricted by the perturbations caused in the cycles of proliferating cells (Ehmann et al . 1975, Marz et al . 1977, Pollack et al .…”

Section: Discussionmentioning

confidence: 95%

Effects of Heavy Ions on Rabbit Tissues: Analysis of Low Levels of DNA Damage in Retinal Photoreceptor Cells

Keng

Bergtold

Lett

1983

International Journal of Radiation Biology and Related Studies

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When suitable precautions are taken, sedimentation of DNA through reoriented alkaline sucrose gradients in zonal rotors can be used to determine small amounts of DNA damage in mammalian cells without resorting to radioactive precursors. Hence, the method is especially useful for studying the efficacies of DNA repair mechanisms in the neurons of the central nervous system (CNS) and the accumulation of DNA damage in the ageing CNS. Here we describe the technique as it has been used to examine the DNA damage occurring in the photoreceptor cells of the retina of the New Zealand white rabbit during the course of natural ageing or after exposure to heavy ions. This article is an integral part of a series of reports of the latter studies (Lett et al. 1980, Keng and Lett 1981, Cox et al. 1981, 1982, Keng et al. 1982). With the same analytical technique, very low levels of radioactive DNA precursors can be used to advantage in investigations of proliferating cells.

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“…A, unperturbed HPBL; B, 3 h 3H-TdR-labeled; C, 6 h 3H-TdR-labeied; D, 12 h 3H-TdRlabeled; E, 18 h 3H-TW-labeled; F, 24 h 3H-TdR-labeled. In this experiment G1Q was located on channel 17 DNA and the bottom of G2A was located on channel 18 protein.…”

Section: Cell Kinetic Effects Of 3h-tdr In the Presence Of Colcemidmentioning

confidence: 99%

“…More recently, flow cytometric analysis of DNA content per cell has permitted the rapid approximation of cell kinetic effects from radiation exposure (1,16,17,20,21,29). The radiation-induced changes in the relative numbers of cells in G2 +M reflect, in a semiquantitative manner, the relative numbers of cells slowed or arrested in these phases.…”

mentioning

confidence: 99%

Cell kinetic effects of incorporated ³H‐thymidine on proliferating human lymphocytes: Flow cytometric analysis using the DNA/nuclear protein method

et al. 1985

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Phytohemagglutinin-stimulated human peripheral blood lymphocytes incorporating high concentrations of 3H-thymidine accumulate in 6 2 and show a consequent reduction in the number of cells entering M (division delay). The simultaneous flow cytometric analysis of DNA content (propidium iodide fluorescence) and nuclear protein content (fluorescein isothiocyanate fluorescence) allows for the accurate quantitation of these events; 6 2 and M are separated in the bivariate distributions. A good correlation was observed between mitotic indices, quantitated by manually counting mitotic cells, and integration of the M area in DNNnuclear protein histograms. Moreover, significant differences in 6 2 nuclear protein levels were found between untreated and 3H-thymidine-treated lymphocytes. In order to characterize this effect, 6 2 was empirically divided into low nuclear protein (G2A) and high nuclear protein (G2B) compartments. 3H-thymidine caused an initial accumulation of lymphocytes in GZA, followed within 3-6 h by a gradual movement of some cells into GZB, with a subsequent accumulation of cells in G2B. The results suggest that the distribution of cells in 6 2 (G2A and G2B), the average nuclear protein content of G2B cells, and the proportion of cells in M are parameters that when used in combination provide a unique description of radiobiological effects.Key terms: Flow cytometry, DNA, nuclear protein, tritiated thymidine incorporation, human lymphocytesIn the past, the measurement of the inhibitory effects of incorporated 3H-thymidine (3H-TdR), or external ionizing radiation, on cell proliferation has involved timeconsuming procedures such as the counting of colony formation (colony count inhibition) and the quantitation of mitotic indices (mitotic delay) (11,13,15,23,27). More recently, flow cytometric analysis of DNA content per cell has permitted the rapid approximation of cell kinetic effects from radiation exposure (1,16,17,20,21,29). The radiation-induced changes in the relative numbers of cells in G2 +M reflect, in a semiquantitative manner, the relative numbers of cells slowed or arrested in these phases. While single parameter DNA histogram analysis represents a significant advance over older methods, preliminary data suggested that the simultaneous flow cytometric analysis of DNA and nuclear protein would describe in much greater detail cell kinetic changes in response to irradiation.Recently, Roti Roti et al. (25) and Pollack et al. (22) described evidence that G2 is separated from M in the bivariate distributions resulting from simultaneous measurements of DNA and nuclear protein. Our initial characterization of the DNNnuclear protein method included an experiment in which proliferating human peripheral blood lymphocytes (HPBL) were treated with a concentration of 3H-TdR that caused extensive G2 blockade. The preliminary results suggested that the increase in the proportion of irradiated cells in G2 and the consequent reduction in the proportion of cells in M could be quantitated simultaneously. In addition,...

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G₂ + M arrest of cultured mammalian cells after incorporation of tritium‐labeled nucleosides

Cited by 32 publications

References 11 publications

Replication of Lactate Dehydrogenase-elevating Virus in Macrophages: 1. Evidence for Cytocidal Replication

Replication of Lactate Dehydrogenase-elevating Virus in Macrophages: 1. Evidence for Cytocidal Replication

Effects of Heavy Ions on Rabbit Tissues: Analysis of Low Levels of DNA Damage in Retinal Photoreceptor Cells

Cell kinetic effects of incorporated ³H‐thymidine on proliferating human lymphocytes: Flow cytometric analysis using the DNA/nuclear protein method

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G2 + M arrest of cultured mammalian cells after incorporation of tritium‐labeled nucleosides

Cited by 32 publications

References 11 publications

Replication of Lactate Dehydrogenase-elevating Virus in Macrophages: 1. Evidence for Cytocidal Replication

Replication of Lactate Dehydrogenase-elevating Virus in Macrophages: 1. Evidence for Cytocidal Replication

Effects of Heavy Ions on Rabbit Tissues: Analysis of Low Levels of DNA Damage in Retinal Photoreceptor Cells

Cell kinetic effects of incorporated 3H‐thymidine on proliferating human lymphocytes: Flow cytometric analysis using the DNA/nuclear protein method

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G₂ + M arrest of cultured mammalian cells after incorporation of tritium‐labeled nucleosides

Cell kinetic effects of incorporated ³H‐thymidine on proliferating human lymphocytes: Flow cytometric analysis using the DNA/nuclear protein method