The use of stimulated primary spleen cell cultures in evaluating cell cycle response to toxicant insult

Anson, Jeanne A.; Hinson, William G.; Schol, Henry M.; Pipkin, James L.

doi:10.1007/bf01834629

Cited by 20 publications

(4 citation statements)

References 7 publications

(2 reference statements)

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“…The cell pellet was resuspended in less than 1 .0 ml of cold PBS. Vindelov's propidium iodide (PI) stain [Vindelov, 19771, as modified by Anson et al [1983], was added to the cell suspension at a concentration of 4.0 ml per 1 X lo6 cells. After centrifugation, the supernatant was removed, and the nuclei were resuspended in PI stain at a concentration of 1.0 x Intact stained nuclei were sorted with the FACStar Plus (Becton-Dickinson, Mountain View, CA) flow cytometer on the basis of DNA fluorescence.…”

Section: Nuclear Protein Phosphorylationmentioning

confidence: 99%

Effect of interleukin‐2 on cell proliferation, sister‐chromatid exchange induction, and nuclear stress protein phosphorylation in Pha‐stimulated fischer 344 rat spleen lymphocytes: Modulation by 2‐mercaptoethanol

Morris

Aidoo

Domon

et al. 1990

Environ and Mol Mutagen

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The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.

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Section: Nuclear Protein Phosphorylationmentioning

confidence: 99%

Effect of interleukin‐2 on cell proliferation, sister‐chromatid exchange induction, and nuclear stress protein phosphorylation in Pha‐stimulated fischer 344 rat spleen lymphocytes: Modulation by 2‐mercaptoethanol

Morris

Aidoo

Domon

et al. 1990

Environ and Mol Mutagen

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“…The cell monolayer was rinsed twice with HBSS, exposed to trypsin-EDTA, triturated and enumerated. The cell suspension was then subjected to gentle centrifugation, the supernatant aspirated, and the cell pellet resuspended in propidium iodide staining solution (Anson et al, 1983;Vindelov, 1977), at a concentration of 1 x 106 cells per ml. The suspension of intact, stained nuclei was maintained in an ice bath under an aluminum foil light shield for a minimum of 30 min before analysis.…”

Section: Flow Cytometrymentioning

confidence: 99%

Effect of bromodeoxyuridine on the proliferation and growth of ethyl methanesulfonate-exposed P3 cells: Relationship to the induction of sister-chromatid exchanges

et al. 1992

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Although sister-chromatid exchange (SCE) analysis is recognized as an indicator of exposure to DNA-damaging agents, the results of these analyses have been confounded by the use of bromodeoxyuridine (BrdUrd) to differentially label the sister chromatids. Not only does BrdUrd itself induce SCE, it also modulates the frequency of SCE induced by certain DNA-damaging agents. In order to examine this effect of BrdUrd on SCE frequency, an indirect method which lends itself to measurements both with and without BrdUrd was employed. Human teratocarcinoma-derived (P3) cells were exposed to ethyl methanesulfonate (EMS) and cultured with increasing concentrations of BrdUrd for lengths of time corresponding to one, two, and three generations of cell growth. At each time point, the distribution of nuclei among the phases of the cell-cycle and cell growth were evaluated for each concentration and chemical. A statistical model was employed which tested both for the main effects of chemicals and culture times and for interactions between these factors. Both EMS and BrdUrd significantly affected the percentages of nuclei within the cell-cycle. Exposure to EMS resulted in decreases in the percentages of nuclei in G0 + G1 and increases in the G2 + M compartment. Exposure to BrdUrd affected the size of the G0 + G1 compartment as well as the percentage of S-phase nuclei. Cell growth was reduced as a consequence of increasing EMS concentration and as a function of BrdUrd concentration; the effects of these chemicals were more readily apparent at the later time points. Most importantly, for both the cell-cycle kinetics data and the cell growth data, no evidence of an interaction between the effects of EMS and the effects of BrdUrd was detected statistically. These results may be interpreted to mean that while both EMS and BrdUrd affect the induction of SCE, under the conditions of this experiment, the effects are additive rather than interactive.

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“…by centrifugation, the media were aspirated, and the cell pellet was resuspended in propidium iodide (PI) [Vindelov, 19771 staining solution, as modified by Anson et al [1983], at a concentration of 1 x lo6 cells per ml. The cell-cycle position of the intact, stained nuclei was determined on a FACScan (Becton-Dickinson, Sunnyvale, CA) flow cytometer.…”

Section: Flow Cytometric Analysismentioning

confidence: 99%

“…These characteristics, especially the observation of karyotypic stability, indicated that the P3 cell line might lend itself to flow cytometric analysis of cellular proliferation. This type of analysis relies on the accurate determination of cellular DNA content [Anson et al, 1983;Bagwell et al, 19791 by stoichiometric binding of fluorescent, DNA-specific dyes [Vindelov, 19771. In cell lines in which the karyotype is not stable, the addition or loss of chromatin could easily confound the analysis. The feasibility of flow cytometric analyses in the P3 cell line was confirmed in preliminary studies [McGarrity et al, 19881. Coupled with the demonstration that SCE, AGT, and relative cloning ability could easily be measured in P3 cells and responded to carcinogen exposure in a concentration-dependent manner [Moms et al, 19881, we chose this cell line for our experiments.…”

Section: Introductionmentioning

confidence: 99%

Flow cytometric analysis of bromodeoxyuridine‐induced inhibition of cell proliferation in the human teratocarcinoma‐derived cell line, p3

Morris

McGarrity

Domon

et al. 1989

Environ and Mol Mutagen

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We have developed methods in our laboratory whereby the effects of toxicant exposure on cell proliferation can be evaluated flow cytometrically. We sought to relate the flow cytometric analyses to other biological response measurements. Thus, we exposed P3 cells to increasing concentrations of bromodeoxyuridine (BRdU) and measured sister-chromatid exchange (SCE) frequency, average generation time (AGT), and relative cloning ability. Each of these is well documented (see introduction) to respond to BRdU exposure in a concentration-dependent manner. In this study, SCE frequency remained constant between the concentrations of 2.5 and 10 microM of BRdU. However, a small, but significant, increase in SCE frequency was observed between the concentrations of 10 microM and 50 microM BRdU. A significant increase in AGT was noted in 50 microM BRdU-exposed cells. Relative cloning efficiency decreased in a concentration-dependent manner when cells were cultured for 24, 48, or 72 hours with BRdU. When cell proliferation was assessed by flow cytometric analysis in cells exposed to 0, 10, or 50 microM BRdU, a statistically significant delay in the cell-cycle was observed in BRdU-exposed cells. These results may be interpreted to mean that inhibition of cell proliferation is detected by this type of analysis at toxicant concentrations that induce other biological endpoints. The inclusion of flow cytometric analysis in a test battery to evaluate toxicant effects is warranted.

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The use of stimulated primary spleen cell cultures in evaluating cell cycle response to toxicant insult

Cited by 20 publications

References 7 publications

Effect of interleukin‐2 on cell proliferation, sister‐chromatid exchange induction, and nuclear stress protein phosphorylation in Pha‐stimulated fischer 344 rat spleen lymphocytes: Modulation by 2‐mercaptoethanol

Effect of interleukin‐2 on cell proliferation, sister‐chromatid exchange induction, and nuclear stress protein phosphorylation in Pha‐stimulated fischer 344 rat spleen lymphocytes: Modulation by 2‐mercaptoethanol

Effect of bromodeoxyuridine on the proliferation and growth of ethyl methanesulfonate-exposed P3 cells: Relationship to the induction of sister-chromatid exchanges

Flow cytometric analysis of bromodeoxyuridine‐induced inhibition of cell proliferation in the human teratocarcinoma‐derived cell line, p3

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