Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non‐stoichiometric Hoechst fluorochrome binding

Kubbies, Manfred

doi:10.1002/cyto.990110309

Cited by 23 publications

(14 citation statements)

References 32 publications

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“…Cell killing induced by CDDP is indicated by the nuclear/ cellular decay lanes shown in Figure 1 (lower left part of the cytograms of panels c, e and g) (Kubbies, 1990b). After addition of GSH to CDDP treated PBL's these nuclear/cellular decay lanes are significantly decreased (panels d, f and h), and are almost identical to the control cultures (panels a and b).…”

mentioning

confidence: 83%

“…However, none of the analytical techniques used were sufficient to reveal the cellular effects induced at the single cell level in heterogenous responding in vitro cell populations. In this study we used a novel BrdU/ Hoechst flow cytometric analysis to evaluate the multiple cell kinetic effects of CDDP and GSH (Kubbies et al, 1989;Kubbies et al, 1990a;Kubbies, 1990b (Kubbies et al, 1989;Kubbies et al, 1990a;Kubbies, 1990b (Kubbies et al, 1987;Kubbies et al, 1989). As shown in Figure 1 the BrdU/Hoechst flow cytometric analysis displays as many as three consecutive cell cycles, and reveals the proliferative heterogeneity of PHA-activated PBL's at a single time-point after CDDP and GSH treatment.…”

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confidence: 99%

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Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes

Kubbies

Göller²,

Schetters³

et al. 1991

Br J Cancer

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Summary Cis-platinum (CDDP) induces severe inhibition of cell activation and cell cycle progression in PHA-stimulated human PBL's. Applying the novel BrdU/Hoechst flow cytometric technique for high resolution cell cycle analysis we show that CDDP induced multiple cell kinetic disturbances occur simultaneously comprising GO/Gl-arrest, and slow down and arrest of cells in S and G2/M-phase. We investigated whether the administration of reduced glutathione (GSH) might rescue cells from proliferative disturbances induced by CDDP. GSH at 0.15 mg ml-' only partially restored normal cell activation and cell cycle progression. However, at 1.5mgml-' a complete normal proliferation pattern was obtained. At the highest GSH dose rescue from inhibition of cell activation (GO/G1-phase arrest) as well as of cell cycle progression (S-and G2/M-phase arrest) was also present after delayed addition of GSH (1, 4 and 20 h) to CDDP treated PBL's. In addition cell viability of CDDP exposed PBL's is restored after GSH treatment. Our in vitro experiments give evidence that an increase of WBC found in CDDP/GSH treated patients has a real underlying cellular physiological mechanism protecting human peripheral lymphocytes from CDDP toxicity.

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confidence: 83%

mentioning

confidence: 99%

Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes

Kubbies

Göller²,

Schetters³

et al. 1991

Br J Cancer

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“…Many such investigations have used human peripheral blood lymphocytes (PBLs), normally in GO phase of the cell cycle, which were stimulated into cycle by the addition of a mitogen such as phytohaemagglutinin (PHA) (14,(16)(17)(18)(21)(22)(23)(24)(25)(36)(37)(38)(39)(40). Another approach has been to render fibroblasts quiescent by the removal of serum and then to stimulate them by the re-addition of serum (5,6,19,21,35,37,41).…”

Section: Data Analysis Synchronised Cellsmentioning

confidence: 99%

“…If the resolution of the cytogram and the concentration of BrdUrd is sufficient, three rounds of replication can be observed. The entry and exit kinetics of the cells into successive S, GYM, and G1 phases can be measured and the effect of various growth factors (6,14,16,19,20,39) Figure 2 shows typical data from initially synchronous GO/Gl phase PBLs stimulated with PHA. BrdUrd was added at the onset of PBL culture.…”

Section: Data Analysis Synchronised Cellsmentioning

confidence: 99%

“…The continuous BrdUrd labelling technique applying the BrdUrd/Hoechst quenching effect has been used to examine the cell cycle kinetics of quiescent cells which have been stimulated into cycle (6,14,16,19,21,(23)(24)(25)31,32,36,37). The method can also be applied to asynchronously dividing cells (10,11,13,29,30); this has the advantage that it does not require artificial cell synchronisation with possible interference in the process of cell activation and cell cycle progression.…”

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Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst‐propidium iodide stain

Ormerod

Kubbies²

1992

Cytometry

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Continuous labelling of cells with deoxybromouridine (BrdUrd) followed by staining with a bis-benzimidazole (Hoechst 33258) and a phenanthridinium (propidium iodide or ethidium bromide) allows the cells to be separated by flow cytometry according to the extent of their DNA replication. This BrdUrd-Hoechstl PI method has been used mainly to observe perturbations of the cell cycle in synchronously growing cells. In this paper we demonstrate that, when the method is applied to asynchronously dividing cells, more extensive information can be derived about the effects of cytotoxic a n d other treatments on the kinetics of the cell cycle. The interpretation of the data is explained, the effects of different types of cytotoxic agent are described, and the method is compared briefly to other methods for following cell cycle kinetics. o 1992 Wiley-Liss, Inc.Key terms: Cell cycle kinetics, DNA, counterstaining Knowledge of the proliferative characteristics of cells is important in many areas of biology and oncology and, over the years, many flow cytometric methods have been developed for the study of cell cycle kinetics. The more powerful of these methods have employed 5-bromodeoxyuridine (BrdUrd), which is incorporated into DNA in place of thymidine. Detection of BrdUrd in the cellular DNA enables the fraction of cells in the S phase of their cycle to be enumerated and, using multiparametric flow cytometry, also gives detailed information about the kinetics of the cell cycle.There are three approaches to these measurements. Monoclonal antibodies which react specifically with BrdUrd have been used to provide an analysis of the kinetics of cells pulse-labelled with BrdUrd both in vitro (9,121 and in vivo (1,27,42). Counterstaining with propidium iodide (PI) is used to display cell cycle dependent anti-BrdUrd labelling. The second approach makes use of the observation by Latt (26) that the fluorescence of bis-benzimidazoles (Hoechst 33258 and 33342) bound to DNA is quenched by BrdUrd, the degree of quenching depending on the amount of BrdUrd incorporated (2,211. By staining cells with a combination of Hoechst 33258 and an intercalating dye such as PI or ethidium bromide (EB) whose fluorescence is unaffected by BrdUrd, the resolution of the different compartments of the cell cycle in cells continuously labelled with BrdUrd is greatly improved (3,4,13,20,21,31,32,37). The third technique, which detects BrdUrd in pulse-labelled cells, takes advantage of the decrease of Hoechst and increase of mithramycin fluorescence in the presence of BrdUrd (7). This technique requires more complex data recording due to the subtraction of fluorescence signals in real time.The continuous BrdUrd labelling technique applying the BrdUrd/Hoechst quenching effect has been used to examine the cell cycle kinetics of quiescent cells which have been stimulated into cycle

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Anabolic steroids induce injury and apoptosis of differentiated skeletal muscle

et al. 1997

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Apoptosis is an active form of cellular death, or suicide, which plays an important physiologic role during organ development and in cellular turnover in differentiated tissues. Apoptosis has also been demonstrated to occur in several organs in response to hypoxic/ischemic, oxidative, or drug‐induced injury and is thus involved in disease pathogenesis. However, it is generally assumed that apoptosis does not occur in differentiated skeletal muscle. Apoptosis has been demonstrated in differentiated myocardial muscle, neonatal skeletal muscle, and skeletal myoblasts in response to injury. We therefore studied differentiated murine C2 skeletal muscle cells that have been injured by supraphysiologic doses (>10 μM) of an anabolic steroid, stanozolol. Stanozolol‐injured muscle cells exhibited pathologic features suggestive of apoptosis: cytoplasmic shrinkage and chromatin condensation. Muscle cells also showed positive in situ nick‐end labeling of nuclear chromatin, indicating DNA strand breakage. Staining with the DNA‐binding dye 33342 (bisbenzimide) also showed chromatin changes characteristic of apoptotic nuclei. Total protein levels measured at 4 and 24 hr post‐stanozolol injury was not significantly decreased, indicating absence of cell lysis. Cellular ATP levels (nmol ATP/mg protein) of stanozolol‐injured muscle cells, measured 4 and 24 hr postinjury, also did not change significantly. In contrast, necrotic muscle cells, injured by the calcium ionophore A23187 (2 μM), showed a progressive decline in total protein and ATP levels. This study supports two other histologic studies that showed evidence of apoptosis in differentiated skeletal muscle fibers. Our data further suggest that during the early stages of apoptosis, but not necrosis, cellular energy metabolism is preserved. J. Neurosci. Res. 47:186–197, 1997. © 1997 Wiley‐Liss, Inc.

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Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non‐stoichiometric Hoechst fluorochrome binding

Cited by 23 publications

References 32 publications

Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes

Glutathione restores normal cell activation and cell cycle progression in cis-platinum treated human lymphocytes

Cell cycle analysis of asynchronous cell populations by flow cytometry using bromodeoxyuridine label and Hoechst‐propidium iodide stain

Anabolic steroids induce injury and apoptosis of differentiated skeletal muscle

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