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. 2004 Sep;63(9):1135–1144. doi: 10.1136/ard.2003.011452

Extranuclear detection of histones and nucleosomes in activated human lymphoblasts as an early event in apoptosis

C Gabler 1, N Blank 1, T Hieronymus 1, M Schiller 1, J Berden 1, J Kalden 1, H Lorenz 1
PMCID: PMC1755110  PMID: 15308524

Abstract

Objective: To evaluate the presence of histones and nucleosomes in cell lysates of freshly isolated peripheral blood mononuclear cells (PBMC), fully activated lymphoblasts, or lymphoblasts after induction of apoptosis.

Methods: Each histone class (H1, H2A, H2B, H3, and H4) was detected by western blot analysis with specific antibodies. Annexin V/propidium iodide (PI) staining was performed for each treatment to distinguish viable, early, and late apoptotic, and necrotic cells. DNA degradation was evaluated by PI staining in a hypotonic buffer.

Results: All five histones were detected in cell lysates of activated lymphoblasts in higher concentrations than in lysates of PBMC. An additional significant increase of H2A, H2B, H3, H4, and complete nucleosomes in cell lysates of lymphoblasts was found during interleukin (IL)2 deprivation for 8 or 24 hours. The content of these histones or nucleosomes in cell lysates decreased after delayed IL2 readdition. H1 content in cell lysates was not affected by IL2 deprivation or addition. Quantities of H2A, H2B, H3, and H4 in cell lysates correlated significantly with signs of early apoptosis. UV-B light exposure or etoposide treatment of lymphoblasts resulted in a distinct increase for each histone class in cell lysates compared with standard curves. No bands for post-translationally modified histones were detected in cell lysates in contrast with signals in nuclear preparations.

Conclusion: Extranuclear accumulation of histones and nucleosomes is an early event of apoptosis in human lymphoblasts. Dysregulation of early apoptosis might support the induction of autoimmunity against nuclear components.

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Figure 1.

Figure 1

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 Experimental design showing times of IL2 withdrawal and readdition. Black bars represent the time of IL2 deprivation (med = medium R10); grey bars represent the time of the treatment with 10 U/ml IL2.

Figure 5.

Figure 5

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 Densitometric analysis of histone H1 (A), H2A (B), H2B (C), H3 (D), and H4 (E) in cell lysates of lymphoblasts after induced apoptosis. Cells were lysed, and 40 µg of cell lysates was separated using SDS-PAGE as described in "Material and methods". Western blot was performed with monoclonal antibodies directed against distinct histones. Band intensities were scanned and analysed by densitometry. The corresponding integrated absorbance (mean (SEM); n = 6 normal healthy donors) is depicted in relation to the band intensity of the histone band at 8 hours of cytokine withdrawal (8 hours med). A representative example of histone bands is illustrated. ETO, etoposide.

Figure 2.

Figure 2

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 Densitometric analysis of histone H1 (A), H2A (B), H2B (C), H3 (D), and H4 (E) in cell lysates of PBMC (first column) and lymphoblasts (all other columns). Cells were lysed, 40 µg of cell lysates were separated using SDS-PAGE as described in "Material and methods". Western blot was performed with monoclonal antibodies directed against distinct histones. Band intensities were scanned and analysed by densitometry. The corresponding integrated absorbance (mean (SEM); n = 6 normal healthy donors) is depicted in relation to the band intensity of the histone band at time 8 (B, D, E) or 24 hours (A, C) of cytokine withdrawal. A representative example of histone bands is illustrated. *Indicates significant difference (p<0.05) between the different times. In addition, the amount of each histone class in cell lysates of PBMC was significantly lower (p<0.001) than in cell lysates of lymphoblasts at all times.

Figure 3.

Figure 3

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 Relative contents of nucleosomes in cell lysates of apoptotic lymphoblasts compared with lymphoblasts at t0. Nucleosome ELISA was performed as described in "Material and methods". Absorption values (405 nm) were referred to lymphoblasts at t0 (value of 1). Data are mean (SEM) of six normal healthy donors. *Indicates significant difference (p<0.05) between the different times.

Figure 4.

Figure 4

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 Correlation of integrated optical density (ODxmm2) of histone bands H1 (A), H2A (B), H2B (C), H3 (D), and H4 (E) with the percentage of early apoptotic cells. The normalised values of the band intensities for each histone after each treatment were correlated with the percentage of early apoptotic cells (AxVposPIneg) after identical treatment within the identical experiment. The p values and the correlation coefficients r are given.

Figure 6.

Figure 6

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 Western blots for the detection of the following post-translationally modified histones: (A) acetylated H4; (B) phosphorylated H1; and (c) phosphorylated H3. Cells were lysed and 40 µg of cell lysates or 1/10 of the nuclear preparation from 2x106 lymphoblasts was separated using SDS-PAGE as described in "Material and methods". Western blot was performed with antibodies directed against the distinct post-translational modifications of the specific histone. As controls, the identical cell lysates were separated in a different gel. Western blot was performed with monoclonal antibodies directed against H4 (A; KM2), H1 (B; MRA12), or H3 (C; LG 2.1.).

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