The effect of oxidative stress on histone acetylation and IL-8 release

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Abstract

Acetylation of histone residues regulates the expression of inflammatory genes and is controlled by the activities of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Analysis of histone acetylation in human cells is limited by the large numbers needed to perform activity assays or Western blotting. We have used flow cytometry to investigate changes in HAT and HDAC activities at the single cell level and to investigate the effect of hydrogen peroxide (H2O2) on histone H4 acetylation and cell-cycle progression. Using an anti-acetylated histone H4 antibody we show that H2O2 induced a time-dependent increase in histone acetylation that was maintained for 12 h. This was associated with increased IL-8 production. H2O2 also affected cell-cycle progression. HAT activity was found to be highest in G2/M and equivalent in G0/G1 and S phases of the cell cycle. These data show that detection of acetylated histone residues at the single cell level using FACs may be a powerful new tool for the analysis of modulation of cell proliferation and gene transcription.

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Materials and methods

Cell culture. SV-40 transformed bronchial epithelial cells (BEAS-2B) were obtained from the American Type Culture Collection (Manassas, VA). Cells (passages 32–70) were cultured in serum-free keratinocyte basal medium (Sigma, Poole, UK) supplemented with epithelial growth factor (0.2 ng/ml; Gibco-BRL, Paisley, UK) and 20 g/ml bovine pituitary extract (Gibco-BRL). Cells were deprived of growth factors or FCS for 48 h before stimulation.

Immunohistochemical detection of acetylated histone residues.

Hyperacetylation of histone H4 by TSA and H2O2 in BEAS-2B cells

We initially confirmed that pretreatment of BEAS-2B cells with TSA (10 ng/ml, 1 h) induced hyperacetylation at histone H4 lysine residues by immunocytochemistry. TSA induced hyperacetylation of lysines in all nuclei (Fig. 1). This effect persisted up to 16 h after treatment (data not shown). H2O2 (200 μM, 1 h) also induced a persistent acetylation of histone H4 lysines (Fig. 1).

H2O2 and TSA induce IL-8 release in BEAS-2B cells

Stimulation of BEAS-2B cells for 4 h with H2O2 (200 μM) enhanced IL-8 release (167±14 versus 78±12 pg/ml). IL-8 release was

Discussion

Histone acetylation and deacetylation are linked to cell-cycle progression, and have been correlated with repair and recombination events, as well as with gene transcription [3], [14]. Highly acetylated nucleosomes are associated with transcriptionally active chromatin, while hypoacetylated histones are found in inactive chromatin. Overexpression of the enzymes controlling histone acetylation and deacetylation confirms that acetylation of histones is an important step in transcription [3], [4].

Acknowledgements

This work was funded by the Clinical Research Committee (Royal Brompton Hospital) and GlaxoSmithKline plc (UK).

References (22)

  • A. Clement et al.

    Role of cyclins in epithelial response to oxidants

    Am. J. Respir. Crit. Care Med.

    (2001)

    • Cited by (0)

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