Histone deacetylase inhibitor induces DNA damage, which normal but not transformed cells can repair

Abstract

Histone deacetylase inhibitors (HDACi) developed as anti-cancer agents have a high degree of selectivity for killing cancer cells. HDACi induce acetylation of histones and nonhistone proteins, which affect gene expression, cell cycle progression, cell migration, and cell death. The mechanism of the tumor selective action of HDACi is unclear. Here, we show that the HDACi, vorinostat (Suberoylanilide hydroxamic acid, SAHA), induces DNA double-strand breaks (DSBs) in normal (HFS) and cancer (LNCaP, A549) cells. Normal cells in contrast to cancer cells repair the DSBs despite continued culture with vorinostat. In transformed cells, phosphorylated H2AX (gammaH2AX), a marker of DNA DSBs, levels increased with continued culture with vorinostat, whereas in normal cells, this marker decreased with time. Vorinostat induced the accumulation of acetylated histones within 30 min, which could alter chromatin structure-exposing DNA to damage. After a 24-h culture of cells with vorinostat, and reculture without the HDACi, gammaH2AX was undetectable by 2 h in normal cells, while persisting in transformed cells for the duration of culture. Further, we found that vorinostat suppressed DNA DSB repair proteins, e.g., RAD50, MRE11, in cancer but not normal cells. Thus, the HDACi, vorinostat, induces DNA damage which normal but not cancer cells can repair. This DNA damage is associated with cancer cell death. These findings can explain, in part, the selectivity of vorinostat in causing cancer cell death at concentrations that cause little or no normal cell death.

Fig. 1.

Vorinostat induces accumulation of acetylated α-tubulin, histone H3, and γH2AX in normal (HFS) and transformed (LNCaP, A549) cells. Cells were cultured with 5 μM vorinostat for indicated times. Immunoblots are of acetylated α-tubulin (A), acetylated histone H3 and γH2AX (B), immunoblots of γH2AX and acetylated histones H3 and H4 at early time points (C), GAPDH, and total histone H3 are indicated as the loading controls. Graphs, expressed as a ratio to the loading control, of the accumulation of acetylated α-tubulin (D), acetylated histone H3 (E), and γH2AX (F). Graphs were prepared by quantifying immunoblot bands by using TINA 2.0 software (Ray Tests).

Fig. 2.

Vorinostat induced accumulation of γH2AX foci in normal (HFS) and transformed (LNCaP, A549) cells. Cells were cultured with 5 μM vorinostat for indicated times and probed with γH2AX antibody and DAPI (nuclei). Representative images were observed by immunofluorescence, γH2AX, DAPI, and merged images of γH2AX and DAPI fluorescence staining. (A) HFS cells. (B) LNCaP cells. (C) A549 cells. (Scale bars: 10 μm.)

Fig. 3.

Normal cells recover from vorinostat induced DNA DSBs after removal of HDACi but not transformed cells. Washout experiments were performed as described in Materials and Methods. (A) Graphs represent cell viability after replacing medium with or without vorinostat, after 24-h culture with vorinostat. Each time point is the mean of three independent experiments; each bar is ±SD. (B) Immunoblots of acetylated tubulin and acetylated peroxiredoxins (prx1). Total α-tubulin is the loading control. (C) Immunoblots of acetylated histone H3 and γH2AX. Total histone H3 is the loading control. Cell cycle distribution of HFS (D), LNCaP (E), and A549 cells (F) at times indicated.

Fig. 4.

Vorinostat can suppress expression of DNA DSB repair proteins in transformed, but not normal, cells. Cells were cultured with 5 μM vorinostat for indicated times. Immunoblots were probed with antibodies as described in Materials and Methods. DNA damage repair proteins assayed: RAD50, MRE11, 53BP1, KAP1, TIP60, p-ATM, and ATM.