Cancer Res Treat.  2005 Apr;37(2):122-128.

A Histone Deacetylase Inhibitor, Trichostatin A, Enhances Radiosensitivity by Abrogating G2/M Arrest in Human Carcinoma Cells

• Kim IA
• Kim JH
• Shin JH
• Kim IH
• Kim JS
• Wu HG
• Chie EK
• Kim YH
• Kim BK
• Hong S
• Park SW
• Ha SW
• Park CI

Abstract

PURPOSE
Histone deacetylase inhibitors (HDIs) are emerging as potentially useful components in anticancer therapy. In this study, we tried to confirm the radiosensitizing effect of trichostatin A (TSA) on a panel of human carcinoma cell lines and elucidate its mechanism of interaction. MATERIALS AND METHODS: A549, HeLa and Caski cells were exposed to TSA for 18 hr prior to irradiation, and the cell survival then measured using a clonogenic assay. Western blot and flow cytometric analyses, for histone acetylation, and cell cycle and apoptosis, respectively, were also performed. RESULTS: TSA increased the acetylation of histone H3. The pretreatment of TSA consistently radiosensitized all three cell lines. The SF2 (surviving fraction at 2 Gy) of TSA-treated cells was significantly lower than that of mock treated cells. The SER (sensitizer enhancement ratio) increased in all 3 cell lines, in concentration dependent manners. The TSA treated cells showed abrogation of radiation-induced G2/M arrest, in a concentration dependent manner. CONCLUSION: The pretreatment of TSA enhanced the radiosensitivity of a panel of human carcinoma cells, which was attributed, in part, to the abrogation of radiationinduced G2/M arrest.

Keyword

Trichostatin A; Histone deacetylase inhibitor; Radiosensitization; G2/M arrest

MeSH Terms

Acetylation
Apoptosis
Blotting, Western
Cell Cycle
Cell Line
Cell Survival
Histone Deacetylase Inhibitors*
Histone Deacetylases*
Histones*
Humans*
Radiation Tolerance*
Radiation-Sensitizing Agents
Histone Deacetylase Inhibitors
Histone Deacetylases
Histones
Radiation-Sensitizing Agents

Figure

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  Fig. 1 TSA increased hyperacetylation of histone H3. Cells were treated 200 nM TSA for 18 hr, the proteins harvested, separated on SDS-PAGE and electroblotted. The membrane was probed with anti-histone H3 antibody. α-tubulin was used as a loading control. The same amount of drug carrier (DMSO) treated cells were used as a mock control. (A) A549; (B) HeLa.

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  Fig. 2 Survival curves for cells treated with trichostatin A (TSA) prior to irradiation. Cells were pretreated with 0, 50 or 200 nM TSA for 18 hr, and then irradiated with 4 MV X-rays after the removal of the TSA from the culture media. The survival was measured using a clonogenic assay. Each point on the survival curves represents the mean surviving fraction from at least three dishes. Experiments were repeated a minimum of three times. The error bars represent the standard errors. (A) A549; (B) HeLa; (C) Caski.

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  Fig. 3 Surviving fraction of cells pretreated with trichostatin A (TSA) prior to 2 Gy (SF2) of irradiation. Cells were pretreated with 0, 50, 100 or 200 nM TSA for 18 hr, and then irradiated with 4 MV X-rays after the removal of the TSA from the culture media. The survival was measured using a clonogenic assay. The SF2s of the TSA-treated cells were compared with those of mock treated cells using the t test. The error bars represent the standard errors.

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  Fig. 4 Sensitizer enhancement ratio (SER) of trichostatin A (TSA). The SER was defined as the ratio of the radiation dose required to obtain a surviving fraction (SF) of 0.5, without TSA pretreatment, to that required to obtain the same SF after TSA pretreatment. Cells were pretreated with 0, 50, 100 or 200 nM TSA for 18 hr, and then irradiated with 4 MV X-rays after the removal of the TSA from the culture media. The survival was measured using a clonogenic assay.

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  Fig. 5 Cell cycle analysis for A549 cells treated with trichostatin A (TSA). Cells were treated with 0~1,000 nM TSA for 18 hr, and collected, fixed and stained with propidium iodide, and then analyzed using flow cytometry.

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  Fig. 6 Sub-G0/G1 proportion in A549 cells pretreated with trichostatin A (TSA). Cells were treated with 0~1,000 nM TSA for 6~48 hr, and the cell cycle distribution then determined using flow cytometry. The proportions of cells with DNA contents less than that in the G0/G1 phase cells were defined as the sub-G0/G1 proportions.

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  Fig. 7 The change in the G2/M proportions of cells following the combination of TSA and irradiation. A549 cells were pretreated with 0 nM (-○-), 200 nM (-□-) and 400 nM(-△-) TSA for 18 hr. The TSA was then removed from the media, and the cells irradiated with 4 MV X-rays at 0 Gy (A), 2 Gy (B) or 8 Gy (C) at 0 hr. After irradiation, the cells were collected, fixed and stained with propidium iodide, and then analyzed using flow cytometry at 2, 4, 6, 8, 12, 16, 20 and 24 hr.

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