International Journal of Radiation Oncology*Biology*Physics
Clinical investigation: biology contributionSusceptibility and radiosensitization of human glioblastoma cells to trichostatin A, a histone deacetylase inhibitor
Introduction
The organization of chromatin is crucial for the regulation of gene expression. Chromatin is composed of nucleosomes, which consist of 146 bp DNA tightly wrapped around an octameric histone core. The core histone octamers consist of two H2A, two H2B, two H3, and two H4 histones, which are subject to a variety of enzyme-catalyzed posttranslational modifications. Concerning these modifications, the acetylation status of core histones is associated with transcriptional regulation and is determined by two classes of enzymes: histone acetyl transferases (HATs) and histone deacetylases (HDACs) (1). The acetylation of histones is thought to loosen the histone-DNA contact by neutralizing the positively charged lysine residues in the core histones. This loosening leads to a more open DNA conformation, allowing transcription factors and transcription apparatus to gain access to DNA and express specific genes (2). Nonhistone proteins, such as p53, have been known to serve as substrates of HAT and HDAC in vitro and in vivo, and the acetylation status of cellular proteins is thought to modulate the activity of target proteins 3, 4. In addition, aberrant acetylation has been reported to be associated with carcinogenesis in multiple organs (5).
So far, several HDAC-Is have been discovered or synthesized (6). HDAC-Is, in addition to deacetylase activity, show various biologic effects—for example, morphologic change (7), transcriptional change 8, 9, cell differentiation (10), cell cycle arrest 11, 12, antiangiogenesis (13), and apoptosis 12, 14, 15, 16. In addition, HDAC-Is have in vitro and in vivo antitumor activity against transformed cells of various histologic origins 17, 18, 19, 20, 21, 22, 23, 24. Several HDAC-Is have been synthesized and tested for toxicity and antitumor effect in clinical trials 25, 26, 27, 28, 29, 30. Several Phase I/II clinical trials using HDAC-Is are under way.
Although research on HDAC-Is has progressed to the clinical trial stage, studies on the interaction between HDAC-Is and ionizing radiation are lacking. Recently, two studies reported the in vitro radiosensitizing effect of HDAC-Is in human nasopharyngeal (31) and colon carcinoma cells (32).
In this study, we investigated the effect of trichostatin A (TSA), the most potent HDAC-I discovered so far, on the radiosensitivity of human glioblastoma cells, and found that treatment with TSA at nanomolar concentrations sensitizes human glioblastoma cells to radiation-induced cellular lethality.
Section snippets
Cell culture
The U373MG and U87MG, human glioblastoma cell lines, were purchased from the Korean Cell Line Bank (33). Cells were grown as attached monolayers in 25-cm2 flasks in RPMI 1640 media (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) and 12.5 μg/mL gentamicin (Gibco). Cells were incubated at the exponential growth phase in humidified 5% CO2/95% air atmosphere at 37°C.
Assay of intrinsic TSA cytotoxicity
Cells were trypsinized from exponentially growing monolayer cultures, and 2 × 105 cells per 25-cm2 flask
Intrinsic TSA cytotoxicity
The clonogenicities of U373MG and U87MG cells were not significantly influenced by treating cells with 50–100 nM TSA for up to 24 h (Fig. 1). However, exposure to 200 nM TSA showed significant cytotoxicity in two cell lines. The cytotoxicity of 200 nM TSA showed time-dependence. We chose to test the radiosensitizing effect of TSA at 50, 100, and 200 nM, because significant intrinsic toxicity was expected at TSA concentration of 200 nM or higher.
TSA effect on radiosensitivity
SF2s of untreated U373MG and U87MG were 0.694 ±
Discussion
Histone deacetylase inhibitors have diverse biologic effects, such as transcriptional change, differentiation induction, cell cycle arrest, and apoptosis, and are known to inhibit the growth of various transformed cells. To date, several HDAC-Is have been discovered, and TSA has the most potent deacetylase activity among these. The low bioavailability of TSA has led to the development of several synthetic HDAC-Is 6, 19, 21, 34, 35. Some of the synthetic HDAC-Is selectively inhibit specific
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