The histone deacetylase inhibitor, trichostatin A, enhances radiation sensitivity and accumulation of gammaH2A.X

Abstract

Histone deacetylase inhibitors have been shown to induce numerous biologic effects including, altering cell cycle distribution, cytostasis and in certain cases apoptosis. Given their ability to disrupt critical biological processes in cancer cells, these agents are emerging as potential therapeutics for cancer. Recently, it has been identified that histone deacetylase inhibitors can also efficiently enhance the radiation sensitivity of cells, both in vitro and in vivo. In this study, we investigated whether the potent histone deacetylase inhibitor, Trichostatin A, modulates the radiation sensitivity of human erythroleukemic K562 cells.The endpoints we used were clonogenic survival, apoptosis and γH2AX immunoprecipitations of soluble chromatin. The findings from clonogenic survival assays indicated that incubation with Trichostatin A 24 hours prior to irradiation enhances the radiation sensitivity of K562 cells. The dose modification factors ranged from 1.1 when cells were incubated with 0.1 µM Trichostatin A to 2.3 at 1 µM Trichostatin A. Similarly, caspase-3 and caspase-7 assays indicated that Trichostatin A potentiates radiation-induced apoptosis in K562 cells, in a concentration dependent manner. Our results suggest the modulation of radiation effects observed at the lower Trichostatin A concentrations was associated with histone hyperacetylation and changes in phosphorylated γH2A.X formation on euchromatin. In contrast, at the higher Trichostatin A concentrations mechanisms such as drug-mediated cytotoxicity and G 1 cell cycle arrest, contributed to the sensitization effect. More generally, our findings are consistent with those from recent studies and support the development of histone deacetylase inhibitors for use as radiation sensitizers, particularly for targeting radioresistant cancers.

Histone deacetylase inhibitors and genomic instability

“…In mouse xenograft models, the combination of HDACI treatment (MS-275 [42], valproic acid [43], LBH589 [44], LAQ824 [45]and AN-9 [46]) with radiation therapy, results in a greater delay in tumor growth than that accounted for by a simple additive effect of the corresponding individual treatments. HDAC inhibition by LBH589 [44], TSA [47], depsipeptide [48] and SAHA [49] stabilizes and enhances IR-induced phosphorylated histone H2AX nuclear foci, classical markers of DSBs, suggesting that HDACIs inhibit DSB repair and/or render DNA more susceptible to IR-induced damage. HDACIs facilitate radiation-induced killing in tumor cells and at the same time appear to protect healthy tissues from cutaneous radiation syndrome.…”

Clinical Potential of Histone Deacetylase Inhibitors as Stand Alone Therapeutics and in Combination with other Chemotherapeutics or Radiotherapy for Cancer

“…When high concentrations of HDAC inhibitors are used, relatively large dose modification factors (DMF; ratio of radiation doses in HDAC inhibitor treated and untreated cells that result in the same level of cytotoxicity) are achieved (DMFs as high as ~2). 85 It is evident that drug-mediated cell-cycle redistribution, particularly G 1 -phase arrest, inhibition of DNA synthesis and induction of apoptosis are responsible for the radiosensitizing effect at the relatively high inhibitor concentrations. [82][83][84][85] The synergistic effects at HDAC inhibitor concentrations that are not toxic and do not affect cell cycle distribution may, at least in part, be the result of histone-hyperacetylation induced changes in chromatin structure per se.…”

“…85 It is evident that drug-mediated cell-cycle redistribution, particularly G 1 -phase arrest, inhibition of DNA synthesis and induction of apoptosis are responsible for the radiosensitizing effect at the relatively high inhibitor concentrations. [82][83][84][85] The synergistic effects at HDAC inhibitor concentrations that are not toxic and do not affect cell cycle distribution may, at least in part, be the result of histone-hyperacetylation induced changes in chromatin structure per se. 85,93 It has been shown that incubation of cells with HDAC inhibitors prior to irradiation results in increased levels of γH2AX-the phosphorylated form of the histone variant H2A, which forms nuclear foci in response to DNA damage and is a sensitive marker of ionizing radiation induced double strand breaks (DSB) 94,95 -indicating that the change in chromatin conformation results in increased DSB formation.…”

“…[82][83][84][85] The synergistic effects at HDAC inhibitor concentrations that are not toxic and do not affect cell cycle distribution may, at least in part, be the result of histone-hyperacetylation induced changes in chromatin structure per se. 85,93 It has been shown that incubation of cells with HDAC inhibitors prior to irradiation results in increased levels of γH2AX-the phosphorylated form of the histone variant H2A, which forms nuclear foci in response to DNA damage and is a sensitive marker of ionizing radiation induced double strand breaks (DSB) 94,95 -indicating that the change in chromatin conformation results in increased DSB formation. 85,93 In addition, studies indicate that the ability of HDAC inhibitors to regulate the transcription of genes involved in DSB repair pathways, may account for the synergistic effects.…”

“…79 Although, the early studies did not directly correlate the radiosensitizing effect with histone hyperacetylation, they appear to have prompted the more detailed investigation of HDAC inhibitors as potential radiation sensitizers. Numerous studies have now shown that HDAC inhibitors including, traditional drugs such as sodium butyrate, [75][76][77] phenylbutyrate, 78 tributiryn, 80 TSA, [81][82][83][84][85] valproic acid, 86 MS-275 [87][88][89] and SAHA 83,84,90 as well as novel compounds such as the cyclic tetrapeptide analogue bicyclic tetrapeptide 87 and numerous hydroxamic acid analogues such as M344, 83,84 can enhance the sensitivity of various cell-lines to the effects of ionizing radiation. The molecular basis (although not yet fully understood), for the radiosensitizing properties of HDAC inhibitors has been recently extensively (reviewed in refs.…”