Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)

Abstract

UV-induced apoptosis is a protective mechanism that is primarily caused by DNA damage. Cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts are the main DNA adducts triggered by UV radiation. Because the formation of DNA lesions in the chromatin is modulated by the structure of the nucleosomes, we postulated that modification of chromatin compaction could affect the formation of the lesions and consequently apoptosis. To verify this possibility we treated human colon carcinoma RKO cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposure to UV radiation. Our data show that pre-treatment with TSA increased UV killing efficiency by more than threefold. This effect correlated with increased formation of CPDs and consequently apoptosis. On the other hand, TSA treatment after UV exposure rather than before had no more effect than UV radiation alone. This suggests that a primed (opened) chromatin status is required to sensitize the cells. Moreover, TSA sensitization to UV-induced apoptosis is p53 dependent. p53 and acetylation of the core histones may thus contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin.

Introduction

Living cells are constantly exposed to different sources of DNA damaging agents. In order to maintain their genomic integrity and survive, cells have evolved a complex response that either leads to apoptosis or cell cycle arrest. Several genes that are activated by stress responsive protein kinases regulate these two cellular events. In general, the genes that are activated by the stress response play a protective role against the cellular insults [1]. UV radiation is a major source of DNA damage that can activate the stress response. In addition of causing the formation of DNA lesions such as cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PPs), UV radiation can also trigger plasma membrane and mitochondria events that can lead to apoptosis [2], [3]. However, the main source of UV-induced apoptosis is the formation of DNA lesions [4]. This was best illustrated in cells that are deficient for nucleotide excision repair (NER), the mechanism responsible for the repair of UV-induced DNA damage. These cells are dramatically hypersensitive to UV-induced apoptosis, which indicates that DNA damage is the major stimulus for the apoptotic response [5]. The capacity to induce apoptosis following exposure to UV radiation is believed to be a protective mechanism that allows the elimination of severely damaged cells that could potentially lead to malignant transformation.

Even though primary DNA lesions such as CPDs and 6-4PPs are the predominant initiators of UV-induced apoptosis, they do not cause apoptosis by themselves [6]. These lesions need to be converted into more critical secondary lesions, probably DNA double strand breaks (DSBs), through replication. This is supported by the fact that electroporation of the restriction enzyme PvuII into mouse fibroblasts is sufficient to induce apoptosis [7]. Formation of DNA strand breaks is also sufficient to activate the tumor suppressor p53 [8]. p53 is the most mutated gene in human cancers and sits at an important decisional fork to regulate both cell cycle arrest and apoptosis in response to DNA damage [9]. In response to low levels of DNA damage, p53 can activate the transcription of regulatory genes such as p21 to arrest the cell cycle and presumably allow time to repair the DNA. On the other hand, if damage is beyond repair, p53 can trigger an apoptotic response to eliminate the damaged cells and prevent transmission of potentially mutagenic material to the next generation of cells. In response to UV radiation, p53 can mediate an apoptotic response but its role is rather complex and seems to be cell type specific. For example, lymphoblastoid cells that are wild type for p53 are more sensitive to UV-induced apoptosis than their p53 mutated counterparts while p53 is not required to induce apoptosis in response to UV radiation in fibroblasts [6]. This may reflect differing basal patterns of expression of pro- and anti-apoptotic regulators that are under the control of p53 in these cells [10].

Another possible mechanism by which p53 could affect UV-induced apoptosis is through its recently described chromatin relaxation activity in response to UV radiation [11]. p53 mediates this effect by recruiting the histone acetylase p300 to allow acetylation of histone H3 at sites of NER [11]. Post-translational modifications, especially acetylation, of core histone have been associated with a looser, more open, chromatin structure that facilitates accessibility to transcription, replication, and repair machinery [12]. Acetylation of the amino-terminal tails of the core histones occurs on lysine residues and is mediated by histone acetyltransferases (HATs). However, acetylation is a reversible process that is catalyzed by the histone deacetylases (HDACs) enzymes. Deacetylation of the core histone thus results in a repressed (compacted) chromatin structure that is inhibitory to most cellular processes [12]. The formation of UV-induced DNA lesions is thus likely to be affected by the structure of the chromatin (compact vs. open) that is dictated by the nucleosomes acetylation status. This is because the formation of the lesions is not random on the chromatin. 6-4PPs are predominantly formed in linker DNA while CPDs occur at sites where the DNA minor grooves face the outside of the nucleosome cores [13].

To verify the possibility that modulation of chromatin through inhibition of histone deacetylases could affect UV-induced apoptosis, we treated human colon carcinoma cells with the histone deacetylase inhibitor trichostatin A (TSA) prior to exposing the cells to UV radiation. Our data show that pre-treatment with TSA decreased the survival of colon cancer cells exposed to UV radiation by more than threefold. This effect correlates with increased formation of CPDs and apoptosis as measured by TUNEL assay and DNA ladder formation. Moreover, the increased apoptosis was not observed in cells expressing reduced p53 levels thus indicating that the TSA effect is p53 dependent. Because TSA treatment after rather than before UV radiation did not result in a similar synergy, we conclude that a primed (opened) chromatin structure is required to sensitize the cells to UV radiation. Collectively these data suggest that p53 and acetylation of the core histone could contribute to UV-induced apoptosis by modulating the formation of DNA lesions on chromatin.