Sensitization to UV-induced apoptosis by the histone deacetylase inhibitor trichostatin A (TSA)
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.
Section snippets
Cell culture and treatments
The human colon carcinoma RKO and RKO-E6 cells, the human breast cancer cells MCF-7, and the normal human skin fibroblasts Malme-3 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). RKO, RKO-E6, and MCF-7 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum in the absence of antibiotics. The Malme-3 cells were grown in DMEM:Ham's F-12 containing 10% Chelex-treated horse serum. UV radiation was performed with a UVC Philips 30-W germicidal lamp
Results
The molecular mechanisms underlying UV-induced apoptosis are fairly complex and involved the induction of several key regulatory proteins. To verify the molecular response of the human colon carcinoma RKO cells to UV radiation we exposed the cells to different (0–40 J m−2) UV doses and performed Western blot analyses. The data shown in Fig. 1 indicate that as expected p53 protein levels increase in response to UV doses [15]. The protein levels of the growth arrest and DNA damage inducible
Discussion
The initiation of apoptosis following UV exposure is determined by several factors including DNA damage, the activation of the tumor suppressor p53, triggering of cell death receptors, mitochondrial damage, and cytochrome c release [4]. How these different cellular events work together to orchestrate the apoptotic response is still ill defined but DNA damage appears to be the predominant factor determining whether a cell undergoes apoptosis or not. The two major classes of DNA lesions produced
Acknowledgment
This work was supported by a Departmental Research Initiative Fund from the School of Medicine, University of Maryland, Baltimore.
References (38)
- et al.
Repair capability and the cellular age response for killing and mutation induction after UV
Mutat. Res.
(1982) DNA damage-triggered apoptosis: critical role of DNA repair, double-strand breaks, cell proliferation and signaling
Biochem. Pharmacol.
(2003)- et al.
p53: 25 years after its discovery
Trends Pharmacol. Sci.
(2004) - et al.
Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A
J. Biol. Chem.
(1990) - et al.
A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia–telangiectasia
Cell
(1992) - et al.
Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis
J. Biol. Chem.
(1998) - et al.
Caspase activation involves the formation of the aposome, a large (approximately 700 kDa) caspase-activating complex
J. Biol. Chem.
(1999) - et al.
Phosphoinositide 3-OH kinase (PI3K) and PKB/Akt delay the onset of p53-mediated, transcriptionally dependent apoptosis
J. Biol. Chem.
(1999) - et al.
Functional genomics as a window on radiation stress signaling
Oncogene
(2003) - et al.
Nuclear and cell membrane effects contribute independently to the induction of apoptosis in human cells exposed to UVB radiation
Proc. Natl. Acad. Sci. U. S. A.
(1999)
DNA damage, death receptor activation and reactive oxygen species contribute to ultraviolet radiation-induced apoptosis in an essential and independent way
Oncogene
Molecular mechanisms of UV-induced apoptosis
Photodermatol., Photoimmunol. Photomed.
DNA double-strand breaks trigger apoptosis in p53-deficient fibroblasts
Carcinogenesis
DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways
Mol. Cell. Biol.
Live or let die: the cell's response to p53
Nat. Rev., Cancer
p53 is a chromatin accessibility factor for nucleotide excision repair of DNA damage
EMBO J.
Chromatin dynamics at DNA replication, transcription and repair
Eur. J. Biochem.
Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair
EMBO J.
Inhibition of histone deacetylase increases cytotoxicity to anticancer drugs targeting DNA
Cancer Res.
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Present address: Johns Hopkins University, Baltimore, MD 21205, USA.