Generally, histone deacetylase (HDAC) inhibitor-induced p21Waf1/Cip1 expression is thought to be p53 independent. Here we found that an inhibitor of HDAC, depsipeptide (FR901228), but not trichostatin A (TSA), induces p21 Waf1/Cip1 expression through both p53 and Sp1/Sp3 pathways in A549 cells (which retain wild-type p53). This is demonstrated by measuring relative luciferase activities of p21 promoter constructs with p53 or Sp1 binding site mutagenesis and was further confirmed by transfection of wild-type p53 into H1299 cells (p53 null). That p53 was acetylated after depsipeptide treatment was tested by sequential immunoprecipitation/Western immunoblot analysis with anti-acetylated lysines and anti-p53 antibodies. The acetylated p53 has a longer half-life due to a significant decrease in p53 ubiquitination. Further study using site-specific antiacetyllysine antibodies and transfection of mutated p53 vectors (K319/K320/K321R mutated and K373R/K382R mutations) into H1299 cells revealed that depsipeptide specifically induces p53 acetylation at K373/K382, but not at K320. As assayed by coimmunoprecipitation, the K373/K382 acetylation is accompanied by a recruitment of p300, but neither CREB-binding protein (CBP) nor p300/CBP-associated factor (PCAF), to the p53 C terminus. Furthermore, activity associated with the binding of the acetylated p53 at K373/K382 to the p21 promoter as well as p21Waf1/Cip1 expression is significantly increased after depsipeptide treatment, as tested by chromatin immunoprecipitations and Western blotting, respectively. In addition, p53 acetylation at K373/K382 is confirmed to be required for recruitment of p300 to the p21 promoter, and the depsipeptide-induced p53 acetylation at K373/K382 is unlikely to be dependent on p53 phosphorylation at Ser15, Ser20, and Ser392 sites. Our data suggest that p53 acetylation at K373/K382 plays an important role in depsipeptide-induced p21 Waf1/Cip1 expression.

In addition to its demethylating function, 5-aza-2-deoxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR induces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations (0.01-5 M) induces inhibition of cell proliferation as well as increased p53/p21Waf1/Cip1 expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21 As demethylating agents, 5-aza-cytidine and 5-aza-2Ј-deoxycytidine (5-aza-CdR) 1 have been extensively used for epigenetic research (1-4). Both demethylating agents are incorporated into DNA where they bind DNA methyltransferase (DNMT) in an irreversible, covalent manner, thus sequestering the enzyme and preventing maintenance of the methylation state (5-7). Consequently, silenced genes induced by hypermethylation are re-expressed after treatment with these demethylating agents.Originally, 5-aza-cytidine and 5-aza-CdR were developed as anticancer agents (5, 8) and have been shown to have significant cytotoxic and antineoplastic activities in many experimental tumors (9 -12). 5-Aza-CdR is reported to be noncarcinogenic and incorporates into DNA but not RNA or protein (13,14). 5-Aza-CdR has been found empirically to have more potent therapeutic effects than 5-aza-cytidine in cell culture and animal models of human cancers. However, 5-aza-CdR-induced cytotoxicity may not be linked to its demethylating function (3,(15)(16)(17). In addition, the therapeutic effects of 5-aza-CdR in the treatment of different human cancer cells are conflicting. 5-Aza-CdR appears to be beneficial in the treatment of human leukemias (9,18,19), myelodysplastic syndromes (20, 21), and hemoglobinopathies (22, 23). On the other hand, there has been less positive experience in the effectiveness of 5-aza-CdR for the treatment of human solid tumors (10, 24). Therefore, it is possible that one or more critical factors may be involved in regulating the cellular response to 5-aza-CdR treatment that vary in different cell types.p53 is a very important tumor suppressor gene and is reported to be abnormal in more than 50% of human cancers (25). Chemotherapeutic agents frequently act through the mechanism of DNA damage, and p53 plays an important role in the induction of cell cycle arrest and apoptosis in response to DNA damage (26). 5-Aza-CdR has also shown anticancer activity that may be related to its ability to induce DNA damage (15,27). Based on the scenario mentioned above, it is hypothesized that 5-aza-CdR may induce DNA damage, thereby activating p53, which in turn increases p21Waf1/Cip1 expression, leading to the inhibition of cell proliferation.To confirm the role of p53 in the 5-aza-CdR-induced inhibition of cell proliferation, human lung cancer cells with different p53 status were selected as the targets for this study. As an important downstream target of p53 activation, p21Waf1/Cip1 plays a critical role in inhibit...

Histone deacetylase inhibitor (HDACi) has been shown to demethylate the mammalian genome, which further strengthens the concept that DNA methylation and histone modifications interact in regulation of gene expression. Here, we report that an HDAC inhibitor, depsipeptide, exhibited significant demethylating activity on the promoters of several genes, including p16, SALL3, and GATA4 in human lung cancer cell lines H719 and H23, colon cancer cell line HT-29, and pancreatic cancer cell line PANC1. Although expression of DNA methyltransferase 1 (DNMT1) was not affected by depsipeptide, a decrease in binding of DNMT1 to the promoter of these genes played a dominant role in depsipeptide-induced demethylation and reactivation. Depsipeptide also suppressed expression of histone methyltransferases G9A and SUV39H1, which in turn resulted in a decrease of di-and trimethylated H3K9 around these genes' promoter. Furthermore, both loading of heterochromatin-associated protein 1 (HP1␣ and HP1␤) to methylated H3K9 and binding of DNMT1 to these genes' promoter were significantly reduced in depsipeptide-treated cells. Similar DNA demethylation was induced by another HDAC inhibitor, apicidin, but not by trichostatin A. Our data describe a novel mechanism of HDACi-mediated DNA demethylation via suppression of histone methyltransferases and reduced recruitment of HP1 and DNMT1 to the genes' promoter.

5-aza-2′-deoxycytidine (5-aza-CdR) is used extensively as a demethylating agent and acts in concert with histone deacetylase inhibitors (HDACI) to induce apoptosis or inhibition of cell proliferation in human cancer cells. Whether the action of 5-aza-CdR in this synergistic effect results from demethylation by this agent is not yet clear. In this study we found that inhibition of cell proliferation was not observed when cells with knockdown of DNA methyltransferase 1 (DNMT1), or double knock down of DNMT1-DNMT3A or DNMT1-DNMT3B were treated with HDACI, implying that the demethylating function of 5-aza-CdR may be not involved in this synergistic effect. Further study showed that there was a causal relationship between 5-aza-CdR induced DNA damage and the amount of [3H]-5-aza-CdR incorporated in DNA. However, incorporated [3H]-5-aza-CdR gradually decreased when cells were incubated in [3H]-5-aza-CdR free medium, indicating that 5-aza-CdR, which is an abnormal base, may be excluded by the cell repair system. It was of interest that HDACI significantly postponed the removal of the incorporated [3H]-5-aza-CdR from DNA. Moreover, HDAC inhibitor showed selective synergy with nucleoside analog-induced DNA damage to inhibit cell proliferation, but showed no such effect with other DNA damage stresses such as γ-ray and UV, etoposide or cisplatin. This study demonstrates that HDACI synergistically inhibits cell proliferation with nucleoside analogs by suppressing removal of incorporated harmful nucleotide analogs from DNA.

Most agents that damage DNA act through posttranslational modifications of p53 and activate its downstream targets. However, whether cellular responses to nucleoside analogue-induced DNA damage also operate through p53 posttranslational modification has not been reported. In this study, the relationship between p53 activation and its posttranslational modifications was investigated in the human cancer cell lines A549 and HCT116 in response to 5-aza-2-deoxycytidine (5-aza-CdR) or cytarabine treatment. The p53 tumor suppressor stands at the cross-roads of cellular responses to various stresses (1, 2). Under normal conditions, p53 is maintained at a low level through its interaction with MDM2 (3, 4), Pirh2 (5), COP1 (6), and ARF-BP1 (7), which mediate both ubiquitination and proteasome-dependent degradation of p53. However, in response to DNA damage, both the quantity and activity of p53 are greatly increased. As a transcription factor, depending on the nature of the stress, p53 can induce expression of many different downstream genes including p21 Waf1/Cip1