Autophagy is characterized by the sequestration of bulk cytoplasm, including damaged proteins and organelles, and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity, the mechanism is not yet clear. Here we report that cytosolic FoxO1, a forkhead O family protein, is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation, but this process was independent of the transcriptional activity of FoxO1. In response to stress, FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2), a NAD(+)-dependent histone deacetylase, and the acetylated FoxO1 bound to Atg7, an E1-like protein, to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy.

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.

DNA methylation in the promoter of certain genes is associated with transcriptional silencing. Methylation affects gene expression directly by interfering with transcription factor binding and/or indirectly by recruiting histone deacetylases through methyl-DNA-binding proteins. In this study, we demonstrate that the human lung cancer cell line H719 lacks p53-dependent and -independent p21Cip1 expression. p53 response to treatment with gamma irradiation or etoposide is lost due to a mutation at codon 242 of p53 (C3W). Treatment with depsipeptide, an inhibitor of histone deacetylase, was unable to induce p53-independent p21Cip1 expression because the promoter of p21Cip1 in these cells is hypermethylated. Although a strong correlation between promoter methylation and gene silencing has been extensively demonstrated (5,24,35), the molecular mechanisms of this methylation-modulated gene inactivation remains unclear. Two hypotheses have been proposed to explain transcriptional inactivation from promoter methylation. One of them is based on the finding that methyl-CpG-binding proteins (MBPs), such as MeCP2, specifically bind to symmetrically methylated DNA through a methyl-CpG-binding domain (11,41). MBPs then recruit transcriptional repressors such as Sin3, NuRD, and histone deacetylases (HDACs) through its transcriptional-repression domain (25,32,54). Since Sin3 and HDACs are known transcriptional repressors (2, 50), methylated DNA may repress gene expression indirectly through MeCP2 and other MBPs. In addition, deacetylation of histones results in a net increase in positively charged lysines and arginines at the N-terminal tail of the histones (18, 21), thus inducing a tighter noncovalent linkage between the positively charged histones and the negatively charged DNA (3, 47). Consequently, transcription factors have difficulty accessing their DNA-binding sites (4, 29, 47), with a reduction or silencing of gene transcription. This hypothesis, based on the interaction between DNA methylation and histone acetylation status, has been extensively supported by accumulated experimental evidence (7,16,37,40). For example, trichostatin A (TSA), an inhibitor of HDAC, induces a robust reexpression of silenced genes when used with minimal doses of the demethylating agent, 5-aza-2Ј-deoxycytidine (5-azaCdR), although TSA or 5-aza-CdR alone do not lead to gene reexpression (7). Our previous data also show a link between histone acetylation status and DNA methylation, such that 5-aza-CdR significantly enhances acetylation of histones H3 and H4 induced by a HDAC inhibitor, depsipeptide. Related to this, depsipeptide-induced apoptosis is dramatically increased in cells pretreated with 5-aza-CdR (56). In addition, p19 INK4D expression is greatly enhanced when human lung cancer cells are treated with depsipeptide and 5-aza-CdR together compared to treatment with each agent alone (55). These studies support the notion that methylation and histone acetylation work cooperatively to influence gene expression and other biological processes.A...

Autophagy is a dynamic and highly regulated process of self-digestion responsible for cell survival and reaction to oxidative stress. As oxidative stress is increased in uremia and is associated with vascular calcification, we studied the role of autophagy in vascular calcification induced by phosphate. In an in vitro phosphate-induced calcification model of vascular smooth muscle cells (VSMCs) and in an in vivo model of chronic renal failure, autophagy was inhibited by the superoxide dismutase mimic MnTMPyP, superoxide dismutase-2 overexpression, and by knockdown of the sodium-dependent phosphate cotransporter Pit1. Although phosphate-induced VSMC apoptosis was reduced by an inhibitor of autophagy (3-methyladenine) and knockdown of autophagy protein 5, calcium deposition in VSMCs was increased during inhibition of autophagy, even with the apoptosis inhibitor Z-VAD-FMK. An inducer of autophagy, valproic acid, decreased calcification. Furthermore, 3-methyladenine significantly promoted phosphate-induced matrix vesicle release with increased alkaline phosphatase activity. Thus, autophagy may be an endogenous protective mechanism counteracting phosphate-induced vascular calcification by reducing matrix vesicle release. Therapeutic agents influencing the autophagic response may be of benefit to treat aging or disease-related vascular calcification and osteoporosis.