Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1).

Generally, histone deacetylase (HDAC) inhibitor-induced p21(Waf1/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 p21(Waf1/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.

Impact of donepezil use in routine clinical practice on health care costs in patients with Alzheimer's disease and related dementias enrolled in a large medicare managed care plan: a case-control study.

BACKGROUND: Clinical studies have shown efficacy of cholinesterase inhibitors (eg, donepezil) in mild to moderate Alzheimer's disease (AD). However, there are limited studies examining the impact on health care costs of cholinesterase inhibitors prescribed in routine clinical practice. OBJECTIVE: The purpose of this study was to estimate the impact of donepezil use on health care costs and utilization in patients with mild to moderate AD and related dementias. METHODS: This case-control study was conducted using data from the Health Insurance Plan of Greater New York (New York, New York). Data from patients with predominantly mild to moderate AD and related dementias who were enrolled in this Medicare managed care plan from January 1, 1999, to December 31, 2002, were included. The health care costs and utilization of patients who had received donepezil prescribed in routine clinical practice were compared with those of patients who had never received donepezil or other cholinesterase inhibitors (control group). The 2 study groups were matched for age, sex, number of comorbid conditions, and presence of complications of late-stage dementia. Regression analysis was used to estimate the impact of donepezil use on health care costs and utilization during a 12-month follow-up period, controlling for characteristics associated with the outcomes. The analyses did not use a direct measure of disease severity but instead used proxy measures of severity based on medical conditions associated with late-stage dementia. RESULTS: Data from 687 patients were included in the study. The donepezil group comprised 229 patients (140 women, 89 men; mean age, 79.6 years); the control group, 458 patients (280 women, 178 men; mean age, 80.0 years). The mean costs of medical services per year in the donepezil group were US $2500 (95% CI, $300-$4671) less than those in the control group (P = 0.024). Lower medical costs in the donepezil group ($3325; 95% CI, $1163-$5486; P < 0.003 vs controls) were largely attributable to the lower costs of services performed in the hospital ($2594; 95% CI, $846-$4341; P < 0.004 vs controls) and postacute skilled nursing facility (SNF) ($1012; 95% CI, $444-$1579; P < 0.001 vs controls), which were partially offset by $1241 in higher prescription, physician's office, and outpatient hospital costs. Patients receiving donepezil had shorter mean lengths of stay in the hospital (3.00 vs 5.43 days; 95% CI, 0.66-4.19; P < 0.008) and postacute SNF (0.42 vs 3.40 days; 95% CI, 1.28-4.69; P < 0.001) but a higher mean number of physician's office visits (10.91 vs 7.91 visits; 95% CI, 1.63-4.36; P < 0.001) compared with controls. CONCLUSIONS: In this case-control study in patients with predominantly mild to moderate AD and related dementias, donepezil therapy prescribed in routine clinical practice was associated with reduced health care costs to the Medicare managed care plan studied. The findings support previous pharmacoeconomic studies with larger sample sizes obtained over a longer period of time, and with improved case-matching criteria.

The transcription factor c-Fos coordinates with histone lysine-specific demethylase 2A to activate the expression of cyclooxygenase-2.

Cyclooxygenase-2 (COX-2) is overexpressed in a variety of human epithelial cancers, including lung cancer, and is highly associated with a poor prognosis and a low survival rate. Understanding how COX-2 is regulated in response to carcinogens will offer insight into designing anti-cancer strategies and preventing cancer development. Here, we analyzed COX-2 expression in several human lung cancer cell lines and found that COX-2 expression was absent in the H719 and H460 cell lines by a DNA methylation-independent mechanism. The re-expression of COX-2 was observed after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment in both cell lines. Further investigation found that H3K36 dimethylation was significantly reduced near the COX-2 promoter because histone demethylase 2A (KDM2A) was recruited to the COX-2 promoter after TPA treatment. In addition, the transcription factor c-Fos was found to be required to recruit KDM2A to the COX-2 promoter for reactivation of COX-2 in response to TPA treatment in both the H719 and H460 cell lines. Together, our data reveal a novel mechanism by which the carcinogen TPA activates COX-2 expression by regulating H3K36 dimethylation near the COX-2 promoter.

Histone deacetylase inhibitor depsipeptide activates silenced genes through decreasing both CpG and H3K9 methylation on the promoter.

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 (HP1alpha and HP1beta) 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.

An ATM- and Rad3-related (ATR) signaling pathway and a phosphorylation-acetylation cascade are involved in activation of p53/p21Waf1/Cip1 in response to 5-aza-2'-deoxycytidine treatment.

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. 5-Aza-CdR induces p53 posttranslational modifications through activation of an ATM- and Rad3-related (ATR) signaling pathway, and 5-aza-CdR-induced association of replication protein A with chromatin is required for the binding of ATR to chromatin. Upon treatment with 5-aza-CdR, ATR activation is clearly associated with p53 phosphorylation at Ser(15), but not at Thr(18), Ser(20), or Ser(37). This specific p53 phosphorylation at Ser(15) in turn results in acetylation of p53 at Lys(320) and Lys(373)/Lys(382) through transcriptional cofactors p300/CBP-associated factor and p300, respectively. These p53 posttranslational modifications are directly responsible for 5-aza-CdR induced p21(Waf1/Cip1) expression because the binding activity of acetylated p53 at Lys(320)/Lys(373)/Lys(382) to the p21(Waf1/Cip1) promoter, as well as p21(Waf1/Cip1) expression itself are significantly increased after 5-aza-CdR treatment. It is of interest that p53 phosphorylation at Ser(15) and acetylations at Lys(320)/Lys(373)/Lys(382) mutually interact in the 5-aza-CdR induced p21(Waf1/Cip1) expression shown by transfection of artificially mutated p53 expression vectors including S15A, K320R, and K373R/K382R into p53-null H1299 cells. These data taken together show for the first time that 5-aza-CdR activates the ATR signaling pathway, which elicits a specific p53 phosphorylation-acetylation cascade to induce p21(Waf1/Cip1) expression.

HDAC inhibitors act with 5-aza-2'-deoxycytidine to inhibit cell proliferation by suppressing removal of incorporated abases in lung cancer cells.

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 [(3)H]-5-aza-CdR incorporated in DNA. However, incorporated [(3)H]-5-aza-CdR gradually decreased when cells were incubated in [(3)H]-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 [(3)H]-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 gamma-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.

Do after school programs reduce delinquency?

After school programs (ASPs) are popular and receive substantial public funding. Aside from their child-care and supervision value, ASPs often provide youth development and skill-building activities that might reduce delinquent behavior. These possibilities and the observation that arrests for juvenile crime peak between 2 p.m. and 6 p.m. on school days have increased interest in the delinquency prevention potential of ASPs. This study examined effects of participation in ASPs conducted in Maryland during the 1999--2000 school year and the mechanism through which such programs may affect delinquent behavior. Results imply that participation reduced delinquent behavior for middle-school but not for elementary-school-aged youths. This reduction was not achieved by decreasing time spent unsupervised or by increasing involvement in constructive activities, but by increasing intentions not to use drugs and positive peer associations. Effects on these outcomes were strongest in programs that incorporated a high emphasis on social skills and character development.

5-aza-2'-deoxycytidine activates the p53/p21Waf1/Cip1 pathway to inhibit cell proliferation.

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 microm) induces inhibition of cell proliferation as well as increased p53/p21(Waf1/Cip1) expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21(Waf1/Cip1) expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21(Waf1/Cip1) expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21(Waf1/Cip1) expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.