p21(WAF1/CIP1) induction by 5-azacytosine nucleosides requires DNA damage - PubMed (original) (raw)

p21(WAF1/CIP1) induction by 5-azacytosine nucleosides requires DNA damage

A Jiemjit et al. Oncogene. 2008.

Abstract

Decitabine (DAC) and 5-azacitidine have recently been approved for the treatment of myelodysplastic syndrome. The pharmacodynamic effects of DAC and 5-azacitidine outside their known activity as inhibitors of DNA methyltransferases (DNMTs) require further investigation. The purpose of this study was to investigate the effect of DAC on the expression of p21(WAF1/CIP1), a gene with a putative CpG island surrounding its promoter region. Promoter methylation analysis of p21(WAF1/CIP1) in leukemia cells revealed the absence of CpG methylation. However, DAC upregulated p21(WAF1/CIP1) expression in a dose-dependent manner (ED(50)=103.34 nM) and induced G2/M cell cycle arrest in leukemia cells. Sequential application of DAC followed by different histone deacetylase inhibitors induced expression of p21(WAF1/CIP1) synergistically. Upregulation of p21(WAF1/CIP1) paralleled DAC-induced apoptosis (ED(50)=153 nM). Low doses of DAC induced gamma-H2AX expression (ED(50)=16.5 nM) and upregulated p21(WAF1/CIP1) in congenic HCT 116 colon cancer cells in a DNMT-independent and p53-dependent fashion. Inhibition of p53 transactivation by pifithrin-alpha or the kinase activity of ATM by either the specific ATM inhibitor KU-5593 or caffeine abrogated p21(WAF1/CIP1) upregulation, indicating that DAC upregulation of p21(WAF1/CIP1) was p53- and ATM-dependent in leukemia cells. In conclusion, DAC upregulates p21(WAF1/CIP1) in DNMT-independent manner via the DNA damage/ATM/p53 axis.

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Figures

Figure 1

Decitabine (DAC)induces p21WAF1/CIP1 expression, G2/M arrest, apoptosis and DNA damage in leukemia cells. (a)ML-1 cells were treated with graded doses of DAC for 48 h, and p21WAF1/CIP1 expression was measured by western blotting and signals quantified by densitometry. Results represent the mean±s.d. for three independent experiments. Closed circle indicates MS275 (1 μM) as a positive control. The inset shows a representative blot. (b)Synchronize d ML-1 cells (serum starvation)were treated with different concentrations of DAC for 48 h for cell cycle analysis by propidium iodide staining. Results represent the mean±s.d. for three independent experiments. G2/M values are shown above each bar. (c)ML-1 cells were treated with different concentrations of DAC for 72 h, and apoptosis was measured as described under Materials and methods. Results represent the mean±s.d. for three independent experiments. (d)ML-1 cells were treated with different concentrations of DAC for 48 h, and the expression of the DNA damage marker γ-H2AX was determined by western blotting. A representative blot of three independent experiments is shown. Actin was used as a loading control. (e)ML-1 cells were sequentially treated with different concentrations of DAC (48 h)followed by MS-275 or trichostatin A (TSA)for 24 h, and the expression of the DNA damage marker γ-H2AX was determined by western blotting. Actin was used as a loading control.

Figure 2

Sequential treatment of decitabine (DAC) and histone deacetylase (HDAC) inhibitors synergistically upregulates p21WAF1/CIP1 expression. ML-1 cells were incubated with different concentrations of DAC (0.0125–0.05 μM)for 48 h prior to addition of sodium phenylbutyrate (NaPB; 0.25–1mM), MS-275 (0.25–1 μM), trichostatin A (TSA; 0.08–0.33 μM)or valproic acid (VPA; 0.25–1mM)for 24 h. p21WAF1/CIP1 expression was analysed by western blotting and densitometry. The mean signal intensity from three independent experiments was used for the median effect analysis. The combination index (CI)as a function of fraction affected was plotted for the combination of DAC with HDAC inhibitors in ML-1 at fixed ratios. Representative graphs for the combination of DAC with MS-275 and TSA are shown along with the calculated CI values for each combination. CI < 1 indicates a synergistic interaction.

Figure 3

Sequential treatment with decitabine (DAC)and histone deacetylase (HDAC)inhibitors enhances HDAC inhibitor-induced apoptosis. (a)ML-1 cells were treated with DAC (1 μM)for 48 h then treated with trichostatin A (TSA; 0.33 μM), MS-275 (1 μM), sodium phenylbutyrate (NaPB; 1mM)or valproic acid (VPA; 1mM)for 24 h. Apoptosis was measured as described under Materials and methods. A representative experiment is shown. (b)Apoptosis data for three independent experiments are plotted as the mean±s.d. for the different treatments.

Figure 4

Decitabine (DAC)-induced p21WAF1/CIP1 upregulation and DNA damage are DNA methyltransferase (DNMT) independent and p53 dependent. Congenic HCT116 cells were used to investigate the effect of DAC (1 μM)treatment for 72 h on p21WAF1/CIP1 (a)and γ-H2AX (b)by western blotting. DKO indicates double knockout cells (DNMT1 and DNMT3b). Actin was used as a loading control. The figure is representative of four independent experiments. Numerical values above each blot represent the signal intensity measured by densitometry.

Figure 5

Decitabine (DAC)-induced p21WAF1/CIP1 upregulation is p53 dependent. DAC (1 μM)was used to treat p53-wild-type (WT) ML-1 cells (a)and p53-null HL-60 cells (b)for 24 and 48 h. The expression of p21WAF1/CIP1 was measured by western blotting. MS-275 (1 μM), phorbol 12-myristate 13-acetate (PMA; 10–25 nM)and trichostatin A (TSA; 330 nM)were used as positive controls. Actin was used as a loading control. The figure is a representative of three independent experiments. (c)ML-1 cells pretreated with 20 μM pifithrin-α (PF)for 24 h followed by DAC (1 μM)for 72 h. p21WAF1/CIP1 expression was measured by western blotting. Actin was used a loading control.

Figure 6

Decitabine (DAC)-induced G2/M arrest is p53 dependent and p21WAF1/CIP1 upregulation is ATM dependent. (a)Synchronize d HL-60 cells were treated with DAC (0.5, 1 μM)for 48 h and cell cycle analysis was performed as described under Materials and methods. Results represent the mean±s.d. for three independent experiments. (b)HL-60 and ML-1 cells were treated with DAC (500, 1000 nM)for 72 h. Apoptosis was measured as described under Materials and methods. Results represent the mean±s.d. for three independent experiments. (c)ML-1 cells were treated with DAC (1, 2 μM), caffeine (1mM), MS-275 (1 μM)or cotreated with DAC + caffeine or MS-275 + caffeine for 48 h. The expression of p21WAF1/CIP1 was measured by western blotting. Actin was used as a loading control. Numerical values above each blot represent the signal intensity measured by densitometry. (d)ML-1 cells were treated with DAC (1 μM), caffeine (1mM), MS-275 (1 μM), KU-5593 (20 μM)alone or in combination for 48 h. Caffeine was used as a cotreatment, while KU-5593 was used as a pretreatment for 1 h.

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p21(WAF1/CIP1) induction by 5-azacytosine nucleosides requires DNA damage - PubMed (original) (raw)