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Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Thus, HDAC inhibitors are candidate drugs for differentiation therapy of cancer. Here, we show that the well‐tolerated antiepileptic drug valproic acid is a powerful HDAC inhibitor. Valproic acid relieves HDAC‐dependent transcriptional repression and causes hyperacetylation of histones in cultured cells and in vivo. Valproic acid inhibits HDAC activity in vitro, most probably by binding to the catalytic center of HDACs. Most importantly, valproic acid induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia patients.
More over, tumor growth and metastasis formation are significantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy. Relief of transcriptional repression by VPA Induction of peroxisomal proliferation and activation of a glucocorticoid receptor (GR)–PPARδ hybrid receptor by VPA pointed towards PPARδ as a potential target of VPA ( Werling et al., 2001). Activation of PPARδ by VPA could be caused by activation of the PPARδ ligand‐binding domain and subsequent recruitment of coactivators ( Xu et al., 1999).
Alternatively, VPA could release PPARδ‐dependent transcriptional repression and allow at least partial activation of reporter gene expression probably in conjunction with low levels of endogenous PPARδ ligands. To discriminate between these possibilities, synergism of VPA was tested together with either a bona fide ligand of PPARδ (cPGI 2) or HDAC inhibitors. The PPARδ ligand cPGI 2 and VPA both activate the GR–PPARδ hybrid receptor. Since cPGI 2 and HDAC inhibitors, such as TSA or butyrate, synergistically activate the GR–PPARδ hybrid receptor, an agonistic ligand alone is insufficient for full activation and derepression. VPA at concentrations of 1 or 2 mM acts synergistically with cPGI 2 but not with TSA or butyrate. Highly synergistic activation of the reporter gene by VPA together with cPGI 2 and lack of synergism with TSA or butyrate indicates that VPA does not act like a ligand to PPARδ but rather like an inhibitor of repression. No synergism is found with receptors such as the GR or a PPARα fusion protein (data not shown) which do not recruit corepressor‐associated HDACs.
HDAC inhibitor‐like activation of PPARδ by VPA. ( A) A cell line expressing the ligand‐binding domain of PPARδ fused to the DNA‐binding domain of the glucocorticoid receptor (GR) together with a GR‐controlled reporter gene was treated for 40 h with the PPARδ ligand cPGI 2 (5 μM), VPA or the HDAC inhibitors sodium butyrate (But) and TSA (300 nM). Reporter gene activity was monitored by enzymatic assay for alkaline phosphatase. Values were normalized between experiments according to cPGI 2‐induced activity. ( B) A cell line overexpressing full‐length GR was tested as a control.
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Dexamethasone (1 μM) was used as a GR‐specific ligand. Values are means ± SD from duplicate determinations in 2–5 independent experiments.
HDAC inhibitor‐like activation of PPARδ by VPA. (A) A cell line expressing the ligand‐binding domain of PPARδ fused to the DNA‐binding domain of the glucocorticoid receptor (GR) together with a GR‐controlled reporter gene was treated for 40 h with the PPARδ ligand cPGI2 (5 μM), VPA or the HDAC inhibitors sodium butyrate (But) and TSA (300 nM). Reporter gene activity was monitored by enzymatic assay for alkaline phosphatase. Values were normalized between experiments according to cPGI2‐induced activity. (B) A cell line overexpressing full‐length GR was tested as a control.
Dexamethasone (1 μM) was used as a GR‐specific ligand. Values are means ± SD from duplicate determinations in 2–5 independent experiments. A direct assay for transcriptional repressor activity is based on repression of a high‐baseline promoter. Many transcription factors including thyroid hormone receptor (TR), PPARδ and the corepressors N‐CoR or mSin3 repress transcription of a promoter containing UAS elements when bound as fusion proteins with the heterologous DNA‐binding domain of the Gal4 protein ( Heinzel et al., 1997).
In the absence of the Gal4 fusion proteins, the reporter has a high basal transcriptional activity due to the presence of binding sites for other transcription factors in the thymidine kinase promoter. The Gal4 fusion proteins strongly repress this activity. VPA at a concentration of 1 mM induces relief of this repression by Gal4 fusions of N‐CoR, TR or PPARδ , ETO or Mad1 (data not shown) as efficiently as established HDAC inhibitors. This relief of repression is also found after partial activation of nuclear receptors with their respective ligands. Moreover, oncogenic RAR fusion proteins such as PML–RAR repress RAR‐dependent reporter gene expression after transient transfection.
This repression is relieved efficiently by VPA. Thus, VPA affects the activity of several transcriptional repressors, suggesting that it acts on a common factor in gene regulation such as corepressor‐associated HDACs rather than on individual transcription factors or receptors. HDAC inhibition by VPA We tested whether VPA can lead to HDAC inhibition by analyzing the degree of histone acetylation in vitro and in vivo with an antibody specific for hyperacetylated histones H3 or H4. Only minute amounts of acetylated histones are detected in untreated F9 teratocarcinoma or HeLa cells.
Treatment with VPA at concentrations as low as 0.25 mM increases the amount of acetylated histone H4, and massive acetylation is found with 2 mM VPA. This acetylation level is similar to that induced by 5 mM butyrate and slightly less than that by 100 nM TSA. Maximum bulk histone acetylation appears ∼12–16 h after addition of VPA.
VPA treatment of mice also induces hyperacetylation of histones in spleen. To test whether VPA directly inhibits HDAC activity, 3H‐labeled acetylated histones were deacetylated using anti‐N‐CoR, anti‐mSin3 or anti‐HDAC2 immunoprecipitates from HEK293T and F9 ( and data not shown) cell extracts as a source of HDAC enzymatic activity. Immunoprecipitates typically contain 25–30% (N‐CoR) or 15–20% (mSin3) of the HDAC activity of whole‐cell extracts. Already at a concentration of 0.5 mM, VPA inhibits N‐CoR‐associated HDAC activity almost as efficiently as TSA (300 nM) or sodium butyrate (1 mM, data not shown).
As no further washing which could remove any component is performed after the addition of VPA, and since VPA does not induce dissociation of HDAC3 from the N‐CoR immunoprecipitate (data not shown), enzyme inhibition is most likely to be due to direct effects on HDACs rather than disintegration of the complex. HDAC activities precipitated from both F9 and HEK293T cells with antibodies directed against mSin3 or HDAC2 are also inhibited by VPA, although slightly higher concentrations appear to be required. VPA induces accumulation of hyperacetylated histone and inhibits HDAC activity. ( A) HDAC inhibitors induce the accumulation of hyperacetylated histones H3 and H4. Both the time course and the required concentration for VPA‐induced hyperacetylation were determined by western blot analysis of whole‐cell extracts from F9 cells treated with VPA (1 mM if not indicated otherwise) in comparison with TSA (100 nM) and sodium butyrate (NaBu, 5 mM).
Treatment was for 12 h or as indicated. Equal loading was confirmed by Coomassie Blue staining. Experiments were performed three times with similar results also in HeLa cells. ( B) Histone hyperacetylation in vivo was determined by western blot analysis of histones H3 and H4 from mouse splenocyte nuclear extracts. Three mice each were injected i.p. With 25 ml/kg body weight of 155 mM solutions of NaCl or sodium valproate. Due to the short half‐life of VPA in rodents, another dose (50%) was readministered after 5 h.
Extracts were prepared 10 h after the initial dose. ( C) HDAC activity was determined by the release of 3Hacetate from hyperacetylated radiolabeled histones. Activities were determined in the presence of the indicated compounds in immune precipitates from HEK293T cell extracts with antibodies directed against N‐CoR, mSin3 or HDAC2.
The HDAC activity which precipitated with a non‐related immune serum (NI) was determined for control. Values are presented relative to the activity in the absence of HDAC inhibitors.
The 100% values normalized for ∼1 mg of extract in representative experiments correspond to 1000 (N‐CoR), 500 (mSin3) and 300 c.p.m. Data are means ± SD from three independent experiments. ( D) HDAC activity was determined in immune precipitates from F9 cell extracts with antibodies directed against N‐CoR and in N‐CoR‐depleted extracts. Efficiency of N‐CoR depletion was assessed by western blot for N‐CoR in the IP pellet as well as in equivalent amounts of whole‐cell extracts before and after depletion (data not shown). ( E) IC 50 values were calculated as those concentrations required for 50% inhibition of 3Hacetate release.
HDAC assays were performed using immune precipitates from F9 cell extracts with antibodies directed against HDACs 2, 5 or 6. HDACs 5 or 6 were precipitated from extracts which had been depleted with antibodies directed against N‐CoR, mSin3 and HDACs 1–3. VPA induces accumulation of hyperacetylated histone and inhibits HDAC activity. (A) HDAC inhibitors induce the accumulation of hyperacetylated histones H3 and H4. Both the time course and the required concentration for VPA‐induced hyperacetylation were determined by western blot analysis of whole‐cell extracts from F9 cells treated with VPA (1 mM if not indicated otherwise) in comparison with TSA (100 nM) and sodium butyrate (NaBu, 5 mM).
Treatment was for 12 h or as indicated. Equal loading was confirmed by Coomassie Blue staining. Experiments were performed three times with similar results also in HeLa cells.
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(B) Histone hyperacetylation in vivo was determined by western blot analysis of histones H3 and H4 from mouse splenocyte nuclear extracts. Three mice each were injected i.p. With 25 ml/kg body weight of 155 mM solutions of NaCl or sodium valproate. Due to the short half‐life of VPA in rodents, another dose (50%) was readministered after 5 h. Extracts were prepared 10 h after the initial dose. (C) HDAC activity was determined by the release of 3Hacetate from hyperacetylated radiolabeled histones.
Activities were determined in the presence of the indicated compounds in immune precipitates from HEK293T cell extracts with antibodies directed against N‐CoR, mSin3 or HDAC2. The HDAC activity which precipitated with a non‐related immune serum (NI) was determined for control. Values are presented relative to the activity in the absence of HDAC inhibitors. The 100% values normalized for ∼1 mg of extract in representative experiments correspond to 1000 (N‐CoR), 500 (mSin3) and 300 c.p.m. Data are means ± SD from three independent experiments. (D) HDAC activity was determined in immune precipitates from F9 cell extracts with antibodies directed against N‐CoR and in N‐CoR‐depleted extracts.
Efficiency of N‐CoR depletion was assessed by western blot for N‐CoR in the IP pellet as well as in equivalent amounts of whole‐cell extracts before and after depletion (data not shown). (E) IC50 values were calculated as those concentrations required for 50% inhibition of 3Hacetate release. HDAC assays were performed using immune precipitates from F9 cell extracts with antibodies directed against HDACs 2, 5 or 6. HDACs 5 or 6 were precipitated from extracts which had been depleted with antibodies directed against N‐CoR, mSin3 and HDACs 1–3. A significant difference between HEK293T and F9 cells is found when the sensitivity to VPA of N‐CoR‐depleted supernatants is analyzed. Those HDACs which remain in the N‐CoR‐depleted supernatant of HEK293T cells are inhibited by VPA as efficiently as those in the precipitate (data not shown). The N‐CoR‐depleted supernatant from F9 cell extracts, however, is only inhibited.
HDAC inhibition by compounds related to VPA. ( A) VPA and the related compounds EHXA, and R‐ and S‐4‐yn‐VPA at the indicated concentrations were tested for HDAC inhibitory activity. Addition of TSA (100 nM) to the reaction served as a control. The assays were performed with N‐CoR immunoprecipitates from HEK293T cells in duplicate (untreated enzyme activity 2205 c.p.m. Precipitates of a pre‐immune serum served as a negative control.
( B) Accumulation of hyperacetylated histones H3 and H4 in F9 cells treated with compounds related to VPA was determined as described in the legend to. Cells were treated for 12 h with 1 mM of VPA, R‐ or S‐EHXA, R‐ or S‐4‐yn‐VPA or VPD. One representative out of two similar experiments is shown. HDAC inhibition by compounds related to VPA. (A) VPA and the related compounds EHXA, and R‐ and S‐4‐yn‐VPA at the indicated concentrations were tested for HDAC inhibitory activity. Addition of TSA (100 nM) to the reaction served as a control.
The assays were performed with N‐CoR immunoprecipitates from HEK293T cells in duplicate (untreated enzyme activity 2205 c.p.m. Precipitates of a pre‐immune serum served as a negative control. (B) Accumulation of hyperacetylated histones H3 and H4 in F9 cells treated with compounds related to VPA was determined as described in the legend to Figure 3. Cells were treated for 12 h with 1 mM of VPA, R‐ or S‐EHXA, R‐ or S‐4‐yn‐VPA or VPD. One representative out of two similar experiments is shown.