Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells
2001; Springer Nature; Volume: 20; Issue: 24 Linguagem: Inglês
10.1093/emboj/20.24.6969
ISSN1460-2075
Autores Tópico(s)Epigenetics and DNA Methylation
ResumoArticle17 December 2001free access Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells Martin Göttlicher Corresponding Author Martin Göttlicher Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Saverio Minucci Saverio Minucci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy Search for more papers by this author Ping Zhu Ping Zhu Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Oliver H. Krämer Oliver H. Krämer Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany Search for more papers by this author Annemarie Schimpf Annemarie Schimpf Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany Search for more papers by this author Sabrina Giavara Sabrina Giavara European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy Search for more papers by this author Jonathan P. Sleeman Jonathan P. Sleeman Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Francesco Lo Coco Francesco Lo Coco Department of Cellular Biotechnology and Hematology, University of Rome ‘La Sapienza’, I-00161 Rome, Italy Search for more papers by this author Clara Nervi Clara Nervi Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, I-00161 Rome, Italy Search for more papers by this author Pier Giuseppe Pelicci Pier Giuseppe Pelicci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy Search for more papers by this author Thorsten Heinzel Corresponding Author Thorsten Heinzel Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany Search for more papers by this author Martin Göttlicher Corresponding Author Martin Göttlicher Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Saverio Minucci Saverio Minucci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy Search for more papers by this author Ping Zhu Ping Zhu Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Oliver H. Krämer Oliver H. Krämer Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany Search for more papers by this author Annemarie Schimpf Annemarie Schimpf Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany Search for more papers by this author Sabrina Giavara Sabrina Giavara European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy Search for more papers by this author Jonathan P. Sleeman Jonathan P. Sleeman Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Francesco Lo Coco Francesco Lo Coco Department of Cellular Biotechnology and Hematology, University of Rome ‘La Sapienza’, I-00161 Rome, Italy Search for more papers by this author Clara Nervi Clara Nervi Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, I-00161 Rome, Italy Search for more papers by this author Pier Giuseppe Pelicci Pier Giuseppe Pelicci European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy Search for more papers by this author Thorsten Heinzel Corresponding Author Thorsten Heinzel Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany Search for more papers by this author Author Information Martin Göttlicher 1, Saverio Minucci3, Ping Zhu1, Oliver H. Krämer2, Annemarie Schimpf2, Sabrina Giavara3, Jonathan P. Sleeman1, Francesco Lo Coco4, Clara Nervi5, Pier Giuseppe Pelicci3 and Thorsten Heinzel 2 1Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-Helmholtz-Platz 1, D-76344 Eggenstein, Germany 2Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt, Germany 3European Institute of Oncology, Department of Experimental Oncology, Via Ripamonti 435, 20141 Milan, Italy 4Department of Cellular Biotechnology and Hematology, University of Rome ‘La Sapienza’, I-00161 Rome, Italy 5Department of Histology and Medical Embryology, University of Rome ‘La Sapienza’, I-00161 Rome, Italy *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2001)20:6969-6978https://doi.org/10.1093/emboj/20.24.6969 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Local remodeling of chromatin and dynamic changes in the nucleosomal packaging of DNA are key steps in the regulation of gene expression, consequently affecting proper cell function, differentiation and proliferation. One of the most important mechanisms in chromatin remodeling is the post-translational modification of the N-terminal tails of histones by acetylation, which apparently contributes to a ‘histone code’ determining the activity of target genes (Strahl and Allis, 2000). Acetylation of histones and possibly other substrates is mediated by enzymes with histone acetyltransferase (HAT) activity. Conversely, acetyl groups are removed by histone deacetylases (HDACs). Both HAT and HDAC activities are recruited to target genes in complexes with sequence-specific transcription factors and their cofactors, e.g. corepressors such as N-CoR and SMRT, and coactivators (Chen and Evans, 1995; Hörlein et al., 1995; Xu et al., 1999). Nuclear receptors were the main examples of transcription factors recruiting HAT- and HDAC-associated cofactors depending on their status of activation by an appropriate ligand (Alland et al., 1997; Heinzel et al., 1997; Nagy et al., 1997; Glass and Rosenfeld, 2000). Other transcription factors such as Mad-1, BCL-6 and ETO have also been shown to assemble HDAC-dependent transcriptional repressor complexes (Laherty et al., 1997; Dhordain et al., 1998; Gelmetti et al., 1998; Lutterbach et al., 1998; Wang et al., 1998). Inappropriate repression of genes required for cell differentiation has been linked to several forms of cancer, particularly to acute leukemia. In acute promyelocytic leukemia (APL) patients, retinoic acid receptor (RAR) fusion proteins (e.g. PML–RAR or PLZF–RAR) resulting from chromosomal translocations can interact with components of the corepressor complex (Grignani et al., 1998; Guidez et al., 1998; He et al., 1998; Lin et al., 1998). The hypothesis that corepressor-mediated aberrant repression may be causal for pathogenesis in APL is supported by the finding that the differentiation block in cells transformed by PLZF–RAR is overcome by combined treatment with retinoic acid (RA) and trichostatin A (TSA), which inhibits HDAC activity. Furthermore, a PML–RAR patient who had experienced multiple relapses after RA therapy has been treated with the HDAC inhibitor phenylbutyrate, resulting in complete remission (Warrell et al., 1998). HDAC inhibitors also appear to induce differentiation and/or apoptosis of transformed cells of non-hematopoietic origin without evidence for aberrant repression. Several structurally diverse HDAC inhibitors have been identified and their activity in transformed cells in vitro renders them prime candidates for cancer therapy (Marks et al., 2000; Krämer et al., 2001). However, some HDAC inhibitors (e.g. trapoxin) are of limited therapeutic use due to poor bioavailability in vivo as well as toxic side effects at high doses. Butyrate and phenylbutyrate are degraded rapidly after i.v. administration and therefore require high doses exceeding 400 mg/kg/day (Warrell et al., 1998). Furthermore, these compounds are not specific for HDACs as they also inhibit phosphorylation and methylation of proteins as well as DNA methylation (Newmark and Young, 1995). Valproic acid (VPA, 2-propylpentanoic acid) is an established drug in the long-term therapy of epilepsy. Although VPA is well tolerated by patients, it can induce birth defects such as neural tube closure defects and other malformations when administered during early pregnancy (DiLiberti et al., 1984; Nau et al., 1991). Teratogenicity and antiepileptic activity appear to require different mechanisms of action because modifications of the molecule generate selective compounds with either teratogenic or antiepileptic activity (Nau et al., 1991). However, neither of the mechanisms of action is well understood at present (Löscher, 1999). During the search for mediators of VPA teratogenicity, attention had been drawn to the peroxisome proliferator-activated receptors (PPARs) because VPA induces proliferation of peroxisomes in the rodent liver (Horie and Suga, 1985; Göttlicher et al., 1992; Kliewer et al., 1994; Kersten et al., 2000). PPARδ is activated by VPA and its teratogenic derivatives (Lampen et al., 1999; Werling et al., 2001). The failure to show binding of VPA to PPARδ raised the possibility that VPA and its teratogenic derivatives activate the receptor by a mechanism distinct from binding as a ligand. Here we show that VPA inhibits corepressor-associated HDACs at therapeutically employed concentrations and acts as a potent inducer of differentiation in several types of transformed cells. Results 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 (Figure 1A) 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 (cPGI2) or HDAC inhibitors (Figure 1A). The PPARδ ligand cPGI2 and VPA both activate the GR–PPARδ hybrid receptor. Since cPGI2 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 cPGI2 but not with TSA or butyrate (Figure 1A). Highly synergistic activation of the reporter gene by VPA together with cPGI2 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 (Figure 1B) or a PPARα fusion protein (data not shown) which do not recruit corepressor-associated HDACs. Figure 1.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. Download figure Download PowerPoint 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 (Figure 2). VPA at a concentration of 1 mM induces relief of this repression by Gal4 fusions of N-CoR, TR or PPARδ (Figure 2A), 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 (Figure 2A). 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 (Figure 2B). 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. Figure 2.VPA relieves HDAC-mediated transcriptional repression. (A) HeLa cells were co-transfected with a UAS-TK-luciferase reporter, an SV40 β-Gal control reporter and vectors expressing either the GAL4 DNA-binding domain (amino acids 1–147) or GAL4 fusions of N-CoR (amino acids 1–2453) or the ligand-binding domains of TRβ (amino acids 165–456) with or without TRIAC, or PPARδ (amino acids 138–440) with or without cPGI2, respectively. At 24 h after transfection, cells were treated with trapoxin (10 nM), TSA (100 nM) or VPA (1 mM) for 16–20 h. Subsequently, cells were harvested and luciferase and β-galactosidase activities were measured. Results are represented as fold repression relative to GAL4. Values are means ± SD from triplicate determinations in two independent experiments. (B) Phoenix cells were co-transfected with the indicated expression vectors, using a luciferase reporter based on the human RARβ2 promoter (de Thé et al., 1990). VPA (1 or 3 mM) was added 18 h after transfections, and cells were analyzed for reporter activity after an additional 24 h. Download figure Download PowerPoint 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 (Figure 3). 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 (Figure 3A). 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 (Figure 3B). 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 (Figure 3C) and F9 (Figure 3D 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 (Figure 3C). Figure 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. (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 [3H]acetate 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. (HDAC2). 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 [3H]acetate 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. Download figure Download PowerPoint 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 by <40% even in the presence of 5 mM VPA, although TSA inhibits this HDAC activity completely (Figure 3D). This finding suggests that the spectrum of HDACs which are not tightly associated with N-CoR differs between the two cell types and that not all HDAC isoforms may be inhibited with equal efficiency by VPA. Significant levels of class II HDACs 5 and 6 were detectable in depleted F9 but not in HEK293T cell extracts (data not shown), suggesting that in F9 cells a substantial fraction of HDACs 5 and 6 is not associated with N-CoR. The IC50 concentrations for HDACs 5 and 6 precipitated from F9 cell extracts depleted with N-CoR, mSin3 and HDAC 1–3 antibodies were determined as 2.8 and 2.4 mM VPA, respectively (Figure 3E). Nevertheless, TSA inhibits the HDACs 5 and 6 precipitates with efficiency similar to the class I HDAC precipitates. These results suggest that these class II HDACs may be significantly less susceptible to inhibition by VPA than class I enzymes, whereas many other HDAC inhibitors do not discriminate between isoenzymes. To investigate further the mechanism of HDAC inhibition, we performed binding studies with radiolabeled VPA. [3H]VPA binds to protein complexes precipitated with antibodies against N-CoR, mSin3 or HDAC2 in vitro (Figure 4). TSA has been shown to bind directly to the active site of a deacetylase (Finnin et al., 1999). Since TSA efficiently competes for binding of [3H]VPA, both compounds not only exert similar effects on HDAC activity but also appear to share identical or overlapping binding sites. Therefore, we speculate that the mechanism of HDAC inhibition by VPA involves blocking of substrate access to the catalytic center of the enzyme. Figure 4.Binding of VPA to corepressor or HDAC2 immunoprecipitates. Antibodies directed against N-CoR, mSin3 and HDAC2 co-precipitate substantial HDAC activity, most probably in the form of multiprotein complexes (Figure 3C). Immunoprecipitates from HEK293T cell extracts were incubated with 2 μCi of 3H-labeled VPA in the presence or absence of TSA (100 nM). Radioactivity retained after washing is shown. Values are means ± SD from three independent experiments carried out in duplicate. Download figure Download PowerPoint VPA has several activities which are unlikely to follow the same mechanisms of action. The antiepileptic activity and teratogenic side effects can be dissociated by appropriate modifications of the VPA molecule. Both stereoisomers of 4-yn-VPA (2-propinyl-pentanoic acid) exhibit identical low antiepileptic activity, although only one of them (S-4-yn-VPA) is teratogenic (Nau et al., 1991). Similarly, only one of the stereoisomers of the chemically related 2-ethylhexanoic acid (R-EHXA) is teratogenic, whereas S-EHXA is not (Hauck et al., 1990). Valpromide (VPD) is an even more potent antiepileptic drug than VPA but lacks substantial teratogenicity (Löscher and Nau, 1985; Radatz et al., 1998). Isomers of 4-yn-VPA and EHXA as well as VPA and VPD were used to test whether HDAC activity is associated with one of the previously known activities. HDAC activity in vitro is only inhibited by the teratogenic compounds S-4-yn-VPA, racemic EHXA (Figure 5A) and VPA (Figure 3 and data not shown). Also, accumulation of hyperacetylated histones H3 and H4 is induced only by the teratogenic stereoisomers of 4-yn-VPA and EHXA. Histone acetylation is not induced in cells treated with the non-teratogenic isomers or by VPD (Figure 5B). In summary, we identified several VPA-related compounds which clearly separate antiepileptic properties from HDAC inhibitory activity, whereas a strict correlation of HDAC inhibition and teratogenic activity was observed. Figure 5.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. = 100%). 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. Download figure Download PowerPoint Differentiation of transformed cells in vitro and in vivo HDAC inhibitors apparently affect cells of hematopoietic and non-hematopoietic origin by inducing cellular differentiation and/or apoptosis. Thus several cell lines of epithelial origin were tested for responsiveness to VPA. In F9 teratocarcinoma cells, VPA induces a specific type of differentiation characterized by reduced proliferation, morphological alterations, marker gene expression and particularly the accumulation of the AP-2 transcription factor as a potential marker of neuronal or neural crest cell-like differentiation (Werling et al., 2001). This type of differentiation is indistinguishable from TSA-induced differentiation by morphological criteria and by accumulation of AP-2 (Figure 6A), suggesting that it is caused by the HDAC inhibitory properties of VPA or TSA. VPA impairs cell proliferation or survival as indicated by decreased incorporation of [3H]thymidine in F9 and P19 teratocarcinoma cells (Werling et al., 2001), HT-29 colon and MT-450 breast carcinoma cells (Figure 6B). In MT-450 cells, an initial delay in proliferation is followed by the appearance of apoptotic cells after 3–6 days of VPA treatment (Figure 6C). Figure 6.VPA induces cell differentiation and apoptosis in carcinoma cell lines and inhibits tumor growth and metastasis formation in the rat. (A) F9 teratocarcinoma cells were treated for 48 h with TSA (30 nM) or VPA (1 mM). The appearance of AP-2 protein as a specific marker of the VPA-induced type of F9 cell differentiation was as described (Werling et al., 2001). One out of two experiments with similar results is shown. (B) Thymidine incorporation into cultures of HT-29 colonic cancer or MT-450 breast carcinoma cells was tested as a parameter for cell proliferation. Cells were cultured for 72 h in the absence or presence of the indicated concentrations of VPA prior to analysis of [3H]thymidine incorporation. The graph shows the means ± SD from quadruple determinations. Similar results were obtained in two additional independent experiments. (C) The appearance of apoptotic cells in VPA-treated cultures of MT-450 cells was analyzed by staining of cell surface-exposed phosphatidylserine by FITC-conjugated annexin V after exclusion of necrotic cells by means of propidium iodide uptake (lower right quadrant of the graphs). Similar results were obtained in a second experiment. (D) Subcutaneous tumor growth and lung metastasis of MT-450 breast cancer cells were analyzed in rats treated with VPA or saline, respectively. Tumor volume was determined at day 21 (onset of VPA treatment) and day 43 (termination of experiment). Lung metastasis visible from the organ surface was scored. Scores: 0, no metastasis; 1, <50 metastases or all metastatic nodules 2 mm in diameter (upper right frame); 3, most of the lung's surface consists of metastatic nodules (upper left frame). Values are means ± SD and significance of differences was calculated by Student t-test. (E) Pictures of two preparations each representative for control or VPA-treated rats are shown. The original height of the frames is 25 mm. Download figure Download PowerPoint These results prompted us to study the effects of VPA on tumor growth and metastasis in the MT-450 rat breast cancer model. VPA delayed growth of the primary tumors. All rats of the control group showed metastasis and most of the lungs were full with metastases (Figure 6D). Metastasis was also found in VPA-treated rats. However, the size and number of metastases was much smaller compared with NaCl-treated rats (Figure 6E). In a dose finding experiment, our dosage protocol led to high initial serum levels (e.g. 3.6 mM at 1 h after i.p. injection) which rapidly dropped to levels (e.g. 0.25 mM at 4 h after i.p. injection) below those maintained during therapy of epilepsy in humans. In summary, this experiment shows that even though VPA serum levels cannot be maintained at the optimal effective range of >0.5 mM in rodents, VPA treatment nevertheless substantially decreases primary tumor growth and lung metastasis. Aberrant recruitment of HDACs by several acute myeloid leukemia (AML) fusion proteins is required for their capacity to block myeloid differentiation (Minucci et al., 2001). Since AML cells are known to respond to treatment with HDAC inhibitors alone, or in synergistic combination with differentiating agents such as retinoids (Ferrara et al., 2001), we analyzed the effect of VPA on the AML Kasumi-1 cell line expressing the t(8;21) translocation product AML1/ETO, and on primary hematopoietic cells expressing the t(15;17) translocation product PML–RAR. Kasumi-1 cells are differentiated by RA as determined by the appearance of nitroblue tetrazolium (NBT)-positive cells. VPA induces partial differentiation of Kasumi-1 cells on its own and remarkably enhances the differentiating effect exerted by RA. VPA is as efficient as TSA in inducing Kasumi-1 cell differentiation (Figure 7A). Murine hematopoietic progenitor (lin−) cells in culture show myeloid differentiation, as indicated by expression of th
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