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. Moreover, tumor growth and metastasis formation are signi®cantly reduced in animal experiments. Therefore, valproic acid might serve as an effective drug for cancer therapy. Keywords: cancer therapy/HDAC inhibitor/histone deacetylase/leukemia/valproic acid IntroductionLocal 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 modi®cation 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 sequencespeci®c transcription factors and their cofactors, e.g. corepressors such as N-CoR and SMRT, and coactivators (Chen and Evans, 1995;Ho Èrlein 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 ®nding that the differentiation block in cells transformed by PLZF±RAR is overc...

DNA methylation of tumor suppressor genes is a frequent mechanism of transcriptional silencing in cancer. The molecular mechanisms underlying the specificity of methylation are unknown. We report here that the leukemia-promoting PML-RAR fusion protein induces gene hypermethylation and silencing by recruiting DNA methyltransferases to target promoters and that hypermethylation contributes to its leukemogenic potential. Retinoic acid treatment induces promoter demethylation, gene reexpression, and reversion of the transformed phenotype. These results establish a mechanistic link between genetic and epigenetic changes during transformation and suggest that hypermethylation contributes to the early steps of carcinogenesis.

The molecular pathogenesis of diffuse large-cell lymphoma (DLCL), the most frequent and clinically relevant type of lymphoma, is unknown. A gene was cloned from chromosomal translocations affecting band 3q27, which are common in DLCL. This gene, BCL-6, codes for a 79-kilodalton protein that is homologous with zinc finger-transcription factors. In 33 percent (13 of 39) of DLCL samples, but not in other types of lymphoid malignancies, the BCL-6 gene is truncated within its 5' noncoding sequences, suggesting that its expression is deregulated. Thus, BCL-6 may be a proto-oncogene specifically involved in the pathogenesis of DLCL.

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Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed 430 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: o100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.