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...

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate PPAR (peroxisome proliferator-activated receptor), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from the rat that is homologous to that from the mouse [Issemann, I. & Green, S. (1990) Unsaturated fatty acids induce peroxisomal proliferation and lower blood triglyceride levels (1-3). Similar effects are evoked by a number of man-made compounds which are either considered for therapy of hyperlipidemia-e.g., clofibric acid, nafenopin, WY-14,643, or sulfur-substituted fatty acids-or are in use as industrial plasticizers (4-6). The steroid dehydroepiandrosterone (DHEA) also induces peroxisomal proliferation but increases blood triglyceride and cholesterol levels (7,8). Two main hypotheses have been developed to explain the complex response of peroxisomal proliferation to this wide variety of inducers. According to one theory the intracellular accumulation of fatty acids is the key stimulus for triggering peroxisomal proliferation (4, 9). The other theory postulates the involvement of a receptor protein (4, 10) and an as-yet-unknown intracellular messenger-e.g., the ligand for this receptor.The latter idea gained substantial support from the discovery of mouse peroxisome proliferator-activated receptor (mPPAR), a member of the steroid nuclear receptor superfamily (11,12). The gene encoding PPAR belongs to a number of genes cloned in the last few years by means of their homology with steroid receptors. In general, ligands or physiologically occurring activators have been identified for only a few of these so-called orphan receptors (13-15). In the case of mPPAR, transactivation studies using chimeric proteins composed of the putative ligand-binding domain of the novel receptor and DNA-binding domains of known steroid receptors showed that mPPAR could be activated by peroxisome proliferators (11). However, the identity ofthe ultimate ligand of the receptor protein, the nature of physiological activators, and how the receptor might relate to the concept of fatty acids as inducers of peroxisomal proliferation remain unclear.We now describe the cloning from rat liver of a gene homologous to that encoding mPPAR.

contributed equally to this work Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as ef®cient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin±proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2.

Inappropriate transcriptional repression involving histone deacetylases (HDACs) is a prominent cause for the development of leukemia. We now identify faulty expression of a specific mediator of transcriptional repression in a solid tumor. Loss of the adenomatosis polyposis coli (APC) tumor suppressor induces HDAC2 expression depending on the Wnt pathway and c-Myc. Increased HDAC2 expression is found in the majority of human colon cancer explants, as well as in intestinal mucosa and polyps of APC-deficient mice. HDAC2 is required for, and sufficient on its own to prevent, apoptosis of colonic cancer cells. Interference with HDAC2 by valproic acid largely diminishes adenoma formation in APC(min) mice. These findings point toward HDAC2 as a particularly relevant potential target in cancer therapy.