Valproic acid (VPA), a well-established therapy for seizures and bipolar disorder, has recently been shown to inhibit histone deacetylases (HDACs). Similar to more widely studied HDAC inhibitors, VPA can cause growth arrest and induce differentiation of transformed cells in culture. Whether this effect of VPA is through inhibition of HDACs or modulation of another target of VPA has not been tested. We have used a series of VPA analogs to establish a pharmacological profile for HDAC inhibition. We find that VPA and its analogs inhibit multiple HDACs from class I and class II (but not HDAC6 or HDAC10) with a characteristic order of potency in vitro. These analogs also induce hyperacetylation of core histones H3 and H4 in intact cells with an order of potency that parallels in vitro inhibition. VPA and VPA analogs induce differentiation in hematopoietic cell lines in a p21-dependent manner, and the order of potency for induction of differentiation parallels the potencies for inhibition in vitro, as well as for acetylation of histones associated with the p21 promoter, supporting the argument that differentiation caused by VPA is mediated through inhibition of HDACs. These findings provide additional evidence that VPA, a well-tolerated, orally administered drug with extensive clinical experience, may serve as an effective chemotherapeutic agent through targeting of HDACs.

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The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMPmediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/ PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.Cyclic adenosine 3Ј,5Ј-monophosphate (cAMP) is produced as an intracellular second messenger in response to a variety of extracellular signals, including hormones, growth factors, and neurotransmitters. cAMP regulates a wide range of important biological processes, which, alongside cell metabolism, include cell division, growth, differentiation, secretion, memory, and neoplastic transformation. For many years, major intracellular effects of cAMP in mammalian cells were believed to be mediated by cAMP-dependent protein kinase (PKA).1 The regulation of PKA is achieved via a unique three-component signaling system in which PKA is composed of two separate subunits, the catalytic (C) and regulatory (R) subunits that interact to form an inactive holoenzyme complex (1). Although phosphorylation of Thr 197 in the activation loop of the C subunit is necessary for the maturation and optimal catalytic activity of PKA (2, 3), unlike most other kinases whose activity is regulated by dynamic phosphorylation/dephosphorylation of the activation loop this phosphorylation step does not seem to be a regulatory mechanism for PKA in vivo. Once phosphorylated, PKA is fully active in its catalytic potential and the Thr 197 phosphate does not turn over readily (4). The activation of PKA is achieved by binding of the second messenger cAMP to the R subunit, which consequently induces a conformational change in the R subunit and leads to the dissociation of the holoenzyme into its constituent subunits (1). The free active C subunit can then affect a range of diverse cellular events by phosphorylating an array of cytoplasmic and nuclear protein substrates, including enzymes and transcription factors (5).The effect of ...

Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, ␤-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-L-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.Mammalian Rap proteins Rap1a/b and Rap2a/b/c are members of the Ras superfamily of small GTPases. Rap proteins are active when bound to GTP and inactive when bound to GDP. Cellular Rap activity is regulated by the concerted action of guanine nucleotide exchange factors that activate Rap (RapGEFs) and Rap-specific GTPaseactivating proteins (RapGAPs) that inactivate Rap (reviewed in reference 10). The Rap1GAP family is composed of several members, including Rap1GAP, Rap1GAPII, Spa-1/SIPA1, and E6TP1/SIPA1L1. Several lines of evidence suggest that RapGAPs function as tumor and/or invasion suppressors. Downregulation of E6TP1 by human papillomavirus protein E6 contributes to cervical cancer (20, 21), and Spa-1 deficiency in mice induces a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (26). The SPA1 gene was identified as a candidate for the metastasis efficiency modifier locus in mice (38). Although the relevance of this observation to humans is not yet clear, single-nucleotide polymorphisms in the SPA1 gene in human breast tumors have been associated with lymph node involvement and poor survival (15). Intriguingly, Spa-1 interacts with Brd4 (18) and Rrp-1b (13), the protein products of genes associated with patterns of extracellular matrix protein gene expression characteristic of metastatic tumors (14).The RAP1GAP gene maps to 1p35-36, a chromosomal region subject to copy number alterations in human tumors (36, 49). Rap1GAP protein levels are decreased in pancreatic adenocarcinomas (53), pap...

Although abundant in well-differentiated rat thyroid cells, Rap1GAP expression was extinguished in a subset of human thyroid tumor-derived cell lines. Intriguingly, Rap1GAP was downregulated selectively in tumor cell lines that had acquired a mesenchymal morphology. Restoring Rap1GAP expression to these cells inhibited cell migration and invasion, effects that were correlated with the inhibition of Rap1 and Rac1 activity. The reexpression of Rap1GAP also inhibited DNA synthesis and anchorage-independent proliferation. Conversely, eliminating Rap1GAP expression in rat thyroid cells induced a transient increase in cell number. Strikingly, Rap1GAP expression was abolished by Ras transformation. The downregulation of Rap1GAP by Ras required the activation of the Raf/MEK/extracellular signal-regulated kinase cascade and was correlated with the induction of mesenchymal morphology and migratory behavior. Remarkably, the acute expression of oncogenic Ras was sufficient to downregulate Rap1GAP expression in rat thyroid cells, identifying Rap1GAP as a novel target of oncogenic Ras. Collectively, these data implicate Rap1GAP as a putative tumor/invasion suppressor in the thyroid. In support of that notion, Rap1GAP was highly expressed in normal human thyroid cells and downregulated in primary thyroid tumors. Rap1GAP (30,33) is a member of a family of GTPaseactivating proteins (GAPs) for Rap1/2 GTPases that includes the splice variant Rap1GAPII, SPA-1, and E6TP1. Rap1GAP shares structural similarities with the RhebGAP tuberin. Tuberin is subject to mutational inactivation and loss in tuberous sclerosis, a disease syndrome associated with the formation of multiple benign tumors (15,19,22,37). The downregulation of E6TP1 by human papillomavirus E6 protein is believed to contribute to cervical cancer (10, 11), and an SPA-1 deficiency in mice results in a spectrum of myelodysplastic disorders similar to chronic myelogenous leukemia (13). The rap1GAP gene has been mapped to 1p35-36, a chromosomal region subject to deletion in a variety of human tumors including breast (28) and endocrine (41) neoplasia. Recently, decreased expression and loss of heterozygosity for Rap1GAP were reported for human oropharyngeal squamous cell (43) and pancreatic (21, 42) carcinomas.Rap1GAP is abundant in rat thyroid epithelial cells, where thyroid-stimulating hormone (TSH) regulates Rap1GAP protein stability. The stable overexpression of Rap1GAP in thyroid cells impaired DNA synthesis and the growth rate, and based on this, we suggested that Rap1GAP might function as a tumor suppressor (34). We now provide further support for this idea. Eliminating Rap1GAP expression in differentiated rat thyroid cells induced a transient increase in cell proliferation. Moreover, while highly expressed in normal thyroid follicular cells, Rap1GAP expression was downregulated in primary thyroid tumors and in thyroid carcinoma cell lines. In vitro, decreased expression of Rap1GAP was observed selectively in thyroid carcinoma cell lines that exhibited migratory and inva...