Autotaxin (ATX), an exo-nucleotide pyrophosphatase and phosphodiesterase, was originally isolated as a potent stimulator of tumor cell motility. In order to study whether ATX expression a ects motility-dependent processes such as invasion and metastasis, we stably transfected full-length ATX cDNA into two nonexpressing cell lines, parental and ras-transformed NIH3T3 (clone7) cells. The e ect of ATX secretion on in vitro cell motility was variable. The ras-transformed, ATX-secreting subclones had enhanced motility to ATX as chemoattractant, but there was little di erence in the motility responses of NIH3T3 cells transfected with atx, an inactive mutant gene, or empty vector. In Matrigel TM invasion assays, all subclones, which secreted enzymatically active ATX, demonstrated greater spontaneous and ATX-stimulated invasion than appropriate controls. This di erence in invasiveness was not caused by di erences in gelatinase production, which was constant within each group of transfectants. In vivo studies with athymic nude mice demonstrated that injection of atx-transfected NIH3T3 cells resulted in a weak tumorigenic capacity with few experimental metastases. Combination of ATX expression with ras transformation produced cells with greatly ampli®ed tumorigenesis and metastatic potential compared to ras-transformed controls. Thus, ATX appears to augment cellular characteristics necessary for tumor aggressiveness.

We previously reported that apicidin arrested human cancer cell growth through selective induction of p21 WAF1/Cip1 . In this study, the apoptotic potential of apicidin and its mechanism in HL60 cells was investigated. Treatment of HL60 cells with apicidin caused a decrease in viable cell number in a dose-dependent manner and an increase in DNA fragmentation, nuclear morphological change, and apoptotic body formation, concomitant with progressive accumulation of hyperacetylated histone H4. In addition, apicidin converted the procaspase-3 form to catalytically active effector protease, resulting in subsequent cleavages of poly-(ADP-ribose) polymerase and p21 WAF1/Cip1 . Incubation of HL60 cells with z-DEVD-fmk, a caspase-3 inhibitor, almost completely abrogated apicidin-induced activation of caspase-3, DNA fragmentation, and cleavages of poly-(ADP-ribose) polymerase and p21 WAF1/Cip1 . Moreover, these effects were preceded by an increase in translocation of Bax into the mitochondria, resulting in the release of cytochrome c and cleavage of procaspase-9. The addition of cycloheximide greatly inhibited activation of caspase-3 by apicidin by interfering with cleavage of procaspase-3 and DNA fragmentation, suggesting that apicidin-induced apoptosis was dependent on de novo protein synthesis. Consistent with these results, apicidin transiently increased the expressions of both Fas and Fas ligand. Preincubation with NOK-1 monoclonal antibody, which prevents the Fas-Fas ligand interaction and is inhibitory to Fas signaling, interfered with apicidin-induced translocation of Bax, cytochrome c release, cleavage of procaspase-3, and DNA fragmentation. Taken together, the results suggest that apicidin might induce apoptosis through selective induction of Fas/Fas ligand, resulting in the release of cytochrome c from the mitochondria to the cytosol and subsequent activation of caspase-9 and caspase-3.

A growing number of studies have demonstrated that physiological factors can influence the progression of several cancers via cellular immune function, angiogenesis and metastasis. Recently, stress-induced catecholamines have been shown to increase the expression of various cancer progressive factors, including vascular endothelial growth factor (VEGF), matrix metalloproteinases and interleukins. However, a detailed mechanism remains to be identified. In this study, we investigated the role of adrenergic receptors and hypoxia-inducible factor (HIF)-1a protein in catecholamine-induced VEGF expression and angiogenesis. Treatment of the cells with norepinephrine (NE) or isoproterenol induced VEGF expression and HIF-1a protein amount in a dose-dependent manner. Induction of VEGF expression by NE was abrogated when the cells were transfected with HIF-1a-specific siRNA. Similarly, adenylate cyclase activator forskolin and cyclic AMP-dependent protein kinase A inhibitor H-89 enhanced and decreased HIF-1a protein amount, respectively. More importantly, conditioned medium of NE-stimulated cancer cells induced angiogenesis in a HIF-1a protein-dependent manner. In addition, pretreatment of cells with propranolol, a b-adrenergic receptor (AR) blocker, completely abolished induction of VEGF expression and HIF-1a protein amount by NE in all of the tested cancer cells. However, treatment with the a1-AR blocker prazosin inhibited NE-induced HIF-1a protein amount and angiogenesis in SK-Hep1 and PC-3 but not MDA-MB-231 cells. Collectively, our results suggest that ARs and HIF-1a protein have critical roles in NE-induced VEGF expression in cancer cells, leading to stimulation of angiogenesis. These findings will help to understand the mechanism of cancer progression by stress-induced catecholamines and design therapeutic strategies for cancer angiogenesis.Researchers have shown that stress and other behavioral conditions are involved in cancer progression.

Autotaxin (ATX)1 is a 125-kDa glycoprotein secreted by the human melanoma cell line A2058. ATX stimulates both random and directed motility in its producer cells (1), and its recent cloning and sequencing (2) has revealed homology with the active site of bovine intestinal 5Ј-nucleotide PDE (EC 3.1.4.1) (4) and extensive homology with the ectoprotein PC-1 (5), the brain-type PDE I-nucleotide pyrophosphatase gene 2 (6), and the rat neural differentiation antigen gp130 RB13-6 (7). ATX contains two tandem somatomedin B regions, the loop region of an EF-hand and a type I PDE catalytic site, and possesses 5Ј-nucleotide PDE activity (2) .Early studies on digestive enzymes responsible for RNA degradation identified a class of enzymes characterized by their reaction product, a 5Ј-monophosphate nucleotide, and their activity toward p-nitrophenyl-thymidine monophosphate (⌽-TMP) (8). This type I PDE activity has also been detected in a variety of mammalian tissues, their plasma membranes, and cell surfaces (9 -11). The unifying features of these activities, in addition to the reaction product, are the broad specificity for substrates and competitive inhibitors, the alkaline pH optimum, and the ability to hydrolyze the phosphodiester bond between the ␣-and ␤-phosphates in nucleoside polyphosphates. ATX possesses type I PDE activity and also induces a known biological response, the potent stimulation of cellular locomotion; thus it is possible to investigate the role of this enzyme reaction center in extracellular signal transduction.The reaction mechanism for type I PDE has been described as involving formation of nucleotidylated threonine as a covalently bound reaction intermediate (4), and PC-1 can be autophosphorylated on this threonine at the PDE catalytic center using [␥-32 P]ATP (12). Previous studies from this laboratory on ATX with point mutations at the PDE active site showed that the corresponding threonine in ATX (Thr 210 ) is required for its chemotactic, 5Ј-nucleotide PDE and threonine phosphorylation activities, and that phosphorylation-deficient, 5Ј-nucleotide PDE-competent ATX (K209L) is fully active in the stimulation of cellular motility (3). These findings suggested that the dephosphorylated state of ATX is a biologically active form and prompted us to investigate the relationship between the phosphorylation state and the catalytic properties of ATX. These earlier studies had also shown that phospho-ATX contains the ␥-and not the ␣-phosphate from ATP but addressed neither the stability of this construct nor the fate of the ␤-phosphate. In addition, unanswered questions remained concerning the nucleotide reaction products, the ability of ATX to use substrates other than ATP, and the possibility that the phosphorylation of ATX was due to the presence of a co-purifying protein kinase. We have resolved these issues by characterizing the enzymatic activities of ATX using homogeneously pure recombinant ATX (rATX) derived from the human teratocarcinoma cell line N-tera2D1 (13) and partially purified ATX (A2058 AT...

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Abstract. Histone deacetylases (HDAcs) are associated with the development and progression of cancer, but it is not known which of the HDAc isoforms play important roles in breast cancer metastasis. This study identified the specific HDAC isoforms that are necessary for invasion and/or migration in human breast cancer cell lines. MDA-MB-231 cells were significantly more invasive and expressed higher levels of matrix metalloproteinase-9 (MMp-9) compared to McF-7 cells. We compared the expression of HDAc isoforms between McF-7 and MDA-MB-231 cells and found greater expression of HDAc4, 6 and 8 in MDA-MB-231 cells by rt-pcr and Western blot analyses. In addition, apicidin, a histone deacetylase inhibitor, was shown to attenuate the invasion, migration and MMP-9 expression in MDA-MB-231 cells. Using specific sirnAs directed against HDAc1, 4, 6 and 8, we show that inhibition of HDAc1, 6 and 8, but not HDAc4, are responsible for invasion and MMp-9 expression in MDA-MB-231 cells. We analyzed the invasiveness of McF-7 cells overexpressing HDAc1, 4, 6 or 8 and found that overexpression of HDAc1, 6 or 8 increased invasion and MMp-9 expression. By developing HDAc isoforms as potential biomarkers for breast cancer metastasis, the present study can be extended to developing therapies for breast cancer invasion.

Autotaxin, a potent human tumor cell motility-stimulating exophosphodiesterase, was isolated and cloned from the human teratocarcinoma cell line NTera2D1. The deduced amino acid sequence for the teratocarcinoma autotaxin has 94% identity to the melanoma-derived protein, 90% identity to rat brain phosphodiesterase I/nucleotide pyrophosphatase (PD-I alpha), and 44% identity to the plasma cell membrane marker PC-I. Utilizing polymerase chain reaction screening of the CEPH YAC library, we localized the autotaxin gene to human chromosome 8q23-24. Northern blot analysis of relative mRNA from multiple human tissues revealed that autotaxin mRNA steady state expression is most abundant in brain, placenta, ovary, and small intestine.

We previously reported that apicidin, a novel histone deacetylase inhibitor, inhibited the proliferation of tumor cells via induction of p21 WAF1/Cip1 . In this study, we determined the molecular mechanisms by which apicidin induced the p21 WAF1/Cip1 gene expression in HeLa cells. Apicidin induced p21 WAF1/Cip1 mRNA independent of the de novo protein synthesis and activated the p21 WAF1/Cip1 promoter through Sp1-3 site located at ؊82 and ؊77 relative to the transcription start site. This transcriptional activation appears to be mediated by protein kinase C (PKC), because calphostin C, a PKC inhibitor, significantly attenuated the activation of p21 WAF1/Cip1 promoter via Sp1 sites, which was accompanied by a marked suppression of p21 WAF1/Cip1 mRNA and protein expression induced by apicidin. Consistent with the transcriptional activation of p21 WAF1/Cip1 promoter by apicidin, apicidin treatment led to the translocation of PKC⑀ from cytosolic to particulate fraction, which was reversed by pretreatment with calphostin C, indicating the involvement of PKC in the transcriptional activation of p21 WAF1/Cip1 via Sp1 sites by apicidin. However, the PKC-mediated transcriptional activation of p21 WAF1/Cip1 by apicidin appears to be independent of the histone hyperacetylation, because apicidin-induced histone hyperacetylation was not affected by calphostin C. Furthermore, a PKC activator, phorbol 12,13-dibutyrate, alone induced the transcriptional activation of p21 WAF1/Cip1 promoter, p21 WAF1/Cip1 mRNA, and protein expression without induction of the histone hyperacetylation, suggesting that the transcriptional activation of p21 WAF1/Cip1 by apicidin might have been mediated by a mechanism other than chromatin remodeling through the histone hyperacetylation. Taken together, these results suggest that the PKC signaling pathway plays a pivotal role in the transcriptional activation of the p21 WAF1/Cip1 gene by apicidin.