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Background: Aspartyl (asparaginyl) β-hydroxylase (AAH) is an α-ketoglutarate-dependent dioxygenase that hydroxylates aspartate and asparagine residues in EGF-like domains of proteins. The consensus sequence for AAH β-hydroxylation occurs in signaling molecules such as Notch and Notch homologs, which have roles in cell migration.Aim: This study evaluated the potential role of AAH in cell migration using cholangiocarcinoma cell lines as models due to their tendency to widely infiltrate the liver.Methods: Five human cholangiocarcinoma cell lines established from human tumors were examined for AAH expression and motility. The effect of antisense oligodeoxynucleotide inhibition of AAH on cholangiocarcinoma cell migration was investigated.Results: Western blot analysis detected the ∼86 kDa AAH protein in all five cholangiocarcinoma cell lines, and higher levels of AAH in cell lines derived from moderately or poorly differentiated compared with well-differentiated tumors. Immunocytochemical staining and fluorescence activated cell sorting analysis revealed both surface and intracellular AAH immunoreactivity. Using the phagokinetic non-directional migration assay and a novel ATPLite luminescence-based directional migration assay, we correlated AAH expression with motility. Correspondingly, antisense and not sense or mutated antisense AAH oligodeoxynucleotides significantly inhibited AAH expression and motility in cholangiocarcinoma cells.Conclusions: AAH over-expression may contribute to the infiltrative growth pattern of cholangiocarcinoma cells by promoting motility. [Copyright &y& Elsevier]

Background/Aims: We measured aspartyl (asparaginyl)-β-hydroxylase (AAH) gene expression in human hepatocelluar carcinoma and surrounding uninvolved liver at both the mRNA and protein level and examined the regulation and function of this enzyme. Methods: Since growth of HCC is mediated by signaling through the insulin-receptor substrate, type 1 (IRS-1), we examined—if AAH is a downstream gene regulated by insulin and IGF-1 in HCC cells. In addition, IRS-1 regulation of AAH was examined in a transgenic (Tg) mouse model in which the human (h) IRS-1 gene was over-expressed in the liver, and an in vitro model in which a C-terminus truncated dominant-negative hIRS-1 cDNA (hIRS-ΔC) was over-expressed in FOCUS HCC cells. The direct effects of AAH on motility and invasiveness were examined in AAH-transfected HepG2 cells. Results: Insulin and IGF-1 stimulation increased AAH mRNA and protein expression and motility in FOCUS and Hep-G2 cells. These effects were mediated by signaling through the Erk MAPK and PI3 kinase-Akt pathways. Over-expression of hIRS-1 resulted in high levels of AAH in Tg mouse livers, while over-expression of hIRS-ΔC reduced AAH expression, motility, and invasiveness in FOCUS cells. Finally, over-expression of AAH significantly increased motility and invasiveness in HepG2 cells, whereas siRNA inhibition of AAH expression significantly reduced directional motility in FOCUS cells. Conclusions: The results suggest that enhanced AAH gene activity is a common feature of human HCC and growth factor signaling through IRS-1 regulates AAH expression and increases motility and invasion of HCC cells. Therefore, AAH may represent an important target for regulating tumor growth in vivo. [Copyright &y& Elsevier]

Background/Aims: Cytochromes P4502A6 (CYP2A6) and P4501A2 (CYP1A2) were described as hepatic autoantigens in the autoimmune polyglandular syndrome type-1 (APS-1). We evaluated the significance of anti-CYP2A6 and anti-CYP1A2 in several hepatic diseases in the absence of APS-1.Methods: A radioligand assay (RLA) based on immunoprecipitation of [35S]-methionine-labeled CYP2A6 and CYP1A2 was used. Four hundred and thirty subjects with chronic viral hepatitis (n=185), autoimmune liver diseases (n=181), autoimmune rheumatic diseases (ARD, n=31) and healthy (n=33) were tested.Results: Seven out of 366 patients with liver diseases were anti-CYP2A6 positive. Neither healthy nor ARD patients showed anti-CYP2A6. One out of 181 patients with autoimmune liver diseases tested anti-CYP2A6 positive. A significantly higher prevalence of anti-CYP2A6 (P<0.05) was detected with six out of seven patients positive in the viral hepatitis group. The latter were infected by flaviviruses (1 HGV/GBVC, 5 HCV). 4/5 HCV/anti-CYP2A6 positive sera were positive for anti-LKM-1 by immunofluorescence and for anti-CYP2D6 by RLA. None of the 430 sera recognized CYP1A2.Conclusions: For the first time CYP2A6 is reported as a hepatic autoantigen in patients with viral hepatitis caused by flaviviruses and in particular in HCV/anti-LKM-1 positive patients. Multicenter studies are needed in order to investigate the clinical importance of this novel finding. This study further supports that anti-CYP2A6 in the absence of flavivirus is rather limited to APS-1. [Copyright &y& Elsevier]