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The expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is deregulated in human cancer cells with tumor inhibiting or promoting functions. Due to less knowledge on the role of UCHL1 in melanoma progression, the expression pattern and function of UCHL1 as well as the deregulated signaling pathways were characterized. A large number of melanoma cell lines, tissue microarrays of melanoma lesions and control tissues were analyzed for UCHL1 expression using PCR, Western blot and/ or immunohistochemistry. The analysis revealed that melanocyte cultures, 24 of 331 melanoma lesions, two of 18 short-term cultures and two of 19 melanoma cell lines tested, respectively, heterogeneously expressed UCHL1. The low frequency of UCHL1 expression in melanoma cells was due to gene silencing by promoter DNA hypermethylation. Using different transfection models an enzyme activity-dependent growth promoting function of UCHL1 via the activation of the mitogen-activated protein kinase signaling pathway was found in melanoma cells. Under oxygen stress a dose-dependent effect of UCHL1 was detected, which was mediated by a dynamic modification of the PI3K-Akt signaling. Thus, the aberrant UCHL1 expression in melanoma cells is linked to dynamic changes in growth properties and signal transduction cascades suggesting that UCHL1 provides a novel marker and/or therapeutic target at least for a subset of melanoma patients.Ubiquitination plays a key role in the post-translational modification of proteins and regulates in combination with phosphorylation and other post-translational alterations a number of cellular processes such as differentiation, proliferation, apoptosis and neoplastic transformation. The ubiquitination of proteins is controlled by so called deubiquitinating enzymes (DUB), which revert the binding between ubiquitin (ub) and their substrates, 1 protect proteins from proteasomal degradation and recycle ub-molecules of poly-ub chains. The DUB family is categorized into five different groups: (i) the ubiquitin carboxyl-terminal hydrolases (UCH), (ii) the ubiquitinspecific proteases (USP), (iii) the ovarial tumor proteases, (iv) the protein domain proteases of the Machado-Josef-disease and (v) the Jab1/MPN domain associated metalloproteinases. 2 The UCH family member UCHL1, also known as PGP9.5, GAD or PARK5, is a cysteine protease with a molecular weight of approximately 25 kDa. Next to its hydrolase activity UCHL1 also possesses ligase activity, 3,4 thereby the protein repertoire is multivalent regulated by its involvement in the cellular protein degradation and stabilization processes as well as in the homeostasis of the ub balance. Although UCHL1 is mainly expressed in neuronal and neuroendocrine tissues and represents about 2% of the total protein content of the brain, 3 it is also found in the tubule epithelium of the kidney, 5,6 in ovary 7 as well as in testis. 8 In neurodegenerative diseases such as Alzheimer's (AD) or Parkinson's disease (PD), an altered expression of UCHL1 could be indicated 9,10 suggesting that...

Neutral endopeptidase (NEP/CD10) is a cell surface zinc metalloprotease cleaving peptide bounds on the amino terminus of hydrophobic amino acids and inactivating multiple physiologically active peptides. Loss or decrease in NEP/CD10 expression have been reported in many types of malignancies, but the role of NEP/ CD10 in pancreatic carcinoma has not yet been identified. Using real-time RT-PCR, flow cytometry as well as immunohistochemistry, NEP/CD10 expression was quantified in both pancreatic carcinoma cell lines and in tumor specimens obtained from patients with primary pancreatic carcinomas. Three out of 8 pancreatic carcinoma cell lines exhibit heterogeneous NEP/CD10 expression levels: PATU-8988T expressed the highest NEP/CD10 levels, whereas HUP-T4 and HUP-T3 cells showed a moderate to low NEP/CD10 expression. NEP/CD10 immunoreactivity was found in 6 of 24 pancreatic ductal adenocarcinomas, but also in 3 of 6 tissues of patients with chronic pancreatitis. NEP/CD10 expression in pancreatic tumor lesions and cell lines was not associated with tumor grading and staging. Treatment of PATU-8988T cells with the histone deacetylase inhibitors sodium butyrate and valproic acid induced an increase of NEP/CD10 expression. This was accompanied by a reduced cell proliferation rate of PATU-8988T cells, which was increased by the addition of the enzyme activity inhibitors phosphoramidon and thiorphan. Thus, NEP/CD10 is differentially expressed in pancreatic tumors and might be involved in the proliferative activity of pancreatic cancer cells. However, further studies are needed to provide more detailed information of the role of NEP/CD10 under physiological and pathophysiological conditions of the pancreas. ' 2007 Wiley-Liss, Inc.Key words: neutral endopeptidase (NEP/CD10); pancreatic carcinoma; histone deacetylase inhibitors Cancer of the exocrine pancreas accounts for 2-3% of all cancers, but is the fourth most frequent cause of cancer deaths. 1 Pancreatic cancer is more common among males than females with the peak incidence occurring at age 60. The 5 years survival rate of patients with pancreatic cancer is less than 5%. Although the etiology of this disease remains unclear, cigarette smoking and alcohol abuse have been related with an increased incidence of pancreatic cancer.Neutral endopeptidase (NEP/CD10, EC 3.4.24.11) is a cell-surface metallopeptidase that is normally expressed by numerous tissues including prostate, kidney, breast, intestine, endometrium, adrenal glands and lung. This enzyme disintegrates peptide bonds on the amino terminus of hydrophobic amino acids and inactivates a variety of physiologically active peptides like atrial natriuretic factor, substance P, bradykinin, oxytocin, Leu-and Met-enkephalins, neurotensin, bombesin, calcitonin gene-related peptide, endothelin-1 and bombesin-like peptides. [2][3][4][5][6][7][8][9] NEP/CD10 reduces the local concentration of peptides available for receptor binding and signal transduction. The biological function of NEP/CD10 appears to be organ specific. In...

Aminopeptidase N (APN)/CD13 as ubiquitously expressed membrane peptidase exerts important functions in diverse cellular processes, such as proliferation, migration and differentiation. Previously, a role of APN in the invasiveness of melanoma cells has been demonstrated, but the underlying molecular mechanisms controlling APN expression are not understood. The present study demonstrates that lack of APN expression in primary and established melanoma cells was directly associated with a high-grade DNA methylation status of the myeloid APN promoter. Demethylation by 5-aza-2'-desoxycytidine not only induced constitutive and cytokine-regulated APN protein expression but also resulted in an increased APN-dependent migration of melanoma cells. Furthermore, its heterogeneous expression was inversely correlated to the expression of melanocytic marker proteins in established as well as in short-term cultured human melanoma cells. Staining of tissue microarrays generated from a large series of melanoma samples and control tissues demonstrated a higher APN expression in primary melanoma lesions when compared with nevi and metastases, which was neither associated with clinico-pathological parameters nor with the patients' outcome. Thus, the heterogeneous APN expression pattern in melanoma cells is epigenetically controlled and directly associated with an altered migration capacity but not of clinical significance in our study group.

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