Phosphatase of regenerating liver (PRL)-3, encoding a 22-kD low molecular weight tyrosine phosphatase, has been reported to be associated with metastasis of colorectal carcinoma. We assessed the levels of PRL-3 mRNA expression to know whether its up-regulation was involved in progression and metastasis of gastric carcinoma. Levels of PRL-3 expression in 94 human gastric adenocarcinomas and 54 matched lymph node metastases were detected by in situ hybridization and compared with clinicopathological characteristics including prognosis. High PRL-3 expression was detected in 36.2% of primary gastric carcinoma (with nodal metastasis, 55.6%; without nodal metastasis, 10%; P < 0.001) and in 74.1% of lymph node metastases. The incidence of high PRL-3 expression in lymph node metastasis was significantly higher than in primary tumors (P < 0.044). Moreover, high expression of PRL-3 was closely associated with tumor size, lymphatic invasion, venous invasion, extent of lymph node metastasis, and tumor stage. These results suggest that high PRL-3 expression may participate in the progression and metastasis of gastric carcinoma. PRL-3 might be a novel molecular marker for aggressive gastric cancer.

Phosphatase of regenerating liver-3 (PRL-3) is a member of the PRL protein tyrosine phosphatase family and has been proposed to promote the invasiveness and metastastic capability of colorectal cancers (CRCs); however, the underlying mechanisms and target molecules of PRL-3 protein remain unknown. On the basis of the biological significance of PRL-3 phosphatase activity confirmed by the catalytically inactive PRL-3 mutant (C104S) and a PRL-3 inhibitor in CRC-derived SW480 cells, we performed protein expression profiling to search for PRL-3-mediated effector proteins. By a comparative study of phosphorylated proteins that differentially expressed in wild type and C104S mutant PRL-3-transfected SW480 cells; the cytoskeletal intermediate filament keratin 8 (KRT8) was identified as a physiological PRL-3-interacting protein. Indeed, treatment with the PRL-3 inhibitor effectively suppressed the phosphorylation of KRT8 at S73 and S431. Moreover, we detected the physiological interaction between PRL-3 and KRT8 and their colocalization at cellular lamellipodias and ruffles in vivo. In CRC tissue samples, tumor cells with high PRL-3 expression showed reduction or loss of phosphorylated KRT8 expression, particularly at the invasive front and in the liver metastases. In conclusion, our results indicate that PRL-3 may play an important role for the promotion of CRC cell migration and metastatic potential through direct KRT8 dephosphorylation. ' 2008 Wiley-Liss, Inc. Key words: phosphatase of regenerating liver-3 (PRL-3); keratin 8 (KRT8); migration; metastasis; colorectal carcinoma (CRC)Metastasis is the leading cause of death in cancer patients, and understanding the molecular mechanism of cancer metastasis is therefore one of the urgent problems. Phosphatase of regenerating liver-3 (PRL-3) was identified as the only gene that is consistently overexpressed in liver metastases derived from colorectal cancer (CRC), whereas it is undetectable in normal colorectal epithelia and shows intermediate expression in advanced primary cancer. 1 Increasing evidence suggests that PRL-3 plays multiple roles in cancer migration and metastasis: PRL-3 expression is elevated in most metastatic lesions from various human malignancies including those of the colorectum, 2,3 stomach, 4 mammary gland 5 and ovary. 6 In the in vivo cancer-metastasis models, overexpression of PRL-3 in Chinese hamster ovary (CHO) cells promoted the incidence of metastasis, 7,8 and knockdown of PRL-3 expression in CRC-derived DLD-1 cells by RNA interference effectively abrogated the activities of cancer cell motility in vitro and hepatic colonization in vivo. 3

BACKGROUND: Scirrhous-type gastric carcinoma (SGC) exhibits an extensive submucosal fibrosis and extremely poor patient prognosis. We investigated the importance of the cancer -stromal interaction in the histogenesis of SGC. METHODS: Gastric fibroblasts NF-25 and intestinal fibroblasts NF-j2 were co-cultured with SGC-derived (HSC-39) or non-SGCderived (HSC-57 and HSC-64) cells. To identify genes that are up-or downregulated in NF-25, complementary DNA (cDNA) microarray analysis was performed. The antibody against vascular-cell adhesion molecule-1 (VCAM-1) was used for cell growth test and immunohistochemistry. Moreover, the impact of interaction with NF-25 fibroblasts on HSC-39 cells was investigated using western blot and reverse transcription-polymerase chain reaction. RESULTS: HSC-39 cells stimulated growth of NF-25 but not NF-j2 when co-cultured. Induction of VCAM-1 in NF-25 fibroblasts was identified, which was specific when co-cultured with HSC-39 but not with non-SGC-derived HSC-57 and HSC-64 cells. Neutralising antibody to VCAM-1 suppressed NF-25 growth in dose-dependent manners. In tissue samples, positive immunoreactivity of VCAM-1 in SGC-derived fibroblasts was significantly higher than that in non-SGC-derived fibroblasts. Furthermore, interaction with NF-25 fibroblasts not only induced the epithelial -mesenchymal transition-like change, but also expressions of matrix metalloproteinaserelated genes in HSC-39 cells. CONCLUSION: Direct interaction between SGC cells and gastric fibroblasts establishes the tumour microenvironment and reinforces the aggressiveness of SGC.

Objective: Tricellulin plays a central role in the sealing of epithelia at tricellular contacts. We examined the effects of Snail, an epithelial-mesenchymal transition (EMT)-related transcription factor, on the regulation of tricellulin expression in human gastric carcinoma (GC)-derived cells. Method: Six human GC-derived cell lines were used in this study. Expression and localization of tricellulin was analyzed by reverse transcription (RT)-PCR and immunohistochemistry. Also, a Snail expression vector was transfected into HSC-45 cells to examine altered mRNA levels of tricellulin,E-cadherin, vimentin, N-cadherin and several EMT transcription factors by quantitative real-time RT-PCR. Results: Abundant tricellulin expression was detected in all GC-derived cells examined. In HSC-45 cells, transduction of Snail decreased the expression levels of tricellulin and E-cadherin but increased vimentin and N-cadherin, which was accompanied by induction of EMT transcription factors such as Twist1, Twist2 and Slug. In normal gastric mucosa, tricellulin protein was localized at the tricellular tight junction; however, in HSC-45 cells, tricellulin protein was distributed in the cytoplasm. In GC tissues, tricellulin expression at the cellular membrane was retained in a subset of EMT-negative GCs, and it disappeared in EMT-positive GCs. Conclusions: The findings in the present study suggest that repression of tricellulin expression may be related to Snail-induced EMT in human GCs.

Background: The efficient manufacture of recombinant Der p 1 and Der f 1 has been an important bottleneck in the study of house dust mite allergies and the development of applications for allergen engineering. While Der f 1 has only one N-glycosylation motif in the mature sequence, Der p 1 has two motifs, one in the prosequence and the other in the mature sequence. To test whether inefficient maturation of a recombinant Pro-Der p 1 versus Pro-Der f 1 is due to N-glycosylation, the maturation speed of N-glycosylation motif mutants was compared. Methods: Expression vectors for the mutants, in which the motif in the Der p 1 prodomain was disrupted or a motif was created within the Der f 1 prodomain, were constructed by site-directed mutagenesis of preproforms with or without the motif within the mature portion. Culture supernatants of yeast Pichia pastoris transfectant cells containing proforms were buffer exchanged by gel filtration and incubated for maturation. Samples from the reactions were collected every 20 min and subjected to electrophoresis. The maturation speed was compared based on the band densities of the pro- and mature forms. Results: Disruption of the motif in the mature portion decreased the productivity and accelerated the maturation. Maturation was also accelerated by disruption of the other motif in the Der p 1 prodomain and slowed down by introduction of the motif into the Der f 1 prodomain. Conclusions: Maturation systems using Pro-Der p 1 without the prodomain glycosylation are useful for the efficient preparation of a recombinant mature allergen. In addition, these results demonstrated that the maturation of cysteine protease could be controlled through glycosylation of the prodomain.