Coffey, unpublished observations)

Coffey, unpublished observations). highly expressed in giant trophoblasts and the spongiotrophoblast layer in mouse placenta (2). Subsequently, mRNA expression was Prazosin HCl observed in myeloid and lymphoid cells, as well as in epithelial cells in the lung and intestine (3). transcripts are upregulated in immortalized mouse colonocytes transformed by combined mutant and (7). Knockdown of endogenous in these cells and in a human colorectal cancer Prazosin HCl (CRC) cell line, HT-29, reduces growth of xenografts in nude mice (7). Although PLAC8 has important functions in normal physiology, and a possible role in CRC, its molecular function(s) and the cellular distribution of endogenous protein have not been studied. Epithelial-to-mesenchymal transition (EMT) is usually a complex developmental process that drives key morphogenetic events, such as gastrulation and neural crest migration. During the process of EMT, cohesive epithelial cells undergo a loss of apicobasal polarity and cell-cell contact, while acquiring mesenchymal characteristics, enabling them to move as individual cells (8C11). EMT is usually thought to be a transcriptional program involving repression of and concomitant induction of and mesenchymal genes like (12C14). In carcinoma, there appears to be inappropriate activation of the EMT program, whereby tumor cells become mesenchymal-like, enabling them to delaminate from the primary tumor and invade locally (15C18). Herein, by combined analysis of zebrafish and human tissues, we show that PLAC8 protein is present in normal intestine, where it localizes to the apical domain name of differentiated intestinal epithelium. However, PLAC8 is usually upregulated and cytosolic in medullary and mucinous CRC, and cytosolic PLAC8 correlates with tumor progression and tumor grade. Overexpression of PLAC8 in a human CRC cell line, HCA-7, results in Prazosin HCl morphological, molecular, and functional features of EMT. Unlike in classical EMT, there is post-transcriptional reduction in cell surface CDH1 and no change in expression increases in immortalized mouse colonocytes transformed by mutant AOM and knockdown reduces tumor growth in xenografts (7). However, how PLAC8 contributes to colonic neoplasia is usually unknown. To address the role of PLAC8 in CRC, we examined its distribution in both normal and neoplastic human colon by immunofluorescence Prazosin HCl using a commercial PLAC8-specific polyclonal antibody. PLAC8 was found exclusively at the apical domain name of fully differentiated normal colonic epithelium in both colonocytes (Physique ?(Physique1,1, A and B) and goblet cells (Supplemental Physique 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI71103DS1). We observed abrupt loss of PLAC8 immunoreactivity in epithelial cells deeper in the crypt (Physique ?(Physique1,1, A and B) and absence of PLAC8 staining at the crypt base (Supplemental Physique 1, C and D). Staining was also observed in some scattered mononuclear cells in the stroma (A. Powell and R. Coffey, unpublished observations). Open in a separate window Physique 1 PLAC8 immunofluorescence in normal and neoplastic human colon.(A and B) In normal colon, PLAC8 immunofluorescence (red) localizes to the apical domain name of the differentiated colonic epithelium at the top of crypts. The boxed region in A is usually magnified in B. Epithelial cells are layed out by CDH1 immunofluorescence (green). (D) In a typical moderately differentiated adenocarcinoma (H&E-stained sections at lower magnification are shown in C), PLAC8 also localizes to the apical domain name, but immunoreactivity extends deeper into the neoplastic crypts. (F and H) PLAC8 immunofluorescence is largely detected in the cytoplasm of medullary (F) and mucinous (H) adenocarcinoma. (C, E, and G) Serial H&E-stained sections at lower magnification correspond to comparable areas in D, F, and H. In all immunofluorescent panels, DAPI (blue) marks nuclei. Scale bars: 100 m. Cytosolic PLAC8 is usually correlated with tumor grade and linked to medullary and mucinous CRC. We next analyzed PLAC8 expression in CRC using a tissue microarray (TMA) that contains.