The present paper describes the primary structure, glycosylation and tissue localization of fetal antigen 1 (FA1) isolated from second‐trimester human amniotic fluid. FA1 is a single‐chained, heterogeneous glycoprotein of 225–262 amino acid residues. FA1 has six well conserved epidermal‐growth‐factor motifs and contains up to ten O ‐glycosylation and N ‐glycosylation sites, six of which are differentially glycosylated. Alignment to the translated sequences of Mus. musculus dlk and human dlk revealed 86% and 99% identity, respectively, to a 259‐amino‐acid residue overlap, and this high similarity extends with minor corrections to the human adrenal‐specific mRNA, pG2 as well. Immunohistochemical analysis demonstrated the presence of FA1 in 10 out of 14 lung tumors containing neuroendocrine elements, and in the placental villi where FA1 was exclusively seen in stromal cells in close contact to the vascular structure. In the pancreas, FA1 co‐localized with insulin in the insulin secretory granules of the β cells within the islets of Langerhans. Our findings suggest that FA1 is synthesized as a membrane anchored protein and released into the circulation after enzymic cleavage, and that circulating FA1 represents the post‐translationally modified gene product of human dlk which, in turn, is identical to human adrenal‐specific mRNA pG2.
The trimeric extracellular matrix molecule laminin-5 and its constituent chains (alpha 3, beta 3, gamma 2) are normally not detectable intracellularly in intestinal epithelial cells but the laminin gamma 2 chain can be detected in cancer cells at the invasive front of a subset of colon carcinomas. These cells are subjected to cytokines such as transforming growth factor beta 1 (TGF-beta 1) and hepatocyte growth factor (HGF), produced by the tumour cells or by the surrounding stromal cells. The purpose of the present work was to investigate whether TGF-beta 1 and HGF, known to stimulate the LAMC2 gene encoding the laminin gamma 2 chain, might synergize to activate the LAMC2 promoter, and to identify the promoter elements involved. We find evidence for synergy between TGF-beta and HGF with respect to laminin gamma 2 chain expression and promoter activation and demonstrate that this requires the 5' activator protein-1 (AP-1) element of the promoter and an additional upstream element which is also responsive to co-expression of the Smad3 protein from the TGF-beta signalling pathway. The transcripts encoding the other laminin-5 chains are not synergistically activated by HGF and TGF-beta. Thus the synergistic activation of the LAMC2 gene is mediated via different cis-elements and results in an overproduction of the laminin gamma 2 chain relative to the other laminin-5 constituent chains. This difference may explain why laminin gamma 2 chains accumulate in the cells at the invasive front of colon carcinomas.
// Susanne Holck 1 , Hans Jørgen Nielsen 2 , Niels Pedersen 2 and Lars-Inge Larsson 1,3 1 Department of Pathology, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark 2 Department of Surgical Gastroenterology, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark 3 Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Copenhagen, Denmark Correspondence to: Lars-Inge Larsson, email: // Keywords : ERK, radiochemotherapy, rectal carcinoma, immunohistochemistry, phosphorylation Received : July 23, 2015 Accepted : September 05, 2015 Published : September 21, 2015 Abstract Locally advanced rectal adenocarcinoma is treated with radiochemotherapy (RCT) before surgery. The response to RCT is heterogeneous and consensus regarding reliable predictors is lacking. Since the ERK pathway is implicated in radioprotection, we examined pretreatment biopsies from 52 patients by immunohistochemistry for phosphorylated ERK (pERK). Immunostaining for pERK was considerably enhanced by use of alkaline demasking. Nuclear staining occurred in both cancer cells and stromal cells. Blind-coded sections were scored by 2 independent investigators. In patients showing no residual tumor after RCT (TRG1), staining for pERK in cancer, but not stromal, cell nuclei was significantly weaker than in patients showing a poor RCT response (TRG1 vs TRG4: p = 0.0001). Nuclear staining for pERK predicted poor responders, as illustrated by receiver operating characteristic curves with an area under curve of 0.86 ( p = 0.0007) and also predicted downstaging (area under curve: 0.76; p = 0.01). A number of controls documented the specificity of the optimized staining method and results were confirmed with another pERK antibody. Thus, staining for pERK in cancer cell nuclei can predict the response to RCT and may help spare poor responders this treatment. These results also raise the question whether inhibitors of ERK activation may serve as response modifiers of RCT.
A complete and balanced overview of all aspects of immunocytochemistry is presented providing a clear understanding of their impact on experiment. All available techniques and many diagnostic and research applications are included, as well as practical step-by-step instructions for carrying out recommended methods. Intended for the novice as well as the experienced researchers.
