86 Chapter 4 ditions of stress resulting in an intracellular proapoptotic 40 kDa fragment (p40β) (19, 20). On the other hand, they were explained by MET ectodomain shedding (21) and presenilin-dependent regulated intramembrane proteolysis (22). Ectodomain shedding is the release of substantial parts of the extracellular domain of proteins located at the membrane facilitated by proteinases such as ADAMs (23, 24). The remaining C-terminal transmembrane-associated fragment for type 1 proteins like MET, may be internalized by endocytosis or might undergo further cleavage such as regulated intramembrane proteolysis. During presenilin-dependent regulated intramembrane proteolysis, the extracellular domain of MET is shed from the cell and the remaining 55 kDa membrane-anchored C-terminal fragment—referred to as p55β—is cleaved by the γ-secretase complex at the membrane (25). The resulting 50 kDa protein fragment, being the intracellular domain of MET and referred to as p50β, is released into the cytosol and degraded by the proteasome (22). It has been described that both internalized C-terminal fragments and intracellular domains alter cellular functions (26) and that MET ectodomain shedding unleashes the aggressive nature of the MET oncogene (27). The goal of this study was to investigate whether MET ectodomain shedding occurs in oral squamous cell carcinoma cell lines and tissues (fresh frozen and formalin-fixed paraffin-embedded) and consider whether its presence is of predictive clinical value using 156 formalin-fixed paraffin-embedded oral squamous cell carcinoma sampled on a tissue microarray. To achieve this, two reliable antibodies directed against the C- or N-terminus of MET were used in parallel, e.g., D1C2 (18) and A2H2-3 (28). Materials & Methods Cell lines MET antibody validation cell line panel The reliability of A2H2-3 and its performance in relation to D1C2 was investigated using a previously described cell line panel (HT-29, DU145, DU145#Sh167, PC3, HeLa, LNCaP, and SK-BR-3). Details regarding handling of the cells have been described (18). Oral squamous cell carcinoma cell line panel A series of six oral squamous cell carcinoma cell lines (SCC-4, SCC-9, SCC-25, UMSCC-14C, BICR-16, and CAL-27) was used to examine whether MET ectodomain shed-
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