Profiling Biomarkers For Diagnosis Of Pancreatic Cancer Using MSILAC Technology & Cloning, Expression And Function Analysis Of A Novel Gene Nlel

1. Profiling biomarkers For Diagnosis of Pancreatic Cancer Using mSILAC technologyProteins with high turnover rates are often considered to be the source of biomarkers in clinic diagnosis of pancreatic cancer. Few effective cancer biomarkers though have been discovered in serum or plasma from cancer patients because of the complexity of serum or plasma. The presence of highly abundant proteins (e.g. albumin) in plasma or serum makes it difficult to discover the subtle changes of secreted proteins with low abundance. Stable isotope 15N was incorporated in newly synthesized proteins of pancreatic cancer cells cultured with 15N labeled amino acids. A well established algorithm was used to determine protein turnover rates. mSILAC is a modified SILAC combined with our new algorithm. In this thesis, in order to discover potential biomarkers for pancreatic cancer diagnosis, mSILAC was used in conjunction with serum-free medium to profile secreted proteins with high turnover rates in pancreatic cancer cells.MIA PaCa-2 cells were first cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum, then in serum-free modified DMEM with or without 50% 15N amino acid mixture. The effect of oxythiamine chloride on secreteome was studied. Secreteome from cell culture media was analyzed by 2-dimensional (2D) gel electrophoresis. Differentially expressed proteins were detected and identified. Protein turnover rates were calculated according to the newly established algorithm. Western blot and enzyme linked immuno-sorbent assay (ELISA) were used to validate identified proteins. Among the 14 differentially expressed proteins after oxythiamine treatment, tissue inhibitor of metalloproteases-1 (TIMP-1) and cytokeratin-10 were identified as 2 newly synthesized secreted proteins caused by substantial 15N incorporation with a turnover rate no less than 90%. The expression of TIMP-1 in MIA PaCa-2 cells by oxythiamine treatment was further validated by Western blotting and ELISA analyses. To determine the relevance to human pancreatic cancer, ELISAs of TIMP-1 and CA19-9, a marker that is currently in clinical trial as a pancreatic cancer marker, were carried out in plasma samples from patients with pancreatic adenocarcinoma and samples from control subjects. Compared with CA19-9, TIMP-1 has a high sensitivity and a moderate specificity as a potential biomarker of pancreatic cancer. In conlusion, mSILAC technology in conjunction with depleted serum allows the identification of actively secreted low abundant proteins from pancreatic cancer cells. Our approach is generally applicable and reliable in profiling of serum biomarker of diseases. 2. Cloning, expression and function analysis of a novel gene Nle1Heart development involves an evolutionarily conserved network precisely orchestrated of expression of transcription factors and their interactions that connect signaling pathways which regulate the whole developmental process. Notch signaling pathway has an essential role in heart development. Studies indicate that mutations in Notch signaling elements give rise to heart abnormalities. Drosophila Notchless binds to the cytoplasmic domain of Notch and causes a suppression of wing notching. Notchless activity increases Notch activity. We cloned Nlel, a human homolog of Drosophila Notchless, and demonstrated with luciferase report assay that Nle1 also binds to the cytoplasmic domain of Notch and suppresses Notch activity. Report assay analysis also showed that overexpression of Nlel inhibits the transcriptional acitivities of SRE and AP-1, suggesting that Nlel may act as a suppressor of MAPK signaling pathway to mediate cellular functions.