| Pancreatic carcinoma is the second most common gastrointestinal malignancy and the fifth leading cause of adult deaths from cancer. Nonspecific risk factors and symptoms prevent early detection and lead to diagnosis in the late stages of the disease. Detection of cancer-derived gene products from biologic fluids is an important emerging approach to the diagnosis of malignant diseases. Due to the lack of specific and sensitive markers for the early diagnosis of pancreatic cancer, the focus of this project was to detect genes specifically expressed in pancreatic tumors and to examine their efficacy as tumor markers. PCR-mediated techniques, differential display and amplification of protease family cDNAs with degenerate oligonucleotides, were utilized for comparison of mRNAs from normal and malignant pancreatic ductal epithelia. Using differential display PCR we identified down-regulated in adenoma (DRA) and the GABAA receptor pi subunit which encode anion channels capable of chloride transport. The differential expression of both anion channels in pancreatic tumors compared to normal duct and chronic pancreatitis tissue was confirmed by RT-PCR and by immunohistochemical analysis of DRA. Protease PCR amplification of cDNAs from the serine protease and astacin metalloendopeptidase families revealed two proteases that were overexpressed in pancreatic tumors, stratum corneum chymotryptic enzyme (SCCE) and meprin β. The expression of the serine protease, SCCE, and meprin β, and astacin family member, were analyzed in tumors at progressing stages of differentiation by RT-PCR. Both proteases were overexpressed in all of the tumors analyzed. Immunohistochemical analysis of pancreatic carcinoma tissue sections verified their tumor expression. Stable transfectants expressing meprin β were generated to examine the effect of meprin β expression on the invasiveness of two carcinoma cell lines. In vitro invasion assays demonstrated the ability of meprin β to increase the invasiveness of HeLa cells but not HepG2 cells. However HepG2 cells expressing meprin β exhibited an altered morphology compared to untransfected HepG2s which may be indicative of decreased adhesion molecules on those cells. The characterization of these four genes that are overexpressed in pancreatic cancer may thus be used in the development of specific diagnostic and treatment strategies. |
