Mapping and expression pattern analysis of key components of the major histocompatibility complex class I antigen processing and presentation pathwayin a representative human renal cell carcinoma cell line

Renal cell carcinoma (RCC) represent approximately 5% of all cancer deaths. At the time of presentation, over 50% of the patients have already develop ed locally advanced or metastatic disease with five-year survival rates of less than 20%. Although relative resistant to conventional regimens, RCC ar e partially susceptible to T cell-based immunotherapy. To further develop t his treatment modality, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied for both the mapping of the key components of the ma jor histocompatibility complex (MHC) class I antigen processing and present ation machinery (APM) and the characterization of the constitutive and cyto kine-regulated protein expression profiles in a representative human RCC ce ll line. The latter aspect is based on the fact, that the expression level of some of the APM components can be altered in response to interferon (IFN )-gamma treatment. Total cell lysates from untreated and IFN-gamma -treated tumor cells were separated on 2-D PAGE gels using broad range immobilized pH gradient (IPG) strips. Serial Western blot analyses using sets of APM-sp ecific antibodies were performed to target the relevant protein spots. Prot ein verification was mostly accomplished via peptide mass fingerprinting us ing matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS ). To date, the majority of the APM-related components have been identified and mapped. In addition, the different protein expression profiles of untr eated and IFN-gamma -treated RCC cells are under investigation.