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

Abstract

Renal cell carcinoma (RCC) represent approximately 5% of all cancer deaths. At the time of presentation, over 50% of the patients have already developed locally advanced or metastatic disease with five-year survival rates of less than 20%. Although relative resistant to conventional regimens, RCC are partially susceptible to T cell-based immunotherapy. To further develop this treatment modality, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied for both the mapping of the key components of the major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) and the characterization of the constitutive and cytokine-regulated protein expression profiles in a representative human RCC cell 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-specific antibodies were performed to target the relevant protein spots. Protein verification was mostly accomplished via peptide mass finger-printing using 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 untreated and IFN-gamma-treated RCC cells are under investigation.

Altered Detoxification Status and Increased Resistance to Oxidative Stress by K-Ras Transformation

“…After washing, the protein pellet was air dried and then solubilized in 500 AL lysis buffer [7 mol/L urea, 2 mol/L thiourea, 0.2 mol/L dimethylbenzylammonium propane sulfonate (NDSB, ICN Biomedicals GmbH), 1% (w/v) DTT, 4% (w/v) CHAPS, 2% IPG buffer pH 6-11 (Amersham Biosciences) supplemented with traces of bromophenol blue]. The protein concentration was determined by the method of Bradford as described previously (13).…”

“…The excised protein spots from silver gels were destained and digested according to Gharahdaghi and colleagues (16). Protein spots from preparative gels were processed as previously described (13). Tryptic digests were cocrystallized with an equal volume of matrix solution [10 mg/mL a-cyano-4-hydroxy cinnamic acid (Bruker); 50% (v/v) acetonitrile/0.1% (v/v) trifluoroacetic acid] and analyzed by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (Voyager-DE PRO Biospectrometry Workstation, Applied Biosystems).…”

Identification of 14-3-3β Gene as a Novel miR-152 Target Using a Proteome-based Approach

“…The cell lines MZ1257RC, MZ1795RC, and MZ1851RC as well as buf1088, FM82, and WM1862 were established from patients with renal cell carcinoma (RCC) or metastatic melanoma, respectively, and have been described recently (17)(18)(19)(20). With the exception of JEG-3 cells, which were maintained in RPMI 1640 medium (Invitrogen), all other cell lines were cultured in DMEM (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FCS) (PAA; Pasching, Austria), 2 mM L-glutamine (Lonza, Basel, Switzerland), and 1% (v/v) penicillin/streptomycin (PAA).…”

“…The lysate was sonicated using two cycles of five impulses (0.5 s/impulse) at 100% power (Bandelin UW 2070 sonicator, MS 73 needle; Bandelin, Berlin, Germany) and then cleared by centrifugation (18,000 ϫ g, 90 min, 15°C). Total protein concentration was determined as described previously (19). Samples (500 g of protein in a volume of 350 l of lysis buffer) were applied to IPG strips (pH 3-10 NL, 18 cm, GE Healthcare) by in-gel rehydration and covered with 450 l of Immobiline DryStrip Cover Fluid (GE Healthcare).…”

“…In-gel Digestion and MS-Digestion with trypsin and subsequent spotting of peptide solutions onto the MALDI targets were performed as described previously (19) with slight modifications. For protein identification, the proteins were excised from stained two-dimensional gels using the Herolab spot hunter (Herolab GmbH, Wiesloch, Germany) and destained by addition of 50% (v/v) ACN.…”