Rudolf Lichtenfels

Identification of metabolic enzymes in renal cell carcinoma utilizing PROTEOMEX analyses.

PROTEOMEX, an approach which combines conventional proteome analysis with serological screening, is a powerful tool to separate proteins and identify immunogenic components in malignant diseases. By applying this approach, we characterized nine metabolic enzymes which were differentially expressed in renal cell carcinoma (RCC) cell lines and compared their expression profiles to that of normal kidney epithelium cells. Four of these proteins, superoxide dismutase (SODC), triosephosphatase isomerase (TPIS), thioredoxin (THIO) and ubiquitin carboxyl-terminal hydrolase (UBL1) were further analysed for both their constitutive and interferon (IFN)-gamma inducible protein expression pattern in cell lines or tissue specimens derived from RCC or normal kidney epithelium using Western blot analysis and immunohistochemistry, respectively. With the exception of the RCC cell line MZ1940RC, which completely lacks the expression of UBL1, a heterogeneous and variable expression pattern of the different metabolic enzymes was detected in RCC and normal renal epithelium. The highest differences in the expression levels were found for THIO in the RCC cell lines, which was 2-fold upregulated when compared to autologous normal kidney epithelium. Moreover, IFN-gamma treatment did not influence the constitutive expression of these metabolic enzymes. Thus, PROTEOMEX represents a valuable approach for the identification of metabolic enzymes which might be used as markers for the diagnosis of RCC.

Heat shock protein expression and anti-heat shock protein reactivity in renal cell carcinoma.

Heat shock proteins (HSP) are families of highly conserved proteins which are induced in cells and tissues upon exposure to extreme conditions causing acute or chronic stress. They exhibit distinct functions and have been implicated in the pathogenesis of a number of diseases, including cancer. A causal relationship between HSP expression and immunogenicity has been demonstrated in murine and human tumors and is also associated with the immune response. In order to investigate the correlation of HSP expression and their immunogenic potential in renal cell carcinoma (RCC), we here analyzed (i) the protein expression profile of various members of the HSP family in untreated and interferon (IFN)‐γ treated RCC cell lines as well as normal kidney epithelium, and (ii) the anti‐heat shock protein reactivity in sera derived from RCC patients and healthy controls using proteomics‐based techniques. A heterogeneous expression pattern of members of the HSP families was demonstrated in RCC cell lines and in cells representing normal renal epithelium. In some cases the expression rate is moderately altered by IFN‐γ treatment. In addition, a distinct anti‐heat shock protein reactivity could be detected in autologous and allogeneic sera from RCC patients and healthy controls. These data suggest that HSP play a role in the immunogenicity of RCC and thus might be used for the design of immunization strategies to induce a potent antitumor response in this disease.

Rapid and Sensitive Identification of Major Histocompatibility Complex Class I-associated Tumor Peptides by Nano-LC MALDI MS/MS

Identification of major histocompatibility complex (MHC)-associated peptides recognized by T-lymphocytes is a crucial prerequisite for the detection and manipulation of specific immune responses in cancer, viral infections, and autoimmune diseases. Unfortunately immunogenic peptides are less abundant species present in highly complex mixtures of MHC-extracted material. Most peptide identification strategies use microcapillary LC coupled to nano-ESI MS/MS in a challenging on-line approach. Alternatively MALDI PSD analysis has been applied for this purpose. We report here on the first off-line combination of nanoscale (nano) LC and MALDI TOF/TOF MS/MS for the identification of naturally processed MHC peptide ligands. These peptides were acid-eluted from human leukocyte antigen (HLA)-A2, HLA-A3, and HLA-B/-C complexes separately isolated from a renal cell carcinoma cell lysate using HLA allele-specific antibodies. After reversed-phase HPLC, peptides were further fractionated via nano-LC. This additional separation step provided a substantial increase in the number of detectable candidate species within the complex peptide pools. MALDI MS/MS analysis on nano-LC-separated material was then sufficiently sensitive to rapidly identify more than 30 novel HLA-presented peptide ligands. Peptide sequences contained perfect anchor amino acid residues described previously for HLA-A2, HLA-A3, and HLA-B7. The most promising candidate for a T-cell epitope is an HLA-B7-binding nonamer peptide derived from the tumor-associated gene NY-BR-16. To demonstrate the sensitivity of our approach we characterized peptides binding to HLA-C molecules that are usually expressed at the cell surface at approximately only 10% the levels of HLA-A or HLA-B. In fact, multiple renal cell carcinoma peptides were identified that contained anchor amino acid residues of HLA-Cw5 and HLA-Cw7. We conclude that the nano-LC MALDI MS/MS approach is a sensitive tool for the rapid and automated identification of MHC-associated tumor peptides.

Heat shock proteins in renal cell carcinomas.

Renal cell carcinoma (RCC) represents one of the most common cancer types in the Western World. One third of the RCC patients had metastasis at presentation with a poor 5-year survival. Nephrectomy is the most important treatment modality of this disease, since most of the RCCs are resistant to cytotoxic chemotherapy and radiation therapy. Recent immunotherapeutic approaches have been shown to improve the survival rate of RCC patients. Thus, RCC appears to have an immunogenic basis, and therefore represents an attractive target for immunotherapies. So far, only a few RCC-associated antigens have been characterized. However, with the implementation of ome-based technologies, an increasing number of tumor-associated antigens and tumor markers has been identified that includes various heat shock proteins (HSPs). RCC lesions demonstrate heterogeneous expression patterns for HSPs. In most cases overexpression of certain HSPs, such as HSP27, HSP70 and HSP72, has been detected both in RCC cell lines as well as in the tumor lesions when compared to normal kidney epithelium. Furthermore, HSPs play an important role in apoptotic cell death, in the regulation of cell proliferation and in the augmentation of lysis of RCC by HLA class I-restricted cytotoxic T lymphocytes. In this context, it is noteworthy that wild-type and mutated HSPs have been identified to act as tumor-associated antigens, which consequently resulted in the first clinical phase I and II trials using HSP for vaccination of RCC patients. In this chapter we will briefly present the relevance of HSPs in the pathomechanisms of RCC.

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.

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)‐γ treatment. Total cell lysates from untreated and IFN‐γ‐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 fingerprinting 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‐γ‐treated RCC cells are under investigation.