PCR was performed as described. pVHL targets proteins for ubiquitin-mediated degradation. 10 Binding to fibronectin contributes to GSK256066 2,2,2-trifluoroacetic acid the ability of VHL protein to suppress tumor growth. 8 Two biologically active gene products with molecular masses of 30 kd (pVHL30) and 19 kd (pVHL19) have been described. 13 The shorter variant of pVHL protein, pVHL19, appears to arise as a result of alternative translation initiation. Few studies have determined pVHL expression in normal and neoplastic human tissue. 14-17 A comprehensive expression analysis of pVHL in RCC has not yet been performed. To study prevalence and prognostic relevance of pVHL expression in a large number of various tumors, we used our recently developed tissue microarray (TMA) technology. 18 The TMA technology allows examination of the immunohistochemical profile of hundreds of individual tumors at a time. 19-21 A recently developed polyclonal antibody raised against both pVHL isoforms (pVHL19 and pVHL30) 9 and a commercially available monoclonal antibody (Ig33) against the pVHL30 protein was used in this study. Loss of heterozygosity (LOH) at the locus was also assessed to analyze the association between pVHL expression and allelic deletion. Materials and Methods Renal-Tumor TMAs Renal-tumor TMAs were used for immunohistochemical analysis of pVHL expression. The construction of a renal-tumor TMA has been recently described. 21 The TMA contained 404 clear-cell RCC, 64 papillary RCC, 25 chromophobe RCC, and 17 oncocytomas. All tumors were reviewed by one pathologist (H.M.). Histological grading and tumor staging were done according to Thoenes 22 and the International Union against Cancer (UICC). 23 Rabbit polyclonal to MAPT Tumor-specific survival data were obtained by reviewing the hospital records, by direct communication with the attending physicians, and from the Cancer Registry of Basel. To minimize false-negative immunohistochemical staining results due to tumor heterogeneity, four copies of a renal cancer TMA were constructed. Tissue cylinders for the replicate TMAs were taken from carefully selected morphologically representative regions of different tumor areas. A second GSK256066 2,2,2-trifluoroacetic acid renal-cancer TMA was constructed to determine the relevance of candidate marker proteins for the metastatic process. This array contained 166 tissue cylinders; there were 50 primary tumors with 60 corresponding metastases of different sites. Five of the primary tumors were papillary RCC, 2 primary tumors GSK256066 2,2,2-trifluoroacetic acid were unclassified, and 43 were clear-cell RCC. In addition, tissues of 56 metastases from clear-cell RCC without available tissue of the primary tumors were included in this TMA. Immunohistochemistry Two antibodies were used for the detection of pVHL: a recently generated polyclonal rabbit anti-pVHL antibody 10 and a commercially available mouse monoclonal antibody (Ig33; Neomarkers, Fremont, CA). Our affinity-purified polyclonal rabbit anti-pVHL recognizes both the long (pVHL30) and the short forms (pVHL19) of pVHL. 9 Specificity of the antibody for both pVHL isoforms was demonstrated in human U2-OS cells by Western blotting. 9 The mouse monoclonal antibody Ig33 (Neomarkers, Fremont, CA) was raised against amino acids 1C54 and recognizes exclusively human pVHL30. Standard indirect immunoperoxidase procedures were used for immunohistochemistry (VECTASTAIN Elite ABC kit; Vector Laboratories, Burlingame, CA). The optimal titer for pVHL expression was defined as the dilution that gave clearly identifiable cytoplasmic and/or nuclear staining and negligible background on conventional histological samples of normal renal tissue and breast cancer tissue. Breast cancer was used as positive control in accordance with previous reports, showing frequent pVHL expression in breast cancer tissue. 17 GSK256066 2,2,2-trifluoroacetic acid Primary antibodies were omitted in negative controls. After determination of the optimal antibody dilution on large tissue sections, the immunohistochemical procedure was adopted on renal TMA sections. The mouse monoclonal antibody Ig33, which we termed anti-pVHL30, was used at a dilution of 1 1:100 after microwave pretreatment. The polyclonal anti-pVHL30/pVHL19 antibody was used at a dilution of 1 1:200 after heat pretreatment (steam). Determination of pVHL expression of renal tumors was done on all four replicates of the renal cancer TMA and on the renal cancer metastases TMA. Five-m-thick sections were cut from the TMAs. Each TMA contained normal renal tissue as positive internal control. Tumors were considered pVHL-positive if cytoplasmic and/or nuclear expression was found in at least one of the four tumor samples. Nuclear and cytoplasmic pVHL expression were independently assessed. Loss of Heterozygosity (LOH) A subset of 113 consecutive formalin-fixed, paraffin-embedded clear-cell RCC was selected for locus-specific LOH analysis to compare pVHL expression with deletion. Clinicopathological data of this tumor set have been previously published. 24 Tumor tissue was defined on the basis of hematoxylin and.