Interferon gamma regulates a unique set of proteins in fresh human bladdertransitional cell carcinomas

Poly(A) mRNA was isolated from human placental trophoblast cells stimulated with 100 U/mL of interleukin-2 and 5 mu g/mL of phytohemagglutinin and rev erse-transcribed. The cDNA coding for the mature interferon-gamma (IFN-gamm a) protein was amplified using specific primers, cloned into the pGEX-4T2 v ector, and expressed in Escherichia coil. Treatment of four fresh bladder t ransitional cell carcinoma (TCC) biopsies (TCCs 845-1, grade II, Ta; TCC 92 5-1, grade II, Ta; TCC 919-1, grade III, T1;TCC 950-1, grade III, T1) with the purified recombinant trophoblast IFN-gamma (50 U/mL, 20 h), followed by proteome analysis using two-dimensional gel electrophoresis, revealed seve ral major proteins whose level of expression were affected by this cytokine . Of these, five (tryptophanyl-tRNA synthetase, the interferon gamma-induci ble protein gamma 3, mangase superoxide dismutase, and two unknown proteins of apparent molecular masses of 35.8 and 11.2 kDa, respectively) were upre gulated in at least 75% of the tumors analyzed while one was downregulated (aldose reductase). Proteins were identified using a combination of techniq ues that included microsequencing, two-dimensional polyacrylamide gel elect rophoresis (2-D PAGE) immunoblotting and comparison with the transitional c ell carcinoma isoelectric focusing (IEF) database (http://biobase.dk/ cgi-b in/celis). Proteome profile analysis of primary cultures from a low-grade l esion (TCC 846-1, Grade II, Ta) labeled in the presence and absence of IFN- gamma showed that all of the proteins disregulated in vivo were also affect ed in the cultures. The cultured cells, on the other hand, exhibited additi onal changes that were not detected in vivo and that may reflect adaptation to the culturing conditions. Taken together, the results provide a first g lance at the effect of IFN-gamma on the protein expression profiles of TCCs , and in due course may form the basis for more comprehensive studies aimed at evaluating the usefulness of this cytokine in bladder cancer management .