Cytotoxic activated macrophages secrete soluble factors that have a variety of effects on tumor cells. Included in the tumor cell response are the inhibition of mitochondrial respiration and inhibition of DNA synthesis. A monokine called RIF, which was found to reproduce these effects in vitro, has been characterized and the amino acid sequence of the amino-terminus determined. The isolation and verification of the cDNA for RIF is the primary focus of this project. Based on the sequence obtained from the 80kD protein purified in the RIF preparations, a synthetic peptide was constructed and antibodies against the peptide were produced. This antibody probe and an antiserum against a preparation of RIF protein will be used to screen a lambda expression library in order to isolate a cDNA clone corresponding to the RIF protein. The cDNA will be fully characterized and expression studies will be initiated. The availability of recombinant protein and cDNA clones will permit a second phase of studies which will entail expression studies in mammalian systems, animal- tumor model studies of recombinant proteins, and mechanism studies of the recombinant protein.
