ISOLATION, INVESTIGATION OF BIOLOGICAL-ACTIVITY, AND CHARACTERIZATIONOF IMMUNOSUPPRESSIVE LIVER FACTOR CAUSING APOPTOSIS OF THYMOMA EL-4 CELLS IN-VITRO

A factor (ISFnp) suppressing proliferation and viability of thymoma EL -4 cells in vitro was isolated from mouse liver, Screening based on me asuring MTT-tetrazolium bioconversion to MTT-formazan by mitochondrial enzymes of viable cells was used to obtain the factor as an individua l chromatographic fraction. This enabled preparation of rabbit polyclo nal antibodies. The protein peak was immunochemically homogeneous, sin ce in the double immunodiffusion reaction the antiserum obtained forme d a single precipitation line both with the purified factor preparatio n and partly purified fractions containing ISFnp. The antiserum reacte d with the ISFnp proper, since the antibody-carrying immunosorbent eli minated biological activity from the factor-containing fractions. Doub le immunodiffusion, gel filtration, and SDS-PAGE followed by immunoblo tting have shown that the factor is orgoanospecifically localized in l iver and consists of 40 and 42 kDa subunits that form. dimers of appar ent mel. mass 70-80 kDa. In thymoma EL-4 cells the factor causes oligo nucleosomal DNA fragmentation similar to DNA degradation in thymocyte apoptosis caused by dexametasone. The fragmentation precedes EL-4 cell lysis detected by Cr-51 release into the growth medium. This is a str ict confirmation of the involvement of apoptosis in the mechanism of t hymoma cell death. ISFnp practically completely inhibits blast-cell tr ansformation in MLC response to a mutant MHC class 2 molecule. This ef fect is not due to the death of normal T-cell antigen-reactive clones, since in cultures treated with ISFnp during the first four days of cu ltivation the factor removal is accompanied by the blast-cell transfor mation, along with complete preservation of cell viability and the abi lity for secondary proliferation response in MLC to the same antigen.