ISOLATION, INVESTIGATION OF BIOLOGICAL-ACTIVITY, AND CHARACTERIZATION OF 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 measuring MTT-tetrazolium bioconversion to MTT-formazan by mitochondrial enzymes of viable cells was used to obtain the factor as an individual chromatographic fraction. This enabled preparation of rabbit polyclonal antibodies. The protein peak was immunochemically homogeneous, since in the double immunodiffusion reaction the antiserum obtained formed a single precipitation line both with the purified factor preparation and partly purified fractions containing ISFnp. The antiserum reacted with the ISFnp proper, since the antibody-carrying immunosorbent eliminated biological activity from the factor-containing fractions. Double immunodiffusion, gel filtration, and SDS-PAGE followed by immunoblotting have shown that the factor is orgoanospecifically localized in liver and consists of 40 and 42 kDa subunits that form. dimers of apparent mel. mass 70-80 kDa. In thymoma EL-4 cells the factor causes oligonucleosomal 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 strict confirmation of the involvement of apoptosis in the mechanism of thymoma cell death. ISFnp practically completely inhibits blast-cell transformation in MLC response to a mutant MHC class 2 molecule. This effect 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 cultivation the factor removal is accompanied by the blast-cell transformation, along with complete preservation of cell viability and the ability for secondary proliferation response in MLC to the same antigen.