Preparation of monoclonal antibodies for monitoring radiation effects

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The aims of the project were (1) to characterize the appearance of a protein after X-irradiation in radiosensitive human T cell leukemia MOLT-4 cells, and (2) to prepare monoclonal antibodies against the protein.By two-dimensional electrophoresis and silver staining, a new spot was detected after irradiation. The location was 41 kDa in the mass and 4.0 of pI.The protein (p41) was identified from its partial sequences as a set gene product, whose gene translocation was previously found in a case of acute undifferentiated leukemia. Polyclonal rabbit antibodies against p41 were prepared ; the antibodies detected p41 and a 42kDa protein (p42). p42 was present also in non-irradiated control cells. Photoimage analyzes of the silver stained gels and Western blots indicated that appearance of p41 was time dependent, starting 4 hrs after irradiation and reaching a plateau by 10 hrs, and dose dependent between 1 and 10 Gy.By the following evidences, p41 was suggested to be derived from p42 through post-translational modification ; (1) p41 appeared in the presence of cycloheximide, a protein synthesis inhibitor, (2) radioactivity was not found in p41 after post-irradiation labeling with ^<35>S-amino acids, and (3) in cells which had been pre-labeled with ^<35>S-amino acids, the radioactivity was found in p41 and p42 after irradiation. Suppression of p41 appearance after irradiation by vanadate, an inhibitor of protein phosphatase, suggested that phosphorylation/dephosphorylation would be involved in the post-translational modification.The anti-p41 polyclonal antibodies were prepared and have been successfully utilized, as described above, for the analyzes of p41/p42. The preparation of monoclonal antibodies against p41 and p42 has been under way.