Use of a P815-derived line with an amplified adenosine deaminase gene: an improved target for cellular cytotoxicity.
We describe a cytotoxic T lymphocyte-mediated cytotoxicity assay in which the release of a cytoplasmic enzyme, adenosine deaminase (ADA), instead of the widely used radioactive chromium is a measure of target lysis. In this enzyme-release assay the target is a mastocytoma P815-derived cell line, noted P815 ADA++, isolated by applying a selection procedure devised to specifically amplify the ADA gene. Gene amplification in P815 ADA++ was indeed demonstrated. Routine measurement of ADA activity from numerous supernatants is performed using a specific and sensitive colorimetric assay. The use of 96-well microtiter plates as well as of an automatic Multiscan spectrophotometer makes this measurement rapid and convenient. We show that this ADA-release assay is significantly more sensitive than the classical chromium-release test because of its consistently lower (5 to 10-fold) spontaneous release in 4 h, short-term cytotoxicity experiments. We also found that it is especially suited for the rapid detection, by visual screening, of rare, active killer clones among large, heterogeneous cytotoxic T lymphocyte populations. The assay could easily be adapted to other tumor targets (EL4, YAC-1, K562) of common use in studies involving immune lysis; indeed, the procedure of amplifying the ADA gene used in the isolation of the P815 ADA++ hyperactive line may be generally applied to these targets.
D000243:Adenosine Deaminase / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic; D005784:Gene Amplification; D007694:Killer Cells, Natural / Q000276:immunology; D012515:Mast-Cell Sarcoma / Q000276:immunology; D051379:Mice; D009700:Nucleoside Deaminases
D002860:Chromium Radioisotopes; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase