A HIGHLY SENSITIVE CYTOTOXICITY ASSAY BASED ON THE RELEASE OF REPORTER ENZYMES, FROM STABLY TRANSFECTED CELL-LINES

The well-established methods of generating stably transfected cell lin es, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotox icity assays. In these assay systems, the detection of released enzyme s was used to quantitate the leakage of intracellular proteins after m embrane disintegration. Target cell lines were transfected with a luci ferase reporter gene under the control of a strong eucaryotic promoter . Release of the intracellular expressed enzyme into the culture super natant occurred after membrane perforation and was measured as an indi cator of cellular death. The quantitation of released enzyme was a rel iable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially e stablished with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intrac ellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial P-galactosida se to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than lucifer ase. In a direct comparison between the standard Cr-51 release and bet a-galactosidase release, the enzyme release showed a much higher signa l-to-noise ratio, i.e., low background and high induced release if spo ntaneous release and detergent induced maximal lysis were measured. Si nce a wide range of human and murine cell lines can be stably transfec ted and several reporter genes are available, the system should provid e an alternative for conventional cytotoxicity assays. The detection o f released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of lu minometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular dea th using transgenic animals feasible.