| One of the potential mechanisms by which cytolytic cells may kill tumor target cells involves granule exocytosis upon recognition and binding of susceptible target cells. A major component of these granules is a protein with cytolytic activity called perforin. To further study the role of perforin in cell-mediated cytotoxicity, we transfected the human natural killer-like cell line YT-INDY with an antisense perforin plasmid in order to inhibit perforin protein synthesis. We observed a 30% reduction in perforin protein levels in these transfected cells as compared to control cells. More importantly, we observed that YT-INDY cells transfected with the antisense perforin construct showed lytic activity of less than 10% of that displayed by control cells. From these data we infer that perforin plays a vital role in YT-INDY cell-mediated cytotoxicity. In addition, this laboratory has previously reported that perforin mRNA rapidly disappears in cytolytic cells exposed to sensitive target cells. The mechanisms regulating this phenomenon, however, were unclear. Target cell-induced perforin mRNA disappearance was shown to be accelerated mRNA turnover specific for perforin and other cytolytic protein messages, as mRNA levels of the constitutive gene beta actin are stable following target cell exposure. We next sought to determine the relative location of the sequence(s) within the perforin transcript responsible for mediating the turnover event. The perforin cDNA was divided into three fragments; each of these cDNA fragments was subcloned into the 3... |
