PERFORIN-DEPENDENT NUCLEAR ENTRY OF GRANZYME-B PRECEDES APOPTOSIS, AND IS NOT A CONSEQUENCE OF NUCLEAR-MEMBRANE DYSFUNCTION

Killer lymphocytes utilize the synergy of a membranolytic protein, per forin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces t he redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has n ot been demonstrated. In the present study, we used confocal laser sca nning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptos is. Two distinct and mutually exclusive cellular responses were observ ed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FIT C-grB in the nucleus (maximal at 7 min; t(1/2) Of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cy toplasm, and escaped apoptosis, Under these conditions, DNA fragmentat ion was not observed for at least 13 min, indicating nuclear accumulat ion of grB preceded the execution phase of apoptosis, Furthermore, nuc lear import of grB proceeded through an intact nuclear membrane, as th e nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dext ran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB, These findings indicated that perfor in-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not lar ge enough to permit a 13 kDa protein (yeast cdk p13(suc)) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equ ilibrate between the cytosol and the exterior. Therefore, transmembran e pores large enough to allow passive diffusion of grB (32 kDa) into t he cell are not necessary for apoptosis, Rather, a perfor-independent signal results in a redistribution of grB from the cytoplasm to the nu cleus, where it may contribute to the nuclear changes associated with apoptosis.