GRANZYME-B (GRAB) AUTONOMOUSLY CROSSES THE CELL-MEMBRANE AND PERFORININITIATES APOPTOSIS AND GRAB NUCLEAR-LOCALIZATION

Granzyme B (GraB) induces apoptosis in the presence of perforin. Perfo rin polymerizes in the cell membrane to form a nonspecific ion pore, b ut it is not known where GraB acts to initiate the events that ultimat ely lead to apoptosis. It has been hypothesized that GraB enters the t arget cell through a perforin channel and then initiates apoptosis by cleaving and activating members of the ICE/Ced-3 family of cell death proteases. To determine if GraB can enter the cell, we treated YAC-1 o r HeLa cells with FITC-labeled GraB and measured intracellular fluores cence with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm withi n 10 min. Uptake was inhibited at low temperature (4 degrees C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug that both blocks microfilament formation, and FITC-GraB rema ined on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant increase in cytopla smic fluorescence was observed over that found in cells treated only w ith FITC-GraB. However, FITC-GraB was now detected in the nucleus of a poptotic cells labeling apoptotic bodies and localized areas within an d along the nuclear membrane. The ability of GraB to enter cells in th e absence of perforin was reexamined using anti-GraB antibody immunogo ld staining of ultrathin cryosections of cells incubated with GraB. Wi thin 15 min, gold particles were detected both on the plasma membrane and in the cytoplasm of cells with some gold staining adjacent to the nuclear envelope but not in the nucleus. Cells internalizing GraB in t he absence of perforin appeared morphologically normal by Hoechst stai ning and electron microscopy. GraB directly microinjected into the cyt oplasm of B16 melanoma cells induced transient plasma membrane blebbin g and nuclear coarsening but the cells did not become frankly apoptoti c unless perforin was added. We conclude that GraB can enter cells aut onomously but that perforin initiates the apoptotic process and the en try of GraB into the nucleus.