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From the * Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Manitoba,
R3EOV9, Canada; and Granzyme B (GraB) induces apoptosis in the presence of perforin. Perforin polymerizes in the
cell membrane to form a nonspecific ion pore, but it is not known where GraB acts to initiate
the events that ultimately lead to apoptosis. It has been hypothesized that GraB enters the target
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 or HeLa cells with FITC-labeled GraB and measured intracellular fluorescence
with a high sensitivity CCD camera and image analyzer. GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min. Uptake was inhibited at low temperature
(4°C) and by pretreatment with metabolic inhibitors, NaF and DNP, or cytochalasin B, a drug
that both blocks microfilament formation, and FITC-GraB remained on the cell membrane localized in patches. With the simultaneous addition of perforin and FITC-GraB, no significant
increase in cytoplasmic fluorescence was observed over that found in cells treated only with
FITC-GraB. However, FITC-GraB was now detected in the nucleus of apoptotic cells labeling apoptotic bodies and localized areas within and along the nuclear membrane. The ability of
GraB to enter cells in the absence of perforin was reexamined using anti-GraB antibody immunogold staining of ultrathin cryosections of cells incubated with GraB. Within 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 the absence of perforin appeared morphologically normal by Hoechst staining and electron microscopy. GraB directly microinjected into the cytoplasm of B16 melanoma cells induced
transient plasma membrane blebbing and nuclear coarsening but the cells did not become
frankly apoptotic unless perforin was added. We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.
Austin Hospital Research Institute, Heidelberg, Victoria, Australia 3084
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