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Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization.

Shi L, Mai S, Israels S, Browne K, Trapani JA, Greenberg AH - J. Exp. Med. (1997)

Bottom Line: GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min.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.We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.

ABSTRACT
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 degrees 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.

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GraB is inactive  against YAC-1 target cells with  detergent permeabilized plasma  membrane. (A) GraB was titered  against a constant dose of perforin (40 ng/ml). (Filled triangles)  Increasing amounts of Triton  X-100 were added to cells with  either a constant dose of GraB  (1 μg/ml) (filled circles) or medium control (filled squares). (B)  GraB titered with perforin as in  Fig. 1 A (filled triangles). GraB  (1 μg/ml) was incubated with  increasing amounts of digitonin  (filled circles) and compared to incubation with digitonin only  (filled squares).
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Figure 7: GraB is inactive against YAC-1 target cells with detergent permeabilized plasma membrane. (A) GraB was titered against a constant dose of perforin (40 ng/ml). (Filled triangles) Increasing amounts of Triton X-100 were added to cells with either a constant dose of GraB (1 μg/ml) (filled circles) or medium control (filled squares). (B) GraB titered with perforin as in Fig. 1 A (filled triangles). GraB (1 μg/ml) was incubated with increasing amounts of digitonin (filled circles) and compared to incubation with digitonin only (filled squares).

Mentions: A second method of determining if cytoplasmic introduction of GraB was sufficient to induce apoptosis was to solubilize the plasma membrane with the detergents digitonin or Triton X-100 to allow free entry of the granzyme into the cell. We determined if the detergents over a wide concentration range could support GraB induced apoptosis of YAC-1 cells. At none of the doses of either detergent were we able to see evidence of DNA damage measured by 125IUdR release compared to perforin in the same experiment (Fig. 7, A and B).


Granzyme B (GraB) autonomously crosses the cell membrane and perforin initiates apoptosis and GraB nuclear localization.

Shi L, Mai S, Israels S, Browne K, Trapani JA, Greenberg AH - J. Exp. Med. (1997)

GraB is inactive  against YAC-1 target cells with  detergent permeabilized plasma  membrane. (A) GraB was titered  against a constant dose of perforin (40 ng/ml). (Filled triangles)  Increasing amounts of Triton  X-100 were added to cells with  either a constant dose of GraB  (1 μg/ml) (filled circles) or medium control (filled squares). (B)  GraB titered with perforin as in  Fig. 1 A (filled triangles). GraB  (1 μg/ml) was incubated with  increasing amounts of digitonin  (filled circles) and compared to incubation with digitonin only  (filled squares).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196167&req=5

Figure 7: GraB is inactive against YAC-1 target cells with detergent permeabilized plasma membrane. (A) GraB was titered against a constant dose of perforin (40 ng/ml). (Filled triangles) Increasing amounts of Triton X-100 were added to cells with either a constant dose of GraB (1 μg/ml) (filled circles) or medium control (filled squares). (B) GraB titered with perforin as in Fig. 1 A (filled triangles). GraB (1 μg/ml) was incubated with increasing amounts of digitonin (filled circles) and compared to incubation with digitonin only (filled squares).
Mentions: A second method of determining if cytoplasmic introduction of GraB was sufficient to induce apoptosis was to solubilize the plasma membrane with the detergents digitonin or Triton X-100 to allow free entry of the granzyme into the cell. We determined if the detergents over a wide concentration range could support GraB induced apoptosis of YAC-1 cells. At none of the doses of either detergent were we able to see evidence of DNA damage measured by 125IUdR release compared to perforin in the same experiment (Fig. 7, A and B).

Bottom Line: GraB was internalized and found diffusely dispersed in the cell cytoplasm within 10 min.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.We conclude that GraB can enter cells autonomously but that perforin initiates the apoptotic process and the entry of GraB into the nucleus.

View Article: PubMed Central - PubMed

Affiliation: Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg, Canada.

ABSTRACT
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 degrees 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.

Show MeSH
Related in: MedlinePlus