Limits...
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.

Show MeSH

Related in: MedlinePlus

Cell associated GraB induces apoptosis with the addition of  perforin. (A) 125IUdR-labelled YAC-1 cells were preincubated in GraB  (2 μg/ml) for 15 min, then washed and perforin added at increasing concentrations and the percent apoptotic cells calculated after incubation for  3 h. (B) GraB (2 μg/ml) was incubated with YAC-1 cells for 15 min,  washed, and then incubated for increasing periods of time as shown. Cells  were then washed again and perforin added for an additional 3 h. (Filled  circles) 60 ng/ml, (filled squares) 30 ng/ml, (filled triangles) 15 ng/ml.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196167&req=5

Figure 3: Cell associated GraB induces apoptosis with the addition of perforin. (A) 125IUdR-labelled YAC-1 cells were preincubated in GraB (2 μg/ml) for 15 min, then washed and perforin added at increasing concentrations and the percent apoptotic cells calculated after incubation for 3 h. (B) GraB (2 μg/ml) was incubated with YAC-1 cells for 15 min, washed, and then incubated for increasing periods of time as shown. Cells were then washed again and perforin added for an additional 3 h. (Filled circles) 60 ng/ml, (filled squares) 30 ng/ml, (filled triangles) 15 ng/ml.

Mentions: To determine if the cell-associated GraB detected by fluorescence was sufficient to induce apoptosis, YAC-1 cells were labeled with [125I]UdR then incubated with GraB (2 μg/ml) for 15 min and then washed to remove extracellular protease. This was immediately followed by the addition of perforin at increasing concentrations. These cells underwent apoptosis efficiently and in a dose-dependent manner (Fig. 3 A). If the cells were first treated with GraB as above and then washed and incubated for increasing times, the addition of perforin became gradually less efficient at inducing DNA damage suggesting that the cell associated GraB had been progressively eliminated from the cells (Fig. 3 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)

Cell associated GraB induces apoptosis with the addition of  perforin. (A) 125IUdR-labelled YAC-1 cells were preincubated in GraB  (2 μg/ml) for 15 min, then washed and perforin added at increasing concentrations and the percent apoptotic cells calculated after incubation for  3 h. (B) GraB (2 μg/ml) was incubated with YAC-1 cells for 15 min,  washed, and then incubated for increasing periods of time as shown. Cells  were then washed again and perforin added for an additional 3 h. (Filled  circles) 60 ng/ml, (filled squares) 30 ng/ml, (filled triangles) 15 ng/ml.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Cell associated GraB induces apoptosis with the addition of perforin. (A) 125IUdR-labelled YAC-1 cells were preincubated in GraB (2 μg/ml) for 15 min, then washed and perforin added at increasing concentrations and the percent apoptotic cells calculated after incubation for 3 h. (B) GraB (2 μg/ml) was incubated with YAC-1 cells for 15 min, washed, and then incubated for increasing periods of time as shown. Cells were then washed again and perforin added for an additional 3 h. (Filled circles) 60 ng/ml, (filled squares) 30 ng/ml, (filled triangles) 15 ng/ml.
Mentions: To determine if the cell-associated GraB detected by fluorescence was sufficient to induce apoptosis, YAC-1 cells were labeled with [125I]UdR then incubated with GraB (2 μg/ml) for 15 min and then washed to remove extracellular protease. This was immediately followed by the addition of perforin at increasing concentrations. These cells underwent apoptosis efficiently and in a dose-dependent manner (Fig. 3 A). If the cells were first treated with GraB as above and then washed and incubated for increasing times, the addition of perforin became gradually less efficient at inducing DNA damage suggesting that the cell associated GraB had been progressively eliminated from the cells (Fig. 3 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