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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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Cytoplasmic uptake of FITC-GraB. (A, left) Apoptosis of  YAC-1 cells comparing GraB (filled triangles) to FITC-labeled GraB (filled  circles)with a constant amount of perforin (125 ng/ml). Apoptosis was  measured by the release of 125IUdR and the results are expressed as percentage of total label incorporated into the target cells. (Right) Apoptosis  of YAC-1 cells by increasing concentrations of FITC-GraB in the presence of perforin (125 ng/ml) incubated for 10 min (filled circles) or 30 min  (filled triangles) as indicated. Apoptosis was measured by the condensation  of chromatin visualized by Hoechst staining. (B) Quantitation of fluorescence label in YAC-1 cells treated with FITC-GraB (GraB) with or without perforin (Perf). Cells were incubated in 1 μg/ml of FITC-GraB for 10  or 30 min then fixed and visualized with a CCD camera. Intracellular fluorescence was quantitated using an IPLabs Spectrum H-SU2 image analysis program. At least 30 cells were measured for each condition and the  difference between extracellular and a maximum plateau of intracellular  fluorescence calculated and scored. Data is expressed as the mean and  SEM. The experiment was repeated several times with YAC-1 or HeLa  cells with the same result. (C) Fluorescence of two YAC-1 cells treated  for 30 min with FITC-GraB and analyzed using an IPLabs Spectrum image analysis program. Intracellular fluorescence was calculated for each cell by the  difference between the background surrounding the cell and the mean peak of intracellular fluorescence on the y-Axis. (D) Image analysis of two cells  treated with FITC-GraB and perforin for 30 min. (E) Image analysis of the autofluorescence of two YAC-1 cells used as background.
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Figure 1: Cytoplasmic uptake of FITC-GraB. (A, left) Apoptosis of YAC-1 cells comparing GraB (filled triangles) to FITC-labeled GraB (filled circles)with a constant amount of perforin (125 ng/ml). Apoptosis was measured by the release of 125IUdR and the results are expressed as percentage of total label incorporated into the target cells. (Right) Apoptosis of YAC-1 cells by increasing concentrations of FITC-GraB in the presence of perforin (125 ng/ml) incubated for 10 min (filled circles) or 30 min (filled triangles) as indicated. Apoptosis was measured by the condensation of chromatin visualized by Hoechst staining. (B) Quantitation of fluorescence label in YAC-1 cells treated with FITC-GraB (GraB) with or without perforin (Perf). Cells were incubated in 1 μg/ml of FITC-GraB for 10 or 30 min then fixed and visualized with a CCD camera. Intracellular fluorescence was quantitated using an IPLabs Spectrum H-SU2 image analysis program. At least 30 cells were measured for each condition and the difference between extracellular and a maximum plateau of intracellular fluorescence calculated and scored. Data is expressed as the mean and SEM. The experiment was repeated several times with YAC-1 or HeLa cells with the same result. (C) Fluorescence of two YAC-1 cells treated for 30 min with FITC-GraB and analyzed using an IPLabs Spectrum image analysis program. Intracellular fluorescence was calculated for each cell by the difference between the background surrounding the cell and the mean peak of intracellular fluorescence on the y-Axis. (D) Image analysis of two cells treated with FITC-GraB and perforin for 30 min. (E) Image analysis of the autofluorescence of two YAC-1 cells used as background.

Mentions: To determine whether GraB entered cells, we examined the localization of FITC-labeled human GraB using a high-sensitivity CCD camera and image analysis to detect and quantify intracellular fluorescence. We first evaluated the apoptotic activity of FITC-GraB to determine whether the fluorescenation of the granzyme had ablated its biological activity. YAC-1 cells were incubated with increasing concentrations of FITC-GraB (0.0625–2.0 μg/ml) in the presence of a constant amount of perforin (125 ng/ml). After 10 min of incubation a low level of activity was detected on the basis of Hoechst staining of apoptotic nuclei and by 30 min ∼70% of the cells had undergone apoptosis by the highest GraB concentrations (Fig. 1 A, right). The apoptosis produced by FITC-GraB was equivalent to that induced by unlabeled GraB and perforin (Fig. 1 A, left).


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)

Cytoplasmic uptake of FITC-GraB. (A, left) Apoptosis of  YAC-1 cells comparing GraB (filled triangles) to FITC-labeled GraB (filled  circles)with a constant amount of perforin (125 ng/ml). Apoptosis was  measured by the release of 125IUdR and the results are expressed as percentage of total label incorporated into the target cells. (Right) Apoptosis  of YAC-1 cells by increasing concentrations of FITC-GraB in the presence of perforin (125 ng/ml) incubated for 10 min (filled circles) or 30 min  (filled triangles) as indicated. Apoptosis was measured by the condensation  of chromatin visualized by Hoechst staining. (B) Quantitation of fluorescence label in YAC-1 cells treated with FITC-GraB (GraB) with or without perforin (Perf). Cells were incubated in 1 μg/ml of FITC-GraB for 10  or 30 min then fixed and visualized with a CCD camera. Intracellular fluorescence was quantitated using an IPLabs Spectrum H-SU2 image analysis program. At least 30 cells were measured for each condition and the  difference between extracellular and a maximum plateau of intracellular  fluorescence calculated and scored. Data is expressed as the mean and  SEM. The experiment was repeated several times with YAC-1 or HeLa  cells with the same result. (C) Fluorescence of two YAC-1 cells treated  for 30 min with FITC-GraB and analyzed using an IPLabs Spectrum image analysis program. Intracellular fluorescence was calculated for each cell by the  difference between the background surrounding the cell and the mean peak of intracellular fluorescence on the y-Axis. (D) Image analysis of two cells  treated with FITC-GraB and perforin for 30 min. (E) Image analysis of the autofluorescence of two YAC-1 cells used as background.
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Figure 1: Cytoplasmic uptake of FITC-GraB. (A, left) Apoptosis of YAC-1 cells comparing GraB (filled triangles) to FITC-labeled GraB (filled circles)with a constant amount of perforin (125 ng/ml). Apoptosis was measured by the release of 125IUdR and the results are expressed as percentage of total label incorporated into the target cells. (Right) Apoptosis of YAC-1 cells by increasing concentrations of FITC-GraB in the presence of perforin (125 ng/ml) incubated for 10 min (filled circles) or 30 min (filled triangles) as indicated. Apoptosis was measured by the condensation of chromatin visualized by Hoechst staining. (B) Quantitation of fluorescence label in YAC-1 cells treated with FITC-GraB (GraB) with or without perforin (Perf). Cells were incubated in 1 μg/ml of FITC-GraB for 10 or 30 min then fixed and visualized with a CCD camera. Intracellular fluorescence was quantitated using an IPLabs Spectrum H-SU2 image analysis program. At least 30 cells were measured for each condition and the difference between extracellular and a maximum plateau of intracellular fluorescence calculated and scored. Data is expressed as the mean and SEM. The experiment was repeated several times with YAC-1 or HeLa cells with the same result. (C) Fluorescence of two YAC-1 cells treated for 30 min with FITC-GraB and analyzed using an IPLabs Spectrum image analysis program. Intracellular fluorescence was calculated for each cell by the difference between the background surrounding the cell and the mean peak of intracellular fluorescence on the y-Axis. (D) Image analysis of two cells treated with FITC-GraB and perforin for 30 min. (E) Image analysis of the autofluorescence of two YAC-1 cells used as background.
Mentions: To determine whether GraB entered cells, we examined the localization of FITC-labeled human GraB using a high-sensitivity CCD camera and image analysis to detect and quantify intracellular fluorescence. We first evaluated the apoptotic activity of FITC-GraB to determine whether the fluorescenation of the granzyme had ablated its biological activity. YAC-1 cells were incubated with increasing concentrations of FITC-GraB (0.0625–2.0 μg/ml) in the presence of a constant amount of perforin (125 ng/ml). After 10 min of incubation a low level of activity was detected on the basis of Hoechst staining of apoptotic nuclei and by 30 min ∼70% of the cells had undergone apoptosis by the highest GraB concentrations (Fig. 1 A, right). The apoptosis produced by FITC-GraB was equivalent to that induced by unlabeled GraB and perforin (Fig. 1 A, left).

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