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A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

Trapani JA, Sutton VR, Thia KY, Li YQ, Froelich CJ, Jans DA, Sandrin MS, Browne KA - J. Cell Biol. (2003)

Bottom Line: Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays.Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it.Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au

ABSTRACT
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

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K44A-dynamin–overexpressing HeLa cells are not protected from granzyme B–induced cell death. (a) 51Cr-labeled HeLa cells overexpressing either K44A- or wild-type (WT) dynamin were incubated with sublytic perforin (Pfp) or pneumolysin (PLO) either alone or together with granzyme B (50 nM) for 90 min at 37°C. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments. (b) Clonogenic survival of K44A- or wild-type (WT) dynamin-overexpressing HeLa cells after incubation with sublytic perforin (Pfp) and various concentrations of granzyme B. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 HeLa colonies became evident when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods).
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fig3: K44A-dynamin–overexpressing HeLa cells are not protected from granzyme B–induced cell death. (a) 51Cr-labeled HeLa cells overexpressing either K44A- or wild-type (WT) dynamin were incubated with sublytic perforin (Pfp) or pneumolysin (PLO) either alone or together with granzyme B (50 nM) for 90 min at 37°C. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments. (b) Clonogenic survival of K44A- or wild-type (WT) dynamin-overexpressing HeLa cells after incubation with sublytic perforin (Pfp) and various concentrations of granzyme B. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 HeLa colonies became evident when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods).

Mentions: We next determined whether K44A-overexpressing cells are protected from cell death mediated by granzyme B. We initially measured specific 51Cr release in response to granzyme B delivered either by sublytic quantities of perforin or an alternative lytic agent, pneumococcal pneumolysin (PLO), which has been shown to closely mimic perforin's delivery of granzyme B (Browne et al., 1999). In this context, 51Cr release is a measure of plasma membrane permeability during apoptosis and is a reliable short term measure of cell survival in response to granzyme B (Sutton et al., 1997; Trapani et al., 1998). Addition of granzyme B (50 nM) with either perforin or PLO induced equivalent 51Cr release from K44A- and wild-type dynamin-overexpressing cells over 4 h (Fig. 3 a). This result is reminiscent of an experiment of Motyka et al. (2000) who showed that MPR-overexpressing and MPR- L cells released equivalent 51Cr when they were attacked by intact CTL. The authors attributed the surprisingly high 51Cr release in cells lacking the MPR/clathrin pathway to perforin-mediated lysis and ruled out the effect as being caused by granzyme B. However, our experiment performed with the same cell lines and purified perforin and granzyme B clearly showed that, even in the absence of MPR, granzyme B is able to synergize with perforin or PLO to induce 51Cr release. In this setting, the release of 51Cr therefore reflects the loss of plasma membrane integrity as the result of granzyme B–induced apoptosis, not necrosis (lysis), in response to perforin. We also confirmed that K44A- and wild-type dynamin-expressing cells were equally susceptible to granzyme B/perforin in assays of clonogenic survival. There was an equivalent dose-related reduction in colony numbers as the concentration of granzyme B increased, irrespective of whether the MPR/dynamin pathway was functional or not (Fig. 3 b).


A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

Trapani JA, Sutton VR, Thia KY, Li YQ, Froelich CJ, Jans DA, Sandrin MS, Browne KA - J. Cell Biol. (2003)

K44A-dynamin–overexpressing HeLa cells are not protected from granzyme B–induced cell death. (a) 51Cr-labeled HeLa cells overexpressing either K44A- or wild-type (WT) dynamin were incubated with sublytic perforin (Pfp) or pneumolysin (PLO) either alone or together with granzyme B (50 nM) for 90 min at 37°C. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments. (b) Clonogenic survival of K44A- or wild-type (WT) dynamin-overexpressing HeLa cells after incubation with sublytic perforin (Pfp) and various concentrations of granzyme B. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 HeLa colonies became evident when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods).
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Related In: Results  -  Collection

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fig3: K44A-dynamin–overexpressing HeLa cells are not protected from granzyme B–induced cell death. (a) 51Cr-labeled HeLa cells overexpressing either K44A- or wild-type (WT) dynamin were incubated with sublytic perforin (Pfp) or pneumolysin (PLO) either alone or together with granzyme B (50 nM) for 90 min at 37°C. Specific 51Cr release is shown as the mean of triplicate data points ± standard error. The assay is representative of three similar experiments. (b) Clonogenic survival of K44A- or wild-type (WT) dynamin-overexpressing HeLa cells after incubation with sublytic perforin (Pfp) and various concentrations of granzyme B. Results are shown as the percentage inhibition of colony growth (mean of three cultures ± standard error at each granzyme concentration) compared with untreated cells. Typically, 75–150 HeLa colonies became evident when the cells were untreated. The experiment shown is representative of four similar experiments (as described in Materials and methods).
Mentions: We next determined whether K44A-overexpressing cells are protected from cell death mediated by granzyme B. We initially measured specific 51Cr release in response to granzyme B delivered either by sublytic quantities of perforin or an alternative lytic agent, pneumococcal pneumolysin (PLO), which has been shown to closely mimic perforin's delivery of granzyme B (Browne et al., 1999). In this context, 51Cr release is a measure of plasma membrane permeability during apoptosis and is a reliable short term measure of cell survival in response to granzyme B (Sutton et al., 1997; Trapani et al., 1998). Addition of granzyme B (50 nM) with either perforin or PLO induced equivalent 51Cr release from K44A- and wild-type dynamin-overexpressing cells over 4 h (Fig. 3 a). This result is reminiscent of an experiment of Motyka et al. (2000) who showed that MPR-overexpressing and MPR- L cells released equivalent 51Cr when they were attacked by intact CTL. The authors attributed the surprisingly high 51Cr release in cells lacking the MPR/clathrin pathway to perforin-mediated lysis and ruled out the effect as being caused by granzyme B. However, our experiment performed with the same cell lines and purified perforin and granzyme B clearly showed that, even in the absence of MPR, granzyme B is able to synergize with perforin or PLO to induce 51Cr release. In this setting, the release of 51Cr therefore reflects the loss of plasma membrane integrity as the result of granzyme B–induced apoptosis, not necrosis (lysis), in response to perforin. We also confirmed that K44A- and wild-type dynamin-expressing cells were equally susceptible to granzyme B/perforin in assays of clonogenic survival. There was an equivalent dose-related reduction in colony numbers as the concentration of granzyme B increased, irrespective of whether the MPR/dynamin pathway was functional or not (Fig. 3 b).

Bottom Line: Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays.Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it.Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Cancer Immunology Laboratory, Peter MacCallum Cancer Institute, Melbourne 8006, Australia. j.trapani@pmci.unimelb.edu.au

ABSTRACT
The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR- L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.

Show MeSH
Related in: MedlinePlus