Cancer Res. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the intact transcription factors role in mediating protection from the ganglioside. their ganglioside accumulation and apoptogenicity for co-incubated T-cells(3). Our present work focuses on the mechanism by which gangliosides induce T-cell apoptosis. Current evidence implies that gangliosides mediate their pro-apoptotic effects by directly activating the intrinsic apoptotic pathway. In separate reports, Garcia-Ruiz et al.(17) and Rippo et al.(18) demonstrated that the ganglioside GD3 could stimulate a burst of ROS and mitochondrial permeability in purified mitochondria, leading to the release of apoptogenic factors such as cytochrome-c and apoptosis inducing factor (AIF). Later studies showed that mitochondria are targeted even when intact cells are exposed to gangliosides, as GD3-treated hepatocytes underwent apoptosis in association with ROS production, Pitolisant MPT, cytochrome-c release and activation of caspase-9(19, 20). The means by which tumor-derived gangliosides stimulate the apoptosis of T-cells remains undefined, however. Garcia-Ruiz et al. showed that in response to TNF, endogenous GD3 redistributes from the outer leaflet of hepatocyte membranes to mitochondria via Rab5-and Rab7-positive endosomes, where it induces the same series of pro-apoptotic events observed when mitochondria are treated with the same ganglioside(20). Studies pertaining to ganglioside transport in Niemann-Pick disease indicate that even exogenous gangliosides can be internalized and targeted to the Golgi complex within Rab-expressing vesicles(21), potentially localizing to the mitochondria where they can induce toxic levels of ROS in glutathione-depleted cells, as described previously for the transported, endogenous gangliosides(20). The notion that exogenous gangliosides may also stimulate T-cell apoptosis in a mitochondrial-dependent manner is suggested by the ability of the Bcl-2 transgene to protect CEM lymphoma cells from GD3-induced caspase-9 activation and death(18). The experiments described below suggest that such a scenario may in fact be the mechanism by which GD3 kills T-lymphocytes: we find that GD3 specifically induces the apoptosis of activated but not resting T-cells. Our results indicate that activated T-cells internalize abundant levels of exogenously administered GD3 within 90 minutes, causing ROS production, the mitochondrial permeability transition and cytochrome-c release by 24h and apoptosis by 48h. GD3-mediated apoptosis additionally involves p53 stabilization and induction of Bax, and is amplified by the caspase-dependent degradation of NFB-inducible anti-apoptotic proteins. Resting T-cells, which fail to internalize appreciable levels of the ganglioside, do not undergo any of these pro-apoptotic changes within the time frame examined, thus explaining the comparative resistance of that lymphocyte population to GD3-mediated destruction. Materials and Methods Antibodies and Reagents Anti-Bcl-xL, anti-Bcl-2, anti-CIAP-2, anti-p53, anti-Bax, anti-RelA and horseradish peroxidase-conjugated donkey anti-goat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-XIAP was from BD Transduction Laboratories (San Diego, CA), and anti-Actin (AC-15) was from Novus Biologicals (Littleton, CO). Anti-pro-caspase-9 and procaspase-8 antibodies were from Oncogene Research Products (Boston, MA), and anti-cytochrome-c and anti-GD3 were from BD PharMingen (San Diego, CA). HRP-conjugated sheep anti-mouse and donkey anti-rabbit secondary antibodies were from Amersham (Arlington Heights, IL), and Caspase inhibitor III , Caspase-9 Inhibitor II, and Caspase-8 inhibitor I (IETD-CHO) were all obtained from Calbiochem (La Jolla, CA). Anti-CD3 antibody (OKT3, Ortho Biotech, Raritan, NJ) and anti-CD28 antibody (Becton Dickenson Immunocytometry Systems, San Jose, CA) were used for the stimulation of T-lymphocytes. Human recombinant interleukin-2 (IL-2) (CHIRON Corporation, Emeryville, CA) was used at 20U/ml to maintain the viability of activated T-cells. Cell lines and Tissue Culture Conditions Wild type Jurkat cells, caspase-8-negative Jurkat cells(22) (gift from Dr. John Blenis (Harvard University), HA-RelA over-expressing Jurkat cells(1), primary RCC lines(23), the long-term renal cell carcinoma lines SK-RC-45 Rabbit Polyclonal to hnRNP L and SK-RC-26b(3) (gift of Dr.Neil Bander, The New York Hospital, Cornell University Medical College), and the normal kidney epithelial (NKE) cell line(24) were derived and maintained as described previously. The caspase-9 D/N Jurkat cell line was generated using a cDNA expression vector encoding the active site mutation cysteine-287 to alanine (25). A well characterized, long-term glioblastoma cell line U87 was obtained courtesy of Dr. Michael Vogelbaum (Cleveland Clinic) and CCF52 was a short-term GBM line sub-cultured from a fresh glioma by our Pitolisant Brain Tumor Registry. Immunofluorecence Tumor cells were immunostained to assess GD3 expression levels. RCC tumor cells, NKE control cells and glioblastoma cells were fixed, blocked and stained with 2g/ml anti-GD3 antibodies as described previously(5). After washing, the cells were incubated with an Alexa 594-labeled secondary antibody (Molecular Probes Eugene, OR), washed and counterstained with DAPI to visualize the nuclei. Resting and activated T-cells were also assessed for.It is also possible that only RelA is cleaved(1), thus inhibiting the transcription of these NFB-dependent, anti-apoptotic molecules; indeed, caspase inhibitors coincidentally rescued both RelA and the anti-apoptotic proteins in GD3-treated T cells. susceptibility. Pitolisant Resting T-cells did not internalize appreciable levels of GD3, and did not undergo any of the proapoptotic changes that characterize activated T-lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the intact transcription factors role in mediating protection from the ganglioside. their ganglioside accumulation and apoptogenicity for co-incubated T-cells(3). Our present work focuses on the mechanism by which gangliosides induce T-cell apoptosis. Current evidence implies that gangliosides mediate their pro-apoptotic effects by directly activating the intrinsic apoptotic pathway. In separate reports, Garcia-Ruiz et al.(17) and Rippo et al.(18) demonstrated that the ganglioside GD3 could stimulate a burst of ROS and mitochondrial permeability in purified mitochondria, leading to the release of apoptogenic factors such as cytochrome-c and apoptosis inducing factor (AIF). Later studies showed that mitochondria are targeted even when intact cells are exposed to gangliosides, as GD3-treated hepatocytes underwent apoptosis in association with ROS production, MPT, cytochrome-c release and activation of caspase-9(19, 20). The means by which tumor-derived gangliosides stimulate the apoptosis of T-cells remains undefined, however. Garcia-Ruiz et al. showed that in response to TNF, endogenous GD3 redistributes from the outer leaflet of hepatocyte membranes to mitochondria via Rab5-and Rab7-positive endosomes, where it induces the same series of pro-apoptotic events observed when mitochondria are treated with the same ganglioside(20). Studies pertaining to ganglioside transport in Niemann-Pick disease indicate that even exogenous gangliosides can be internalized and targeted to the Golgi complex within Rab-expressing vesicles(21), potentially localizing to the mitochondria where they can induce toxic levels of ROS in glutathione-depleted cells, as described previously for the transported, endogenous gangliosides(20). The notion that exogenous gangliosides may also stimulate T-cell apoptosis in a mitochondrial-dependent manner is suggested by the ability of the Bcl-2 transgene to protect CEM lymphoma cells from GD3-induced caspase-9 activation and death(18). The experiments described below suggest that such a scenario may in fact be the mechanism by which GD3 kills T-lymphocytes: we find that GD3 specifically induces the apoptosis of activated but not resting T-cells. Our results indicate that activated T-cells internalize abundant levels of exogenously administered GD3 within 90 minutes, causing ROS production, the mitochondrial permeability transition and cytochrome-c release by 24h and apoptosis by 48h. GD3-mediated apoptosis additionally involves p53 stabilization and induction of Bax, and is amplified by the caspase-dependent degradation of NFB-inducible anti-apoptotic proteins. Resting T-cells, which fail to internalize appreciable levels of the ganglioside, do not undergo any of these pro-apoptotic changes within the time frame examined, thus explaining the comparative resistance of that lymphocyte population to GD3-mediated destruction. Materials and Methods Antibodies and Reagents Anti-Bcl-xL, anti-Bcl-2, anti-CIAP-2, anti-p53, anti-Bax, anti-RelA and Pitolisant horseradish peroxidase-conjugated donkey anti-goat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-XIAP was from BD Transduction Laboratories (San Diego, CA), and anti-Actin (AC-15) was from Novus Biologicals (Littleton, CO). Anti-pro-caspase-9 and procaspase-8 antibodies were from Oncogene Research Products (Boston, MA), and anti-cytochrome-c and anti-GD3 were from BD PharMingen (San Diego, CA). HRP-conjugated sheep anti-mouse and donkey anti-rabbit secondary antibodies were from Amersham (Arlington Heights, IL), and Caspase inhibitor III , Caspase-9 Inhibitor II, and Caspase-8 inhibitor I (IETD-CHO) were all obtained from Calbiochem (La Jolla, CA). Anti-CD3 antibody (OKT3, Ortho Biotech, Raritan, NJ) and anti-CD28 antibody (Becton Dickenson Immunocytometry Systems, San Jose, CA) were used for the stimulation of T-lymphocytes. Human recombinant interleukin-2 (IL-2) (CHIRON Corporation, Emeryville, CA) was used at 20U/ml to maintain the viability of activated T-cells. Cell lines and.
