Bettina Wagenknecht

Proteasome inhibitor-induced apoptosis of glioma cells involves the processing of multiple caspases and cytochrome c release

Abstract: The proteasome is a multiprotein complex that is involvedin the intracellular protein degradation in eukaryotes. Here, we show thathuman malignant glioma cells are susceptible to apoptotic cell death inducedby the proteasome inhibitors, MG132 and lactacystin. The execution of theapoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9.Apoptosis is inhibited by ectopic expression of X‐linked inhibitor ofapoptosis (XIAP) and by coexposure to the broad‐spectrum caspase inhibitor,benzoyl‐VAD‐fluoromethyl ketone (zVAD‐fmk), but not by the preferentialcaspase 8 inhibitor, crm‐A. It is interesting that specific morphologicalalterations induced by proteasome inhibition, such as dilated roughendoplasmic reticulum and the formation of cytoplasmic vacuoles and densemitochondrial deposits, are unaffected by zVAD‐fmk. Apoptosis is alsoinhibited by ectopic expression of Bcl‐2 or by an inhibitor of proteinsynthesis, cycloheximide. Further, cytochrome c release anddisruption of mitochondrial membrane potential are prominent features ofapoptosis triggered by proteasome inhibition. Bcl‐2 is a stronger inhibitor ofcytochrome c release than zVAD‐fmk. XIAP and crm‐A fail to modulatecytochrome c release. These data place cytochrome c releasedownstream of Bcl‐2 activity but upstream of XIAP‐ and crm‐A‐sensitivecaspases. The partial inhibition of cytochrome c release by zVAD‐fmkindicates a positive feedback loop that may involve cytochrome crelease and zVAD‐fmk‐sensitive caspases. Finally, death ligand/receptorinteractions, including the CD95/CD95 ligand system, do not mediate apoptosisinduced by proteasome inhibition in human malignant glioma cells.

Processing of Immunosuppressive Pro-TGF-β1,2 by Human Glioblastoma Cells Involves Cytoplasmic and Secreted Furin-Like Proteases

TGF-β is a putative mediator of immunosuppression associated with malignant glioma and other types of cancer. Subtilisin-like proprotein convertases such as furin are thought to mediate TGF-β processing. Here we report that human malignant glioma cell lines express furin mRNA and protein, exhibit furin-like protease (FLP) activity, and release active furin into the cell culture supernatant. FLP activity is not modulated by exogenous TGF-β or neutralizing TGF-β Abs. Exposure of LN-18 and T98G glioma cell lines to the furin inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethylketone, inhibits processing of the TGF-β1 and TGF-β2 precursor molecules and, consequently, the release of mature bioactive TGF-β molecules. Ectopic expression of PDX, a synthetic antitrypsin analog with antifurin activity, in the glioma cells inhibits FLP activity, TGF-β processing, and TGF-β release. Thus, subtilisin-like proprotein convertases may represent a novel target for the immunotherapy of malignant glioma and other cancers or pathological conditions characterized by enhanced TGF-β bioactivity.

CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release

Death ligands such as CD95 ligand (CD95L) or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) induce apoptosis in radiochemotherapy-resistant human malignant glioma cell lines. The death-signaling TRAIL receptors 2 (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/DR4 were expressed more abundantly than the non-death-inducing (decoy) receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the absence of a protein synthesis inhibitor, cycloheximide (CHX). Three of the 12 cell lines were still TRAIL/Apo2L-resistant in the presence of CHX. TRAIL-R2 expression predicted sensitivity to apoptosis. Coexposure to TRAIL/Apo2L and cytotoxic drugs such as topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, CCNU) or temozolomide resulted in synergistic killing. Synergistic killing was more often observed in cell lines retaining wild-type p53 activity (U87MG, LN-229) than in p53 mutant cell lines (LN-18, T98G, U373MG). Drug exposure resulted in enhanced TRAIL-R2 expression, but decreased TRAIL-R4 expression in U87MG cells. Ectopic expression of dominant-negative p53V135A abrogated the drug-induced changes in TRAIL-R2 and TRAIL-R4 expression, but had no effect on synergy. Thus, neither wild-type p53 function nor changes in TRAIL receptor expression were required for synergy. In contrast, synergy resulted possibly from drug-induced cytochrome c release from mitochondria, serving as an amplifier of the TRAIL/Apo2L-mediated cascade of caspase activation. These data provide novel insights into the role of the TRAIL/Apo2L system in malignant gliomas and illustrate that TRAIL/Apo2L-based immunochemotherapy may be an effective therapeutic strategy for these lethal neoplasms.

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-XL. These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.

Taxol-mediated augmentation of CD95 ligand-induced apoptosis of human malignant glioma cells: association with bcl-2 phosphorylation but neither activation of p53 nor G2/M cell cycle arrest.

The anti-tumour alkaloid taxol shows strong cytotoxic and antiproliferative activity in two human malignant glioma cell lines, T98G and LN-229. CD95 (Fas/APO-1) ligand is a novel cytotoxic cytokine of the tumour necrosis factor (TNF) family that exerts prominent antiglioma activity. At clinically relevant taxol concentrations of 5-100 nM, taxol and CD95 ligand showed significant synergistic cytotoxicity and growth inhibition. High concentrations of taxol induced G/M cell cycle arrest in both cell lines. The synergy of taxol and CD95 ligand was independent of cell cycle effects of taxol as synergy was achieved at much lower taxol concentrations than G2/M arrest and as cell cycle effects of taxol were unaffected by co-exposure to CD95 ligand. Similarly, high concentrations of taxol were required to induce p53 activity in the p53 wild-type cell line LN-229. This effect was not modulated by CD95 ligand, suggesting that synergy is also independent of p53 activation. However, taxol induced a mobility shift of the bcl-2 protein on immunoblot analysis, indicative of bcl-2 phosphorylation. Bcl-2 phosphorylation on serine was confirmed by immunoprecipitation and phosphoserine immunoblot analysis. Considering (1) that phosphorylation of bcl-2 interferes with its heterodimerization with bax and (2) the inhibition of CD95-mediated apoptosis by bcl-2, we propose that taxol sensitizes malignant glioma cells to CD95 ligand by increasing the functional bax/bcl-2 rheostat in favour of bax and thus cell death.

Proteasome Inhibitors Induce p53/p21-Independent Apoptosis in Human Glioma Cells

The proteasome is a multiprotein complex involved in the degradation of ubiquitinated proteins. Three proteasome inhibitors, calpain inhibitor I, lactacystin and MG132, induced apoptosis in several human malignant glioma cell lines. Although proteasome inhibitors induced p53 accumulation in a cell line retaining wild-type p53 activity, p53 activity was dispensable for apoptosis since transdominant-negative p53 abrogated p53-dependent p21 induction but did not modulate apoptosis. Further, p21 was induced by higher concentrations of proteasome inhibitors in a p53-independent manner both in p53 wild-type and in p53 mutant cell lines. Although there was a strong G2/M arrest in response to proteasome inhibition in glioma cells, this G2/M arrest was also observed in p21–/– colon carcinoma cells, suggesting that p21 is dispensable for the G2/M arrest associated with proteasome inhibition. Interestingly, the p21–/– cells were more resistant to protease inhibitors than parental p21+/+ cells. In summary, our data indicate that proteasome inhibition induces a p21-independent G2/M arrest and p53-independent apoptosis in human malignant glioma cells.

Death ligand/receptor-independent caspase activation mediates drug-induced cytotoxic cell death in human malignant glioma cells

Death ligand/receptor interactions and caspase activation mediate drug-induced apoptosis in certain cancer cells. The molecular mechanisms responsible for the chemoresistance of human malignant gliomas are largely unknown. Here, we report that malignant glioma cells co-express CD95 and CD95L without undergoing suicidal or fratricidal apoptosis. Glioma cells do not commit CD95/CD95L-dependent suicide or fratricide even when RNA and protein synthesis are inhibited. This is because ectopic expression of the viral caspase inhibitor, crm-A, or exposure to a neutralizing CD95L antibody, block apoptosis induced by exogenous CD95L but not cell death induced by cytotoxic concentrations of inhibitors of RNA and protein synthesis. Although some cytotoxic drugs enhance the expression of CD95 or CD95L, crm-A fails to block drug-induced cytotoxic and clonogenic cell death, suggesting that the drug-induced changes in CD95 and CD95L expression are epiphenomenal. There is also no difference in drug-induced apoptosis between crm-A-transfected and control cells as assessed by electron microscopy, in situ DNA end labeling and DNA fragmentation. Further, glioma cells selected for resistance to CD95L do not acquire cross-resistance to chemotherapy. However, the broad spectrum caspase inhibitor, ZVAD-fmk, inhibits drug-induced cytotoxic cell death, suggesting a role of crm-A-insensitive caspases in drug-induced apoptosis of glioma cells. Thus, drug resistance of malignant glioma cells may involve deficiencies in two interrelated pathways that mediate death in order tumor cell types: (i) death ligand/receptor signalling; and (ii) caspase activation.

Lipoxygenase inhibitors block CD95 ligand-mediated apoptosis of human malignant glioma cells

CD95 ligand is a cytotoxic cytokine that induces apoptosis. Here we report that CD95‐mediated apoptosis of human malignant glioma cells is associated with arachidonic acid (AA) release. Inhibitors of phospholipase A2, phospholipase C or diacylglycerol lipase have minor effects on AA release and fail to modulate apoptosis. Formation of two AA metabolites generated during CD95‐dependent apoptosis is attenuated by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA). NDGA also blocks CD95 ligand‐induced apoptosis. This effect is independent of antioxidant properties of NDGA. Lipoxygenase may thus play a critical role in CD95 ligand‐induced apoptosis of human malignant glioma cells.

Interferon-α enhances CD95L-induced apoptosis of human malignant glioma cells

Abstract CD95 ligand (CD95L)-induced apoptosis is a novel immunotherapeutic approach to malignant glioma. Here, we report that interferon- α (IFN- α ) sensitizes LN-229 and T98G human malignant glioma cells to CD95L-induced apoptosis. In contrast to the effects of IFN- γ and TNF- α which sensitize glioma cells to CD95 antibody-induced apoptosis in part by enhancing CD95 expression, IFN- α has no effect on CD95 expression at the cell surface of LN-229 and T98G cells. To confirm that changes in CD95 expression are not required for the effects of IFN- α , we show that IFN- α enhances CD95L-induced apoptosis even in CD95-transfected LN-308 glioma cells. These LN-308 cells have little endogenous CD95 expression but express high levels of CD95 from a stably integrated CD95 expression plasmid. The sensitizing effects of IFN- α appear to be independent of cell cycle effects of IFN- α and are unaffected by ectopic expression of the bcl -2 proto-oncogene. IFN- α enhances CD95L-induced activation of caspase-3, a critical mediator of CD95L-induced cell death. IFN- α also increases the cytotoxic effects of BCNU, teniposide and cytarabine in both cell lines, and of vincristine in LN-229 cells. Doxorubicin and 5-fluorouracil toxicity are unaffected by IFN- α . IFN- α may be a useful adjunct to novel strategies of immunochemotherapy for malignant gliomas that target CD95-mediated apoptosis.

C2-ceramide signaling in glioma cells: synergistic enhancement of CD95-mediated, caspase-dependent apoptosis

Most human malignant glioma cell lines are susceptible to CD95 ligand (CD95L)-induced apoptosis. Here, we report that glioma cells are also susceptible to the cytotoxic effects of exogenous C2-ceramide. This form of cell death exhibits some morphological features of apoptosis as assessed by electron microscopy, but is unaffected by the broad spectrum caspase inhibitor, zVAD-fmk. Further, CD95L-induced apoptosis is synergistically enhanced by coexposure of the glioma cells to CD95L and C2-ceramide. CD95L-induced caspase 3-like activity, cytochrome c release and cleavage of caspases 3, 8, 9 and poly(ADP-ribose)polymerase (PARP) increase substantially after cotreatment with CD95L and C2-ceramide compared with CD95L treatment alone. None of these events occur in response to cytotoxic concentrations of C2-ceramide alone. C2-ceramide does not alter CD95 expression. Gene transfer-mediated enhancement of CD95 expression results not only in increased susceptibility to CD95L, but also in increased sensitivity to C2-ceramide. We conclude that (i) synergistic induction of apoptosis by C2-ceramide and CD95L depend on a cross-talk between the two signal transduction pathways and that (ii) C2-ceramide, independently of its sensitizing effects on CD95-dependent caspase activation, is also capable of triggering an apoptotic signaling cascade that is unaffected by zVAD-fmk-mediated caspase inhibition, but promoted by high levels of CD95 expression. Cell Death and Differentiation (2001) 8, 595–602