QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rudner, J. Articles by Belka, C. Search for Related Content PubMed PubMed Citation Articles by Rudner, J. Articles by Belka, C.

Wild-type, mitochondrial and ER-restricted Bcl-2 inhibit DNA damage-induced apoptosis but do not affect death receptor-induced apoptosis

¹ Department of Radiation Oncology, University of Tübingen, Hoppe-Seyler Str. 3, D-72076 Tübingen, Germany
² Department of Physiology I, University of Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany
³ Department of Immunology and Cell Biology, Institute of Experimental Dermatology, University of Münster, Röntgenstr. 21, D-48149 Münster, Germany
⁴ Department of Internal Medicine I, University of Tübingen, Ottfried-Müller Str. 10, D-72076 Tübingen, Germany

View larger version (64K): [in a new window]   Fig. 1. Expression of wild-type Bcl-2 and Bcl-2 mutants with restricted subcellular location. Jurkat T-cells were stably transfected with expression constructs targeting Bcl-2 to the endoplasmic reticulum (Bcl-2/ER) or mitochondria (Bcl-2/MT), a construct lacking the transmembrane domain (Bcl-2/TM) or with wild-type Bcl-2. Confocal microscopy was performed to confirm the subcellular localization of Bcl-2 in the appropriate compartment. The pool-transfected cells were co-stained with Bcl-2 (in red) and either with the calcium ATPase SERCA for endoplasmic staining or with cytochrome c for mitochondrial staining (in green). Colocalization of the molecules is indicated by the yellow color in the merged micrographs. In Bcl-2/ER cells (A) Bcl-2 colocalizes with SERCA but not with cytochrome c. In Bcl-2/MT cells (B) Bcl-2 clearly colocalizes with cytochrome c but not with SERCA. By contrast, Bcl-2/TM (C) shows a diffuse expression pattern in the cytoplasm and nucleus. Wild-type Bcl-2 (D) is detectable in the perinuclear region, the ER and the mitochondria.

View larger version (24K): [in a new window]   Fig. 2. Induction of apoptosis by ionizing irradiation. Immunoblot analysis with an anti-Bcl-2 antibody revealed that the transfectants expressed the different forms of Bcl-2 at roughly similar levels (A). Apoptosis induction in control or cells irradiated with 10 Gy was quantified by flow cytometry (B). Overexpression of Bcl-2/MT and Bcl-2/WT strongly protected cells against apoptosis. Similar to Bcl-2/MT, ER-targeted Bcl-2 conferred survival against irradiation-induced cell death. Cells expressing the cytosolic, non-membrane Bcl-2/TM mutant revealed apoptosis to similar levels as vector control cells. The error bars indicate the standard deviations from independent measurements of the same cell batch (five independent experiments).

View larger version (21K): [in a new window]   Fig. 3. Caspase activation after irradiation of Jurkat cells expressing different Bcl-2 mutants. Western blot analyses were performed with cell lysates prepared 14-20 hours after irradiation with 10 Gy. The blots show that activation of caspase-9, caspase-3 and caspase-8 as well as PARP cleavage was abrogated by overexpression of either mitochondrial (Bcl-2/MT), endoplasmic (Bcl-2/ER) or wild-type Bcl-2 (Bcl-2/WT). No differences in caspase activation were detectable when Bcl-2/TM cells were compared with vector control cells.

View larger version (20K): [in a new window]   Fig. 4. Breakdown of the mitochondrial membrane potential after irradiation occurs prior to caspase activation. Prior to irradiation (10 Gy) cells were pretreated with the broad-spectrum caspase inhibitor zVAD (20 µM). No influence on the breakdown of m was detectable (A), although caspase activation indicated by the abrogation of PARP cleavage was blocked (B).

View larger version (22K): [in a new window]   Fig. 5. Loss of mitochondrial membrane potential after irradiation. Cells were irradiated with 10 Gy and the mitochondrial membrane potential m was determined by flow cytometry. Bcl-2 overexpression at mitochondria (Bcl-2/MT), the ER (Bcl-2/ER) or in both compartments (Bcl-2/WT) protected cells from radiation-induced mitochondrial potential breakdown, whereas Bcl-2/TM had no significant effects. The error bars indicate the standard deviations from independent measurements of the same cell batch.

View larger version (20K): [in a new window]   Fig. 6. CD95- and TRAIL-mediated apoptosis in Jurkat cells overexpressing Bcl-2 at different intracellular locations. Cells were stimulated with 100 ng/ml of either anti-CD95 (A) or TRAIL (B). After the indicated times apoptosis was quantified by flow cytometry. The experiments reveal that overexpression of the Bcl-2 mutants had no significant effect on death receptor-mediated apoptosis. The error bars indicate the standard deviations from independent measurements of the same cell batch (five independent measurements).

View larger version (21K): [in a new window]   Fig. 7. Effect of the Bcl-2 mutants on CD95-mediated caspase activation. Cell lysates were prepared 1, 2, 4 and 6 hours after CD95 stimulation (100 ng/ml CH11) and analyzed for caspase processing and PARP cleavage by immunoblotting using antibodies against caspase-8, active caspase-9, active caspase-3 and PARP. The immunoblots show the proforms and p43/41 intermediate cleavage products of caspase-8, the p35 intermediate caspase-9 fragment, the different cleavage products of caspase-3, and the full-length and p85 fragment of PARP. In vector control cells as well as in Bcl-2/TM- and Bcl-2/ER-expressing cells procaspase-8 was almost completely processed, whereas in cells expressing the wild-type and mitochondrial form of Bcl-2 the processing was attenuated. Caspase-9 and caspase-3 activation as well as PARP cleavage was also delayed in Bcl-2/WT and Bcl-2/MT cells.

View larger version (21K): [in a new window]   Fig. 8. Effect of the Bcl-2 mutants on the breakdown of the mitochondrial membrane potential after CD95 (A) or TRAIL (B) stimulation. Cells were stimulated for the indicated time points, and the mitochondrial potential (m) was determined using the mitochondrial potential-specific dye TMRE. The amount of cells with a low m was quantified by FACS analysis. When overexpressed at mitochondria or as wild-type protein, Bcl-2 delayed m breakdown. However, after 24 hours almost all cells displayed a low m irrespective of the Bcl-2 status. Bcl-2/ER influenced the breakdown of m only modestly, whereas Bcl-2/TM had no effect compared with vector control cells. The error bars indicate the standard deviations from independent measurements of the same cell batch.

View larger version (20K): [in a new window]   Fig. 9. Effect of irradiation on caspase-12 processing. The cleavage of caspase-12 was analyzed by immunoblotting using a polyclonal antibody. After irradiation with 10 Gy a fragment of approximately 17 kDa became detectable. Bcl-2 overexpression at the ER the mitochondria, or as wild-type Bcl-2 abrogated caspase-12 processing (A). Pretreatment with the caspase-9 inhibitor LEHD-fmk (50 µM) abrogated the processing of caspase-12 as well as the activation of caspase-9 and caspase-3 (B).

View larger version (20K): [in a new window]   Fig. 10. Schematic model of the effect of Bcl-2 subcellular location in two forms of apoptosis. During death receptor-mediated apoptosis caspase-8 is the most apical caspase. Once activated, caspase-8 in turn triggers the downstream effector cascade either directly through caspase-3 or through engagement of the mitochondrial pathway. In this process, Bid triggers cytochrome c release, which is needed for caspase-9 activation. Bcl-2 located at mitochondria interferes with the activation of this amplification loop, whereas Bcl-2 at the ER has no influence on death receptor-mediated apoptosis. By contrast, the most apical caspase in radiation-induced apoptosis is caspase-9, which is activated in response to mitochondrial damage. A hypothetical crosstalk between mitochondria and the ER, which is upstream of caspase activation and may involve perturbations in calcium homeostasis or other ER-based pro-apoptotic molecules, may essentially control the initial steps of radiation-induced cell death.