«Date:_ Approved: _ Sally Kornbluth, Supervisor _ Donald McDonnell _ Robert Wechsler-Reya _ Gerard Blobe _ Salvatore Pizzo Dissertation submitted in ...»
Understanding the Cellular Response to Cytosolic Cytochrome c
Carrie Elizabeth Johnson
Department of Pharmacology and Cancer Biology
Sally Kornbluth, Supervisor
Salvatore Pizzo Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University
ABSTRACTUnderstanding the Cellular Response to Cytosolic Cytochrome c by Carrie Elizabeth Johnson Department of Pharmacology and Cancer Biology Duke University Date:_______________________
Sally Kornbluth, Supervisor ___________________________
Donald McDonnell ___________________________
Robert Wechsler-Reya ___________________________
Gerard Blobe ___________________________
Salvatore Pizzo An
of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University Copyright by Carrie Elizabeth Johnson Abstract Cytosolic cytochrome c promotes apoptosis by triggering caspase activation. In healthy cells cytochrome c localizes to mitochondria, where it participates in the electron transport chain. Apoptotic stimuli induce permeabilization of the outer mitochondrial membrane and release of cytochrome c. Once cytosolic, cytochrome c binds Apaf-1, inducing the formation of a protein complex that recruits and activates caspases, which serve to dismantle the dying cell. Although the steps of this signaling pathway have been described, many of the regulatory mechanisms influencing the cellular response to cytosolic cytochrome c remain unclear. Using apoptosis assays and microinjection techniques, we investigated the response of several cell-types to cytosolic cytochrome c.
First, we demonstrate that cytosolic cytochrome c kills brain tumor cells but not normal brain tissue. This differential sensitivity to cytochrome c is attributed to high Apaf-1 levels in brain tumors compared with negligible Apaf-1 in brain tissue. These differences in Apaf-1 abundance correlate with differences in E2F1, a previously identified activator of Apaf-1 transcription. Chromatin immunoprecipitation assays reveal that E2F1 binds the Apaf-1 promoter specifically in tumor tissue, suggesting that E2F1 contributes to Apaf-1 expression in brain tumors. These results demonstrate an unexpected sensitivity of brain tumors to cytochrome c and raise the possibility that this phenomenon could be exploited therapeutically to selectively kill brain cancers.
Secondly, we develop a method for monitoring caspase activity in Xenopus laevis oocytes and early embryos. The approach, utilizing microinjection of a near-infrared dye
that emits fluorescence only after its cleavage by active caspases, has enabled the elucidation of subtleties in the apoptotic program. We demonstrate that brief caspase activation is sufficient to cause death. We illustrate the presence of a cytochrome c dose threshold, which is lowered by neutralization of inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that eventual death of these oocytes is caspase-independent. Imaging caspase activity in the embryo suggests that apoptosis in early development is not cell-autonomous. Finally, we believe this method presents a useful screening modality for identifying novel apoptotic regulators as well as pro-apoptotic small-molecules that could be useful in treating brain tumors.
List of Figures
List of Abbreviations
1.1.1 Apoptosis signaling pathways
1.2 Post‐mitochondrial apoptosis signaling
1.2.1 Cytochrome c: structure and function
1.2.2 Cytochrome c binding to Apaf‐1 and apoptosome formation
1.2.3 Recruitment of caspase‐9 and activation of effector caspases
1.2.4 Cell dismantling by activated effector caspases
1.3 Regulation of post‐mitochondrial apoptosis
1.3.1 Regulation of apoptosome formation—Apaf‐1 down‐regulation
1.3.2 Regulation of caspase activity by inhibitor of apoptosis proteins
1.4 Mechanisms of apoptotic evasion in cancer
1.5 Apoptosis regulation in primary brain cancers
1.5.1 Glial cell cancers
1.5.3 Sensitivity of brain cancer cells to cytochrome c‐induced apoptosis................ 35
1.7 Studying apoptosis in Xenopus laevis
1.7.1 The Xenopus egg extract system
1.7.2 Post‐mitochondrial apoptosis in Xenopus oocytes
2. Materials and methods
2.1 Cell culture and microinjection of neurons
2.2 Cell‐free lysate preparation and caspase assays
2.2.1 Preparation of cytosolic lysates (extracts)
2.2.2 Cell‐free caspase assays
220.127.116.11 Mammalian caspase assays
18.104.22.168 Xenopus caspase assays
2.3 Antibodies and immunoblotting
2.4 Real‐time RT‐PCR
2.5 Microarray data analysis
2.6 Chromatin immunoprecipitation
2.7 Cloning, protein expression and mRNA synthesis
2.8 Intermolecular crosslinking and mass spectrometry
2.8.1 Covalent crosslinking and gel analysis of crosslinked proteins
2.8.2 Sample preparation (performed by the Duke Proteomics Facility).................. 51 22.214.171.124 In‐gel digestion
126.96.36.199 In‐solution digestion
2.8.3 LC‐MS/MS (performed by the Duke Proteomics Facility)
2.8.4 MS data analysis (performed in collaboration with the Duke Proteomics Facility)
188.8.131.52 Dead‐end crosslinked products
184.108.40.206 Intrapeptide crosslinked products
220.127.116.11 Interpeptide crosslinked products
2.9 Cell imaging
2.10 Xenopus oocyte isolation, maturation and lysate preparation
2.11 Xenopus in vitro fertilization
2.12 Microinjection of Xenopus oocytes and embryos
2.13 Protein binding assays
3. Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues
3.2.1 Multiple types of neurons become resistant to cytochrome c upon maturation
3.2.2 Cytochrome c induces robust caspase activation in brain tumor cells............. 64 3.2.3 Endogenous mouse models of high‐grade astrocytoma and medulloblastoma demonstrate selective cytochrome c‐induced caspase activation in tumor tissue.... 66 3.2.4 Apaf‐1 expression levels determine the differential sensitivity to cytochrome c in normal and malignant brain tissue
3.2.5 Levels of Apaf‐1 in normal and malignant brain tissue are transcriptionally regulated
3.3.1 Low Apaf‐1 levels offer protection from cytochrome c‐dependent apoptosis in differentiated neurons and neural tissue
3.3.2 Brain tumor susceptibility to cytochrome c‐induced apoptosis
3.3.3 Apoptosome activation as a therapeutic strategy
4. The cytochrome c—Apaf-1 interaction and development of a cytochrome c mimetic 79
4.2.1 Temozolomide‐resistant glioblastoma cells remain sensitive to cytochrome c‐ mediated caspase activaiton
4.2.2 Brain tumor stem cells express Apaf‐1 mRNA
4.2.3 Cytochrome c fragments are incapable of binding Apaf‐1 or mediating caspase activation
4.2.4 Intermolecular crosslinking and mass spectrometry analysis of cytochrome c—Apaf‐1 complexes: can we use this approach to map molecular interaction surfaces?
4.2.5 Expressing active, cytosolic cytochrome c in brain cancer cells
4.2.6 Development of a small‐molecule screen to identify a cytochrome c mimetic
4.3.1 Drug‐resistant brain cancer cells can be targeted for apoptosis with cytosolic cytochrome c
4.3.2 Mapping the cytochrome c—Apaf‐1 binding sites
4.3.3 Developing a therapeutic to activate the apoptosome
5. Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring
5.2.1 A near‐infrared caspase substrate enables the detection of caspase activity in Xenopus oocytes
5.2.2 Caspases are rapidly activated and long‐acting, but are required for a limited period of time to ensure cell death
5.2.3 Inhibitor of apoptosis proteins serve as a brake to apoptosis in the oocyte.. 119 5.2.4 Meiotic oocytes develop resistance to cytochrome c, and death of oocytes arrested in meiosis is caspase‐independent
5.2.5 Apoptosis is not cell‐autonomous in the early Xenopus embryo
6. Conclusions and perspectives
6.1 Sensitivity of brain cancer cells to cytochrome c‐induced apoptosis
6.2 Selectively killing brain tumor cells with cytochrome c
6.3 Cytochrome c‐induced apoptosis in the oocyte and early embryo
6.4 The cellular response to cytosolic cytochrome c
6.5 Concluding remarks
List of Figures Figure 1.1: Morphological features of apoptosis.
Figure 1.2: Caspase activation via the intrinsic and extrinsic pathways of apoptosis....... 5 Figure 1.3: Structure of cytochrome c
Figure 1.4: Apaf‐1 and apoptosome formation
Figure 1.5: Caspase substrates and potential functions of their cleavage products......... 18 Figure 1.6: PHAPI‐mediated sensitivity to cytochrome c‐induced apoptosis in breast cancer.
Figure 1.7: Morphological features of Xenopus oocytes
Figure 3.1: Cytochrome c is incapable of activating caspases and inducing apoptosis in mature neurons
Figure 3.2: Brain cancer cells are hypersensitive to cytochrome c‐induced apoptosis..... 65 Figure 3.3: Brain tumors from mouse models of high‐grade astrocytoma and medulloblastoma display sensitivity to cytochrome c‐mediated apoptosis
Figure 3.4: A marked increase in Apaf‐1 causes the increased sensitivity of brain tumor tissues to cytochrome c
Figure3.5: Transcriptional regulation of Apaf‐1 mRNA levels contributed by E2F1.
...... 71 Figure 4.1: Drug‐resistant glioblastoma cells remain sensitive to cytochrome c.............. 84 Figure 4.2: Brain tumor stem cells express Apaf‐1 mRNA.
Figure 4.3: Critical lysine residues in cytochrome c and diagram of synthesized cytochrome c fragments.
Figure 4.4: Peptide fragments of cytochrome c neither activate nor inhibit caspase activation.
Figure 4.5: Crosslinking strategy to map the cytochrome c—Apaf‐1 binding sites......... 93
Figure 4.6: Immunblot analysis of cytochrome c—Apaf‐1 covalent crosslinking............. 94 Figure 4.7: Intramolecular crosslinking of cytochrome c.
Figure 4.8: Cytosolic expression of cytochrome c and heme lyase.
Figure 4.9: Co‐expression of cytosolic cytochrome c and heme lyase.
Figure 4.10: A small‐molecule screen to identify cytochrome c mimetics.
Figure 5.1: Fluorescence can be detected in oocytes microinjected with the IRDye and cytochrome c.
Figure 5.2: Oocyte fluorescence is due to activation of effector caspases.
Figure 5.3: Caspases are rapidly activated in response to cytochrome c; caspases remain active for hours although their activity is only required for 10 minutes to ensure apoptosis.
Figure 5.4: Inhibitor of apoptosis proteins contribute to setting the cytochrome c threshold in oocytes
Figure 5.5: Progesterone‐induced oocyte maturation decreases sensitivity to cytochrome c
Figure 5.6: Cytochrome c‐induced apoptosis in the early embryo is not cell‐autonomous.
Figure 5.7: Cytoplasmic transfer between blastomeres prevents cell‐autonomous apoptosis.
PARP Poly (ADP-ribose) polymerase PDGF Platelet-derived growth factor PHAPI Putative HLA class II associated protein I PI3K Phosphoinositide-3 kinase RT-PCR Reverse transcriptase polymerase chain reaction
TOM Transporter outer membrane TRAIL Tumor necrosis factor (TNF)-related apoptosis-inducing ligand TR Temozolomide-resistant