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«Date:_ Approved: _ Sally Kornbluth, Supervisor _ Donald McDonnell _ Robert Wechsler-Reya _ Gerard Blobe _ Salvatore Pizzo Dissertation submitted in ...»

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Understanding the Cellular Response to Cytosolic Cytochrome c

by

Carrie Elizabeth Johnson

Department of Pharmacology and Cancer Biology

Duke University

Date:_______________________

Approved:

___________________________

Sally Kornbluth, Supervisor

___________________________

Donald McDonnell

___________________________

Robert Wechsler-Reya

___________________________

Gerard Blobe

___________________________

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    

ABSTRACT

Understanding the Cellular Response to Cytosolic Cytochrome c by Carrie Elizabeth Johnson Department of Pharmacology and Cancer Biology Duke University Date:_______________________

Approved:

___________________________

Sally Kornbluth, Supervisor ___________________________

Donald McDonnell ___________________________

Robert Wechsler-Reya ___________________________

Gerard Blobe ___________________________

Salvatore Pizzo An

Abstract

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.

–  –  –

Contents Abstract

List of Figures

List of Abbreviations

Acknowledgements

1. Introduction

1.1  Apoptosis

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

1.5.2  Medulloblastomas

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

2.2.2.1  Mammalian caspase assays

2.2.2.2  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 2.8.2.1  In‐gel digestion

2.8.2.2  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)

2.8.4.1  Dead‐end crosslinked products

2.8.4.2  Intrapeptide crosslinked products

2.8.4.3  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

3.1  Introduction

3.2  Results

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  Discussion

–  –  –

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

4.1  Introduction

4.2  Results

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  Discussion

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  Results

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

5.3  Discussion

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

References

Biography

–  –  –

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

–  –  –



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