Characterization of cell death in Casp3-deficient mouse embryonic fibroblasts Open Access

Abstract

Caspases are required for the successful implementation of apoptosis. Nonetheless, when apoptosis is not possible, signals that normally trigger apoptosis are still capable of inducing cell death. Caspase-3 is an executioner caspase that is activated downstream of mitochondrial events in intrinsic apoptosis. However, previous work demonstrates that Casp3-deficient mouse embryonic fibroblasts (MEFs) are resistant to mitochondrial-mediated apoptosis and display a delay in the mitochondrial events of apoptosis, including Bax activation, mitochondrial outer membrane permeabilization (MOMP), and cytochrome c release. In the following chapters, we detail work demonstrating that caspase-3 regulates fibronectin secretion, and through this function influences cell morphology, adhesion, migration and the apoptotic threshold of the cell. Furthermore, we show that in the absence of fibronectin, serum withdrawal-induced death occurs by a caspase-independent mechanism. We go on to show that death in this circumstance is autophagy-dependent, and that ablating the cells ability to undergo autophagy results in a blockade of cell death. Taken together our data indicate that Casp3-deficient MEFs are incapable of executing apoptosis triggered by serum withdrawal and are protected from autophagy-dependent death by fibronectin-mediated adhesion. The implications of our research to the cell death field, and consequently diseases involving aberrant cell death, such as cancer, may prove significant. Our work is the first to link autophagy-dependent death to fibronectin adhesion.

Table of Contents

TABLE OF CONTENTS

I. INTRODUCTION ............................................................................................................................ 1

           A. Cell Death ........................................................................................................................... 1

           B. Intrinsic Apoptosis .............................................................................................................. 2

           C. Caspases .............................................................................................................................. 6

           D. Caspase-3............................................................................................................................. 8

           E. Caspase-3 in Cancer ............................................................................................................ 9

           F. Autophagy ........................................................................................................................... 9

           G. Autophagy-dependent Death ............................................................................................. 12

           H. Apoptosis and Autophagy ................................................................................................. 13

           I. Statement of Problem .......................................................................................................... 14

 

II. Procaspase‑3 regulates fibronectin secretion and influences adhesion, migration and survival independent of catalytic function ..................................................................................................... 16 

           A. Summary............................................................................................................................ 17

B. Introduction ....................................................................................................................... 18 

           C. Results ............................................................................................................................... 19

           D. Discussion ......................................................................................................................... 25

E. Materials and Methods ...................................................................................................... 27

F. Acknowledgements ........................................................................................................... 30

G. Figures............................................................................................................................... 32

 

III. Casp3-deficient fibroblasts require fibronectin for protection against autophagy-dependent death.................................................................................................................................................... 46

           A. Abstract ............................................................................................................................. 47

           B. Introduction ....................................................................................................................... 48

           C. Results ............................................................................................................................... 49

           D. Discussion ......................................................................................................................... 57

           E. Materials and Methods ...................................................................................................... 63

           F. Acknowledgements ........................................................................................................... 68

           G. Figures............................................................................................................................... 70

 

IV. DISCUSSION .............................................................................................................................. 87

           A. Implications from studies ................................................................................................. 87

           B. Opportunities for further research .................................................................................... 90 

 

V. REFERENCES ............................................................................................................................. 92

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Cell
Keyword	caspase-3 autophagy-dependent cell death fibronectin autophagy
Committee Chair / Thesis Advisor	Lawrence H. Boise, Emory University
Committee Members	Adam Marcus, Emory University Jing Chen, University of Chicago Shoichiro Ono, Emory University Andrew Kowalczyk, Penn State University

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