The functional role(s) of dual intermediate filament expression in tumor cell migration and invasion. Item Type text; Dissertation-Reproduction (electronic) Authors Chu, Yi-Wen. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 02/10/2021 04:10:29 Link to Item http://hdl.handle.net/10150/186143 INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. 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Contact UMI directly to order. U·M·I University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. M148106-1346 USA 313/761-4700 800:521-0600 Order Number 9322644 The functional role(s) of dual intermediate filament expression in tumor cell migration and invasion Chu, Yi-Wen, Ph.D. The University of Arizona, 1993 V·M·I 300 N. Zccb Rd. Ann Arbor. MI 48106 THE FUNCTIONAL ROLE(S) OF DUAL INTERlVIEDIA TE FILAlv1ENT EXPRESSION IN TUMOR CELL rvnGRA TION AND INVASION by Yi-Wen Chu A Dissertation Submitted to the Faculty of the COMMITTEE ON CANCER BIOLOGY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 199 3 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared by______________________________________ Yi-Wen Chu __ entitled THE FUNCTIONAL ROLE (S) OF DUAL INTERMEDIATE FILA."'1ENT EXPRESSION IN TUMOR CELL HIGRATION AND INVASION and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of _D~o~c~t~o~r_o~f_P~hu.i~lo~s~o~p~h~y~ _____________________ y I r/1.3 DatJ ' tf /q/9'3 Date 1 Cj /~iS Bernstein <___ / ~ ~ ~~ Date' Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: f- ~ ~ 4 ACKNOWLEDGMENTS At this moment, the last stage of my graduate study, I feel grateful to many of the wonderful people I have met in the past several years. First, I would like to express my deepest appreciation to my major advisor, Dr. Mary J.C. Hendrix. She has always shown great confidence in me, and provided support during the most difficult times of my life. She not only provided the guidance for my dissertation research, but also introduced me to many prestigious scientists in this field and gave me the opportunity to collaborate with people. She has spent a tremendous amount of time helping me with my writing and communication skills. With her pleasant personality, she makes me feel there are always alternatives and hopes even at the most frustrating moments. Most importantly, as a woman scientist in the modern world, she demonstrates that with intelligence, enthusiasm and endeavor, we can be what we want to be. She is definitively a world model for me in my future career. To my committee members, I would like to thank them for their valuable comments and suggestions for this work. I am very grateful to Dr. John J. Duffy for the solid training he gave me in Molecular Biology. I would also like to express my deepest gratitude to Dr. Richard B.B. Seftor and Elisabeth Seftor for their expertise and scientific discussions, and their constant encouragement and personal support through my Ph.D. career. I am very lucky to have colleagues like Catherine Frye, Adrian Winter, Hanifa Jones and Padma Sundareshan. I thank them for their help and warm friendship that made everyday of research life filled with joy and laughter. I also would like to thank the Iowa Motility Center staff and faculty, including Drs. David SoIl, Ray Runyan, Karla Daniels, and Michael Solursh, for their help in the analysis of cellular motility and their assistance with confocal microscopy. 5 DEDICATED TO MY PARENTS SHUN CHU AND SHU·CHENG CHENG CHU 6 TABLE OF CONTENTS LIST OF ILLUSTRATIONS ........................................................................ 8 LIST OF TABLE ........................................................................................... 9 ABSTRACT .................................................................................................... 10 I. INTRODUCTION AND OVERVIEW OF LITERATURE ..................... 12 1.l Mechanisms of Tumor Cell Invasion and Metastasis ......................................................................................... 12 l.2 Cytoskeletal Proteins and Neoplasia ......................................... 18 1.3 Keratins and Tumor Transformation .......................................... 24 1.4 Coexpression of IFs in Metastatic Tumor Cells ..... ...... ..... .... 28 l.5 Hypothesis: Dual IF Expression in Highly Metastatic Cells ........ ........ ............... .......... ... ..... ......... ................ .... 30 1.6 Dissertation Format ........................................................................ 32 ll. PRESENT STUDY ........................................................................................ 34 2.1 Disruption of Keratin Filaments in Highly Metastatic Cells ... ..... ........ ............ .... ..... ... .... ..... ............... .......... .... 34 2.2 Mouse L Cell Model: Effects of Dual IF Expression ......... 43 2.3 Transfections of Keratins in Low Metastatic Melanoma Cells ................................................................ .............. 51 2.4 Conclusion ....................................................................................... 57 Ill. APPENDIX A: Transfection of a Deleted CK18 cDNA into a Highly Metastatic Melanoma Cell Line Decreases the Invasive Potential...... .... ... ........... ...... ................... ..... .... ........... ....................................... 63 N. APPENDIX B: Coexpression of Vimentin and Keratins by Human Melanoma Tumor Cells: Correlation with Invasive and Metastatic Potential ....................................................................................... 74 7 TABLE OF CONTENTS - Continued V. APPENDIX C: Expression of Complete Keratin Filaments in Mouse L Cells Augments Cell Migration and Invasion................. ........ ....... ....... 88 VI APPENDIX D: Co expression of Vimentin and Keratins Intermediate Filaments in Human Melanoma Cells Correlates with Increased Motility and Decreased Integrin Localization in Focal Contacts ......................................................................................... 127 VII. REFERENCES ............................................................................................... 149 8 LIST OF ILLUSTRATIONS FIGURE 1, Hypothesis: Dual IF Expression and Cellular Response..... 31 FIGURE 2, Plasmids Used in the Transfection Study ................................ 36 FIGURE 3, Confocal Microscopy of CI070 Cells ...................................... 39 FIGURE 4, Attachment Ability of L Cells on Matrigel...... .......... ... .... ...... 46 FIGURE 5, Fluorescence Localization of F-actin in L Cells. .... ............... 48 FIGURE 6, Plasmids Containing Keratins 8 and 18 cDNAs .................... 52 FIGURE 7, DNA Slot Blot Analysis of Transfected A375P Cells .......... 55 FIGURE 8, Model for Dual IF Expression Augmenting