Differential Expression of Skin Cancer and Hair-Follicle Cycle Regulated Genes in Tumor Susceptible K14-Agouti Mice
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University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2006 Differential Expression of Skin Cancer and Hair-Follicle Cycle Regulated Genes in Tumor Susceptible K14-Agouti Mice Yesim Aydin Son University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Life Sciences Commons Recommended Citation Son, Yesim Aydin, "Differential Expression of Skin Cancer and Hair-Follicle Cycle Regulated Genes in Tumor Susceptible K14-Agouti Mice. " PhD diss., University of Tennessee, 2006. https://trace.tennessee.edu/utk_graddiss/1636 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Yesim Aydin Son entitled "Differential Expression of Skin Cancer and Hair-Follicle Cycle Regulated Genes in Tumor Susceptible K14-Agouti Mice." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Life Sciences. Edward J. Michaud, Major Professor We have read this dissertation and recommend its acceptance: Cymbeline T. Culiat, Mitchel J. Doktycz, Mary Ann Handel, Jay R. Snoddy Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Yesim Aydin Son entitled “Differential Expression of Skin Cancer and Hair-Follicle Cycle Regulated Genes in Tumor Susceptible K14-Agouti Mice.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Life Sciences. Edward J. Michaud _________________________________ Major Professor We have read this dissertation and recommend its acceptance: Cymbeline T. Culiat _________________________ Mitchel J. Doktycz _________________________ Mary Ann Handel _________________________ Jay R. Snoddy _________________________ Accepted for the Council: Anne Mayhew ______________________________ Vice Chancellor and Dean of Graduate Studies (Original signatures are on file with official student records.) DIFFERENTIAL EXPRESSION OF SKIN CANCER AND HAIR-FOLLICLE CYCLE REGULATED GENES IN TUMOR SUSCEPTIBLE K14-AGOUTI MICE A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Yeşim AYDIN SON August, 2006 Copyright © 2006 by Yeşim AYDIN SON All rights reserved. ii DEDICATION This dissertation is dedicated to my family, my father, DR. YUSUF AYDIN, and my husband, DR. ÇAĞDAŞ D. SON, for their endless love and support. iii ACKNOWLEDGMENTS Foremost, I would like to acknowledge my advisor Dr. Edward J. Michaud. Besides introducing me to the world of mouse genetics, I am deeply thankful to him for his guidance, encouragement, positive attitude, always providing constructive criticism, and all the time and energy he put into my project. As members of my doctoral committee I would like to thank to Dr. Cymbeline T. Culiat, Dr. Mitchel J. Doktycz, Dr. Mary Ann Handel, and Dr. Jay R. Snoddy for their invaluable input, advice and support. Also I would like to thank to Dr. Jeffrey M. Becker, former director of Graduate School of Genome Science and Technology Program, for his help, support, and advice on countless occasions. I would like to thank to Dr. Brian O’Nuallain, Dr. Brynn J. Voy, Dr. Peter R. Hoyt, Dr. Erich Baker, and Dr. Roz Miltenberger for their insights, and feedback during my rotations, and research, and all the members of the “Oak Ridge Mouse House” for their technical assistance. My lab partners and friends Sujata Agarwal, Ann Wymore, and Carmen Foster, many thanks to you, for your help and company, which you never hesitated to share, and for your support all through this journey. Also, I would like to thank Michael J. Miller, my former student, for his excellent assistance with the real time qRT-PCR experiments. It was a great pleasure working with you all. Additionally I would like to thank to all my friends in Knoxville and in other cities, for always being there for us. My baby boy Adal, thank you for bringing all the joy to our family, and filling our hearts with love. Last but not least, I would like to thank all my family, and friends in Turkey. Even though you were so far away from us thank you for all your efforts to make us feel that we are always close to home. iv ABSTRACT The mouse agouti protein is transiently expressed in the skin and signals through the melanocortin 1 receptor to switch pigment production of hair-follicle melanocytes from black to yellow. Ubiquitous over-expression of agouti protein in mice carrying the spontaneous dominant mutations Ay and Avy causes a pleiotropic syndrome characterized by solid yellow hair color, obesity, diabetes, and increased susceptibility to carcinogenesis in a wide variety of tissues, including the skin. Over-expression of agouti in the skin of keratin 14 (K14)- Agouti transgenic mice promotes skin carcinogenesis, even in the absence of obesity and diabetes. In this study cDNA microarray and qRT-PCR analyses are used to identify molecular changes in the skin of K14-Agouti mice associated with the promotion stage of carcinogenesis. Histological analysis of the skin revealed that there were no differences in gross morphology or in timing of hair- follicle stages between transgenic and control mice. However, cDNA microarray analysis identified 181 genes with significantly altered expression levels in the skin of transgenic mice. Additionally, qRT-PCR analysis demonstrated that the levels and temporal patterns of expression of 10 genes previously associated with skin and/or other epithelial cancers were significantly altered in the skin of K14- Agouti transgenic mice. Agouti-induced over-expression of these proto- oncogenes in the skin of K14-Agouti mice is proposed to be associated with the increased susceptibility to skin carcinogenesis. A hypothetical model is presented to explain the mechanism of action of the agouti protein as a tumor promoter in skin carcinogenesis. Additionally, strategies for future follow-on experiments to further investigate the role of agouti in tumor promotion and to test aspects of the proposed hypothetical model are discussed. v TABLE OF CONTENTS Chapter Page I. BACKGROUND AND SIGNIFICANCE ………………………………………...1 The Mouse Nonagouti Locus ……………………………………………………….2 Molecular Characterization of the Agouti Gene …………………………….4 Agouti Gene Structure …………………………………………………4 Agouti Protein Structure and Action …………………………………7 Agouti Mutants ………………………………………………………………..11 Dominant Regulatory Mutations of Agouti ………………………...12 Regulation of Pigmentation by Agouti Signaling …………………………..14 Molecular Mechanisms of Obesity and Diabetes in Yellow Mice ………..15 Agouti Signaling in Tumor Promotion …………………………………………...18 Two-Stage Model of Skin Carcinogenesis …………………………………..18 Tumor Susceptibility in Mouse Skin ………………………………………...22 Hair-Follicle Cycle and Tumor Susceptibility ………………………………23 Agouti is a Tumor Promoter …………………………………………………29 II. DIFFERENTIAL EXPRESSION OF SKIN CANCER AND HAIR-FOLLICLE CYCLE REGULATED GENES IN TUMOR SUSCEPTIBLE K14-AGOUTI MICE ……………………………………………………………………………………38 Introduction …………………………………………………………………………39 Material and Methods………………………………………………………………..41 Mice ……………………………………………………………………………..41 RNA Isolation and cDNA Preparation ……………………………………...41 Microarray Analysis …………………………………………………………..42 Statistical Analysis ……………………………………………………………..42 Real-Time qRT-PCR …………………………………………………………43 Synchronization of Hair-Follicle Cycle ……………………………………...43 Histology ……………………………………………………………………….44 Results ……………………………………………………………………………….44 Microarray Analysis of Gene Expression in the Skin of Adult K14-Agouti and Control Mice ……………………………………………….44 qRT-PCR Analysis of the Temporal Expression Profiles of 10 Genes in the Skin of K14-Agouti and Control Mice ……………………….45 Examining Gene Expression Profiles in K14-Agouti Skin at Precisely Determined Stages of the Hair-Follicle Cycle …………………...56 Discussion …………………………………………………………………………...67 vi III. CONCLUSIONS AND FUTURE PERSPECTIVES ………………………….74 Model Signal Transduction Pathways for Agouti-Induced Cellular Proliferation …………………………………………………………………………76 Signal Transducer and Activator of Transcription (STAT) Pathway …….77 Mitogen-Activated Protein Kinase (MAPK) Pathway …………………….82 Protein Kinase C (PKC) Pathway …………………………………………….83 Regulation of Gene Expression through Protein Kinase C ………………..87 Future Perspectives …………………………………………………………………89 LIST OF REFERENCES ………………………………………………………………98 APPENDIX …………………………………………………………………………...116 VITA………………………………………………………………………………...…123 vii LIST OF TABLES Table Page 1. List of 40 genes with known biological functions that are significantly up-regulated in the skin of K14-Agouti mice ………………..47 2. List of 29 genes with known biological functions that are significantly down-regulated in the skin of K14-Agouti mice …………...48 3. Summary of known functions of 10 genes with altered temporal expression