University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 5-2007 Characterization and Comparative Analysis of Gene Expression Profiles of Three Different Human Oral Squamous Carcinoma Cell Lines With Different Invasion and Metastatic Capacities Necip Fazil Erdem University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Medicine and Health Sciences Commons Recommended Citation Erdem, Necip Fazil, "Characterization and Comparative Analysis of Gene Expression Profiles of Three Different Human Oral Squamous Carcinoma Cell Lines With Different Invasion and Metastatic Capacities. " PhD diss., University of Tennessee, 2007. https://trace.tennessee.edu/utk_graddiss/162 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 Necip Fazil Erdem entitled "Characterization and Comparative Analysis of Gene Expression Profiles of Three Different Human Oral Squamous Carcinoma Cell Lines With Different Invasion and Metastatic Capacities." 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 Comparative and Experimental Medicine. David A. Gerard, Major Professor We have read this dissertation and recommend its acceptance: Eric R. Carlson, Jack E. Gotcher, Darryl L. Millis 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 Necip Fazil Erdem entitled “Characterization and comparative analysis of gene expression profiles of three different human oral squamous carcinoma cell lines with different invasion and metastatic capacities”. 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 Comparative and Experimental Medicine David A. Gerard, PhD _ __ _ Major Professor We have read this dissertation and recommend its acceptance: Eric R. Carlson, DMD, MD _ _ Jack E. Gotcher, DMD, PhD _ _ Darryl L. Millis, DVM, MS _ _ Accepted for the Council: _Carolyn Hodges _ Dean of the Graduate School (Original signatures are on file with official student records) Characterization and comparative analysis of gene expression profiles of three different human oral squamous carcinoma cell lines with different invasion and metastatic capacities A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Necip Fazil Erdem MAY, 2007 Acknowledgements I wish to express sincere appreciation to Dr. David Gerard, my major professor, for his valuable and patient guidance and encouragement throughout the planning and accomplishment of this study. Apart from the focus he has given me in my research, I am grateful for his involvement in my studies. My appreciation is also extended to my committee members, Dr. Eric R Carlson, Dr. Jack Gotcher, and Dr. Darryl L Millis for their guidance and positive suggestions they provided. I also would like to thank Dr. Albert Ichiki and Lucy Simpson, Department of Medical Genetics, for their help obtaining the laboratory equipment, Dr. John Wall, Department of Human Immunology and Cancer Program, for assistance with the PET/CT image analysis. Thanks are also extended to Joshua Yuan, Director of Genome Laboratory for his help in cDNA microarray analysis. I am grateful to my wife Ipek Erdem for her support and patience and also my daughter Gamze Nur for her love. I am deeply grateful to my parents and my relatives in Turkey for their love, support, ever-constant encouragement, and understanding. ii Abstract Matrix metalloproteinases (MMP’s) and cathepsins are thought to play key roles in oral squamous cell carcinoma (OSCC) invasion and metastasis. Three OSCC human cell lines, BHY, HN and HSC-3, have been studied based on their reported ability to invade adjacent bone or metastasize to lymph nodes and/or distant organs. The working hypothesis of this study is that OSCC that demonstrate different actions have different proteolytic enzyme and gene expression profiles. Immunocytochemistry and flow cytometry were used to determine the expression levels of certain proteolytic enzymes. Complementary DNA (cDNA) microarray technique was used to determine the gene expression profiles of each of the three OSCC cell lines. The immunocytochemistry and flow cytometry results showed that the BHY cell line expresses MMP- 9 and extracellular matrix metalloproteinase inducer (EMMPRIN). The BHY cell line expressed 139 upregulated genes and 117 downregulated genes. The HN cell line expressed 214 upregulated and 262 downregulated genes. Finally, the HSC- 3 cell line expressed 128 upregulated and 117 downregulated genes. There were 13 genes that were upregulated and 83 genes that were downregulated in all three OSCC cell lines. Eight pathways were upregulated and 3 pathways were downregulated in the BHY cell line. In the HN cell line 10 pathways were upregulated and 9 were downregulated. Finally, in the HSC-3 cell line 3 pathways were upregulated and 6 pathways were downregulated. iii MMP-1, MMP-9 and EMMPRIN appear to play a role in the invasion of OSCC to bone. KIAA, was upregulated in our study in the HN cell line. Upregulation of KIAA may be involved in the late stage of OSSC. PAM gene was the only downregulated gene in the HN cell line. The TM4SF10 gene was downregulated in all three OSCC cell lines. The COL1A2 and COL4A1 genes were upregulated in the HSC-3 cell line and they may cause the lymph node metastasis of OSCC. Based on this study OSCC involves multiple molecular genetic events that take place in many chromosomes and genes. Upregulation of the Jak-STAT signaling pathway may be involved in bone invasion of OSCC. The MAPK signaling pathway was upregulated in all three OSCC cell lines. iv Table of Contents Chapter Page I. Introduction 15 II. Literature Review 19 2.1. Oral squamous cell carcinoma 19 2.2. BHY, HN, HSC-3 and HNEC cell lines 21 2.2.1. BHY cell line 21 2.2.2. HN cell line 21 2.2.3. HSC-3 cell line 22 2.2.4. HNEC cell line 22 2.3. Invasion and metastasis of OSCC 22 2.3.1. The effect of MMPs and EMMPRIN on invasion 25 and metastasis of OSCC 2.3.2. The effect of cathepsins on invasion and 32 metastasis of OSCC 2.4. Microarray studies on OSCC 34 III. Material and Methods 46 3.1. Cell lines and cultures 46 3.1.1. HNEC cell line 46 3.1.2. BHY cell lines 47 3.1.3. HN cell line 48 3.1.4. HSC-3 cell line 48 v Chapter Page 3.2. The invasion and/or metastatic characteristics 49 of OSCC cell lines 3.3. Immunocytochemistry 50 3.4. Flow cytometry 52 3.5. Ultrastructural characterization of three OSCC 53 cell lines 3.6. Total RNA extraction from the cell lines 54 3.7. Checking the quality and quantity of total RNA 55 3.7.1. Synthesis of cDNA from total RNA 55 3.7.2. PCR reaction 56 3.8. Synthesis of mRNA from total RNA 56 3.9. cDNA microarray analysis 57 3.10. Real Time-PCR analysis 59 IV. Results 62 4.1. The invasion and/or metastatic characteristics 62 of OSCC cell lines 4.2. Immunocytochemistry 63 4.3. Flow cytometry 63 4.4. Ultrastructural characterization of three 64 OSCC cell lines 4.5. The quality and quantity of total RNA 64 4.6. cDNA microarray analysis of three OSCC cell lines 65 vi Chapter Page 4.7. Real Time PCR analysis 67 V. Conclusions & Recommendations 69 5.1. The invasion and/or metastasis characteristics of 69 OSCC cell lines 5.2. Characterization of the oral squamous cell 70 carcinoma cells 5.3. cDNA microarray analysis of the three OSCC 73 cell lines and comparison of the results with Real Time PCR analysis 5.3.1. The quality and quantity of total RNA 73 5.3.2. cDNA microarray analysis of three OSCC cell lines 74 5.3.3. Pathway analysis of three OSCC cell lines based 89 on cDNA microarray results 5.3.3.1 BHY cell line 90 5.3.3.2 HN cell line 95 5.3.3.3 HSC-3 cell line 103 5.3.4. Real Time PCR analysis 105 5.4. Recommendations 105 Appendix A Tables 108 Appendix B Figures 156 References 197 Vita 226 vii List of Tables Table Page Table 1: Normal and pathological processes involving MMPs 109 Table 2: Common highly expressed genes in normal oral squamous epithelium 110 Table 3: Common highly expressed genes in OSCC 111 Table 4:16 genes that are highly upregulated in tongue SCC cases 112 Table 5: Upregulated and downregulated gene list of 9 OSCC patients 113 Table 6: Common upregulated genes in head and neck SCC 114 Table 7: Common down regulated genes in head and neck SCC 115 Table 8: List of genes with conflicting expression data in head and neck SCC 116 Table 9: Ontology of altered genes in head and neck carcinoma 117 Table 10: List of proteins with increased and decreased expressions in OSCC 118 Table 11: The expression levels of proteolytic enzymes by using 118 immunocytochemistry. Table 12: The expression levels of proteolytic enzymes in three different 119 OSCC cell lines by using flow cytometry Table 13: 139 upregulated genes in BHY cell line.