LH14E’S EFFECTS ARE MEDIATED THROUGH P21 AND RELATED PROTEINS ____________________________________ A Thesis Presented to the Faculty of California State University, Fullerton ____________________________________ In Partial Fulfillment of the Requirements for the Degree Master of Science in Biology ____________________________________ By Sarah Daoudi Thesis Committee Approval: Nilay V. Patel, Department of Biological Science, Chair Merri-Lynn Casem, Department of Biological Science Catherine Brennan, Department of Biological Science Summer, 2018 ABSTRACT A small library of compounds synthesized at California State University, Fullerton, were screened for their anti-proliferative activity in three human cell lines: HeLa, HUTU80, and MG63. CyQUANT cell proliferation assay also showed some cell line specificity of best performing compounds. This project focuses on identification of the effects of the compound, LH14E, on p21, a cell cycle regulatory protein. Expression of CDKN1A (p21 gene) is regulated by p53 and KLF6, both of which attenuate cell proliferation. Our results show that LH14E increases expression of CDKN1A and the amount of p21 protein. Luciferase reporter gene assay revealed an increase in the transcriptional activity of the p21 promoter upon treatment with LH14E. Western Blot reveals p53 protein levels are not affected by treatment with LH14E, but KLF6 protein increases upon treatment with LH14E. These results suggest that LH14E activates KLF6 expression, and subsequently increases p21 expression, which together may cause cell cycle arrest. These analyses will help identify mechanism of action for the hit compounds in this library of compounds. ii TABLE OF CONTENTS ABSTRACT ................................................................................................................... ii LIST OF TABLES ......................................................................................................... v LIST OF FIGURES ....................................................................................................... vi ACKNOWLEDGMENTS ............................................................................................. vii Chapter 1. INTRODUCTION ................................................................................................ 1 The Cell Cycle ...................................................................................................... 1 The Role of p21 in the Cell Cycle ........................................................................ 3 The Role of p53 in the Cell Cycle ........................................................................ 4 Downstream Targets of p53 .................................................................................. 5 The Interactions Between p21 and p53 ................................................................. 9 Role of KLF6 in Cell Cycle .................................................................................. 9 The Compounds and Cancer ................................................................................. 10 Niclosamide as a Control ...................................................................................... 11 Camptothecin as a Control .................................................................................... 12 Research Design and Project Aim ........................................................................ 13 2. METHODS ........................................................................................................... 14 Cell Culture and Drug Treatment ......................................................................... 14 Compounds ........................................................................................................... 15 CyQUANT Cell Proliferation Assay .................................................................... 17 MitoTracker Staining ............................................................................................ 17 Immunocytochemistry .......................................................................................... 17 RIPA Protein Isolation and BCA Protein Assay .................................................. 18 Western Blot ......................................................................................................... 18 NEON Transfection .............................................................................................. 21 Luciferase Reporter Assay .................................................................................... 21 RNA Isolation and cDNA Synthesis .................................................................... 22 Quantitative Polymerase Chain Reaction (qPCR) and Primer Design ................. 22 iii 3. RESULTS ............................................................................................................. 24 Effects Under Investigation .................................................................................. 24 Effect on Cellular Proliferation............................................................................. 25 Effects on Mitochondrial Membrane .................................................................... 25 Effect on Chromatin Condensation and Mitotic Phase of the Cell Cycle ............ 26 qPCR Analysis of Effects on p53 Target Gene Expression.................................. 30 qPCR Analysis of Effects on p21 Related Gene Expression ................................ 34 Effect on CDKN1A Transcription ......................................................................... 36 Effect on p53 Transcription Activity .................................................................... 38 Effects on Protein Levels using Western Blot Analysis ....................................... 40 Effect on Protein Localization .............................................................................. 44 4. DISCUSSION ....................................................................................................... 46 The Cell Cycle and LH14E ................................................................................... 46 LH14E Reduces Cell Proliferation through Interactions with the Cell Cycle ...... 46 Comparison of LH1E with Niclosamide and Camptothecin ................................ 51 Proposed Mechanism of LH14E ........................................................................... 51 Drug Discovery and LH14E ................................................................................. 53 APPENDICES ............................................................................................................... 54 A. SUPPLEMENTARY CYQUANT FIGURE ................................................. 54 B. SUPPLEMENTARY CYQUANT ASSAY DATA TABLES ...................... 55 REFERENCES .............................................................................................................. 63 iv LIST OF TABLES Table Page 1. Compounds Used ................................................................................................. 18 2. Antibodies Used ................................................................................................... 20 3. qPCR Primers Used ............................................................................................. 23 4. Summary of Results ............................................................................................. 50 v LIST OF FIGURES Figure Page 1. p21 inhibition in varying phases of the cell cycle ................................................ 6 2. Activation of p53 under stress conditions ............................................................ 8 3. Chemical structure of Niclosamide ...................................................................... 12 4. Compounds decrease cell proliferation ................................................................ 27 5. LH14E reduces mitochondrial activity in HUTU80 cells but not HeLa and MG63 cells ........................................................................................................... 28 6. Niclosamide and JF19 reduce phosphor-H3 pSer10 levels relative to DMSO ... 29 7. mRNA expression is impacted for cell-cycle related genes ................................ 33 8. mRNA expression is impacted for p21 related genes .......................................... 35 9. LH14E and Niclosamide increase transcription activity of CDKN1A after treatment .............................................................................................................. 37 10. LH14E increase transcription activity of p53 after treatment .............................. 39 11. p21 protein levels upon treatment with compounds and Niclosamide ................ 41 12. p53 protein levels upon treatment with compounds and Niclosamide ................ 42 13. KLF6 protein levels upon treatment with compounds and Niclosamide ............. 43 14. Camptothecin, LH14E, Niclosamide, JF19, MEY26, KN19 and AV9 increase p21 levels relative to DMSO ............................................................................... 45 15. Proposed mechanism of LH14E .......................................................................... 52 vi ACKNOWLEDGMENTS I would like to especially acknowledge my thesis adviser, Dr. Nilay Patel, for his guidance and patience as well as my other committee members Dr. Casem and Dr. Brennan. I would like to extend my gratitude to Jocelyn Leon, Gustavo Chacon, Lauren Adkins, and Stephanie Youn for their aid with data collection. In addition, I would like to thank