Cx43 Reduces Melanoma Growth Within a Keratinocyte Microenvironment and During Tumorigenesis in vivo

.............................................................................................................................. ii Keywords ........................................................................................................................... iii Co-Authorship Statement .................................................................................................. iiv Acknowledgments ............................................................................................................... v Table of Contents ............................................................................................................... vi List of Figures ................................................................................................................... iix List of Abbreviations ......................................................................................................... ix Chapter 1 ............................................................................................................................. 1 1 Literature Review ........................................................................................................... 1 1.1 Connexins and Gap Junctions .............................................................................. 1 1.2 Gap Junctional Intercellular Communication ...................................................... 4 1.3 GJIC-Independent Roles for Connexins .............................................................. 5 1.4 Cells of the Epidermis ......................................................................................... 6 1.5 Connexins Expressed within the Epidermis ........................................................ 9 1.6 Cancers Derived from the Epidermis ................................................................ 10 1.7 Melanoma as a Disease ...................................................................................... 11 1.8 Melanocyte Transformation and Melanoma Progression .................................. 12 1.9 Current Therapeutic Targets for Melanoma/Personalized Medicine ................ 13 1.10 Connexins in Cancer .......................................................................................... 14 1.11 Connexins in Human and Mouse Melanomas ................................................... 15 1.12 Assessing Melanomas in Complex Environments ............................................ 18 1.13 Hypothesis ......................................................................................................... 19 1.14 Objectives .......................................................................................................... 19 1.15 References ......................................................................................................... 21 vii Chapter 2 ........................................................................................................................... 30 2 Connexin43 reduces melanoma cell growth in a keratinocyte microenvironment and during tumorigenesis in vivo ........................................................................................ 31 2.1 Introduction ........................................................................................................... 32 2.2 Materials and Methods .......................................................................................... 34 2.2.1 Cell culture ................................................................................................ 34 2.2.2 Generation of melanoma cell lines ............................................................ 34 2.2.3 Immunocytochemistry, confocal microscopy and western blotting .......... 35 2.2.4 Microinjection and dye transfer ................................................................. 36 2.2.5 Growth curves and proliferation ................................................................ 36 2.2.6 Anchorage-independent growth ................................................................ 37 2.2.7 Migration scratch assay ............................................................................. 37 2.2.8 Co-culture melanoma cell growth ............................................................. 37 2.2.9 Co-culture microinjections and dye transfer ............................................. 38 2.2.10 In vivo chick chorioallantoic membrane (CAM) assay .............................. 38 2.2.11 Statistics .................................................................................................... 39 2.3 Results ................................................................................................................... 39 2.3.1 Ectopic expression of Cx26 and Cx43 increases gap junction plaques and GJIC in melanoma cells ........................................................................................ 39 2.3.2 Cx43 expression in melanoma cells significantly reduces proliferation ... 42 2.3.3 Cx43-expressing melanoma cells display a reduced malignant phenotype .............................................................................................................................. .42 2.3.4 Cx43 expression reduces melanoma cell growth in an in situ-like microenvironment independent of heterocellular GJIC with keratinocytes ......... 47 2.3.5 Cx43-expressing melanoma cells display reduced primary tumor size in vivo ........................................................................................................................ 54 2.4 Discussion ............................................................................................................. 54 2.5 References ............................................................................................................. 62