Changing gap junctional intercellular communication in mouse epidermal cells during tumorigenesis : a study on underlying processes

Gap junctional intercellular communication (GJIQ plays an important role in the differentiation and growth of cells. Increasing evidence also suggests a role for inhibition of GJIC in the promotion phase of tumor formation. How the level of GJIC is regulated in normal cells, and if this regulation is changed during the process of tumor formation is however not clearly known yet. In the chapters 3 and 4 the studies on the regulation of GJIC by (extracellular and intracellular) Ca 2+ - and cAMP-dependent processes are described for a cell line consisting of initiated cells (3PC), and a carcinoma derived cell line (CA3/7). From these studies it can be concluded that differences exists between the regulation of GJIC in cells representing different stages in the process of tumor formation, and that the short-term regulation of GJIC by intracellular Ca 2+ (Ca 2+ i ) or by cAMP are different routes of regulation. Furthermore it can be concluded that the presence of cell adhesion molecule E-cadherin on the plasma membrane is a prerequested for a high GJIC level, but that E-cadherin is not involved in the short term regulation of GJIC by Ca 2+ i or cAMP. The observed differences between 3PC cells and CA3/7 cells in the regulation of GJIC by intracellular signals suggest that during the process of tumor formation changes have occured in the Ca 2+ i - or calmodulin-dependent regulation of GJIC. These differences may be the result of differences in intracellular concentrations of regulating molecules (such as Ca 2+ i or CaM) or differences in the sensitivity of enzymes which are dependent of Ca 2+ i or CaM. These differences could result in a blocked intercellular communication between cell types representing different stages of tumor formation. For instance, if normal cells with a high GJIC level would come in contact with preneoplastic cells with different intracellular concentrations of calcium, a diffusion of calcium would appear at the moment intercellular gap junctions become functional between the two cell types. This diffusion of calcium will then lead to changed concentrations in the intracellular gap junction regions, which could result in mechanisms closing the gap junction to preserve the cellular homeostasis. Together this might lead to a quickly blocked GJIC between the normal and the preneoplastic cells, which could have a high homoloques GJIC level, as was shown between transformed and non-transformed Balb/c 3T3 cells. Agents which decrease the level of GJIC (including tumor promoters) can play a role in the promotion phase of the process of tumor formation. Several test systems (based on the inhibition of GJIQ for the detection of agents with GJIC inhibiting capacity exist. In these assays however, the target cells for tumor promoters in the process of tumor formation (i.e. initiated cells) are not used. From the work presented in chapter 5 it can be concluded that a cell line consisting of initiated mouse epidermal cells (3PC) is a good model to detect agents with GJIC-inhibiting capacity, and that these cells are more sensitive for inhibition of GJIC compared to carcinomaderived cells. The sensitivity of primary keratinocytes compared to 3PC cells was varying and dependent on the agent used. To determine if the differences between the cell types used for the detection of inhibition of GJIC (chapter 5) are attributable to differences in mechanisms which are thought to play a role in the regulation of GJIC (i.e. connexin amount, connexin phosphorylation, connexin location in the cell, E-cadherin amount, and E-cadherin location in the cell), we studied the effects of tumor promoters on these parameters in the different cell types (chapters 6 and 7). Because calcium plays a role in the regulation of GJIC (chapter 3), we also studied the effect of tumor promoters on Ca 2+ i and the role of Ca 2+ e on these effects (chapter 8). From these studies, the following conclusions can be drawn: 1) The mechanisms involved in the inhibition of GJIC by tumor promoters are agent- and cell type-dependent; 2) The observed differences in the susceptibility of cells for the inhibition of GJIC by tumor promoters can not be associated with effects of the studied agents on one of the studied parameters; and 3) tumor promoters can change [Ca 2+ ] i , but these changes are not associated with inhibition of GJIC. In addition to the results of the studies on the regulation of GJIC (chapters 3 and 4), the studies with tumor promoters showed that, in the mouse epidermal cells used in these studies, only a decreased immunostaining of Cx43 on the plasma membranes of cells can be related to a decreased GJIC level. However, it must be noted that specific amino acid analysis of Cx43 could finally demonstrate if a relationship between inhibition of GJIC and Cx43 phosphorylation exists et all. The mechanisms regulating the transport of connexins to and from the membrane, as well as the mechanisms involved in the formation of connexons are yet unknown. Because several tumor promoters inhibited GJIC in addition to delocation of both Cx43 and E-cadherin, it should be interesting to study the mechanisms involved in the assembly of gap junction proteins or cell adhesion molecules in the plasma membrane. Such study could lead to a better understanding of the mechanisms involved in (chemical-induced) inhibition of GJIC. A study on the time-related changes in tumor promoter-induced Cx43 inummostaining and E-cadherin immunostaining could give more insight in the role of E-cadherin in the regulation of GJIC. For such a study, also cells transfected with E-cadherin or a connexin could be used.