A characteristic feature of most tumor cells is their reduced ability to communicate with each other and with surrounding normal cells through intercellular channels called gap junctions. These membrane channels are vital to tissue homeostasis by mediating communication of signaling molecules between cells: loss of cell cell communication through these channels in tumor cells has led to the hypothesis that gap junctions are important in growth control and neoplastic development. In support of this hypothesis, a wide variety of tumor-promoting chemicals, including organochlorine compounds and phorbol esters, have been shown to suppress gap junction-mediated communication, implicating a role for cell-cell communication in the tumor promotion phase of tumorigenesis. Our recent studies have identified novel anti-tumor compounds that prevent inhibition of gap junction-mediated cell-cell communication by organochlorine and phorbol ester tumor promoters. One of these compounds acts to stabilize the native, functional phosphorylated form of the gap junction protein. In addition, this compound up-regulates anchorage-independent growth and cell-cell communication in Ras transformed cells.
The aims of this proposal are: 1) to understand the mechanisms of action of novel anti-tumor compounds in their ability to combat the inhibitory effects of tumor promoters on cell-cell communication; and 2) to determine their mechanisms of action in up-regulating cell-cell communication and inhibiting growth of Ras-transformed and Src-transformed tumor cells. Methods to achieve these aims include fluorescent dye-transfer assays to monitor cell-cell communication, 1-D and 2-D immunoblot analyses to monitor changes in gap junction protein levels, phosphorylation, and Ras protein levels and membrane association, RNA analyses to measure gene transcript levels, and immunofluorescent staining to visualize gap junction and Ras localization. In addition, kinase activation, inhibition, and expression assays, in combination with transfection of cells with altered Ras pathway genes will be performed to identify cellular signaling pathways mediating the effects of the anti-tumor compounds. The results will provide contributions to our understanding of the role of cell-cell communication in tumorigenesis and will characterize the mechanism of action of novel anti-tumor compounds on Ras- and Src-transformed cells that may prove useful in cancer intervention or prevention.
