The long-term objectives of the project are to determine the role of gap junctional intercellular communication (GJIC) and its control mechanisms in multistage carcinogenesis, and to study how connexin (Cx) genes control cell growth. Dr. Yamasaki now has good evidence that connexin genes form a family of tumor suppressor genes and preliminary results suggest that some human tumors contain mutated connexin genes. He also obtained evidence that mutant connexins inhibit GJIC by a dominant-negative fashion. Although individual cells in vivo usually express two or more specific connexin genes, cultured cells often express only Cx43. Since the data of our own and other laboratories indicate that specific connexin genes exert differential effects, he proposes to use more in vivo models. Based on these results, we propose 1) to study whether cancers have a high prevalence of connexin gene mutations and whether their mutation spectra are associated with exposure to specific carcinogens; 2) to study the dominant-negative effect of mutated connexins on wild-type connexins and its consequence in cell growth control. This will be studied both in cultured cells as well as in rodents by introducing mutated connexin genes driven by a tissue-specific gene promoter. Such dominant-negative transgenes should produce tissue-specific knock-out mice of specific connexin genes; we have already established transgenic mice carrying a mutated Cx32 gene, targeting the liver, and we now plan to target the skin. These mice will be compared with those in which a specific connexin-gene is knocked-out; currently, we have Cx43 knock-out mice, and Cx32 knock-out mice will soon become available. Their response to mutagenic and non-mutagenic carcinogens will be studied; 3) to study whether one connexin species produces heteromeric connexons with another species of connexins and how this may play a role in cell growth control. Most cell types express multiple connexin gene species and our preliminary data suggest that co-expression of the Cx32 gene in HeLa cells, the tumorigenicity of which had been suppressed by Cx26 transfection, resulted in removal of this suppression. Thus, it appears that Cx26/Cx26 connexons are tumor suppressive, but Cx26/Cx32 are not. Dr. Yamasaki proposes to examine the role of multiple connexin gene expression and their heteromeric connexons in cell growth control, employing both in vivo and in vitro models, and 4) to study the mechanisms of cell growth control by connexins. His approaches here include i) the possible role of cell cycle genes known to be associated with cell-cell interaction and ii) the effect on cell adhesion mechanisms; the data suggest that a-catenin expression is associated with connexin gene-mediated growth control.
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