Restoration Of Connexin43 Expression In Human Tumor Lines By Inhibitors Of DNA Methylation And Histone Deacetlyation

Shimabukuro, Kelly Anne
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University of Hawaii at Manoa
Gap junctional intercellular communication (GJIC) mediated by connexin gene expression allows direct communication between cells and the integration of signals within a population. Since a majority of human tumor lines are deficient in GJIC function and/or connexin expression, and restoration of GJIC decreases neoplasia, connexins have been identified as tumor suppressor genes. No inactivating mutations in connexins within tumors have so far been reported. To determine reasons for decreased GJIC, we examined a number of human tumor lines which lack connexin43 (Cx43) expression and functional GJIC. Treatment of a cervical carcinoma (HeLa) cell line which expresses no Cx43 mRNA or protein with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) resulted in expression of the connexin43 gene indicating that the absence of Cx43 may be due directly or indirectly to DNA hypermethylation. Demethylation in these cells resulted in lower saturation density, diminished growth in heterologous culture, and reduced capacity for anchorage-independent growth, however this altered growth phenotype did not generally correspond to Cx43 re-expression. These results suggest the activation of other methylation silenced mechanisms (such as cell cycle regulatory and signaling pathways) and/or a differential capacity for HeLa clones to transmit, receive, and comprehend growth signals. Treatment of these HeLa cells with TrichostatinA (TSA), which inhibits histone deacetylase activity, also resulted in Cx43 reexpression. Additionally, treatment of other Cx43 negative human tumor lines (fibrosarcoma, breast carcinoma, oral carcinoma) with TSA resulted in increased expression of Cx43. These results indicate that diverse tumor cell lines down-regulate Cx43 expression at least in part by histone deacetylation which has in turn been shown to be regulated by DNA methylation. These results implicate epigenetic mechanisms in the suppression of connexin43 gene expression and may explain the apparent lack of tumor-specific mutations in this gene. However, DNA demethylation and inhibition of histone deacetylases were shown to act antagonistically in the re-expression of Cx43 in the HeLa cells surveyed, indicating the presence of more complex processes mediating gene expression within these cells.
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