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Dominant-negative abrogation of connexin-mediated cell growth control by mutant connexin genes
A Duflot-Dancer1, M Mesnil, H Yamasaki
1Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
Abstract:
Connexin genes exert negative growth control when transfected into various types of tumor cell lines. We previously demonstrated that connexin 26 (Cx26) suppresses in vitro and in vivo growth of HeLa cells. In this study, we have examined whether certain Cx26 mutants can abrogate cell growth control and the gap junctional intercellular communication (GJIC) capacity of such Cx26-transfected HeLa cells. For this purpose, we transfected three mutated Cx26 genes (C60F, P87L and R143W) into HeLa cells already containing the wild-type Cx26 gene, which are GJIC-competent and non-tumorigenic. Transfection of P87L and R143W mutants enhanced the tumorigenicity of the HeLa Cx26 cells in nude mice without any change in GJIC capacity. On the other hand, transfection of the C60F mutant reduced the GJIC capacity of HeLa Cx26 cells without affecting their growth in vivo. Immunostaining studies demonstrated that the Cx26 proteins were localized mainly at cell-cell contact areas in the HeLa Cx26 cells both before and after transfection of mutated Cx26 genes. These results suggest that certain mutant Cx26 proteins exert a dominant-negative effect on Cx26-regulated growth of HeLa cells and that such effects may be independent of the effect on GJIC ability. It is proposed that wild-type and mutant Cx26 proteins produce heteromeric connexons and that such heteromeric connexons may exert different effects on growth control from those of homomeric connexons.
Insights
Certain connexin 26 (Cx26) mutants can disrupt tumor growth control in HeLa cells. Some mutants enhance tumor growth, while others affect gap junction communication, suggesting dominant-negative effects independent of GJIC.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Connexin genes, including connexin 26 (Cx26), regulate cell growth.
- Previous studies showed Cx26 suppresses HeLa cell growth in vitro and in vivo.
- The role of Cx26 mutations in abrogating this growth control is not fully understood.
Purpose of the Study:
- To investigate if specific Cx26 mutants can abolish the cell growth control and gap junctional intercellular communication (GJIC) capacity in Cx26-transfected HeLa cells.
- To determine the effects of Cx26 mutants on tumorigenicity and GJIC.
- To explore the mechanism of Cx26-mediated growth regulation.
Main Methods:
- Transfection of three mutated Cx26 genes (C60F, P87L, R143W) into HeLa cells expressing wild-type Cx26.
- Assessment of in vivo tumorigenicity in nude mice.
- Evaluation of gap junctional intercellular communication (GJIC) capacity.
- Immunostaining to localize Cx26 protein expression.
Main Results:
- Transfection of P87L and R143W mutants increased HeLa cell tumorigenicity without altering GJIC.
- Transfection of the C60F mutant decreased GJIC capacity but did not affect in vivo tumor growth.
- Cx26 proteins localized to cell-cell contacts in all transfected cell lines.
- Mutant Cx26 proteins appear to exert dominant-negative effects on growth control, potentially independent of GJIC.
Conclusions:
- Specific Cx26 mutants can override the tumor suppressor function of wild-type Cx26 in HeLa cells.
- The dominant-negative effects of certain Cx26 mutants on cell growth may be independent of their impact on GJIC.
- Heteromeric connexons formed by wild-type and mutant Cx26 proteins might differentially regulate cell growth compared to homomeric connexons.