Inhibitory effect of bisphenol A on gap junctional intercellular communication in an epithelial cell line of rat mammary tissue.

An endocrine disruptor, bisphenol-A (BPA), has been reported to have several short-term actions in various cells and tissues. However, the mechanisms of these actions have not been fully elucidated. In order to assess the effect of BPA on the intercellular communication mediated by gap junctions, we conducted the present study in the rat epithelium-derived BICR-M1Rk cell-line, in which connexin 43 (Cx43) is a major gap junction channel-forming protein. The cytotoxicity of BPA toward the cultured cells was evaluated by using both MTT reduction and LDH leakage assay systems. The results showed no appreciable loss in cell viability in the presence of increasing concentrations of BPA (from 0.1 to 3.2 microM) for 1 h incubation. However, most of cell viability was lost when cells were incubated for 24 hr with the same concentrations of BPA. The BPA acted as an antagonist on gap junction-mediated intercellular communication (GJIC), and the phenomenon was dose-dependent and irreversible. According to the data obtained from scrape-loading dye-transfer experiments, three quarters of normal GJIC was reduced by concentration of 0.4 microM BPA for 1 h incubation. To identify the relevance of this retardation upon BPA treatment, the GJIC to Cx43 synthesis, the mRNA and protein levels of Cx43 were assessed with RT-PCR and Western-blotting, respectively. The total protein level of Cx43 was almost constant in a wide range of BPA concentrations, as well as in Cx43 mRNA level. These results suggest that BPA inhibits GJIC through a modulation of the gating of gap junction channels, not through a genomic modulation of Cx43.

Growth inhibition by connexin26 expression in cultured rodent tumor cells.

The Connexin (Cx) gene family acts as a tumor suppressor. However, it is unclear whether the tumor-suppressing activity acquired by Cx gene transfection is mainly due to the recovery of the gap junction-mediated intercellular communication (GJIC) or to other unknown mechanisms. In order to elucidate the mechanism of the Cx-induced tumor-suppressing activity, we transfected Cx26 cDNA into a rodent mammary tumor cell-line (BICR-M1Rk) in which Cx43 had been normally expressed and a typical pattern of GJIC had been observed. The exogenous Cx26 was mainly localized on the nuclear envelope, whereas most of the endogenous Cx43 resided at the plasma membrane of the transfected BICR-M1Rk. Consistent with the localization of Cx26, GJIC was not increased upon the transfection of Cx26 when it was assessed by a scrape-loading dye transfer technique. A conventional [3H]-thymidine incorporation study showed that the growth rate of the Cx26-transfected cells was significantly decreased (70%), compared to that of the control BICR-M1Rk. Therefore, our results clearly demonstrate that the exogenously expressed Cx26 in the BICR-M1Rk cancer cell-line exerts an anti-proliferate activity in a GJIC-independent manner.

Mutations induced by 1,3-butadiene metabolites, butadiene diolepoxide, and 1,2,3,4-diepoxybutane at the Hprt locus in CHO-K1 cells.

Butadiene (BD) is an important industrial chemical that is classified as a probable human carcinogen. Butadiene diolepoxide (BDE) and 1,2,3,4-diepoxybutane (DEB) are metabolites of carcinogenic BD and contain the DNA-reactive one and two epoxides, respectively. In this study, the mutation frequencies and mutation spectra that are induced by BDE and DEB have been investigated at the hprt locus in CHO-K1 cells. The BDE- and DEB-treated CHO-K1 cells were allowed to grow for several days, then seeded in a medium that contained 6-thioguanine in order to select the hprt mutants. BDE exhibited the mutagenic activity at concentrations that were approximately 100-times higher than DEB. The mutation spectra for BDE and DEB were determined by a reverse transcription-polymerase chain reaction of hprt mRNA, which was followed by automatic DNA sequencing of the PCR products. The mutational spectrum for BDE was exon deletions (16/41), G x C --> A x T transitions (11/41), and A x T --> G x C transitions (5/41). The mutational spectrum for DEB was exon deletions (15/39), G x C --> A x T transitions (11/39), and A x T --> T x A transversions (5/39). The most common base substitution that was induced by both BDE and DEB was G x C --> A x T transitions. The sites of the single base substitutions that were induced by BDE and DEB were guanine and adenine, which was consistent with the DNA adduct profiles. The high frequencies of the exon deletions by each metabolite occurred in the regions of exons 2, 3, or 4. These data indicate that BDE and DEB are mutagenic carcinogens by forming DNA adducts at the site of adenine and guanine, and inducing large exon deletions and single base substitutions.

Growth-suppressing activity of the transfected Cx26 on BICR-M1Rk breast cancer cell line.

There are accumulating evidences suggesting that connexin (Cx), a gap junction channel-forming protein, acts as a growth suppressor in various cancer cells, and this effect is attributed to the gap junction-mediated intercellular communication (GJIC). In order to characterize the relationship between the growth-arresting activity of Cx26 and its cytoplasmic localizations after expression, we linked a nuclear export signal (NES) sequence to Cx26 cDNA before transfecting into a rat breast cancer cell line. A confocal fluorescent microscopic observation revealed that the insertion of NES minimized the nuclear expression of Cx26, and increased its cytoplasmic expression, including plasma membrane junctions. Total cell counting and BrdUrd-labeling experiments showed that the growth of the breast cancer cells was inhibited by 74% upon transfection of Cx26-NES, whereas only 9% inhibition was observed with only Cx26 cDNA.