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Gap junctional intercellular communication (GJIC) has been implicated in homeostasis, development, differentiation, wound healing or regeneration and adaptive responses of differentiated cells. The dysfunction of homologous or heterologous GJIC has been associated with the tumorigenic phenotype. Restoration of growth control and the suppression of the tumorigenic phenotype have been previously associated with the up-regulation of GJIC by various anti-tumorigenic chemicals or transfection of connexin genes into tumor cells. To test the hypothesis that 'tumor suppressor' genes may be associated with the up-regulation of GJIC, we tested clones of tumorigenic HeLa, several non-tumorigenic HeLa-normal human fibroblast somatic cell hybrids and a tumorigenic segregant of one of the non-tumorigenic hybrids for GJIC. The parental HeLa cells (D98 AH.2) had no detectable GJIC but expressed detectable connexin 43 transcripts, while the non-tumorigenic HeLa-human fibroblast hybrids, which contained the chromosome 11 from the normal human fibroblast (CGL-1, CGL-2, ESH15 and EHS15c1), expressed ample connexin 43 transcripts and showed proficient GJIC. The tumorigenic segregant (CGL-3) from the non-tumorigenic HeLa-human fibroblast hybrid showed no GJIC or connexin 43. These results show that the presence of GJIC is closely linked to the suppression of the tumorigenic phenotype in the HeLa-human fibroblast hybrid and further suggest that GJIC may be associated with the mechanisms of tumor suppression. The mechanism by which the tumor suppressor gene(s) on the normal chromosome in the HeLa-human fibroblasts induces the up-regulation of connexin 43 is not yet explained.

We examined gap-junctional intercellular communication (GJIC) in a series of normal and v-raf-, v-myc-, and v-raf/v-myc-transduced rat liver epithelial (RLE) cell lines using the scrape loading-dye transfer and fluorescence-recovery-after-photobleaching (FRAP) assays. Whereas the normal RLE cell line, the control helper virus-transduced cell line, and the v-myc-transduced cell line all showed excellent GJIC, the v-raf-transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v-raf/v-myc-transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. Heterologous GJIC of these oncogene-transduced cell lines was also compared with that in the normal RLE cells. A modified FRAP assay, using fluorescent-microbead labelling to identify the oncogene-transduced cell from surrounding normal cells, was used to quantify the heterologous GJIC. The v-raf/v-myc-transformed RLE cells had no heterologous communication with the normal RLE cells, whereas v-raf- and v-myc-transduced cell lines maintained heterologous GJIC. Northern analysis showed that connexin 43 was the only gap-junction protein message expressed in these cell lines; connexin 32 and connexin 26 were not expressed. The levels of connexin 43 mRNA expression were relatively unchanged in all cell lines, suggesting that the reduction in GJIC was primarily at the posttranslational level. These findings suggest that reduction of homologous GJIC in v-raf- and v-raf/v-myc-transformed RLE cells is linked to their tumorigenic potential. Furthermore, the loss of heterologous GJIC, which we observed only in the v-raf/v-myc-transformed cells, might release such cells from the growth-regulating effects of surrounding normal cells, possibly contributing to their enhanced tumorigenic potential.

Gap junctions are known to mediate cell to cell coupling. This study shows that the potent tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), drastically affects the presence of gap junctions between mouse interfollicular epidermal (IFE) cells. In a time course ultrastructural study after a single application of 1 microgram of TPA the gap junctions between the IFE basal cells begin to decrease by 10 h post-exposure, are totally absent between 18 h and 30 h, and start reappearing at 30 h after TPA application. The potent hyperplasiogen , but very weak tumor promoter, mezerein, produces comparable hyperplasia and wide intercellular spaces but the population of gap junctions remains unchanged.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on epidermal growth factor (EGF)-binding characteristics was studied in a cultured embryonic fibroblast cell line, C3H 10T1/2. At very low concentrations, TCDD was found to cause a persistent decline in EFG binding, the median effective concentration (EC-50) being 10(-12) M. This particular effect was most conspicuous when TCDD was added at the time of medium change with fresh Dulbecco's modified Eagle's medium. Cells at an early stage of confluency were more responsive to TCDD than those at a later stage. Although most reported TCDD-evoked biological changes are recognized to occur slowly during the course of a few days to weeks, the response of C3H 10T1/2 cells to TCDD was swift, showing a sign of decline of EGF binding as early as three hours after TCDD addition. C3H 10T1/2 cells appear to be an excellent in vitro model to study TCDD's biochemical action mechanisms.

We characterized gap junctional communication in the extraembryonic tissues of the 7.5-d gastrulating mouse embryo. At this stage of development, the extraembryonic tissues form a large part of the conceptus, and link the embryo proper to the maternal tissue. Using Lucifer yellow injections, cells in most extraembryonic tissues were observed to be very well dye coupled, the only exception being the peripheral regions of the ectoplacental cone. Of particular interest was the fact that no dye coupling was detected between the three major extraembryonic tissues. Thus, the extraembryonic ectoderm (EEC), the extraembryonic endoderm (EEN), and the ectoplacental cone (EPC) corresponded to separate communication compartments, with the EPC being further subdivided into three compartments. Interestingly, the EEN was observed to exhibit a very low level of dye coupling with the adjacent visceral embryonic endoderm (EN), and consistent with the latter dye coupling results was the finding that the EEN was ionically coupled to the EN, but not with any other extraembryonic tissues. However, in the EPC, ionic coupling studies show that the central region was well coupled ionically to the EEC, but only weakly coupled to the peripheral EPC. These findings, in conjunction with our previous study (1988. J. Cell Biol. 107:241-255), demonstrate that the 7.5-d mouse conceptus is subdivided into at least nine major Lucifer yellow-delineated communication compartments, with ionic coupling across some of these compartments effectively unifying the embryo into two large domains corresponding to the embryo proper and the major extraembryonic tissues.

We characterized the pattern of gap junctional communication in the 7.5-d mouse embryo (at the primitive streak or gastrulation stage). First we examined the pattern of dye coupling by injecting the fluorescent tracers, Lucifer Yellow or carboxyfluorescein, and monitoring the extent of dye spread. These studies revealed that cells within all three germ layers are well coupled, as the injected dye usually spread rapidly from the site of impalement into the neighboring cells. The dye spread, however, appeared to be restricted at specific regions of the embryo. Further thick section histological analysis revealed little or no dye transfer between germ layers, indicating that each is a separate communication compartment. The pattern of dye movement within the embryonic ectoderm and mesoderm further suggested that cells in each of these germ layers may be subdivided into smaller communication compartments, the most striking of which are a number of "box-like" domains. Such compartments, unlike the restrictions observed between germ layers, are consistently only partially restrictive. In light of these results, we further monitored ionic coupling to determine if some coupling might nevertheless persist between germ layers. For these studies, Lucifer Yellow was coinjected while ionic coupling was monitored. The injected Lucifer Yellow facilitated the identification of the impalement sites, both in the live specimen and in thick sections in the subsequent histological analysis. By using this approach, all three germ layers were shown to be ionically coupled, indicating that gap junctional communication is maintained across the otherwise dye-uncoupled "germ layer compartments." Thus our results demonstrate that partially restrictive communication compartments are associated with the delamination of germ layers in the gastrulating mouse embryo. The spatial distribution of these compartments are consistent with a possible role in the underlying development.

A previous paper reports that the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a time-dependent effect on mouse epidermal gap junctions. A single topical application of 1.0 micrograms TPA results in the absence of gap junctions from mouse interfollicular epidermis between 18 and 30 h post-treatment. This paper describes the dose-dependent effect of TPA on mouse epidermis. Observations indicate that only promoting doses of TPA affect the gap junctions. Similarly, while a low dose of the hyperplasiogenic compound mezerein (1.0 microgram) is ineffective, a higher dose (4.0 micrograms) results in a significant reduction in the gap junction number. One and two applications of TPA had identical effects. The potent inhibitor of both stage I and stage II of tumor promotion, Fluocinolone acetonide, used in combination with TPA, completely suppressed the hyperplasiogenic and the gap junction modulating effects of TPA. Retinoic acid, which inhibits only stage II of tumor promotion, did not influence the gap junction eliminating property of TPA. Tosylphenylalanine chloromethyl ketone which is a mild but specific inhibitor of only stage I of tumor promotion counteracted the action of TPA on gap junctions to some extent, which remained present in smaller numbers than in normal tissue at 24 h after the treatment. These results suggest that gap junctions are essential and specifically relevant to stage I tumor promotion.