**5. Non-channel functions of connexin and tumor suppressor genes**

The deficiency or absence of functional GJIC observed in most of solid tumors led Loewenstein (1979) to propose that the GJ would have a role in controlling cell proliferation. At first this function was associated with the functionality of the channels. There was then an effort to identify if GJIC deficiency was related to a specific stage of chemical carcinogenesis.

The assay performed in well established models permitted to demonstrate the tumor promoter's ability in inhibiting the GJIC. Changes in connexin isoform expressed were

Fig. 5. Laser scanning confocal microscope images of HTC cells submitted to

interference contrast microscopy and D) merged channels.

carcinogenesis.

immunofluorescence reaction with anti-connexin43 antibody: A) Nuclei stained with propidium iodide (red); B) Cx43 is presented in the cytoplasm (green); C) differential

**5. Non-channel functions of connexin and tumor suppressor genes** 

The deficiency or absence of functional GJIC observed in most of solid tumors led Loewenstein (1979) to propose that the GJ would have a role in controlling cell proliferation. At first this function was associated with the functionality of the channels. There was then an effort to identify if GJIC deficiency was related to a specific stage of chemical

The assay performed in well established models permitted to demonstrate the tumor promoter's ability in inhibiting the GJIC. Changes in connexin isoform expressed were shown in models of chemical carcinogenesis with compounds that induce liver tumors, such as pesticides and phenobarbital. In some cases it was observed the induction of Cx43 expression, connexin isoform usually not expressed by hepatocytes. The molecular mechanism of this process is not well understood, but alterations in the expression and function of connexin are now consistently associated with the hyperproliferative response. In the earlier 1990s, connexin genes have been proposed as tumor suppressor. This idea was reinforced by experimental data that related functional GJ restoration with exogenous Cx expression. *In vivo* studies clearly showed the relevance of Cx32 in maintenance of hepatic tissue normality and in the prevention of hepatocarcinogenesis (Temme et al., 1997).

Although it is not established if there is specific connexin isoforms acting as tumor suppressor in each histological type of cancer, there are some data linking Cx43 and CX32 to liver cancer. Eghbali et al (1991) transfected Cx32 in Sk-Hep1 cells and did not observe growth inhibition *in vitro*, although they detected lower tumorigenic capacity of these cells when they were injected in animals, compared with non-transfected. Sk-Hep1cell line was established from ascitic fluid of a patient with liver hepatocellular carcinoma but it is, in fact, of endothelial origin according morphological, biochemical and immunological markers. Overexpression of Cx32 reverted to normal the transformed phenotype of a rat liver cell line deficient in GJIC derived from a GJIC-competent parental cell line (WB-344).

We studied the effect of exogenous Cx43 expression in rat hepatocarcinoma cell line (HTC) and observed that transfected cells (HTC-Cx43) presented lower proliferation rate than nontransfect cells. The Cx43 was observed forming clusters at plasma membrane in transfected cells in opposition to non-transfected cells that presented Cx43 mainly localized in cytoplasm. Exogenous Cx43 expression induced morphological changes which were compatible with differentiated phenotype. Despite of Cx43 expressed by HTC-Cx43 cells do reach plasma membrane and form cluster there was not GJIC restoration. Therefore, the exogenous Cx43 expression induced a GJ-independent down-regulation in cell proliferation of HTC cell line. We attributed this effect to changes in phosphorylation pattern of Cx43 which was essential to its delivery to plasma membrane. Cx43 expression profile of HTC-Cx43 cells exhibited P1 band intensity similar to HTC cells, P0 and P2 more intense than HTC cells. All together, the data indicate that the exogenous Cx43 was critical for decreasing growth rate in rat hepatocellular carcinoma cells and contributed to reversion of the transformed phenotype. These events were independent of the functional GJIC restoration (Ionta et al, 2009).

Connexin are able to interact direct or indirectly with several molecules related to the regulation of cell proliferation and cellular compounds such as cytoskeleton elements. These interactions were demonstrated by immunofluorescence or co-precipitation studies, approaches that do not distinguish if the interaction is direct or indirect. In fact, the action of connexin as single proteins would affect the production or activity of cell growth regulators, including p27Kip1, cyclin A, cyclin D1, cyclin D2, cdk5, cdk6, ERK1/2, signal transducer and activator of transcription protein 3, Src, human EGF receptor 2, FGF1 and FGF receptor 3 (review in Vinken et al , 2011). Cx43 seems to have a role in cellular

Gap Junction Intercellular Communication and Connexin

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migration by interacting with actin through the zonula occludens. On the contrary of connexin overexpression experiments, these data were observed in an RNA interference knockdown screen. Interactions between Cx43 and ZO-1 or ZO-2, both zonula occludens proteins, were demonstrated in several studies. They bind to the same region of Cx43 in a cell cycle dynamic process, so Cx43-ZO-1 interaction occurs mainly in G0 and while Cx43- ZO-2 in G0 and S phases.

A different kind of interaction with actin seems to occur in HTC-Cx43/GFP cells treated with geodiamolides, natural peptides from marine sponge usually involved with microfilament disruption. We observed that low geodiamolides concentration for short time (2 or 4 hours) treatment did not alter the organization of actin filaments but induces larger GJ plaques (Rangel et al., 2010). The effect could be related to the stabilization of GJ plaques due to both (i) improvement in the connexon exportation or (ii) inhibition of the degradation pathway. To uncouple events leading to GJ assembly from those related to GJ removal, HTC-CX43/GFP cells were treated with geodiamolides in combination or not with fungal antibiotic BrefeldinA, a drug that disrupts the Golgi and protein trafficking to plasma membrane. BrefeldinA drastically reduced the GJ plaques formation, and the same response was obtained when the cells were treated with BrefeldinA and geodiamolides simultaneously. These data indicate that the peptide affects mainly the delivery pathway of Cx43 protein, suggesting that geodiamolide increases the length of GJ plaques mainly because it improves the delivery pathway of Cx43 protein.
