**4. Modulation of connexin expression, subcellular localization and functional gap junction in HCC cells**

Hepatocytes growing *in vitro* change their morphological features losing the liver-cellspecific functions. As early mentioned, the reversion of differentiated phenotype in hepatocytes growing *in vitro* is accompanied by changes in the expression profile of connexin. Under these new physiological conditions, the cells express mainly Cx43, the major isoform expressed by oval cells, instead of Cx32 or Cx26, isoforms typically expressed by hepatocytes.

Similar event occurs during malignant transformation and hepatocarcinoma cells rarely present mutation in connexin genes. Thus changes in connexin expression profile during hepatocarcinogenesis can be related to epigenetic events. For example, it was demonstrated that the reduction of Cx26 expression in hepatocarcinoma was consequence of its promoter hypermethylation. Furthermore, other events have been related to down regulation of Cx32 expression such as inappropriate phosphorylation pattern and aberrant subcellular localization in HCC cells.

Connexin phosphorylation status is very important and essential to regulate several events including intracellular trafficking, connexon assembly and disassembly, insertion in the plasma membrane, degradation and gating of gap junctional channels (Laird et al., 2005). The majority of connexins are phosphoproteins excepting the Cx26 and concerning phosphorylation process, the Cx43 has been widely studied since this molecule has different phosphorylation sites (21 serine and 2 tyrosine residues that are target of different kinase proteins) (Solan and Lamp, 2005). A number of phosphorylated Cx43 variants have been described with different patterns of electrophoretic mobility in sodium dodecyl sulfatepolyacrylamide gel. At least three forms have been described (P0, non-phosphorylated form; P1 phosphorylated form; and P2 hyper-phosphorylated form).

By comparing two rat derived cell lines, one derived from normal liver (BRL3A) and other from hepatocarcinoma (HTC), we could establish association of different phenotype with the phosphorylation pattern of Cx43. Normal liver cells grow in monolayer present contact inhibition and good cell communication capacity via gap junction channels formed by Cx43 (Figure 4 ). Meanwhile, hepatocarcinoma cell line represents a liver tumor cell line poorly differentiated that grows overlapping, and does not present functional GJIC despite expressing Cx43 (Figure 5). This difference in cell communication capacity is assumed to be due to phosphorylation status of Cx43 and its consequent intracellular localization. The major Cx43 form found in normal cells was the non-phosphorylated (P0) that was correctly inserted in plasma membrane and formed functional GJ. Meanwhile, in hepatocarcinoma cells it was observed predominantly the phosphorylated form of Cx43 (P1) which was concentrated in cytoplasm, unable to form functional gap junction in plasma membrane. These results point out to the importance of phosphorylation status of Cx43 to define its membrane insertion (Ionta et al, 2009).

Although the relationship between GJIC and cell proliferation is well established, its involvement in liver cell apoptosis is not fully understood. It was demonstrated that GJIC is induced in the early phases of apoptosis in serum-deprived rat WB-F344 liver epithelial cells

**4. Modulation of connexin expression, subcellular localization and functional** 

Hepatocytes growing *in vitro* change their morphological features losing the liver-cellspecific functions. As early mentioned, the reversion of differentiated phenotype in hepatocytes growing *in vitro* is accompanied by changes in the expression profile of connexin. Under these new physiological conditions, the cells express mainly Cx43, the major isoform expressed by oval cells, instead of Cx32 or Cx26, isoforms typically expressed

Similar event occurs during malignant transformation and hepatocarcinoma cells rarely present mutation in connexin genes. Thus changes in connexin expression profile during hepatocarcinogenesis can be related to epigenetic events. For example, it was demonstrated that the reduction of Cx26 expression in hepatocarcinoma was consequence of its promoter hypermethylation. Furthermore, other events have been related to down regulation of Cx32 expression such as inappropriate phosphorylation pattern and aberrant subcellular

Connexin phosphorylation status is very important and essential to regulate several events including intracellular trafficking, connexon assembly and disassembly, insertion in the plasma membrane, degradation and gating of gap junctional channels (Laird et al., 2005). The majority of connexins are phosphoproteins excepting the Cx26 and concerning phosphorylation process, the Cx43 has been widely studied since this molecule has different phosphorylation sites (21 serine and 2 tyrosine residues that are target of different kinase proteins) (Solan and Lamp, 2005). A number of phosphorylated Cx43 variants have been described with different patterns of electrophoretic mobility in sodium dodecyl sulfatepolyacrylamide gel. At least three forms have been described (P0, non-phosphorylated form;

By comparing two rat derived cell lines, one derived from normal liver (BRL3A) and other from hepatocarcinoma (HTC), we could establish association of different phenotype with the phosphorylation pattern of Cx43. Normal liver cells grow in monolayer present contact inhibition and good cell communication capacity via gap junction channels formed by Cx43 (Figure 4 ). Meanwhile, hepatocarcinoma cell line represents a liver tumor cell line poorly differentiated that grows overlapping, and does not present functional GJIC despite expressing Cx43 (Figure 5). This difference in cell communication capacity is assumed to be due to phosphorylation status of Cx43 and its consequent intracellular localization. The major Cx43 form found in normal cells was the non-phosphorylated (P0) that was correctly inserted in plasma membrane and formed functional GJ. Meanwhile, in hepatocarcinoma cells it was observed predominantly the phosphorylated form of Cx43 (P1) which was concentrated in cytoplasm, unable to form functional gap junction in plasma membrane. These results point out to the importance of phosphorylation status of Cx43 to define its

parallel to the increased expression and phosphorylation of Cx43.

P1 phosphorylated form; and P2 hyper-phosphorylated form).

membrane insertion (Ionta et al, 2009).

**gap junction in HCC cells** 

by hepatocytes.

localization in HCC cells.

Fig. 4. Scrape loading/dye transfer\* assay performed in BRL3A (A, C and E) and HTC cells and HTC cells (B, D and F).Differential interference contrast microscopy images in A and B, fluorescent images in C and D, and merge in E and F. BRL3A presented good communication capacity because it possible to observe at least 5 rows of fluorescent cells from scrape. HTC cells was deficient in GJIC, the fluorescent cells were visualized only in areas near to scrape.

\*Scrape loading and dye transfer (SL/DT) is a functional assay widely used to evaluate the level of the intercellular communication and it is based in the introduction of the non-permeable fluorescent dye (Lucifer Yellow, MW= 457,2) into cells of monolayer culture through a transient cut in the cell membrane. Lucifer Yellow does not diffuse through intact membrane but it is transferred into adjacent cells via GJ in competent cells. The transference is monitored with fluorescence microscopy.

Gap Junction Intercellular Communication and Connexin

(Temme et al., 1997).

cell line (WB-344).

(Ionta et al, 2009).

Expression Profile in Normal Liver Cells and Hepatocarcinoma 283

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

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

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

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

Fig. 5. Laser scanning confocal microscope images of HTC cells submitted to immunofluorescence reaction with anti-connexin43 antibody: A) Nuclei stained with propidium iodide (red); B) Cx43 is presented in the cytoplasm (green); C) differential interference contrast microscopy and D) merged channels.
