Colette Piccoli

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.

Possible Molecular Mechanism of Loss of Homologous and Heterologous Gap Junctional Intercellular Communication in Rat Liver Epithelial Cell Lines

We have previously characterized a series of rat liver epithelial cell lines that exhibit levels of gap junctional intercellular communication (GJIC) which are inversely related to their levels of expression of transformed phenotypes. Cells of the non-tumorigenic line do not communicate with their tumorigenic counterparts. We have examined the molecular mechanisms involved in this loss of homologous and heterologous GJIC, employing a non-tumorigenic cell line, IAR 20, and a tumorigenic cell line, IAR 6-1. While both cell lines expressed a transcript coding for the gap junction protein, connexin 43 (cx 43), and similar levels of cx 43 protein, they exhibited different phosphorylation states of this protein, revealed by Western analysis. Immunohistochemical analysis showed that the non-tumorigenic IAR 20 cell line, but not the tumorigenic IAR 6-1 cells, was able to incorporate cx 43 gap junction plaques extensively into their plasma membranes. When IAR 20 and IAR 6-1 cells were co-cultured, cx 43 proteins were abundant in IAR 20 cells but IAR 20/IAR 20 cell boundaries were cx 43-positive, while IAR 20/IAR 6-1 boundaries were negative. The different phosphorylation state of cx 43 may partially explain the low GJIC of the IAR 6-1 cells and inability to communicate with their non-tumorigenic counterparts, but other mechanisms such as cell-cell recognition processes may also be involved.

An improved long-term culture of rat hepatocytes to detect liver tumour-promoting agents: results with phenobarbital.

Among various cocultures of hepatocytes with other cell types, we found that mouse embryonal cells (BALB/c 3T3) are more effective in maintaining rat hepatocytes in vitro. Because most human cancers are epithelial in origin, we thought that such a hepatocyte culture system could be used for the detection of tumour-promoting agents, most of which are inhibitors of gap-junctional intercellular communication. We, therefore, have examined the effect of the strong rat liver tumour promoter, phenobarbital, on the gap-junctional intercellular communication capacity of hepatocytes in long-term cultures. A single application of phenobarbital drastically inhibited the gap-junctional intercellular communication between hepatocytes in a coculture for only several hours, but treatment for 3 weeks provoked a constant decrease of gap-junctional intercellular communication (50%) throughout the treatment period. This type of long-term culture of rat hepatocytes may be usable in a rapid in vitro assay to detect tumour-promoting agents.

Long-term connexin-mediated bystander effect in highly tumorigenic human cells in vivo in herpes simplex virus thymidine kinase/ganciclovir gene therapy

Gene therapy via the herpes simplex virus thymidine kinase (tk) gene and ganciclovir (GCV) treatment eliminates experimental tumors. In this approach, cells expressing the tk gene (tk+) and neighboring tumor cells which do not express the gene are killed. We have demonstrated this bystander effect is enhanced in vitro by gap junctional intercellular communication (GJIC). In order to extend our in vitro results into in vivo situations, we injected into nude mice different ratios of tk+/tk− HeLa cells, either lacking or transfected with connexin43 (Cx43), a gene coding for a gap junction protein. When GCV was administered before tumors were palpable, fewer animals developed tumors, even after a longer period, if the injected cells were mixtures of Cx43+-tk+ and Cx43+-tk− while tumor growth was not prevented with mixtures of HeLa cells not expressing Cx43, ie Cx43−-tk+/Cx43−-tk−. When GCV was given after the appearance of tumors, the size of the tumors from Cx43− cells was 30% reduced for 3 weeks if 50% of the injected cells were tk+. However, for cells expressing Cx43, the tumor size was 66% reduced if 10% of the cells were tk+. Such a reduction demonstrates a long-term bystander effect which is dependent on Cx43 expression.

Growth control of 3T3 fibroblast cell lines established from connexin 43–deficient mice

Connexins are considered to be involved in cell growth control, on the basis of studies mainly with tumorigenic cells. To study the role of connexin genes in normal cell growth control, we established fibroblast cell lines from connexin 43 (Cx43)–deficient mice and characterized their growth. Embryonic fibroblasts from wild‐type mice (Cx43+/+) and those with heterozygous (Cx43+/–) and homozygous (Cx43–/–) deficiencies of the Cx43 gene were cultured and passaged by a 3T3 protocol (every 3 d, 3 × 105 cells/60‐mm dish). All cell lines showed a growth crisis during passages 6–15 and then started to grow well. All cell lines grew at similar rates under the 3T3 protocol, but Cx43‐deficient (Cx43–/–) cell lines tended to grow faster when they were plated at 105 cells per dish. Cx43–/– cells did not express Cx43 and showed little gap‐junctional intercellular communication (GJIC), confirming that Cx43 is the major connexin responsible for GJIC of these fibroblasts. While all Cx43+/+ and Cx43+/– cell lines expressed Cx43 protein, some of them showed very little GJIC. Those cell lines with high GJIC showed higher levels of the P2 form of Cx43 protein, and more Cx43 was localized in the plasma membrane than in cell lines with lower GJIC levels. We investigated effects of serum concentration on cell growth in these cell lines. Although different cell lines responded differentially to these agents, there was no clear relationship between Cx43 expression and cell growth stimulation by them. This suggests that Cx43 expression alone is not a strong regulator of mouse fibroblast growth. Mol. Carcinog. 23:121–128, 1998.

Bystander effect from cytosine deaminase and uracil phosphoribosyl transferase genes in vitro: A partial contribution of gap junctions

Among gene therapy strategies elaborated to kill cancer cells, one uses the CodA gene, coding for cytosine deaminase (CD) that converts 5-fluorocytosine (5-FC) into toxic 5-fluorouracil (5-FU). To enhance 5-FC metabolic activation, we prepared a vector carrying CodA and upp (uracil phosphoribosyl transferase) genes which rendered HeLa cells sensitive to 5-FC and enhanced a bystander effect not mediated by gap junctions. However, 1% CD(+)-UPP(+) cells were able to kill 40% of the cell population if the cells were communicating. This suggests that, at very low percentages of CD(+)-UPP(+) cells, CodA and upp induce a bystander effect through gap junction-dependent mechanisms.

Gap junctional intercellular communication and connexin expression in normal and SV 40-transformed human liver cells in vitro.

The gap junction intercellular communication (GJIC) capacity of two normal human liver-derived epithelial cell strains and their SV40 large T oncogene-transformed counterparts was examined. In homologous cultures the GJIC capacity of the transformed cells was considerably less than the parental cells. In heterologous cultures, transformed cells appeared to be able to form GJIC channels with normal cells. Only non-transformed cells expressed connexin (cx) 43 gene and cx 26 or cx 32 transcripts were not detectable in any cell strains tested. When GJIC was assayed in the presence of the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13- acetate (TPA), all four strains showed a marked sensitivity to TPA in inhibitory activity at 1-10 ng/ml. In contrast to a rat liver epithelial cell line, this effect of TPA did not appear to become refractory even after 24 h exposure. These studies demonstrate that GJIC of human liver cells in culture can be decreased by viral oncogene and tumour promoter action. Such disturbance may be an important component of the carcinogenic activity of these agents.

Lack of correlation between the gap junctional communication capacity of human colon cancer cell lines and expression of the DCC gene, a homologue of a cell adhesion molecule (N-CAM)

In many human colorectal cancers, the DCC gene encoding for a homologue of the neural cell adhesion molecule (N‐CAM) is found to be deleted. Previous work suggested that gap Junctional intercellular communication (GJIC) might play an important role in carcinogenesis and could be regulated by the expression of cell adhesion molecules such as E‐cadherin in some epithelial cell systems. In order to examine whether the deletion of the putative cell adhesion molecule DCC is related to the level of GJIC, which might, in turn, be important in human colorectal cancers, we compared levels of expression of the DCC gene with the GJIC capacity of a panel of human colorectal adenocarcinoma cell lines isolated from different stages of tumor progression. While the level of GJIC varied between the cell lines studied, we found no correlation between their communication capacity and DCC expression revealed by a reverse‐transcriptase/polymerase chain reaction method. This lack of correlation suggests that DCC is not a crucial regulator of GJIC.

Use of primary keratinocyte cultures from plucked human hairs for analysis of gap junctional intercellular communication

Hair follicles are a convenient source of human keratinocytes that are easily derived by non-invasive means. In order to test whether such cells could be used for gap junctional intercellular communication (GJIC) studies for the assessment of tumour-promoting activity of environmental chemicals, primary cultures were initiated from plucked hairs of various donors. GJIC measurements were performed when colonies reached a size of 5 mm in diameter, by microinjection of the fluorescent dye Lucifer Yellow CH into single cells at the colony periphery and scoring dye transfer to surrounding cells. The GJIC of cells that had begun to differentiate (appearance of cornified envelopes) can be quantitated more easily by switching cells to a low calcium (0.05-mm) medium. The tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited GJIC in a concentration- and time-dependent manner, resembling the TPA response of mouse primary epidermal keratinocytes. Thus, hair follicle cultures can be readily used for GJIC studies and should be appropriate for other in vitro assays requiring attached cells.

Growth inhibition by expression of connexin 26 in HeLa cells

HeLa cells transfected with cDNAs encoding for various types of connexin (connexins 26, 40 and 43) exhibit different growth properties despite a similar dye-transfer capacity. In particular, a clone expressing connexin 26 was not tumorigenic and its growth properties in vitro were altered. These results suggest that the connexins may induce a specific cell behaviour depending on the cell type in which they are expressed.