Yasufumi Omori

Role of connexin (gap junction) genes in cell growth control and carcinogenesis

Gap junctional intercellular communication (GJIC) is considered to play a key role in the maintenance of tissue independence and homeostasis in multicellular organisms by controlling the growth of GJIC-connected cells. Gap junction channels are composed of connexin molecules and, so far, more than a dozen different connexin genes have been shown to be expressed in mammals. Reflecting the importance of GJIC in various physiological functions, deletion of different connexin genes from mice results in various disorders, including cancers, heart malformation or conduction abnormality, cataract, etc. The possible involvement of aberrant GJIC in abnormal cell growth and carcinogenesis has long been postulated and recent studies in our own and other laboratories have confirmed that expression and function of connexin genes play an important role in cell growth control. Thus, almost all malignant cells show altered homologous and/or heterologous GJIC and are often associated with aberrant expression or localization of connexins. Aberrant localization of connexins in some tumour cells is associated with lack of function of cell adhesion molecules, suggesting the importance of cell-cell recognition for GJIC. Transfection of connexin genes into tumorigenic cells restores normal cell growth, supporting the idea that connexins form a family of tumour-suppressor genes. Some studies also show that specific connexins may be necessary to control growth of specific cell types. We have produced various dominant-negative mutants of Cx26, Cx32 and Cx43 and showed that some of them prevent the growth control exerted by the corresponding wild-type genes. However, we have found that connexins 32, 37 and 43 genes are rarely mutated in tumours. In some of these studies, we noted that connexin expression per se, rather than GJIC level, is more closely related to growth control, suggesting that connexins may have a GJIC-independent function. We have recently created a transgenic mouse strain in which a mutant Cx32 is specifically overexpressed in the liver. Studies with such mice indicate that Cx32 plays a key role in liver regeneration after partial hepatectomy. A decade ago, we proposed a method to enhance killing of cancer cells by diffusion of therapeutic agents through GJIC. Recently, we and others have shown that GJIC is responsible for the bystander effect seen in HSV-tk/ganciclovir gene therapy. Thus, connexin genes can exert dual effects in tumour control: tumour suppression and a bystander effect for cancer therapy.

Intercellular communication and carcinogenesis.

Two types of intercellular communication (humoral and cell contact-mediated) are involved in control of cellular function in multicellular organisms, both of them mediated by membrane-embedded proteins. Involvement of aberrant humoral communication in carcinogenesis has been well documented and genes coding for some growth factors and their receptors have been classified as oncogenes. More recently, cell contact-mediated communication has been found to have an important role in carcinogenesis, and some genes coding for proteins involved in this type of communication appear to form a family of tumor-suppressor genes. Both homologous (among normal or (pre-)cancerous cells) as well as heterologous (between normal and (pre)cancerous cells) communications appear to play important roles in cell growth control. Gap junctional intercellular communication (GJIC) is the only means by which multicellular organisms can exchange low molecular weight signals directly from within one cell to the interior of neighboring cells. GJIC is altered by many tumor-promoting agents and in many human and rodent tumors. We have recently shown that liver tumor-promoting agents inhibit GJIC in the rat liver in vivo. Molecular mechanisms which could lead to aberrant GJIC include: (1) mutation of connexin genes; (2) reduced and/or aberrant expression of connexin mRNA; (3) aberrant localization of connexin proteins, i.e., intracytoplasmic rather than in the cytoplasmic membrane; and (4) modulation of connexin functions by other proteins, such as those involved in extracellular matrix and cell adhesion. Whilst mutations of the cx 32 gene appear to be rare in tumors, cx 37 gene mutations have been reported in a mouse lung tumor cell line. Our results suggest that aberrant connexin localization is rather common in cancer cells and that possible molecular mechanisms include aberrant phosphorylation of connexin proteins and lack of cell adhesion molecules. Studies on transfection of connexin genes into tumor cells suggest that certain connexin genes (e.g., cx 26, cx 43 and cx 32) act as tumor-suppressor genes.

Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild‐type connexin43 in a dominant‐negative manner

Many lines of evidence support the hypothesis that connexins form a family of tumor‐suppressor genes. Transfection of connexin43 (Cx43) into rat C6 glioma cells have revealed that Cx43 functions as a growth‐ and tumor‐suppressor in C6 cells. In previous studies, we and others have reported that several mutant connexins can inhibit gap junctional intercellular communication (GJIC) realized by the wild type in a dominant‐negative manner. We have now examined dominant‐negative effects of Cx43 mutants on cell growth control exerted by wild‐type Cx43 in C6 cells. When 2 Cx43 mutants (L160M and A253V) were transfected into Cx43‐transfected C6 cells, they restored anchorage‐independent growth capacity and reinforced the tumorigenicity of these cells, meaning that these 2 mutants can inhibit growth‐suppressive function of wild‐type Cx43 in a dominant‐negative manner. Neither of the mutants appeared to affect phosphorylation states and subcellular localization of Cx43 proteins. Intriguingly, the mutant A253V did not suppress GJIC capacity, implying a growth‐suppressive pathway mediated by Cx43 may not be related to GJIC.Int. J. Cancer 78:446–453, 1998.

Involvement of gap junctions in tumor suppression: analysis of genetically-manipulated mice.

Accumulating evidence indicates that gap junctions play an important role in the maintenance of normal cell growth, so that genes for the connexin gap junction proteins form a family of tumor-suppressor genes. Although mice from which nine types of connexin gene are deleted have been established, little information from carcinogenesis experiments with these mice is available. We have previously found several mutant forms of connexin 32 (Cx32) to be able to inhibit, in a dominant-negative manner, gap junctional intercellular communication (GJIC) exerted by wild-type Cx32. By introducing a gene for such a dominant-negative Cx32 mutant expressed under the control of a liver-specific albumin gene promoter, we have generated transgenic mouse lines in which the function of Cx32 is down-regulated only in the liver. Although GJIC was diminished in the transgenic liver as expected, the reduced GJIC did not affect viability nor the number of spontaneous liver tumors. Although susceptibility to diethylnitrosamine-induced hepatocarcinogenesis was significantly elevated in the transgenic mice, liver regeneration after partial hepatectomy was delayed compared with wild-type mice, suggesting that gap junctions function not only to suppress excessive cell growth but also to promote cell proliferation when necessary for normal function of tissues. Although the phenotype of Cx32-deficient mice was similar to that of the transgenic mice, the former showed more drastically altered phenotypes, i.e. increased BrdU incorporation in the quiescent liver and development of spontaneous liver tumors. We also established 3T3 fibroblasts from embryos lacking the Cx43 gene and characterized their growth. These fibroblasts showed no difference from the wild type in growth characteristics. From these and other studies, we suggest that gap junctions do not necessarily suppress cell growth but support an optimal growth rate.

Role of blocked gap junctional intercellular communication in non-genotoxic carcinogenesis

Gap junctional intercellular communication mediates the transfer of small molecules from the cytoplasm of one cell to that of neighbouring cells. Connexins are the proteins that form the channels responsible for this type of communication. Aberrant expression and function of connexins are often found in cells exposed to tumor-promoting agents and during carcinogenesis, both in cell culture systems and in tissues freshly removed directly from patients and exposed animals. Transfection of connexin genes into tumorigenic cells often exerts negative growth control, suggesting that connexins act as a family of tumor-suppressor genes. Connexin gene mutations appear to be the cause of two human diseases, i.e. X-linked Charcot-Marie-Tooth syndrome and visceroatrial heterotaxia. Connexin genes are therefore important for the maintenance of homeostasis and thus their dysfunction could lead to various forms of disease.

Connexin Genes and Cell Growth Control

In multicellular organisms, individual cells not only maintain their own functions but also must behave as members of an orderly cellular society. Thus, cell growth is controlled both at the invididual cell level as well as by homeostasis within the cellular society. Known oncogenes and tumor-suppressor genes include not only genes involved in signal transduction and the cell cycle, but also those controlling growth factors and their receptors, which are important for individual cellular functions and cell-cell interaction, respectively.

Role of connexin (gap junction) genes in cell growth control: approach with site-directed mutagenesis and dominant-negative effects

Evidence is accumulating that connexin (Cx) genes form a family of tumor-suppressor genes. Our long-standing study revealed that, in almost all tumors, some abnormality in gap junction is observed, including loss or reduction of expression, aberrant localization of gap junction. In this study, we have examined the dominant-negative effects of mutant (prepared by site-directed mutagenesis) Cx43 constructs in C6 glioma cells, and of mutant Cx26 constructs in HeLa cells, on tumorigenicity. The mutant Cx43 A253V (Ala 253 to Val) inhibited the tumor-suppressive function exerted by wild-type Cx43 in C6 cells. Similarly, the mutant Cx26 P87L (Pro 87 to Leu) manifested dominant-negative inhibition of connexin-mediated cell growth control in HeLa cells. These results suggest that mutations of connexin genes can affect the tumor-suppressive function of gap junction and that gap junctional intercellular communication can be regulated by not only non-genotoxic but also genotoxic activities of environmental carcinogens.

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.