Gap junction proteins connexin32 and connexin43 partially acquire growth-suppressive function in HeLa cells by deletion of their C-terminal tails

Yasufumi Omori and Hiroshi Yamasaki1

Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, 150 cours Albert-Thomas, 69372 Lyon Cedex 08, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our laboratory has previously reported that transfection of a connexin26 (Cx26) gene, but not connexin40 nor connexin43 (Cx43), into HeLa cells expressing no detectable level of connexins suppressed the tumorigenic phenotype of the HeLa cells both in vitro and in vivo, although all of these connexins induced gap junctional intercellular communication in HeLa cells to a similar extent. The most remarkable structural difference between connexin proteins is the length of the C-terminal cytoplasmic tail, Cx26 having almost no tail, while Cx43 and connexin32 (Cx32) have long and intermediate ones, respectively. When Cx32 and Cx43 lose their C-terminal tails, they seem to resemble Cx26 in structure. To examine whether such truncated connexins become tumor suppressive in HeLa cells, we introduced a stop codon into each of the Cx32 and Cx43 cDNAs to remove their C-terminal tails and transfected these constructs ({Delta}Cx) into HeLa cells. Both {Delta}Cx cDNAs induced GJIC as efficiently as the wild-type counterparts. Although none of the truncated connexins affected proliferation rate, the truncated Cx32 and Cx43 proteins suppressed anchorage-independent cell growth in soft agar. Furthermore, when the transfectants were injected into the backs of nude mice, tumor appearance was delayed by 7 days in animals given cells expressing truncated connexins, i.e. tumors became detectable on days 11 and 18 after injection of vector and {Delta}Cx transfectants, respectively. Although throughout these experiments the truncated connexins did not completely eliminate the tumorigenicity of HeLa cells, as Cx26 did, it was evident that deletion of the C-terminal tails gave both Cx32 and Cx43 a capacity for negative growth control, suggesting that the C-terminal tails of these two connexins function as a regulatory region for connexin-mediated growth control in HeLa cells.

Abbreviations: Cx26, connexin26; Cx32, connexin32; Cx43, connexin43; FCS, fetal calf serum; GJIC, gap junctional intercellular communication; LPA, lysophosphatidic acid.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Connexin proteins are components of gap junctions which allow cytoplasmic water-soluble molecules (Mr < 1000), including ions, nucleotides and some physiologically active substances such as cAMP and inositol 1,4,5-triphosphate, to diffuse directly into the cytoplasm of neighboring cells (1). Gap junctional intercellular communication (GJIC) has long been postulated to contribute to the maintenance of tissue homeostasis and coordinated cell growth, controlling the development of small cell populations which manifest aberrant cell growth and thus helping to suppress tumor development (2). Many lines of study have indicated that connexin genes form a family of tumor-suppressor genes (3,4). Abnormalities of gap junctions or of connexin proteins, components of gap junctions, that are commonly seen in tumor tissues include loss or reduction of expression of connexin and aberrant localization of connexin molecules or of connexons, which are connexin hexamers that serve as a hemichannel of a gap junction (5). Moreover, transfection of different connexin genes, which form a multigene family consisting of at least 13 members in mammals, into tumorigenic cells has revealed that connexins suppress cell growth and tumorigenicity in a cell-type-specific manner (3). Our laboratory has previously shown that while transfection of connexin26 (Cx26) suppresses growth of HeLa cells and abolishes tumorigenicity in nude mice, neither connexin40 nor connexin43 (Cx43) show any effect after transfection (6).

All members of the connexin protein family share a very similar tertiary structure, composed of a short intracellular N-terminal region, four membrane-spanning domains, two extracellular loops, a cytoplasmic loop and a C-terminal cytoplasmic tail (7,8). The most remarkable difference in structure between connexin proteins is the length of the C-terminal tails, followed by the amino acid sequences of the cytoplasmic loop. Cx26 is the smallest connexin and has almost no C-terminal tail, while in other connexins the tail is longer in proportion to the molecular size. The C-terminal tail of Cx43 in particular has been intensively analyzed, leading to the conclusion that this domain regulates the function of the protein (1). There are several recognition sites for different protein kinases, including c-Src, MAP kinases, protein kinase C and Cdc2, in the C-terminal tail (911). It has recently been found that Cx43 interacts with a tight junction component ZO-1 (12,13). When connexin32 (Cx32) and Cx43 lose their C-terminal tails, they seem to resemble Cx26 in structure. We hypothesized that Cx32 and Cx43 were not capable of suppressing growth of HeLa cells due to the presence of the C-terminal tail and expected that if this tail could be removed, Cx32 and Cx43 would gain the tumor-suppressive function which was otherwise reserved for Cx26 in HeLa cells. In this study, cDNAs for truncated Cx32 and Cx43 ({Delta}Cx32 and {Delta}Cx43, respectively) were transfected into tumorigenic HeLa cells and the negative effects on different malignant phenotypes of HeLa cells were examined.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Constructs, transfection and cell culture
To generate a construct coding a truncated Cx43 protein, site-directed mutagenesis was carried out as described previously (14). Using pBluescriptII SK+-Cx43 as template, codon 239 CGG (Arg) was replaced with TAG (stop codon Amber). The SpeI–ClaI fragment of the mutated Cx43 cDNA sequence was subcloned into the corresponding sites of the modified pRc/RSV plasmid (Invitrogen, San Diego, CA) (14). This construct was verified for the presence of the desired mutation by DNA sequencing and was named RSV-{Delta}Cx43.

HeLa cells (2x105) were transfected with 2 µg each of supercoiled RSV-{Delta}Cx43 and RSV-wtCx43 (14) as well as with the empty vector pRc/RSV, as a control, using TransIT Transfection Reagents (Pan Vera Corporation, Madison, WI). The following day, selection was started with G418 (1 mg/ml). Independent clones were picked up after 3 weeks of selection. The establishment of wild-type Cx32 and {Delta}Cx32(R220U) transfected HeLa cells was as described previously (15).

All the clones were cultivated in Dulbecco's modified Eagle's medium supplemented with L-glutamine, penicillin/streptomycin and 10% fetal calf serum (FCS). Cell cultures were incubated at 37°C under a humidified 5% CO2 atmosphere and were routinely subcloned by trypsinization with a change of medium twice a week.

To determine cell growth, 5x104 cells were seeded into 60 mm dishes in triplicate in 5 ml of medium with 10% FCS. The cells were grown under the above-described conditions and counted every 3 days with a hemocytometer. Dead cells, as determined by trypan blue staining, were left out of the count.

Dye transfer assay to measure GJIC
To measure GJIC of cultured cells, a 5% (w/v) solution of Lucifer yellow CH in 0.33 M lithium chloride was microinjected into a single cell, as described previously (15). After 10 min, the intercellular transfer of fluorescent Lucifer yellow was estimated under an Olympus IMT-2 phase contrast and fluorescence microscope as described previously (15). For each experimental point, at least 20 microinjections were performed.

A stock solution of lysophosphatidic acid (LPA) (Sigma, St Louis, MO) was made in phosphate-buffered saline containing 1% fat-free bovine serum albumin. The above-described microinjections were performed after a 30 min incubation with LPA or with the vehicle alone.

Anchorage-independent growth assay
This test was carried out in soft agar as described previously (14). Briefly, 104 cells from each clone were seeded in 4 ml of 2x concentrated and complete Dulbecco's modified Eagle's medium containing 0.3% agar on 5 ml of solidified basal layer (0.5%) in 60 mm dishes. Ten days after seeding, colonies containing at least 20 cells in three areas (4 cm2 each) were counted in triplicate plates. Each value was converted to that for a 60 mm dish (28 cm2).

Tumorigenicity assay in nude mice
Suspensions of 105 cells of each clone in 200 µl of phosphate-buffered saline were injected s.c. into the backs of three athymic nude mice per clone (IFFA CREDO, l'Arbesle, France). After injection, each mouse was observed individually and tumor growth was estimated by direct measurement with calipers. Mice bearing tumors were killed and autopsied. Some tumors were analyzed for expression of transgenes.

Immunoblotting analysis
As described previously (14), 15 µg of total protein extract from each clone was loaded onto a 12% SDS–polyacrylamide gel. After electrophoresis, proteins were transferred to PVDF membranes (Bio-Rad). All subsequent procedures were carried out using the ECL western blotting analysis system (Amersham, Little Chalfont, UK). A polyclonal rabbit antibody (14), raised against a synthetic peptide corresponding to amino acids 119–142 located in the cytoplasmic loop of Cx43, was used to detect Cx43 at a final dilution of 1:2000 and another one (16), raised against a synthetic peptide corresponding to amino acids 98–124 located in the cytoplasmic loop of Cx32, was used to detect Cx32 at a final dilution of 1:2000. A horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) was diluted 1:5000 to detect the above two antibodies.

Immunohistochemistry
Six micrometre thick cryosections of tumors were fixed for 5 min in 10% formaldehyde in phosphate-buffered saline (pH 7.4). After overnight blocking in 5% non-fat skimmed milk in phosphate-buffered saline at 4°C, slides were incubated with primary anti-Cx32 or anti-Cx43 polyclonal antibody, described above, diluted 1:5000, followed by a horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma) diluted 1:2000. A positive reaction was revealed with DAB–NiCl2 and subsequent enrichment by silver development (17).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Constructs and transfection
Codon 220 (Arg) of the Cx32 gene and codon 239 (Arg) of the Cx43 gene were substituted with stop codons Umber and Amber, respectively, by site-directed mutagenesis, as described in Materials and methods, such that the resultant constructs would encode connexin proteins having almost no C-terminal tail. The removed region of Cx43 protein is known to contain several phosphorylation sites (S255, Y265, S279 and S282).

These two mutant constructs and their wild-type counterparts, as well as the empty pRc/RSV vector, were transfected into HeLa cells, which express no detectable levels of connexins. After 3 weeks of selection with 1 mg/ml G418, we obtained >10 independent clones from each series of transfections.

Two clones each of {Delta}Cx32 and {Delta}Cx43 HeLa transfectants were used in the present studies; they expressed a similar level of the transgene products to their corresponding wild-type transfectants (Figure 1Go). As shown in Figure 1Go, {Delta}Cx32 and truncated {Delta}Cx43 had higher mobility than their wild-type counterparts. While wild-type Cx43 (wtCx43) showed some phosphorylated forms, {Delta}Cx43 formed only one band, confirming that the C-terminal tail of Cx43 is a target of protein kinases. On the other hand, no additional bands were observed for Cx32 in any transfected HeLa cell line.



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Fig. 1. Immunoblot detection of Cx32 (A) and Cx43 (B) proteins in HeLa cell transfectants. Fifteen micrograms of protein was extracted from each indicated clone. A phosphorylated 45 kDa band is seen only with the wtCx43-transfected clone.

 
Cell–cell communication capacity of transfectants
To examine the GJIC capacity of each clone, Lucifer yellow was microinjected into cells and the number of cells receiving the dye was counted. As shown in Table IGo, both {Delta}Cx32 and {Delta}Cx43 restored GJIC to the same extent as the wild-types, suggesting that the C-terminal tails of connexins are not essential for functional channels.


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Table I. GJIC of HeLa cells transfected with wild-type or mutant Cx32 and Cx43
 
While little is known about the relationship between phosphorylation and function of Cx32 (1), the C-terminal tail of Cx43 is reported to have several consensus MAP kinase recognition sites (18). LPA is a strong MAP kinase activator and is known to inhibit Cx43-mediated GJIC (19). Although truncation of the C-terminal tail did not affect the GJIC capacity of HeLa cells under normal culture conditions, the truncated connexins might respond to MAP kinase differently from wild-types. To examine this possibility, LPA was added to the medium and the GJIC capacity was measured. As previously reported, 25 µM LPA inhibited wtCx43-driven GJIC significantly. However, {Delta}Cx43-driven GJIC was also inhibited by LPA (Table IGo). Moreover, GJIC mediated either by wtCx32 or by {Delta}Cx32 was also inhibited. This result suggests that down-regulation of GJIC by MAP kinase is not due to phosphorylation of the C-terminal tail but to a secondary effect of MAP kinase activation.

Cell growth in vitro
The cell growth rate and saturation density in vitro of each established clone were examined. Cells were plated in 60 mm dishes in triplicate and the cell number was counted with a hemocytometer. As shown by growth curves (Figure 2Go), all the transfectants of the Cx32 series showed similar growth rates and saturation densities. No effects of C-terminal truncation were found. In the Cx43 series, while all the clones showed a similar growth rate, the saturation density of {Delta}Cx43-transfected clones was 75% of that of wtCx43 or vector transfectants.



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Fig. 2. In vitro cell proliferation of HeLa cell transfectants. (A) HeLa cells transfected with wtCx32 or {Delta}Cx43; (B) HeLa cells transfected with wtCx43 or {Delta}Cx43. Cells (5x104) were plated in 60 mm dishes in triplicate. Standard deviations were not large enough to be indicated.

 
Anchorage-independent cell growth
To examine whether truncation of the C-terminal tails of connexins affects the anchorage-independent cell growth capacity of HeLa cells, a colony formation assay in soft agar was performed. While, as previously reported, Cx26-transfected HeLa cells manifested a significant decrease in anchorage-independent cell growth capacity in soft agar (6), wtCx32 and wtCx43 did not affect growth in soft agar (Table IIGo). On the other hand, both {Delta}Cx32 and {Delta}Cx43 decreased the number of colonies formed in soft agar by 20–30%.


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Table II. Anchorage-independent growth of HeLa cells transfected with wild-type or mutant Cx32 and Cx43
 
Tumorigenicity assay in nude mice
Cells (105) of each established clone were injected s.c. into the backs of nude mice after trypsinization and the first appearance and growth of tumors were recorded. All the mice injected with wtCx32-transfected HeLa cells, wtCx43-transfected ones or non-transfectants developed detectable tumors on day 11 after injection and the growth rates of tumors were similar (Figure 3Go). {Delta}Cx32- and {Delta}Cx43-transfected clones could also form tumors in the nude mice with a similar growth rate to those from wtCx32- and wtCx43-transfected clones. However, while the wild-type transfectants of Cx32 and Cx43 formed detectable tumors on day 11, with {Delta}Cx32 and {Delta}Cx43 transfectants appearance of the first tumor was delayed to day 18, suggesting that truncation of the C-terminal tails provided connexin molecules with an ability to block the process by which the dispersed cells develop into a tight mass of cells.



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Fig. 3. In vivo tumorigenicity assay of transfected clones in nude mice. HeLa cells transfected with: (A) empty vector; (B) wtCx32; (C) {Delta}Cx32 (clone 1); (D) {Delta}Cx32 (clone 2); (E) wtCx43; (F) {Delta}Cx43 (clone 1); (G) {Delta}Cx43 (clone 2). The tumor size was estimated at the indicated number of days after s.c. injection of 105 cells into 3 mice/clone.

 
To verify whether the examined clones lost expression of the transgenes during tumor formation, the tumors were examined immunohistochemically to detect transgene products in tumors formed in nude mice. In tumors derived from wtCx32- and wtCx43-transfected HeLa cells, the corresponding proteins were well expressed and localized at cell–cell contact areas. {Delta}Cx32- and {Delta}Cx43-transfected clones also expressed the transgene products at cell–cell contact areas in tumors similarly to the transfectants with the wild-types (Figure 4Go), suggesting that truncation of the C-terminal tail did not affect the mechanism for subcellular localization of connexin proteins.



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Fig. 4. Immunohistochemistry of Cx32 and Cx43 in tumors developed in nude mice. Fixed cryosections were incubated with anti-Cx32 antibody (left) or with anti-Cx43 antibody (right). Tumors from: (A and B) empty vector transfectant; (C) wtCx32 transfectant; (D) wtCx43 transfectant; (E) {Delta}Cx32 transfectant (clone 1); (F) {Delta}Cx43 transfectant (clone 1). Scale bar 75 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study we directed our attention to the fact that Cx32 and Cx43 become structurally similar to Cx26, which has almost no C-terminal tail and is tumor suppressive in HeLa cells, if they lose their C-terminal tails. Thus we examined whether {Delta}Cx32 and {Delta}Cx43 could mimic Cx26 in its tumor-suppressive function in HeLa cells. Both {Delta}Cx32 and {Delta}Cx43 suppressed anchorage-independent growth capacity slightly (Table IIGo) and delayed the first appearance of tumors by 7 days in the tumorigenicity assay (Figure 3Go). However, they did not show total tumor-suppressive activity, as Cx26 did (6).

Truncation of the C-terminal tails of Cx32 and Cx43 did not affect their GJIC capacity, meaning that the C-terminal tail of connexins is not essential for channel formation, including assembly of connexin proteins, membrane insertion and gating. This conclusion is supported by studies with other truncated mutants of Cx43. A mutant truncated at amino acid 257 retained the ability to mediate GJIC in spite of loss of the C-terminal tail, as revealed by electrocoupling of Xenopus oocytes (20). Another mutant truncated at 244 was also functional in the human hepatoma cell line SKHep1 (21). On the other hand, there is strong evidence that the C-terminal tail, which is exposed to intracellular or extracellular signals, functions as a regulatory domain for channels (1), i.e. it is known that Cx43-driven GJIC is inhibited, through an effect on the C-terminal tail, by intracellular acidification (20,22), insulin and insulin-like growth factor (23) and that the C-terminal tail is a determinant of the unitary conductance event of gap junctions composed of Cx43 proteins (21). Our mutants {Delta}Cx43 (R239X) and {Delta}Cx32 (R220U) were, however, still sensitive to MAP kinase activation by LPA. Although several consensus MAP kinase recognition sites (S255, S279 and S282) are present in the C-terminal tail of Cx43 (11,18), MAP kinase-induced down-regulation of GJIC may not be due to phosphorylation of the C-terminal tail by this kinase and it is suggested that some other region(s) can also function as a regulatory domain.

While {Delta}Cx32 did not affect cell growth in vitro (Figure 2AGo), {Delta}Cx43 reduced the saturation density of HeLa cells, but not the growth rate, to 75% of that of vector transfectants (Figure 2BGo). It has been reported that inhibition of Cx43 expression by an antisense oligonucleotide enhances the saturation density of BALB/c 3T3 cells, which predominantly express Cx43, without affecting the growth rate (24). Thus, it appears that Cx43 is prominent in the development of contact inhibition compared with other members of the connexin family. This potential of Cx43 may have been minimized in HeLa cells and may only be revealed in the absence of the C-terminal tail.

{Delta}Cx32 and {Delta}Cx43 failed to show the complete tumor-suppressive action that Cx26 manifests. However, both of these C-terminal-truncated connexin proteins extended the latent period in tumorigenesis in nude mice. Since no difference in tumor growth rate after first appearance was observed between the wild-type and truncated connexins, the C-terminal tails seem to be involved only in regulation of the latency time but not in other stages of tumor formation. Dispersed cells just after injection are likely to be more responsive to the environment provided by host mice than established tumors. Interaction with host cells and some humoral factors from the host are likely to have an adverse effect on the injected cells, to protect the host. Cells expressing {Delta}Cx32 or {Delta}Cx43 might exert a higher level of such activity than those expressing the corresponding wild-types. Thus, it may be that the C-terminal tails of Cx32 and Cx43 play a crucial role in such an interaction with host factors, by mechanisms which perhaps involve phosphorylation of these regions.

Although we initially expected that the C-terminal tail was a determinant of connexin specificity in various functions, {Delta}Cx32 and {Delta}Cx43, which appeared to resemble growth-suppressive Cx26 in structure, did not show complete growth-suppressive activity in HeLa cells, i.e. no effects of truncation were seen on cell growth in vitro or on tumor growth rate in vivo. Thus, tumor and growth suppression by Cx26 seems to occur through two types of mechanism. One is the mechanism common among Cx26, {Delta}Cx32 and {Delta}Cx43 (anchorage-dependent cell growth and extension of the latency time for tumor formation in nude mice), which is negatively regulated by the C-terminal tail in Cx32 and Cx43, and the other is specific to Cx26 (suppression of cell and tumor growth). The intracellular loop of Cx26 may be a candidate for a determinant of this Cx26-specific function, because this domain shows high diversity in amino acid sequences and length between connexin families. It has indeed recently been shown that deletion of the cytoplasmic loop of Cx43 diminishes its tumor-suppressive effect in rat bladder carcinoma cells (25). More accurate analyses of each domain should provide clues about the complex mechanisms of connexin-mediated cell growth control.


    Acknowledgments
 
We are grateful to Dr John Cheney for editing the manuscript and to Mrs Colette Piccoli for her technical assistance. This work was partly supported by a grant from the US National Institutes of Health (R01-CA-40534).


    Notes
 
1 To whom correspondence should be addressed Email: yamasaki{at}iarc.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Bruzzone,R., White,T.W. and Paul,D.L. (1996) Connections with connexins: the molecular basis of direct intercellular signaling. Eur. J. Biochem., 238, 1–27.[Abstract]
  2. Loewenstein,W.R. (1981) Junctional intercellular communication: the cell-to-cell membrane channel. Physiol. Rev., 61, 829–913.[Free Full Text]
  3. Yamasaki,H. and Naus,C.C.G. (1996) Role of connexin genes in growth control. Carcinogenesis, 17, 1199–1213.[ISI][Medline]
  4. Trosko,J.E. and Ruch,R.J. (1998) Cell–cell communication in carcinogenesis. Front. Biosci., 3, D208–D236.[Medline]
  5. Bennett,M.V.L., Barrio,L.C., Bargiello,T.A., Spray,D.C., Hertzberg,E. and Sáez,J.C. (1991) Gap junctions: new tools, new answers, new questions. Neuron, 6, 305–320.[ISI][Medline]
  6. Mesnil,M., Krutovskikh,V., Piccoli,C., Elfgang,C., Traub,O., Willecke,K. and Yamasaki,H. (1995) Negative growth control of HeLa cells by connexin genes: connexin species specificity. Cancer Res., 55, 629–639.[ISI][Medline]
  7. Willecke,K., Hennemann,H., Dahl,E., Jungbluth,S. and Heynkes,R. (1991) The diversity of connexin genes encoding gap junctional proteins. Eur. J. Cell Biol., 56, 1–7.[ISI][Medline]
  8. Kumar,N.M. and Gilula,N.B. (1992) Molecular biology and genetics of gap junction channel. Semin. Cell Biol., 3, 3–16.[Medline]
  9. Lampe,P.D., Kurata,W.E., Warn-Cramer,B.J. and Lau,A.F. (1998) Formation of a distinct connexin43 phosphoisoform in mitotic cells is dependent upon p34cdc2 kinase. J. Cell Sci., 111, 833–841.[Abstract/Free Full Text]
  10. Loo,L.W.M., Berestecky,J.M., Kanemitsu,M.Y. and Lau,A.F. (1995) pp60src-mediated phosphorylation of connexin 43, a gap junction protein. J. Biol. Chem., 270, 12751–12761.[Abstract/Free Full Text]
  11. Warn-Cramer,B.J., Lampe,P.D., Kurata,W.E., Kanemitsu,M.Y., Loo, L.W.M., Eckhart,W. and Lau,A.F. (1996) Characterization of the MAP kinase phosphorylation sites on the connexin43 gap junction protein. J. Biol. Chem., 271, 3779–3786.[Abstract/Free Full Text]
  12. Toyofuku,T., Yabuki,M., Otsu,K., Kuzuya,T., Hori,M. and Tada,M. (1998) Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes. J. Biol. Chem., 273, 12725–12731.[Abstract/Free Full Text]
  13. Giepmans,B.N. and Moolenaar,W.H. (1998) The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein. Curr. Biol., 8, 931–934.[ISI][Medline]
  14. Omori,Y. and Yamasaki,H. (1998) Mutated connexin43 proteins inhibit rat glioma cell growth suppression mediated by wild-type connexin43 in a dominant-negative manner. Int. J. Cancer, 78, 446–453.[ISI][Medline]
  15. Omori,Y., Mesnil,M. and Yamasaki,H. (1996) Connexin32 mutations from X-linked Charcot–Marie–Tooth disease patients: functional defects and dominant negative effects. Mol. Biol. Cell, 7, 907–916.[Abstract]
  16. Krutovskikh,V., Mazzoleni,G., Mironov,N., Omori,Y., Aguelon,A.-M., Mesnil,M., Berger,F., Partensky,C. and Yamasaki,H. (1994) Altered homologous and heterologous gap-junctional intercellular communication in primary human liver tumors associated with aberrant protein localization but not gene mutation of connexin 32. Int. J. Cancer, 56, 87–94.[ISI][Medline]
  17. Merchenthaler,I., Stankovics,J. and Gallyas,F. (1989) A highly sensitive one-step method for silver intensification of the nickel-diaminobenzidine endproduct of peroxidase reaction. J. Histochem. Cytochem., 37, 1563–1565.[Abstract]
  18. Kanemitsu,M.Y. and Lau,A.F. (1993) Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-O-tetradecanoyl 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase. Mol. Biol. Cell, 4, 837–848.[Abstract]
  19. Hii,C.S.T., Oh,S.-Y., Schmidt,S.A., Clark,K.J. and Murray,A.W. (1994) Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade. Biochem. J., 303, 475–479.[ISI][Medline]
  20. Liu,S., Taffet,S., Stoner,L., Delmar,M., Vallano,M.L. and Jalife,J. (1993) A structural basis for the unequal sensitivity of the major cardiac and liver gap junctions to intracellular acidification: the carboxyl tail length. Biophys. J., 64, 1422–1433.[Abstract]
  21. Fishman,G.I., Moreno,A.P., Spray,D.C. and Leinwand,L.A. (1991) Functional analysis of human cardiac gap junction channel mutants. Proc. Natl Acad. Sci. USA, 88, 3525–3529.[Abstract]
  22. Turin,L. and Warner,A. (1977) Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo. Nature, 270, 56–57.[ISI][Medline]
  23. Homma,N., Alvarado,J.L., Coombs,W., Stergiopoulos,K., Taffet,S.M., Lau,A.F. and Delmar,M. (1998) A particle-receptor model for the insulin-induced closure of connexin43 channels. Circ. Res., 83, 27–32.[Abstract/Free Full Text]
  24. Ruch,R.J., Guan,X. and Sigler,K. (1995) Inhibition of gap junctional intercellular communication and enhancement of growth in BALB/c3T3 cells treated with connexin43 antisense oligonucleotides. Mol. Carcinog., 14, 269–274.[ISI][Medline]
  25. Krutovskikh,V.A., Yamasaki,H., Tsuda,H. and Asamoto,M. (1998) Inhibition of intrinsic gap-junction intercellular communication and enhancement of tumorigenicity of the rat bladder carcinoma cell line BC31 by a dominant-negative connexin 43 mutant. Mol. Carcinog., 23, 254–261.[ISI][Medline]
Received May 21, 1999; revised June 18, 1999; accepted June 28, 1999.