Connexin26 expression in brain parenchymal cells demonstrated by targeted connexin ablation in transgenic mice.

Astrocytes are known to express the gap junction forming proteins connexin30 (Cx30) and connexin43 (Cx43), but it has remained controversial whether these cells also express connexin26 (Cx26). To investigate this issue further, we examined immunofluorescence labelling of glial connexins in wild-type vs. transgenic mice with targeted deletion of Cx26 in neuronal and glial cells (Cx26fl/fl:Nestin-Cre mice). The Cx26 antibodies utilized specifically recognized Cx26 and lacked cross reaction with highly homologous Cx30, as demonstrated by immunoblotting and immunofluorescence in Cx26-transfected and Cx30-transfected C6 glioma cells. Punctate immunolabelling of Cx26 with these antibodies was observed in leptomeninges and subcortical brain regions. This labelling was absent in subcortical areas of Cx26fl/fl:Nestin-Cre mice, but persisted in leptomeningeal tissues of these mice, thereby distinguishing localization of Cx26 between parenchymal and non-parenchymal tissue. In subcortical brain parenchyma, Cx26-positive puncta were often co-localized with astrocytic Cx43, and some were localized along astrocyte cell bodies and processes immunolabelled for glial fibrillary acidic protein. Cx26-positive puncta were also co-localized with punctate labelling of Cx47 around oligodendrocyte somata. Comparisons of Cx26 labelling in rodent species revealed a lower density of Cx26-positive puncta and a more restricted distribution in subcortical regions of mouse compared with rat brain, perhaps partly explaining reported difficulties in detection of Cx26 in mouse brain parenchyma using antibodies or Cx26 gene reporters. These results support our earlier observations of Cx26 expression in astrocytes and its ultrastructural localization in individual gap junction plaques formed between astrocytes as well as in heterotypic gap junctions between astrocytes and oligodendrocytes.

Ablation of connexin30 in transgenic mice alters expression patterns of connexin26 and connexin32 in glial cells and leptomeninges.

Expression of connexin26 (Cx26), Cx30 and Cx43 in astrocytes and expression of Cx29, Cx32 and Cx47 in oligodendrocytes of adult rodent brain has been well documented, as has the interdependence of connexin expression patterns of macroglial cells in Cx32- and Cx47-knockout mice. To investigate this interdependence further, we examined immunofluorescence labelling of glial connexins in transgenic Cx30 null mice. Ablation of astrocytic Cx30, confirmed by the absence of immunolabelling for this connexin in all brain regions, resulted in the loss of its coupling partner Cx32 on the oligodendrocyte side of astrocyte-oligodendrocyte (A/O) gap junctions, but had no effect on the localization of astrocytic Cx43 and oligodendrocytic Cx47 at these junctions or on the distribution of Cx32 along myelinated fibres. Surprisingly, gene deletion of Cx30 led to the near total elimination of immunofluorescence labelling for Cx26 in all leptomeningeal tissues covering brain surfaces as well as in astrocytes of brain parenchyma. Moreover northern blot analysis revealed downregulation of Cx26 mRNA in Cx30-knockout brains. Our results support earlier observations on the interdependency of Cx30/Cx32 targeting to A/O gap junctions and further suggest that Cx26 mRNA expression is affected by Cx30 gene expression. In addition, Cx30 protein may be required for co-stabilization of gap junctions or for co-trafficking in cells.

Immortalization of human lymphocytes by fusion with cytoplasts of transformed mouse L cells.

Fusion of mouse L929 cytoplasts with human peripheral blood lymphocytes induced lymphocyte proliferation that gave rise to lymphoid cell lines of B and T cell origin with unlimited growth potential. The immortalized cell lines were routinely grown in standard medium supplemented with fetal calf serum. Furthermore these cell lines could be propagated in chemically defined serum-free media. Each establishment of lymphoid cell lines was preceded by a proliferation phase 2 wk after cytoplast/cell fusion, which appears to be a necessary step in the immortalization process. The immortalized cells have a nearly normal human karyotype, do not form colonies in soft agar medium, and are not tumorigenic in nude mice. Cloned B cell lines produced human immunoglobulins of heavy and light chain types. No cross-reaction with DNA of herpes simplex virus, human cytomegalovirus, human T cell leukemia/lymphoma virus I and II, or polyoma virus was detected in the genome of immortalized cell lines by Southern blot hybridization. Furthermore B and T cell lines were established that appear to be free of Epstein-Barr virus genome.

Major loss of the 28-kD protein of gap junction in proliferating hepatocytes.

There is a reduction in the 28-kD gap junction protein detectable by immunofluorescence in livers of partially hepatectomized rats and in cultured hepatocytes stimulated to proliferate. By the coordinate use of antibodies directed to the hepatic junction protein (HJP28) and the use of a monoclonal antibody that recognizes bromodeoxyuridine (BrdU) incorporated into DNA, we have been able to study the relationship between detectable gap junction protein and cell division. Hepatocytes that label with BrdU in the regenerating liver and in cell culture show a significant reduction of HJP28. Cells that do not synthesize DNA, on the other hand, show normal levels and distribution of immunoreactive gap junction protein. We postulate that the quantitative changes in gap junction expression might play an important role in the control of proliferation in the liver.

Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes.

Affinity-purified antibodies to mouse liver 26- and 21-kD gap junction proteins have been used to characterize gap junctions in liver and cultured hepatocytes. Both proteins are colocalized in the same gap junction plaques as shown by double immunofluorescence and immunoelectron microscopy. In the lobules of rat liver, the 21-kD immunoreactivity is detected as a gradient of fluorescent spots on apposing plasma membranes, the maximum being in the periportal zone and a faint reaction in the perivenous zone. In contrast, the 26-kD immunoreactivity is evenly distributed in fluorescent spots on apposing plasma membranes throughout the rat liver lobule. Immunoreactive sites with anti-21 kD shown by immunofluorescence are also present in exocrine pancreas, proximal tubules of the kidney, and the epithelium of small intestine. The 21-kD immunoreactivity was not found in thin sections of myocardium and adult brain cortex. Subsequent to partial rat hepatectomy, both the 26- and 21-kD proteins first decrease and after approximately 2 d increase again. By comparison of the 26- and 21-kD immunoreactivity in cultured embryonic mouse hepatocytes, we found (a) the same pattern of immunoreactivity on apposing plasma membranes and colocalization within the same plaque, (b) a similar decrease after 1 d and subsequent increase after 3 d of both proteins, (c) cAMP-dependent in vitro phosphorylation of the 26-kD but not of the 21-kD protein, and (d) complete inhibition of intercellular transfer of Lucifer Yellow in all hepatocytes microinjected with anti-26 kD and, in most cases, partial inhibition of dye transfer after injection of anti-21 kD. Our results indicate that both the 26-kD and the 21-kD proteins are functional gap junction proteins.

Enhanced secretion of amylase from exocrine pancreas of connexin32-deficient mice.

To determine whether junctional communication between pancreatic acinar cells contributes to their secretory function in vivo, we have compared wild-type mice, which express the gap junctional proteins connexin32 (Cx32) and connexin26, to mice deficient for the Cx32 gene. Pancreatic acinar cells from Cx32 (-/-) mice failed to express Cx32 as evidenced by reverse transcription-PCR and immunolabeling and showed a marked reduction (4.8- and 25-fold, respectively) in the number and size of gap junctions. Dye transfer studies showed that the extent of intercellular communication was inhibited in Cx32 (-/-) acini. However, electrical coupling was detected by dual patch clamp recording in Cx32 (-/-) acinar cell pairs. Although wild-type and Cx32 (-/-) acini were similarly stimulated to release amylase by carbamylcholine, Cx32 (-/-) acini showed a twofold increase of their basal secretion. This effect was caused by an increase in the proportion of secreting acini, as detected with a reverse hemolytic plaque assay. Blood measurements further revealed that Cx32 (-/-) mice had elevated basal levels of circulating amylase. The results, which demonstrate an inverse relationship between the extent of acinar cell coupling and basal amylase secretion in vivo, support the view that the physiological recruitment of secretory acinar cells is regulated by gap junction mediated intercellular communication.

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung.

From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (alpha subgroup of connexins) than to mouse Cx32 (beta subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lung-similar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two- to fourfold higher than in the corresponding adult tissues. Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +/- 35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (tau = 580 ms at Vj of +/- 50 mV). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into coupling-deficient human HeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.

Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice.

Mice that harbor a targeted homozygous defect in the gene coding for the gap junctional protein connexin26 died in utero during the transient phase from early to midgestation. From day 10 post coitum onwards, development of homozygous embryos was retarded, which led to death around day 11 post coitum. Except for growth retardation, no gross morphological alterations were detected between homozygous connexin26-defective embryos and wild-type littermates. At day 9 postcoitum, when chorioallantoic placenta started to function, connexin26 was weakly expressed in the yolk sac epithelium, between syncytiotrophoblasts I and II in the labyrinth region of the placenta, and in the skin of the embryo. At day 10 post coitum, expression of connexin26 in the placenta was much stronger than at the other locations. To analyze involvement of connexin26 in the placental transfer of nutrients, we have measured embryonic uptake of the nonmetabolizable glucose analogue 3-O-[14C]methylglucose, injected into the maternal tail vein. At day 10 post coitum, viable, homozygous connexin26-defective embryos accumulated only approximately 40% of the radioactivity measured in wild-type and heterozygous littermates of the same size. We conclude that the uptake of glucose, and presumably other nutrients as well, from maternal blood into connexin26-deficient mouse embryos was severely impaired and apparently not sufficient to support the rapid organogenesis during midgestation. Our results suggest that connexin26 gap junction channels likely fulfill an essential role in the transfer of maternal nutrients and embryonic waste products between syncytiotrophoblast I and II in the labyrinth layer of the mouse placenta.

Mouse connexin37: cloning and functional expression of a gap junction gene highly expressed in lung.

The coding sequence (333 amino acids) of a new connexin protein, designated mouse connexin37 (Cx37 or Cx37.6) due to the deduced theoretical molecular mass of 37.600 kD, has been determined from cDNA and genomic clones. As seen in other connexins, its gene has no introns within the coding region and the deduced amino acid sequence is predicted to have similar topology to other connexins that form intercellular channels. The amino acid sequence of mouse Cx37 is most similar to rat connexin43 (59% identity) and Xenopus connexin38 (66% identity) when compared from the NH2 terminus to the end of the fourth putative transmembrane region. When expressed in Xenopus oocytes Cx37 forms functional intercellular channels that exhibit more sensitive and rapid gating in response to voltage than any previously characterized vertebrate gap junction. Under stringent conditions the Cx37 cDNA hybridizes to an mRNA of 1.7 kb that is found highly abundant in lung and to progressively lesser extents in brain, kidney, skin, spleen, liver, intestine, and heart. Embryonic brain, kidney, and skin express two to fivefold higher levels of the Cx37 transcript than the corresponding adult tissues. Cx37 transcripts were also found to increase two to threefold in response to retinoic acid treatment of cultured embryonic carcinoma F9 cells.

Ablation of Cx47 in transgenic mice leads to the loss of MUPP1, ZONAB and multiple connexins at oligodendrocyte-astrocyte gap junctions.

Oligodendrocytes in CNS are linked to astrocytes by heterotypic gap junctions composed of Cx32 and Cx47 in oligodendrocytes and Cx30 and Cx43 in astrocytes. These gap junctions also harbour regulatory proteins, including ZO-1 and ZONAB. Here, we investigated the localization of multi-PDZ domain protein 1 (MUPP1) at these gap junctions and examined accessory proteins and connexins associated with oligodendrocytes in Cx47-knockout mice. In every CNS region tested, punctate immunolabelling for MUPP1 was found on all oligodendrocyte somata in wild-type mice. These MUPP1-positive puncta were colocalized with punctate labelling for oligodendrocytic Cx32 or Cx47, and with astrocytic Cx30 or Cx43 at oligodendrocyte-astrocyte (O/A) gap junctions, but were not found at astrocyte-astrocyte gap junctions. In Cx47-knockout mice, immunolabelling of MUPP1 and ZONAB was absent on oligodendrocytes, whereas some ZO-1-positive puncta remained. In Cx32-knockout mice, MUPP1 and ZONAB persisted at O/A gap junctions. The absence of Cx47 in Cx47-knockout mice was accompanied by a total loss of punctate labelling for Cx30, Cx32 and Cx43 on oligodendrocyte somata, and by a dramatic increase in immunolabelling for Cx32 along myelinated fibers. These results demonstrate MUPP1 at O/A gap junctions and Cx47-dependent targeting of connexins to the plasma membranes of oligodendrocyte somata. Further, it appears that deficits in myelination reported in Cx47-knockout mice may arise not only from a loss of Cx47 but also from the accompanied loss of gap junctions and their regulatory proteins at oligodendrocyte somata, and that loss of Cx47 may be partly compensated for by elevated levels of Cx32 along myelinated fibers.

High incidence of spontaneous and chemically induced liver tumors in mice deficient for connexin32.

Connexins are subunits of gap junction channels, which mediate the direct transfer of ions, second messenger molecules and other metabolites between contacting cells. Gap junctions are thought to be involved in tissue homeostasis, embryonic development and the control of cell proliferation [1,2]. It has also been suggested that the loss of intercellular communication via gap junctions may contribute to multistage carcinogenesis [3-5]. We have previously shown that transgenic mice that lack connexin32 (Cx32), the major gap junction protein expressed in hepatocytes, express lower levels of a second hepatic gap junction protein, Cx26, suggesting that Cx32 has a stabilizing effect on Cx26 [6]. Here, we report that male and female one-year-old mice deficient for Cx32 had 25-fold more and 8-fold more spontaneous liver tumors than wild-type mice, respectively. Incorporation of bromodeoxyuridine (BrdU) into the liver was higher for Cx32-deficient mice than for wild-type mice, suggesting that their hepatocyte proliferation rate was higher. Furthermore, intraperitoneal injection, two weeks after birth, of the carcinogen diethylnitrosamine (DEN) led, after one year, both to more liver tumors in Cx32-deficient mice than in controls, and to accelerated tumor growth. Loss of Cx32 protein from hepatic gap junctions is therefore likely to cause enhanced clonal survival and expansion of mutated ('initiated') cells, which results in a higher susceptibility to hepatic tumors. Our results demonstrate that functional gap junctions inhibit the development of spontaneous and chemically induced tumors in mouse liver.

Reduced cardiac conduction velocity and predisposition to arrhythmias in connexin40-deficient mice.

Intercellular channels of gap junctions are formed in vertebrates by the protein family of connexins and allow direct exchange of ions, metabolites and second messenger molecules between apposed cells (reviewed in [1-3]). In the mouse, connexin40 (Cx40) protein has been detected in endothelial cells of lung and heart and in certain heart muscle cells: atrial myocytes, cells of the atrial ventricular (AV) node and cells of the conductive myocardium, which conducts impulses from the AV node to ventricular myocyctes [3]. We have generated mice homozygous for targeted disruption of the Cx40 gene (Cx40-/-mice). The electrocardiograph (ECG) parameters of Cx40-/- mice were very prolonged compared to those of wild type (Cx40+/+) mice, indicating that Cx40-/- mice have lower atrial and ventricular conduction velocities. For 6 out of 31 Cx40-/- animals, different types of atrium-derived abnormalities in cardiac rhythm were recorded, whereas continuous sinus rhythm was observed for the 26 Cx40+/+ and 30 Cx40+/- mice tested. The expression levels of other connexins expressed in heart (Cx37, Cx43 and Cx45) were the same in Cx40-/- and Cx40+/+ mice. Our results demonstrate the function of Cx40 in the regulation and coordination of heart contraction and show that cardiac arrhythmogenesis can not only be caused by defects in the ion channels primarily involved in cellular excitation but also by defects in intercellular communication through gap junction channels. As the distribution of Cx40 protein is similar in mouse and human hearts, further functional analysis of Cx40 should yield relevant insights into arrhythmogenesis in human patients.

Unique and shared functions of different connexins in mice.

BACKGROUND: Connexins are the protein subunits of intercellular gap junction channels. In mammals, they are encoded by a family of at least 15 genes, which show cell-type-specific but overlapping patterns of expression. Mice lacking connexin43 (Cx43) die postnatally from obstruction of the right ventricular outflow tract of the heart. To discriminate between the unique and shared functions of Cx43, Cx40 and Cx32, we generated two 'knock-in' mouse lines, Cx43KI32 and Cx43KI40, in which the coding region of the Cx43 gene was replaced, respectively, by the coding regions of Cx32 or Cx40. RESULTS: Heterozygous mutants were fertile and co-expressed the wild-type and the corresponding recombinant allele in all tissues analyzed. Heterozygous Cx43KI32, but not Cx43KI40, mutant mothers were unable to nourish their pups to weaning age, possibly reflecting a defect in milk ejection. Homozygous mutant males were sterile because of extensive germ-cell deficiency. The ovaries of homozygous Cx43KI32 neonates exhibited all stages of follicular development and ovulation. The hearts of homozygous Cx43KI32 neonates showed mild morphological defects, but the cardiac morphology of homozygous Cx43KI40 neonates was relatively normal. Spontaneous ventricular arrhythmias were observed in most Cx43KI40 and some Cx43KI32 mutant mice, suggesting increased ventricular vulnerability in these mice. CONCLUSIONS: The postnatal lethality of Cx43-deficient mice was rescued in Cx43KI32 or Cx43KI40 mice, indicating that Cx43, Cx40 and Cx32 share at least some vital functions. On the other hand, Cx43KI32 and Cx43KI40 mice differed functionally and morphologically from each other and from wild-type mice. Thus, these connexins also have unique functions.

Spatial relationships of connexin36, connexin57 and zonula occludens-1 in the outer plexiform layer of mouse retina.

Horizontal cells form gap junctions with each other in mammalian retina, and lacZ reporter analyses have recently indicated that these cells express the Cx57 gene, which codes for the corresponding gap junctional protein. Using anti-connexin57 antibodies, we detected connexin57 protein in immunoblots of mouse retina, and found punctate immunolabeling of this connexin co-distributed with calbindin-positive horizontal cells in the retinal outer plexiform layer. Double immunofluorescence labeling was conducted to determine the spatial relationships of connexin36, connexin57, the gap junction-associated protein zonula occludens-1 and the photoreceptor ribbon synapse-associated protein bassoon in the outer plexiform layer. Connexin36 was substantially co-localized with zonula occludens-1 in the outer plexiform layer, and both of these proteins were frequently located in close spatial proximity to bassoon-positive ribbon synapses. Connexin57 was often found adjacent to, but not overlapping with, connexin36-positive and zonula occludens-1-positive puncta, and was also located adjacent to bassoon-positive ribbon synapses at rod spherules, and intermingled with such synapses at cone pedicles. These results suggest zonula occludens-1 interaction with connexin36 but not with Cx57 in the outer plexiform layer, and an absence of connexin57/connexin36 heterotypic gap junctional coupling in mouse retina. Further, an arrangement of synaptic contacts within rod spherules is suggested whereby gap junctions between horizontal cell terminals containing connexin57 occur in very close proximity to ribbon synapses formed by rod photoreceptors, as well as in close proximity to Cx36-containing gap junctions between rods and cones.

Transforming activity of DNA fragments from normal human lymphocytes results from spontaneous activation of a c-Ha-ras1 gene.

An activated human Ha-ras gene was present in a secondary NIH 3T3 transformant isolated after serial transfection of originally low-molecular-weight DNA fragments from normal human cells. This gene appeared to have acquired its transforming properties by a spontaneous mutation in codon 12 by substitution of a deoxythymidine residue for a deoxyguanosine residue. DNA rearrangements in the flanking sequences of the transferred Ha-ras gene were not involved in the activation of the protooncogene.

Incompatibility of connexin 40 and 43 Hemichannels in gap junctions between mammalian cells is determined by intracellular domains.

Murine connexin 40 (Cx40) and connexin 43 (Cx43) do not form functional heterotypic gap junction channels. This property may contribute to the preferential propagation of action potentials in murine conductive myocardium (expressing Cx40) which is surrounded by working myocardium, expressing Cx43. When mouse Cx40 and Cx43 were individually expressed in cocultured human HeLa cells, no punctate immunofluorescent signals were detected on apposed plasma membranes between different transfectants, using antibodies specific for each connexin, suggesting that Cx40 and Cx43 hemichannels do not dock to each other. We wanted to identify domains in these connexin proteins which are responsible for the incompatibility. Thus, we expressed in HeLa cells several chimeric gene constructs in which different extracellular and intracellular domains of Cx43 had been spliced into the corresponding regions of Cx40. We found that exchange of both extracellular loops (E1 and E2) in this system (Cx40*43E1,2) was required for formation of homotypic and heterotypic conductive channels, although the electrical properties differed from those of Cx40 or Cx43 channels. Thus, the extracellular domains of Cx43 can be directed to form functional homo- and heterotypic channels. Another chimeric construct in which both extracellular domains and the central cytoplasmic loop (E1, E2, and C2) of Cx43 were spliced into Cx40 (Cx40*43E1,2,C2) led to heterotypic coupling only with Cx43 and not with Cx40 transfectants. Thus, the central cytoplasmic loop of Cx43 contributed to selectivity. A third construct, in which only the C-terminal domain (C3) of Cx43 was spliced into Cx40, i.e., Cx40*43C3, showed neither homotypic nor heterotypic coupling with Cx40 and Cx43 transfectants, suggesting that the C-terminal region of Cx43 determined incompatibility.

Abnormal cardiac conduction and morphogenesis in connexin40 and connexin43 double-deficient mice.

Connexin40-deficient (Cx40(-/-)/Cx43(+/+)) and connexin43-heterozygous knockout mice (Cx40(+/+)/Cx43(+/-)) are viable but show cardiac conduction abnormalities. The ECGs of adult double heterozygous animals (Cx40(+/-)/Cx43(+/-)) suggest additive effects of Cx40 and Cx43 haploinsufficiency on ventricular, but not on atrial, conduction. We also observed additive effects of both connexins on cardiac morphogenesis. Approximately half of the Cx40(-/-)/Cx43(+/+) embryos died during the septation period, and an additional 16% died after birth. The majority of the latter mice had cardiac hypertrophy in conjunction with common atrioventricular junction or a ventricular septal defect. All Cx40(-/-)/Cx43(+/-) progeny exhibited cardiac malformations and died neonatally. The most frequent defect was common atrioventricular junction with abnormal atrioventricular connection, which was more severe than that seen in Cx40(-/-)/Cx43(+/+) mice. Furthermore, muscular ventricular septal defects, premature closure of the ductus arteriosus, and subcutaneous edema were noticed in these embryos. Cx40(+/-)/Cx43(-/-) embryos showed the same phenotype (ie, obstructed right ventricular outflow tract) as reported for Cx40(+/+)/Cx43(-/-) mice. These findings demonstrate that Cx43 haploinsufficiency aggravates the abnormalities observed in the Cx40(-/-) phenotype, whereas Cx40 haploinsufficiency does not worsen the Cx43(-/-) phenotype. We conclude that the gap-junctional proteins Cx40 and Cx43 contribute to morphogenesis of the heart in an isotype-specific manner.

Suppression of tumorigenicity in hybrids of tumorigenic Chinese hamster cells and diploid mouse fibroblasts: dependence on the presence of at least three different mouse chromosomes and independence of hamster genome dosage.

Somatic cell hybrids were generated between Chinese hamster cell lines (Cl-4 and TK 17-O) with a near-diploid number of partially abnormal chromosomes and embryonic mouse fibroblasts (BALB/c). Hybrids harboring a near-diploid, near-triploid, and near-tetraploid set of hamster chromosomes plus 22 to 30 mouse chromosomes were analyzed for the expression of the transformed or tumorigenic phenotype, respectively, indicated by their capacity to form colonies in soft agar and by tumor formation after s.c. injection into nude mice. The hybrids showed (partial) suppression of tumorigenicity and of anchorage independence. The minimum number of hybrid cells required to initiate tumor growth in nude mice was 100- to 50,000-fold higher, and the latency period was 3- to 6-fold longer in comparison with the highly tumorigenic parental hamster cells. Suppression of tumorigenicity was also found in intraspecific Chinese hamster hybrids involving tumorigenic cells (E 36-O and TK 17-O) and embryonic hamster fibroblasts. To identify those mouse chromosomes associated with suppression of tumorigenicity, we investigated the expression of mouse isozyme genes and the presence of mouse chromosomes in interspecific suppressed hybrids and their tumorigenic hybrids described previously. No single mouse chromosome, even if present in two copies, and no combination of two different mouse chromosomes was sufficient to suppress tumorigenicity in these hybrids. This conclusion is based on either the presence of these chromosomes in hybrids isolated from tumors or their absence in suppressed hybrids.

Growth inhibition of oncogene-transformed rat fibroblasts by cocultured normal cells: relevance of metabolic cooperation mediated by gap junctions.

We have studied the proliferation of rat 208F cells (a derivative of Rat-1 cells) transformed by activated c-Ha-ras, v-fgr, v-raf, v-fms, or v-src oncogenes during cocultivation with an excess of early passage rat embryonic fibroblasts or immortal 208F cells. The total number and size of foci formed by oncogene-transformed 208F cells were strongly reduced by cocultured normal fibroblasts. The extent of growth suppression of transformed foci appears to be dependent on the type of transforming oncogene and on the type of normal fibroblasts rather than on the extent of gap-junctional communication between transformed and normal cells. Total inhibition of fluorescent dye transfer between normal and transformed cells by the 3 beta-O-hemisuccinate of 18 alpha-glycyrrhetinic acid (18 alpha-carbenoxolone), an inhibitor of gap-junctional communication in human fibroblasts, did not prevent growth inhibition of transformed cells in the cocultivation assay. Since adjacent cells remained electrically coupled in the presence of this inhibitor it is possible that the strongly reduced metabolic cooperation, as indicated by the lack of fluorescent dye transfer, is sufficient for mediating the growth-inhibitory effect of normal fibroblasts. 208F cell-conditioned medium, however, also caused strong growth inhibition of transformed derivatives, suggesting that the effect is in part mediated by release of stable growth inhibitor(s) from 208F cells.