Gap junction channels provide a pathway for exchange of ions and small molecules between coupled cells, and this exchange is believed to be critical for normal tissue growth and development. As a test for a role of gap junction-mediated intercellular communication in control of cell growth, we have compared growth rates of communication-deficient human tumor cells (SKHepl) with. clones stably transfected with cDNA encoding the rat liver gap junction protein connexin 32. In culture, growth rates for parental and transfected clones were similar. However, when sizes oftumors were evaluated following hnuection ofthese clones into athymic nude mice, growth rates for two well-coupled clones were significntly lower than for communication-deficient or poorly coupled clones. This study demonstrates that growth rate ofthese tumor cells in situ is negatively correlated with strength of intercellular communication.A role for gap junctions in normal tissue growth and development has long been postulated because of the widespread expression of gap junctions during embryogenesis (1-3) and the progressive delimitation of communication compartments as ontogenesis proceeds (3-6). This hypothesis has been strengthened by the correlation between tumor promotion and partially reduced coupling induced by various agents (7,8), by the low coupling strength and reduced gapjunction abundance between cells of some tumors, especially in highly metastatic cell populations (9)(10)(11)(12)(13), and by demonstration that oncogene expression reduces coupling in cell lines (14,15 MATERIALS AND METHODS Cell Lines. SKHepl cells were cotransfected with plasmid pcEXV3 containing full-length coding sequence for connexin 32 and/or pSV2neo using the calcium phosphate precipitation technique and selected by antibiotic G418 resistance and subsequently (in the case of the connexin 32 transfectants) on the basis of Lucifer yellow transfer as described (19). Cells were grown in a 370C, 5% CO2 incubator in RPMI medium with 10o added fetal bovine serum to which 0.4-0.5 mg of G418 per ml (GIBCO) was added in the case of transfectants (G418 was withdrawn before growth rate studies were begun). Growth rate was determined in 60-mm tissue culture dishes after seeding from confluent cultures at a density of 5 x 104 cells per dish on day 0. On subsequent days, concentration of cells was determined either by counting the number of cells per unit area of the dish (five areas counted per dish; data not shown) or by counting the number of cells per unit volume using a hemocytometer after cells were dissociated using a mild trypsinization protocol [2 min of exposure to 0.5 mg of trypsin per ml/0.2 mg of EDTA per ml (GIBCO) in RPMI medium]. For each time point in each experiment, two or three dishes of cells were counted and mean values from repeated experiments were compared to determine means and variance.Tumor Studies. BALB/c nude mice derived from breeding stocks were obtained from Frederick Cancer Center (Frederick, MD); they were quarantined for 1 week befo...

The gene family encoding gap junction proteins (connexins) consists of several known members, and multiple connexins are frequently coexpressed by coupled cells. To characterize the channel properties of the major rat liver gap junction protein (connexin 32) in isolation from other gap junction proteins, we have introduced the cDNA encoding it into a human hepatoma cell line (SKHep1) in which we have identified a gap junction deficiency. In this cell line, dye coupling was absent and junctional conductance was near zero. Connexins and connexin 32 mRNA were not detectable by immunocytochemistry and Northern blot analysis. After transfection and selection, cells were strongly coupled with regard to dye and electrical current, and connexin 32 mRNA and punctate connexin 32-immunoreactive membrane contacts were abundant. Functional gap junction channels were still expressed after 19 passages of the cells, indicating stable transfection. When junctional conductance was rendered reversibly low by exposing the cells to agents that uncouple other cell types, currents through single gap junction channels could be observed. The unitary conductance of these expressed channels was about 120-150 pS, a value that is distinctly larger than in heart cells, which express a different gap junction protein.Gap junction channels provide a pathway for exchange of metabolites and second messenger molecules between cells of most tissues and transformed cell lines (see refs. 1 and 2). The cDNAs encoding gapjunction proteins [connexins (3)] in a variety of tissues have recently been sequenced, revealing strong overall sequence homologies and a similar predicted structural motif (3-10). Despite these similarities, the functional properties of gap junctions in various tissues differ (11) and determination of properties specific for each connexin is complicated by coexpression oftwo or more proteins (12)(13)(14). Furthermore, studies of regulation of expression and of the roles played by gap junctions in cellular processes would be aided by controlled manipulation of connexin molecules in a common cell type.One approach to this problem has been the use of the Xenopus oocyte expression system (8,(15)(16)(17) MATERIALS AND METHODSCell Culture. The experiments described here were undertaken with SKHepl cells, which are derived from a highly metastatic human hepatoma (19,20). Cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (GIBCO) and were routinely fed every 5 days and subcultured at confluence.Immunocytochemistry. Cells were plated on glass coverslips and cultured until they approached confluence. Cells were fixed in 70% (vol/vol) ethanol at -20°C for 20 min, rinsed in Dulbecco's phosphate-buffered saline (PBS), and incubated for 1 hr at room temperature in monoclonal antibody R5.21 (ref. 21; 1:50 dilution of culture supernatant). The second incubation was carried out for 1 hr at room temperature in the presence of fluorescein-labeled goat antibody to rat IgG (1:100 dilution). After rinsing in PBS, a drop of p-p...

Pairs of SKHep1 cells, which are derived from a highly metastatic human hepatoma, were studied using the whole cell voltage clamp technique with patch-type electrodes containing CsCl as the major ionic species. In 12 of 81 cell pairs, current flow through junctional membranes was detectable; in the remaining 69 cell pairs, junctional conductance was less than the noise limit of our recording apparatus (worst case: 10 pS). Macroscopic junctional conductance (gj) in the small percentage of pairs where it was detectable ranged from 100 to 600 pS. Unitary junctional conductance (gamma j) determined in the lowest conductance pairs or after reducing conductance with a short exposure to the uncoupling agent halothane was 25-35 pS. To study properties of gap junction channels formed of connexin32, the parental SKHep1 cell line was stably transfected with a plasmid containing cDNA that encodes connexin32, the major gap junction protein of rat liver cells. In 85 of 98 pairs of voltage clamped connexin32-transfected SKHep1 cells, macroscopic gj was greater than 1 nS; gj increased with time after dissociation (from 1.8 +/- 0.6 [mean +/- SE; n = 7] nS at 2 h after plating to 9.3 +/- 2.2 [n = 9] nS, the maximal value, at 24 h). Unitary conductance of gap junction channels between pairs of transfected SKHep1 cells was measured in low conductance pairs and after reducing gj by exposure to halothane or heptanol. Histograms of gamma j values in transfected cells, in 10 experiments where greater than 100 transitions were measurable, displayed two peaks; 120-130 pS and 25-35 pS. The smaller size corresponded to channels that were occasionally detected in the parental cells. We therefore conclude that connexin32 forms gap junctions channels of the 120-130 pS size class.

Communication-deficient cells (the SKHep1 cell line) were stably transfected with a plasmid containing cDNA which encodes the major gap junction protein of rat liver, connexin32. Application of the dual whole-cell voltage clamp technique with patch electrodes to pairs of transfected SKHep1 cells revealed strong sensitivity of junctional conductance (gj) to transjunctional voltages (Vjs) of either polarity, with the ratio of minimal to maximal gj (gmin/gmax) being approximately 0.1 at the highest Vjs. Steady-state gj values as a function of voltages of either polarity were well fit by the Boltzmann equation. V0, the voltage at which gj was reduced by 50%, was approximately 25-30 mV; A, the Boltzmann parameter describing voltage dependence, was approximately 0.06 (corresponding to an energy difference between states of approximately 1 kCal/mol and to approximately 2 gating charges moving through the field). The kinetics of the transjunctional voltage dependence were slow (tau greater than 5 s at 20-40 mV, tau = 2 s at and beyond 70 mV). Voltage sensitivity of the opening rate constant (alpha) was approximately 30% lower than that of the closing rate constant (beta) over the Vj range 0-70 mV; at higher voltages, voltage sensitivity of alpha and beta saturated. The kinetic response of gj to a paradigm in which gj was first rendered low by a prepulse of opposite polarity indicated that the voltage sensors are likely to be arranged in series. Transitions between open and closed states in response to transjunctional voltages of either polarity are single order processes; transitions from one closed state to the other involve passage through the open state.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).