Effective asymmetry in gap junctional intercellular communication between populations of human normal lung fibroblasts and lung carcinoma cells
Zhi-Qian Zhang1,4,
Ying Hu1,
Bing-Jing Wang1,
Zhong-Xiang Lin1,
Christian C.G. Naus2 and
Bruce J. Nicholson3
1 Department of Cell Biology, Beijing Institute for Cancer Research, Beijing University School of Oncology, Da-Hong-Luo-Chang Street, Western District, Beijing 100034, People's Republic of China, 2 Department of Anatomy and Cell Biology, University of British Columbia, Vancouver BC V6T 1Z3, Canada and 3 Department of Biological Sciences, State University of New York, Buffalo, NY 14260, USA
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Abstract
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The dysfunction of homologous and/or heterologous gap junctional intercellular communication (GJIC) has been implicated in tumorigenesis of many kinds of cells. Here we have characterized GJIC and the expression of connexins in six human lung carcinoma cell lines and normal lung fibroblasts (HLF). Compared with HLF, all the carcinoma cells showed reduced or little homologous GJIC. They expressed remarkably reduced connexin(Cx)43 mRNA and variable levels of Cx45 mRNA, but neither Cx43 nor Cx45 protein could be detected. However, using a preloading assay, transfer of calcein was observed between donor HLF cells and first order neighboring recipient tumor cells (recipient cells in 1000-fold excess). Transfer from tumor to HLF cells under the same conditions was not seen, although increasing the ratio of donor tumor cells to recipient HLF cells and plating the cells at low density did reveal weak transfer from tumor cells to HLF. Transfection of Cx43 into giant cell carcinoma PG cells increased homologous communication and eliminated the rectifying behavior of heterologous communication. This indicates that the apparent rectification of dye transfer between normal and tumor cells was a product of low rates of heterologous transfer linked to (i) rapid dilution of the dye to below detectable limits through a very well coupled cell population (tumor to HLF) and (ii) concentration of dye in immediate neighbors in a poorly coupled cell population (HLF to tumor cells). These results suggest that the coupling levels may need to exceed a certain threshold to allow propagation of signals over a sufficient distance to affect behavior of a cell population. We propose that the relative rates of heterologous and homologous coupling of cell populations and the pool size of shared metabolites in tumor cells and the surrounding normal tissue are likely to be very important in the regulation of their growth.
Abbreviations: Cx, connexin; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate; FBS, fetal bovine serum; GFP, green fluorescence protein; GJ, gap junction; GJIC, gap junctional intercellular communication; HLEC, human lung epithelial-like cells; HLF, normal human lung fibroblast; LY, Lucifer yellow; PBS, phosphate-buffered saline
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Introduction
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Tumor cells arise from various genetic alterations (1,2) and in their progression to cancer have to break down the normal social context and establish new cellcell and cellmatrix interactions. Although some progress has been made in the identification of structures and molecules involved in the aberrant interactions between cancer cells and their normal surroundings, the intercellular signaling resulting from these interactions remains largely unknown.
Clusters of intercellular channels, termed gap junctions (GJs), are the only routes for direct intercellular signal exchange by transfer of ions (Ca2+ and H+), metabolites (amino acids), second messengers (inositol 1,4,5-trisphosphate and cAMP) and other hydrophilic molecules of <10002000 Da (3). The proteins composing the channel, termed connexins (Cx), form a multigene family consisting of at least 20 members in mammals (4). Channels formed by different Cxs differ in their gating, permeability and voltage sensitivity properties (58). Loss of or reduced gap junctional intercellular communication (GJIC) and alterations in Cx gene expression have been well documented in various human cancers, such as squamous cell carcinoma (9), gastric carcinoma (10), hepatocellular carcinoma (11,12), glioblastoma (13), rhabdomyosarcoma (14), bladder cancer (15) and prostate cancer (16). Forced expression of Cx genes by transfection in communication-deficient cancer cells (1720) retards cell growth in vivo and/or in vitro, although the effectiveness of different Cx isotypes varies depending on condition and tumor type. Conversely, inhibition of Cx expression by antisense (21,22), dominant negative mutant (2325) or gene knockout (26) methods has resulted in increased malignant phenotype or the incidence of tumor in mice. Hence, it is not surprising that Cxs have been classified among tumor suppressor genes (27).
However, there is contradictory evidence that a number of transformed cells and human cancer cells remain tumorigenic despite robust coupling with one another (2830). Yamasaki and others proposed that the lack of heterologous communication with neighboring normal cells was actually a more significant property of cancer cells (11,29,3133). Recently, there have been several reports that malignant cells can still communicate with some normal cells (30,34) and that this may even enhance invasive potential (35). These results suggest that the roles of GJIC in carcinogenesis are likely to be quite complicated.
The fluorescent dye transfer method has been widely used to evaluate the presence of functional GJ (36,37). Fluorescent dyes such as Lucifer yellow (LY), 4',6-diamidino-2-phenyindol dihydrochloride and propidium iodide can be microinjected (5,27,38) or scrape loaded (39) into donor cells with subsequent visual quantification of dye spread to neighboring cells by fluorescence microscopy. Other dyes, like carboxyfluorescein diacetate (40), calcein (41,42), 2'7'-bis(carboxyethyl)-5-carboxyfluorescein acetoxymethyl ester and 2'7'-bis(carboxyethyl)-6-carboxyfluorescein acetoxymethyl ester (34) are membrane permeant and can be loaded into cells from the extracellular medium. Intracellular cleavage of the ester renders the molecule unable to penetrate the membrane. The labeled cells, as donors, are then mixed with unlabeled recipient cells and plated at confluence onto culture dishes. Dye transfer to the adjacent cells can be captured either by direct observation under a fluorescence microscope or by flow cytometry. The latter is more convenient and objective as a means to screen large numbers of cells for quantitative analysis of homologous or heterologous GJIC.
Despite the high incidence of lung cancer, the characteristics of GJIC in human lung cancer have only recently drawn attention (20,32,43). It has been reported that Cx26, Cx30.3, Cx32, Cx37, Cx40, Cx43, Cx45 and Cx46 are all expressed in normal mouse and human lung tissues and in in vitro lung cell cultures, although the expression pattern varies with cell type, region in the lung tissue and even the culture conditions (4346). Here, we characterize GJIC and expression of the above Cxs in six human non-small cell lung carcinoma cell lines and normal human lung fibroblasts (HLF). All of these carcinoma cells showed reduced or little homologous GJIC compared with HLF. However, limited transfer was detected between HLF and tumor cells in heterologous cultures, although no transfer was evident when tumor cells served as the donors. The nature and basis of this apparent rectification were investigated by changing the pool sizes of donor and recipient and by analysis of a carcinoma cell line in which coupling levels were restored by Cx43 transfection. The significance of this in terms of exchange of signaling molecules between normal and cancer cells in situ are considered.
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Materials and methods
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Cell culture
HLF were established and subcultured in RPMI1640 medium supplemented with 15% (v/v) fetal bovine serum (FBS) (Gibco BRL) as previously described (43). Five human malignant epithelial cell lines (47) derived from non-small cell lung carcinoma, A549, Calu-3, Sklu (adenocarcinoma), Calu-6 (anaplastic carcinoma) and HuAd281 (squamous cell carcinoma), were kindly provided by Dr Klein-Szanto of Fox Chase Cancer Center and cultured in minimal Eagle's medium with 10% FBS. The pulmonary giant cell carcinoma cell line PG (48) (a gift from Dr B.Q.Wu, Department of Pathology, Peking University Medical Center) and the Cx43 transfected PG cell clone PG/C4 (20) were cultivated in RPMI 1640 medium supplemented with 10% FBS. Cell cultures were maintained in a 37°C incubator under a humidified 5% CO2 atmosphere and were routinely subcultured by trypsinization.
Analysis of GJIC capacity
Two complementary methods of monitoring dye transfer, microinjection of LY (5) and preloading of a calcein ester (42), were used to assess homologous communication. Only the latter was used in measuring heterologous GJIC between these tumor cells and HLF.
Microinjection of Lucifer yellow
Cells were grown on glass coverslips to 8090% saturation over 48 h. Single cells were microinjected with 5% LY (Molecular Probes, Eugene, OR) in 0.33 M lithium chloride as described (5,8). Transfer of the dye into adjacent cells was monitored with a Zeiss Axiovert 10 phase/fluorescence microscope 5 min after microinjection. Coverslips were never maintained outside the incubator for longer than 30 min.
Preloading with calcein ester
Donor cells were preloaded with the fluorescent dye calcein/AM (Molecular Probes, Eugene, OR) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR) as described previously (42) with some modifications. Briefly, donor cells growing in 35 mm tissue culture dishes were labeled with a mixture of 5 µM calcein/AM and 10 µM DiI in isotonic glucose solution for 1530 min in a 5% CO2 incubator at 37°C, washed with isotonic glucose, trypsinized and suspended in culture medium. Then the labeled donor cells were mixed with unlabeled recipient cells at a ratio of
1:1000 (donor:recipient) and plated onto 35 mm culture dishes for 45 h in an incubator, by which time most of the cells had attached and spread on the substrate to about 90% confluence. The number of cells positive for calcein (fluorescein channel) but negative for DiI (rhodamine channel) surrounding a double-positive cell were counted under an Olympus BH-2 fluorescence microscope. As noted in Results, the donor: acceptor ratio and plating density were varied in some experiments.
RNA extraction and purification
Total cellular RNA was purified using a single step acid-phenol extraction method (49) with modifications as described (50). In short, exponentially growing cells, briefly rinsed with phosphate-buffered saline (PBS) three times, were collected by scraping into PBS and spinning down. The resulting cell pellet was lysed in a 1:1 mixture of denaturing solution D (4 M guanidinium isothiocyanate) and water-saturated phenol, vortexed, mixed with 1/5 vol of water-saturated chloroform, incubated on ice for 15 min, then centrifuged for 20 min at maximum speed. The upper phase was precipitated with an equal volume of isopropanol at -20°C. The RNA pellet was washed with 70% ethanol and dissolved in diethylpyrocarbonate-treated water. The concentration and quality of RNA were measured using a UV spectrophotometer (Beckman) at 260 and 280 nm and by glyoxal denaturing agarose gel electrophoresis (see below) stained with ethidium bromide.
Connexin probes
Rat Cx43 (in pGEM4Z) and human Cx37 (pBSK), Cx40 (pSP64) and Cx45 (pBSk+) cDNAs were released with EcoRI, PstI, PstI and BglII/PstI, respectively, and then purified using a Geneclean II kit (Bio101 Inc., La Jolla, CA) after separation from the vector on a 0.8% agarose gel. Twenty-five nanograms of each cDNA was labeled with [
-32P]dCTP (NEN Dupont) by random primer extension following the protocol of the Boehringer Mannheim kit. Labeled probes were separated from unincorporated free dNTPs by ethanol precipitation.
Northern blot
One volume of 10 µg total RNA per cell line was denatured with 3 vol of glyoxal denaturation solution (200 µl DMSO, 60 µl deionized formamide, 40 µl 0.1 M phosphate buffer, pH 6.3), separated on a 1.2% agarose gel in 10 mM phosphate buffer (pH 6.3) and capillary transferred onto Hybond N nylon membranes (Amersham) in 20x SSC. After UV crosslinking, the membranes were prehybridized in 5x SSC, 25 mM KH2PO4, 5x Denhardt's, 50% deionized formamide, 0.25% SDS, 10% dextran sulfate and 100 µg/ml salmon sperm DNA for at least 4 h at 42°C and then 32P-labeled probes were added and hybridized overnight. The blots were washed with 2x SSC and 0.1% SDS two or three times at room temperature, followed by 0.2x SSC with 0.1% SDS at 55°C two or three times for a total of
1 h while monitoring with a Geiger counter. The washed blots were exposed to XAR-5 X-ray film (Eastman Kodak) at -70°C with two Lighting Plus intensifying screens (DuPont). To monitor the transfer efficiency and equal loading, the post-transfer gels and autoradiographed membranes were stained with ethidium bromide and methylene blue, respectively (51).
RTPCR
Five micrograms of total RNA were reverse transcribed into first strand cDNA primed with 1 µl Oligo(dT)15 (500 µg/ml; Promega) at 42°C, using 1 µl SuperscriptTM II (Gibco BRL) reverse transcriptase in a 20 µl reaction volume according to the manual accompanying the reverse transcriptase.
An aliquot of 1 µl of the reverse transcribed product was then PCR amplified in a 50 µl reaction volume of 10 mM TrisHCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dATP, dTTP, dGTP and dCTP, 50 pmol each sense and antisense primer and 2.5 U Taq polymerase (Perkin Elmer) using a PE9600 PCR Thermo-Cycler (Perkin Elmer). The primers were designed according to the published Cx sequences of humans using Generunner software (Table I). The PCR reaction mixture was first heated to 94°C for 5 min and amplification was then performed at 94°C for 60 s, 52°C for 90 s and 72°C for 120 s for 30 cycles, followed by an extension of 10 min at 72°C. PCR products were visualized with ethidium bromide after separation on a 1.5% agarose gel. Positive controls of the respective plasmid-borne cDNA and positive tissue RNAs were included for each Cx.
Connexin antibodies
Two Cx43 antibodies were used. One was an affinity-purified rabbit polyclonal antibody (1:250 dilution) raised against a synthetic peptide corresponding to amino acids 252271 of rat Cx43. The other was a monoclonal Cx43 antibody (D-7) (1:20 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA) against a recombinant protein corresponding to amino acids 241254 within the cytoplasmic domain of rat Cx43. The Cx45 antibody (N-19) (1:10 dilution) (Santa Cruz Biotechnology) is an affinity-purified goat polyclonal antibody raised against a peptide mapping near the N-terminus of human Cx45.
Immunofluorescent staining
In the co-culture, DiI could not be used to distinguish different cells on immunostaining because it disappeared after fixation and permeabilization. Green fluoresence protein (GFP) was used instead, by infecting HLF with GFP-pAdeasy adenoviruses. Briefly, adherent HLFs were infected with GFP-pAdeasy adenoviruses at an m.o.i. of 100 and allowed to grow for 24 h. The cells were monitored by fluorescence microscopy and shown to be almost 100% positive for green fluorescence. Labeled HLFs were mixed with the different unlabeled carcinoma cells at a ratio of 1:1000 and plated onto glass coverslips in 35 mm culture dishes.
Cells grown on coverslips to
90% confluence were fixed in 5% acetic acid, 95% ethanol (v/v) for 20 min at -20°C, rinsed with PBS and incubated with connexin antibodies for 1 h at 37°C. After washing with 0.5% Triton X-100 in PBS three times for 10 min each, cells were incubated with affinity-purified fluorescein- or rhodamine-conjugated secondary IgG (Jackson Laboratories, West Grove, PA) for 1 h. This was followed by washing and nuclear staining in 4,6-diamidino-2-phenylindole (Polysciences, Warrington, PA) (0.5 µg/l in 0.9% NaCl). Coverslips were then mounted in 60% glycerol in PBS containing 2.5% 1,4-diazabicyclo(2,2,2)octane (Sigma). Cells were examined with a BH-2 epifluorescence microscope (Olympus) or a Leica DM IRE2 laser scanning confocal microscope.
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Results
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Lack of homologous communication in human lung carcinoma cell lines
The establishment of HLFs has been described in our earlier paper (43). The six human lung carcinoma cell lines, HuAd281, Sklu, PG, A549, Calu-3 and Calu-6, differ in their biological behavior as listed in Table II (47,48). Homologous GJIC in these cell lines was assessed in most cases by both LY microinjection and calcein preloading. HLFs served as a comparison for non-transformed cells. However, as these cells were too thin to allow microinjection, a human epithelial-like lung cell line (HLEC) resulting from primary culture of human fetal lung tissues (Zhang et al., unpublished data) was used here only as a positive control for LY transfer studies.
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Table II. Tumorigenicity and metastatic potential of and homologous communication and Cx gene expression in human lung cells
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While the HLEC showed a high capacity of GJIC with LY transferred to an average of 31 ± 4.0 (SD) neighboring cells (
57 orders of cells) per injected cell (Figure 1a and b), all the tumor cell lines displayed dramatically reduced LY transfer (Figure 1cn). However, comparison among tumor cell lines revealed no consistent correlation between the reported tumorigenicity/malignant behavior of the cells and reduced coupling levels. For example, the poorly metastatic but moderately tumorigenic cell line Calu-3 showed the best coupling, while the least tumorigenic and metastatic cell line, Sklu, showed the poorest coupling, much lower than that seen in the more tumorigenic and metastatic PG and Calu-6 cells (Table II).

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Fig. 1. Representative photograph of homologous GJIC capacity of human lung carcinoma cell lines as determined by the microinjection of LY. For each cell line, the extent of fluorescent dye transfer is shown in the right panel and the corresponding phase contrast area is in the left panel, with the microinjected cell indicated by a star. Note the presence of strong GJIC capacity, with LY transfer to >4 orders beyond the microinjected cell, in the normal HLEC (a and b) and minimal or no LY transfer in all the human lung carcinoma cell lines A549 (c and d), HuAd281 (e and f), Sklu (g and h), PG (i and j), Calu-3 (k and l) and Calu-6 (m and n).
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In calcein preloading experiments, HLF transferred calcein efficiently to the adjacent 57 orders of cells (Figure 2) with an average of 24.2 ± 6.3 (SD) cells per labeled cell. Homologous communication within tumor cell lines A549, PG, Calu-6 and Sklu measured by preloading was again much lower, although it appears that this assay is subject to greater variability than microinjection (Figure 2 and Table II). These results suggest that lack of efficient homologous communication is a common feature in these human lung carcinoma cell lines, but that the actual levels of coupling that are present do not correlate with tumorigenicity.

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Fig. 2. Homologous communication as assayed by preloading with calcein/DiI and calcein transfer in HLF (a, a' and a'') and human lung carcinoma cell lines PG (b, b' and b''), A549 (c, c' and c''), Calu-6 (d, d' and d''), Sklu (e, e' and e''). In HLF, calcein transferred to >5 orders of cells adjacent to the labeled one. All four tumor cell lines showed almost no transfer of calcein.
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Expression of connexins in human lung carcinoma cells
In an effort to understand the basis for the small, but variable, levels of coupling seen in these cell lines, northern blotting and RTPCR were performed to screen for the expression of Cx26, Cx30.3, Cx32, Cx37, Cx40, Cx43, Cx45 and Cx46, which have been reported as the major Cxs expressed in mammalian lung tissues or cultured cells. RTPCR failed to detect any signal for Cx26, Cx30.3, Cx32, Cx37, Cx40 or Cx46 in these lung carcinoma cells or in HLF (data not shown). Northern blots probed for Cx37 and Cx40 also revealed no signal.
In contrast, Cx43 mRNA was readily detectable in HLF by northern blot, although no signal was detected in most of the lung carcinoma cells, PG, HuAd281, Sklu and A549. Reduced, but detectable, Cx43 message was seen in Calu-6 and Calu-3 cells, consistent with the slightly higher coupling these cells displayed. When the more sensitive RTPCR technique was employed, all cell lines, including those showing the least coupling (Sklu and A549), revealed at least trace levels of Cx43 (Figure 3A and B and Table II).

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Fig. 3. Expression of Cx43 and Cx45 mRNAs in normal human lung fibroblasts and human lung carcinoma cells. (A) Northern blot analysis of Cx43 expression. Ten micrograms of total cellular RNA were loaded in each lane. Cx43 was readily detected in HLF, but only trace levels were detected in human lung tumor cell lines Calu-3 and Calu-6. Cx43 was undetectable in other tumor cell lines. One mouse Lewis lung carcinoma cell line (LLC, not included in this study) showed more Cx43 mRNA than other tumor cell lines, but much lower than HLF. The non-specific 28S and 18S rRNA signals (arrows) indicate equal loading in each lane. (B) The more specific RTPCR analysis of Cx43 showed that all the cell lines expressed variable levels of Cx43 mRNA, although only trace levels were detected in HuAd281 and A549 cells. (C) Cx45 northern blot analysis of HLF and tumor cell line PG. Both cell lines express Cx45, although the signal in HLF was slightly stronger. Note that there is a band smaller than Cx45 mRNA in HLF, the nature of which remains to be established. (D) Cx45 expression analysis by RTPCR. Rat heart tissue was included as a positive control. Cx45 was detected in all cell lines, although signals were close to background in the Calu 3 and A549 cell lines. Similar levels to normal HLF cells were detected in two highly metastatic cell lines, PG and Calu-6, and one non-metastatic line, HuAd281. Note that the smaller band seen in the northern blot of HLF is also evident in the PCR products.
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RTPCR revealed that Cx45 was also expressed in all the cell lines, although only at minimally detectable levels in Sklu, A549 and Calu-3. PG, Calu-6 and HuAd281 expressed comparable levels to HLFs. In the case of PG cells this was confirmed by northern blotting. In addition to the expected band at 2.2 kb, a slightly smaller band was also detected in both northern blots and RTPCR products of HLFs (Figure 3C and D and Table II).
The expression of Cx43 and Cx45 proteins from these transcripts was tested by immunocytochemical staining. While abundant Cx43 punctate staining between cells was seen in HLFs, most of the tumor cells showed dispersed punctate staining between only a few cells that was generally indistinguishable from the background. Only A549 cells showed clear punctate staining between many, but not all, cell interfaces (Figure 4). Western blots failed to detect Cx43 in any of these tumor cell lines (data not shown).

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Fig. 4. Cx43 immunofluorescent staining. HLFs were positive for Cx43 protein with abundant punctate staining localized at the interface between cells (A). Much lower levels, near background, were seen in the tumor cells under study, A549 (B), PG (C), Sklu (D), HuAd281 (E) and Calu-6 (F), although some punctate spots were evident in some cell lines (e.g. A549 in B). n, nuclear region. Bar = 40 µm.
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Although HLF and some tumor cells expressed Cx45 mRNA, both western blotting and immunostaining of Cx45 were unable to detect any signal for Cx45 expression in these cells (data not shown), suggesting that this connexin was probably not responsible for the coupling observed. Overall, the levels of Cx43 seemed to correlate best with the recorded dye transfer (summarized above). This latter conclusion is also consistent with previous studies that have shown that Cx45 channels are poorly permeable to dyes the size of calcein, while Cx43 channels are highly permeable to such dyes.
Asymmetric heterologous GJIC between human lung fibroblasts and carcinoma cells
Although all tumor cells showed reduced coupling compared with HLF cells, the lack of a correlation between coupling level of tumor cells and tumorigenic phenotype led us to investigate the hypothesis that loss of heterologous communication between tumor cells and their normal counterparts may correlate better with their uncontrolled growth (29,5255). As fibroblasts are the major cells in the stroma surrounding potential tumor cells in situ, we tested heterologous coupling between four human lung tumor cell lines (A549, Calu-6, PG and Sklu) and HLFs. Using the tumor cells as donors and HLF cells as recipients, preloading experiments revealed no dye transfer to adjacent fibroblasts after 5 h plating (Figure 5A and Table III). This is in good agreement with several previous reports that tumors could not communicate with normal cells (33,41; for reviews see 37,55).

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Fig. 5. Representative photographs of heterologous GJIC between human lung fibroblasts and human lung carcinoma cells as measured by the preloading and dye transfer method. (A) Human lung carcinoma cell lines A549 (a, a' and a''), Sklu (b, b' and b''), PG (c, c' and c'') and Calu-6 (d, d' and d'') were pre-labeled with DiI (a'd') and calcein, co-cultured with HLF for 5 h and examined under a fluorescence microscope. No calcein transfer is observed from these tumor cells to HLF (a''d''). (B) When HLF were prelabeled as donor cells, calcein transfer was detected from HLF to first order human lung carcinoma cell lines A549 (a, a' and a''), Sklu (b, b' and b''), PG (c, c' and c'') and Calu-6 (d, d' and d''). The preloaded HLF cells are marked with asterisks in the calcein photographs a''d''.
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Table III. Heterologous GJIC between human lung carcinoma cells and HLF as assayed by the preloading of calcein and dye transfer method
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However, in the complementary experiment, when HLF cells were used as donors, transfer of calcein to the surrounding tumor cell recipients was seen (Figure 5B and Table III). Calcein transfer was restricted to the adjacent first order cells, but of these 40100% (or 57 cells/donor) received dye. Second order transfer was rarely seen, consistent with the earlier demonstration of restricted homologous communication between these tumor cells.
The results initially suggest that asymmetric heterologous GJIC exists between HLFs and carcinoma cells. The carcinoma cells were unable to transfer dye directly through gap junctions to normal fibroblasts, but first order tumor cells do have the potential to receive dye from adjacent fibroblasts. Clearly, the same could also apply to intracellular growth regulatory signals. Similar cases of apparent rectifying dye transfer have been reported previously (56,57). Two models have been proposed to explain this. The first implicates rectifying heterotypic gap junctions between cell types, although this has been criticized as invalid based on a violation of established physical laws. An alternative model posits that significant differences in the sizes of the donor and recipient compartments could produce apparent rectification of dye transfer through dilution of the dye below detectable levels, even when the two compartments are connected by symmetrical gap junctions.
Lung carcinoma cells transferred calcein to human lung fibroblasts at higher ratios of donor to recipient cells
To test whether the sizes of the donor and recipient compartments could indeed account for the non-reciprocal dye coupling between these cell types, calcein transfer from carcinoma cells to HLFs was re-examined by plating the cells at reduced ratios of acceptors to donors (cf. 1000:1 in the initial experiments). Plating ratios of 5:1 (tumor cell donors to HLF acceptors) gave inconclusive results, as it was difficult to count the weakly fluorescent acceptors on a lawn of very bright donors. However, by significantly reducing the plating density and using an
1:1 ratio of acceptors to donors (i.e. each acceptor often had more than one potential donor), calcein was clearly detected in
5070% of HLFs paired with tumor cells in all four cases tested (i.e. A549, Calu-6, PG and Sklu). Due to the number of donor cells in contact with each acceptor, intensities in the acceptor cells varied for each plate (Figure 6). As expected, transfer from HLFs to tumor cells under these same conditions was now comparable with the reverse direction, appearing in 8090% of pairings observed (data not shown).

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Fig. 6. When plated at a higher donor:acceptor ratio and lower density, lung carcinoma cells PG (a), Sklu (b), A549 (c) and Calu-6 (d) were capable of transferring calcein to HLF, as demonstrated by preloading and dye transfer, although the ratio of recipient to donor dye intensity is quite low.
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This demonstrated that the failure to see dye transfer into HLFs from tumor cells was due to dilution of the dye throughout the well-coupled HLF monolayer and that rectifying junctions between tumor and normal cells do not need to be invoked to explain our observations.
Transfection of Cx43 into PG cells results in symmetric communication between fibroblasts and the transfectants
It could be directly deduced from the above results that the apparent asymmetry of transfer between HLF and tumor cells seen in the original experiments should be eliminated if homologous coupling between tumor cells was increased to levels similar to that seen in HLFs. This prediction was directly tested using PG/C4 cells, a previously established Cx43 transfectant of the PG cell line that expressed high levels of Cx43 mRNA and protein and showed efficient GJIC (20). Despite the lack of correlation between coupling level and tumorigenic potential in the cell lines studied here, the PG/C4 cell line showed enhanced attachment to the substratum and inhibition of colony formation in soft agar. The growth of PG/C4 cells was also retarded both in vitro and in vivo.
The homologous GJIC of PG/C4 cells was checked by the preloading dye transfer method. Consistent with previous scrape loading observations (20), preloading dye transfer experiments with PG/C4 cells revealed a robust transfer of calcein to an average of 17.5 ± 9.0 (SD) recipient cells per labeled donor (Figure 7aa'' and Table IV). Transfer typically occurred to the 3rd and 4th orders of neighboring cells (as high as the 7th order was detected).

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Fig. 7. The assessment of GJIC in Cx43 transfectants PG/C4 by the preloading and dye transfer method. Homologous communication was restored in PG/C4, as demonstrated by calcein transfer from preloaded cells (identified by DiI in a' and marked with asterisks in a'') to 47 orders of adjacent cells (a, a' and a''). When PG/C4 transfectants preloaded with calcein/DiI were mixed with HLF, calcein transferred readily to >5 orders of neighboring HLFs (b, b' and b''). Calcein transfer in the reverse direction (HLF cells as donors) was equally robust (c, c' and c''), demonstrating the loss of any asymmetry of heterologous coupling between these cells.
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Table IV. Heterologous communications between Cx43 transfectants PG/C4 and HLF as assayed by the preloading of calcein method
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As predicted, when heterologous GJIC between HLF and this Cx43 transfectant, PG/C4, was tested, no asymmetry in dye transfer was evident. HLF to PG/C4 transfer reached far beyond first order cells to an average of 24.1 ± 7.1 (SD) acceptor cells per donor, corresponding to 46 orders of neighboring PG/C4 cells per labeled HLF (Figure7cc' and Table IV). PG/C4 transfer to HLF shows similar levels of transfer, with 20.5 ± 9.7 (SD) acceptor cells per donor (Figure 7bb'' and Table IV). Hence, equalizing expression of Cx43 in both cell populations eliminated any asymmetric dye flux between normal and transformed cell populations.
Efficient coupling between HLF and PG/C4 correlates with established Cx43 GJs
To investigate the composition of the junctions that form between HLFs and the various tumor cell lines studied, immunocytochemical staining was performed with antibodies to Cx43 and Cx45, the only Cxs detected in these cells at the RNA level. To distinguish cell types, HLF cells were infected with GFP-pAdeasy adenoviruses. Twenty-four hours after infection,
100% of HLFs were positive for GFP, with a m.o.i. of 100. After an additional 24 h growth, intensity was reduced in
10% of the cells, but even in these cases it was still visible (data not shown) and allowed reliable separation of GFP-negative cells in co-culture.
Although weak heterologous communication was detected between the HLF and lung carcinoma cells, it was difficult to detect any Cx43 or Cx45 labeling above background, either between the tumor cells or at the interface with HLF cells (Figure 8AC). Given the minimal levels of coupling seen in this system, it is likely that these proteins are below the detectable levels.

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Fig. 8. Representative photograph of Cx43 immunocytochemical staining in the co-culture of HLF (green cells, infected with GFP-pAdeasy adenoviruses) and lung carcinoma cells. Only HLF-PG (AC) and HLF-PG/C4 (DF) are shown. Note that Cx43 proteins were not found in the junctions of HLF and PG, while abundant Cx43-positive spots were localized to the borders of HLF and Cx43 transfectant PG/C4.
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In contrast, abundant Cx43 labeling was seen both between PG/C4 cells and at the interface of HLF and PG/C4 cells (Figure 8DF). No Cx45 labeling was detected (data not shown). These observations further reinforce the previous conclusion that dye transfer rates correlate with Cx43 expression.
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Discussion
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Cesen-Cummings et al. (32) extensively analyzed GJIC and Cx expression in several mouse and human lung carcinoma cell lines and found that most, although not all, had much lower levels of dye coupling and Cx43 expression compared with their non-transformed counterparts. Our results extend their findings. While these carcinoma cells consistently showed reduced Cx43 mRNA and protein levels, they express Cx45 mRNA at different levels, with PG and Calu-6 cells containing comparable levels of Cx45 to HLFs. However, the latter protein could not be detected with the available antibodies, suggesting minimal translation of the protein. RTPCR and/or northern blot analysis suggest that other Cxs associated with lung tissue, such as Cx26, Cx30.3, Cx32, Cx37, Cx40 and Cx46, were not significantly transcribed. Whether this reflects a change induced by culture conditions or a property of the transformed phenotype remains unknown. Overall, the expression of Cx43, albeit minimal in most tumor cells, correlated well with the dye coupling observed. For example, Calu-6 and Calu-3 cells show the highest Cx43 RNA expression as well as the best coupling. However, the limited homologous coupling of tumor cells and their malignant behavior did not show any correlation. Given the low levels of homologous coupling, it is not surprising that heterologous coupling with normal HLFs was also minimal and only readily demonstrable in preloading assays from HLF to tumor cells. Transfection of Cx43 into PG cells increased both homologous coupling of the PG cells and heterologous communication between HLF and PG cells. This also resulted in restricted growth both in vitro and in vivo (20). Hence, while Cx43 coupling seems to regulate the transformed phenotype, this only seems true if it exceeds a certain minimum threshold, which correlates with the loss of apparent asymmetry in dye transfer between normal and tumorigenic cells.
Flagg-Newton and Loewenstein (56) first reported asymmetric GJIC in a co-culture system of Balb/c and B fibroblasts. Then, Robinson et al. (57) reported that unidirectional diffusion of dye appeared to occur between different types of neuroglia (astrocytes, oligodendrocytes and Müller cells) in the myelinated band of the intact rabbit retina. Later, Zahs and Newman (58) found similar results in rat retina. However, there has long been controversy about the physical basis for this rectification. All of the reported cases occur between different cell types that often express different connexins (5658). Hence, rectification has often been attributed to the properties of heterotypic gap junction channels, some of which have been shown to display electrical rectifying properties (59). However, electrical coupling is a process driven by external application of a force in the form of a voltage gradient. Dye transfer is a passive, diffusive process that cannot be unidirectional, for the same barriers to transit are encountered in each direction. Even if diffusion rates could differ in the two directions, at equilibrium gradients cannot form without the input of energy (Newton's second law).
An alternative approach is to focus on the properties of the system as a whole, and not just the heterotypic junctions at the interface of the two cell types. For example, in the case studied here, since the tumor cells show much lower levels of Cx43, homologous permeability of the different cell populations is different. Because of the limited expression of Cx43 in the tumor cells, there is also low heterologous coupling capacity between the cell types, although this might be expected to be somewhat greater than homologous coupling between the tumor cells as one partner would contribute higher levels of Cx43 in the heterologous case. Hence, asymmetric transfer of the dye between these two kinds of cells can occur as illustrated in the model in Figure 9. When transfer from HLF to tumor cells is recorded, the dye moves through a poorly coupled interface into an even more poorly coupled population of cells, so the dye that does pass will accumulate in just the first order cells and exceed the detectable limit. In contrast, in the reverse direction, the dye is also moving across a poorly coupled interface, but into a very well coupled recipient population that is in great excess, where the dye is rapidly diluted in the neighboring HLF cells and never accumulates to detectable levels. Hence, this asymmetric transfer is a product of low initial heterologous transfer and the effective sizes of the recipient population, which is restricted in one case (HLF to tumor cells) but not in the other (tumor cells to HLF). This was verified by the observation that calcein transfer from tumor cells to HLF could be detected if the number of recipients was close to the number of donors, thereby eliminating dilution of the dye into neighboring HLF cells. Transfection of tumor cells increases homologous coupling as well as heterologous coupling with HLF. Hence, the initial transfer from donor to recipient is much more efficient, so that even if the dye is diluted among the recipients, it is also flowing into the recipient pool much faster and hence maintains high enough levels for detection. The other claims of unidirectional GJIC (5658) could have been misinterpreted similarly. For example, Robinson (57) observed no spread from a small cell to a large cell, but did see spread from a large cell to a small one, again indicating a dependence on the size of the recipient pool (in this case one cell of different size). Initial quantitative studies in Xenopus oocyte pairs provide no evidence for asymmetric dye transfer through several different types of heterotypic channels if the donor and recipient cells are of similar size (Weber and Nicholson, personal observation).

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Fig. 9. A model of asymmetric transfer of dye between HLFs (squares) and tumor cells (circles) based on the concentration and dilution hypothesis. (A) When the dye is moving from HLF through a poorly coupled interface into a poorly coupled population of cells, the small amount of the dye is concentrated to a level sufficient for detection in first order neighboring cells. (B) Dye transferred across the same poorly coupled heterologous interface from tumor cells to HLF cells is rapidly diluted among well-coupled fibroblasts and falls below the detectable limit.
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The asymmetrical dye transfer between HLF and tumor cells we observed here relates directly to the original hypothesis for growth regulation by dilution through gap junctions proposed by Loewenstein (36,52). Metabolic signals can accumulate to critical levels in first order neighbors of HLF cells through transfer from a well-coupled HLF cell to a poorly coupled tumor cell population (as indicated by detection of fluorescence). However, this will not occur in the reverse direction. This could place the immediate neighboring tumor cells under the growth control of HLF to a certain degree, while preventing oncogenic signals from accumulating in normal cells to levels sufficient for fast malignant transformation, as they would be rapidly diluted into the well-coupled HLF population. This is particularly true when considering natural metabolites that are degraded rapidly within the cells (60,61).
It is generally believed that tumor cells exist widely in healthy people. Our results can explain why they frequently do not form visible tumors. Individual tumor cells would still be subject to regulation by surrounding normal cells. Only when they form a colony of sufficient size such that the tumor cells are removed from contact with normal cells by two or three orders of poorly coupled neighbors would they be released from the growth restriction imposed by normal cells. Our initial observation that the low levels of GJIC recorded between tumor cells did not correlate with the metastatic or tumorigenic phenotype is consistent with the above model if coupling levels must exceed a certain threshold to allow propagation of signals over a sufficient distance. Hence, the tumor suppressive effect was seen once Cx43 was expressed in much higher levels in PG/C4 cells after transfection. Importantly, this study emphasizes the importance of considering the dynamics of the whole system and, particularly, considerations of the pool size of shared metabolites in tumor cells and the surrounding normal tissue.
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Notes
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4 To whom correspondence should be addressed.Email: zqzhang{at}public3.bta.net.cn 
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Acknowledgments
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Thanks are due to Dr Klein-Szanto of the Fox Chase Cancer Center and Dr B.Q.Wu of Peking University Medical Center for providing human lung carcinoma cell lines, and to Dr Weicheng You for critical reading of the manuscript. This study was supported by a UICC-YY Memorial International Cancer Study Grant, a National Major Basic Research Development Program (G2000057002), the National Natural Science Foundation of China (39880016-30270658), the NNSF/China and MRC/Canada Exchange Program, Beijing Municipal Natural Science Foundation (7021001), Peking University Cancer Center and Gene Center for Human Diseases (2000-A-28) and the NIH (CA480490 to B.J.N.).
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Received February 4, 2003;
revised November 12, 2003;
accepted November 17, 2003.