Modulation of homologous gap junctional intercellular communication of human dermal fibroblasts via a paracrine factor(s) generated by squamous tumor cells

Dominik Stuhlmann1,*, Niloofar Ale-Agha1,*, Roland Reinehr2, Holger Steinbrenner1, Maria C. Ramos1, Helmut Sies1 and Peter Brenneisen1,3

1 Institute for Biochemistry and Molecular Biology I and 2 Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Loss of gap junctional intercellular communication (GJIC) is a characteristic of cancer cells. Since a coordinated interaction of epithelial tumor cells with stromal cells is a prerequisite for tumor invasion and metastasis, the present study was designed to test the hypothesis that skin-derived tumor cells may modulate homologous and heterologous GJIC. While homologous GJIC of human dermal fibroblasts as well as epidermal keratinocytes was detected, no communication was measured between SCL-1 cells derived from squamous cell carcinoma of human skin. Interestingly, co-cultures of dermal fibroblasts and SCL-1 tumor cells in serum-containing medium resulted in a 52–70% lowering of the number of communicating fibroblasts. Furthermore, incubation of confluent fibroblast cultures with serum-free supernatant fractions (20–30 kDa) from tumor cells, termed the 20/30 fraction, lowered the homologous gap junction communication of fibroblasts by >90%. This novel aspect of down-regulated homologous GJIC of dermal fibroblasts, which is reversible, was neither mediated by alteration of the expression of connexin43, the major gap junctional protein of dermal fibroblasts, nor by aberrant localization of connexin43 in the plasma membrane. Furthermore, post-translational modifications of connexins, such as phosphorylation, was not measured by mobility shift studies. Tumor cell-mediated GJIC down-regulation between fibroblasts was suppressed using EGTA-containing serum-free tumor cell-derived supernatants suggesting that calcium ions (Ca2+) might mediate the transduction of this effect. The involvement of Ca2+ in down-regulation of homologous GJIC of fibroblasts was supported by an increase in fluorescence intensity of the intracellular calcium-sensitive indicator Fura-2 upon treatment of fibroblasts with the active 20/30 fraction. In conclusion, these data establish homologous GJIC of (stromal) fibroblasts as a parameter modulated by a paracrine acting factor(s) of epithelial tumor cells during tumor–stroma interaction of skin cells.

Abbreviations: CaM, calmodulin; CCD, charge coupled device; CK, cytokeratin; Cx, connexin; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; GJIC, gap junctional intercellular communication; HDF, human dermal fibroblasts; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HRP, horseradish peroxidase; MEK1/2, mitogen-activated protein kinase 1/2; MMP, matrix metalloproteinase; MWCO, molecular weight cut-off; NGS, normal goat serum; NHEK, normal human epidermal keratinocytes; PBS, phosphate-buffered saline; SCC, squamous cell carcinoma; SN(2d) HDF, serum-free conditioned medium harvested after 2 days; SN(2d) SCL-1, serum-free supernatants of SCL-1 cells harvested after 2 days; TPA, 12-O-tetradecanoylphorbol-13-acetate


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The UVB radiation-mediated incidence of non-melanoma skin cancers (photocarcinogenesis), including squamous cell carcinoma (SCC) and basal cell carcinoma, has increased dramatically over recent decades, reaching epidemic proportions (1,2).

(Skin) cancer is generally thought to be the result of disruption of the homeostatic regulation mediated by a complex set of stage-specific genetic and epigenetic changes, subsequently leading to hyperproliferation and loss of terminal differentiation as a result of a lack of growth control (3,4). Homeostasis is based on three major forms of communication, extracellular communication via soluble factors which, secondly, triggers intracellular communication via second messengers and complex signal transductions pathways. These pathways may modulate the third form of communication, gap junctional intercellular communication (GJIC). Disruption of one of these can promote the multistep process of (photo)carcinogenesis (5).

As to cellular cross-talk, the most direct pathway is provided by gap junctions, cell-to-cell channels connecting the cytoplasm of neighboring cells and thereby permitting the direct diffusion of ions and small (<1200 Da) molecules (metabolites, cofactors, second messengers, etc.). Each gap junction channel is comprised of two hemichannels or connexons, each formed by the aggregation of six protein subunits belonging to the connexin (Cx) multigene family (5,6).

In human skin, the two most abundant Cxs are Cx26 and Cx43. While Cx26 is mainly expressed in the epidermal basal layer, Cx43 is expressed in dermal fibroblasts and throughout spinous and granular cell layers of the epidermis, but also focally in the basal layer (7,8). Cx26 and Cx43 are differentially expressed in keratinocytes during development of the epidermis (9). Furthermore, GJIC is beneficial in regulation of epidermal wound repair, facilitating synchronization of the contractile forces within granulation tissue during wound contraction (10). Conversely, in vivo and in vitro studies confirmed the disruption of GJIC in basal and squamous cell carcinomas (1113). The surrounding non-neoplastic cells (stromal cells), if tumor cells invade the underlying connective tissue, were not addressed in these studies.

Tumor–stroma interaction is important in melanoma and skin carcinoma development. Tissue invasion as part of tumor progression requires multiple interactions of the tumor cells with the surrounding stroma (14). In that context, it was shown convincingly that paracrine acting factors resulted in both the expression and activation of distinct members of the serine protease and matrix metalloproteinase (MMP) family and enhancement of angiogenesis as prerequisites of tumor invasion (15,16). However, the mechanisms underlying an increase in or inhibition of intercellular communication of malignant keratinocytes with surrounding stromal cells (heterologous GJIC) have not yet been examined. Recently, an increase in Cx43-derived GJIC of glioma cells and stromal astrocytes was demonstrated to contribute to glioma invasion (17).

In the present study, we address the questions of whether there are direct cellular interactions mediated by gap junctions between a malignant SCC cell line and dermal fibroblasts or whether skin-derived tumor cells may modulate GJIC of these fibroblasts. To explore these possibilities in a controlled and easily accessible model, we developed an in vitro tumor/‘stroma’ system in which the human malignant squamous cell line SCL-1 (18) and fibroblasts were co-cultured and grew as monolayers. Using a combined biochemical and molecular biological approach, we report not only a deficiency of GJIC between the tumor cells and the (stromal) fibroblasts (heterologous GJIC), but, interestingly, also that a paracrine acting factor(s) constitutively secreted by the squamous cell line down-regulates intercellular communication between fibroblasts (homologous GJIC) via an increase in intracellular calcium concentration. These data suggest that inhibition of both homologous and heterologous GJIC may support invasion of cancer cells by blocking growth inhibitory or proapoptotic stimuli from neighboring normal cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Cell culture media [Dulbecco's modified Eagle's medium (DMEM) and keratinocyte-SFM medium plus supplements] and TriFastTM reagent were purchased from Invitrogen GmbH (Karlsruhe, Germany) and peqlab Biotechnologie GmbH (Erlangen, Germany), respectively, and the defined fetal calf serum (FCS gold) was from PAA Laboratories (Linz, Austria). All chemicals and biochemicals were obtained from Sigma unless otherwise indicated. Mitogen-activated protein kinase/extracellular regulated kinase 1/2 (MEK1/2) inhibitor U0126 was supplied by Merck Biosciences (Schwalbach, Germany). The protein assay kit (Bio-Rad DC) was from Bio-Rad Laboratories GmbH (München, Germany). Rabbit polyclonal antibody against human Cx43 and mouse monoclonal anti-human cytokeratin (CK) (Pan) were supplied by Sigma (Deisenhofen, Germany) and Zytomed (Berlin, Germany), respectively. Polyclonal horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) antibody used in western blot analysis was obtained from Dianova (Hamburg, Germany). For immunocytochemistry Alexa 546-coupled goat anti-rabbit IgG (H + L), Alexa 488-coupled goat anti-mouse antibody (H + L) and Alexa 488-coupled phalloidin were from Molecular Probes (MoBiTec GmbH, Göttingen, Germany). The ECL Western Blotting kit used to detect mouse and rabbit primary antibodies on western blots in combination with HRP-conjugated secondary antibodies was supplied by Amersham Biosciences (Freiburg, Germany). Superscript II RNase H-Reverse Transcriptase and HotStarTaq DNA Polymerase were purchased from Invitrogen and Qiagen (Hilden, Germany), respectively. The ultrafiltration devices Centriprep YM-10 [10 kDa molecular weight cut-off (MWCO)] and YM-30 (30 kDa MWCO) were supplied by Millipore GmbH (Schwalbach, Germany), Vivaspin 20 filter unit (20 kDa MWCO) was obtained from Vivascience AG (Hannover, Germany).

Cell culture
Human dermal fibroblasts (HDF) were established by outgrowth from foreskin biopsies of healthy donors (19) with an age of 3–6 years. Cells were used at passages 2–10, corresponding to cumulative population doubling levels of 3–21 (20). Normal human epidermal keratinocytes (NHEK) were prepared from circumcised foreskin based on a protocol described earlier (21) and cultures were maintained in keratinocyte-SFM medium supplemented with 0.09 mM calcium, 35 µg/ml bovine pituitary extract, recombinant epidermal growth factor (0.2 ng/ml) and 5 µg/ml gentamycin. Subconfluent NHEK (~75% confluence) were subcultured as described by the manufacturer and were used for the experiments at passages 2–6. Dermal fibroblasts and the SCC line SCL-1, originally derived from the face of a 74-year-old woman (18) (generously provided by Dr Norbert E.Fusenig, DKFZ Heidelberg, Germany), were maintained in DMEM supplemented with glutamine (2 mM), penicillin (400 U/ml), streptomycin (50 mg/ml), 10% defined fetal calf serum (FCS) in a humidified atmosphere of 5% CO2 and 95% air at 37°C. For co-cultures of dermal fibroblasts and SCL-1 tumor cells, the cells were grown on 3.5 cm diameter tissue culture dishes at ratios (HDF:SCL-1) of 1:1, 2:1 or 3:1. These ratios did not change the morphology of the fibroblasts or the tumor cells.

Gap junctional communication assay (Lucifer Yellow transfer)
In initial experiments, confluent HDF/SCL-1 co-cultures, confluent HDF monolayer cultures and subconfluent (~70% confluence) or confluent SCL-1 cell cultures were maintained in DMEM + 10% FCS throughout the experiments. In subsequent experiments, serum-free supernatants of confluent fibroblasts, called ‘conditioned medium’, and subconfluent SCL-1 cells were harvested after 48 h and put on confluent HDF monolayer cultures which were grown in complete DMEM + 10% FCS. Prior to addition of the supernatants, serum-containing medium was removed and the cells were washed with phosphate-buffered saline (PBS). At different time points thereafter, the cells were used for microinjection. GJIC was measured by microinjection of the fluorescent dye Lucifer Yellow CH (10% in 0.33 M LiCl) into selected fibroblasts or tumor cells by means of a micromanipulator and a microinjector system (Eppendorf, Hamburg).

In co-culture experiments, only those fibroblasts and SCL-1 cells were microinjected which have been surrounded by other cells of the same type. One minute after injection, the number of fluorescent cells around a single cell loaded with the dye were counted. Ten individual cells per dish were injected and medians, 25% quartiles and 75% quartiles were calculated (see below). Images were taken with a Zeiss Axiovert fluorescence microscope with a charge coupled device (CCD) camera (ORCA II; Hamamatsu, Herrsching, Germany).

Immunocytochemistry
For immunocytochemistry, confluent co-cultures, confluent HDF cells, subconfluent NHEK and confluent SCL-1 monolayer cultures were grown in complete medium with FCS or keratinocyte medium supplement on coverslips in 3.5 cm diameter culture dishes before use. Cells were washed with PBS and fixed with methanol for 10 min at -20°C or 3.7% formaldehyde/PBS (F-actin staining) for 10 min at room temperature. Thereafter the cells were incubated for 3 min with 0.1% Triton X-100/PBS (F-actin staining). After washing with PBS, non-specific binding of antibodies was blocked with 3% normal goat serum (NGS) in PBS containing 0.3% (v/v) Triton X-100 for 45 min at room temperature. Cells were incubated with a polyclonal anti-Cx43 antibody or a combination of anti-Cx43 and anti-CK, both diluted 1:1500 in 1% (v/v) NGS/PBS overnight at 4°C. Thereafter, cells were washed three times with PBS and incubated with an Alexa 546-coupled goat anti-rabbit IgG (1:800 diluted in PBS) and/or Alexa 488-conjugated goat anti-mouse IgG (1:1000 diluted in PBS) for 45 min at 37°C. For double staining of Cx43 and F-actin, cells were incubated with an Alexa 546-coupled goat anti-rabbit IgG and Alexa 488-phalloidin (1:80 diluted in PBS, 200 U/ml stock), respectively, for 30 min at 37°C. For DAPI staining, cells were incubated for 10 min at room temperature with 1:1000 diluted DAPI solution (stock solution 0.5 mg/10 ml H2O) in McIlvaine's buffer (100 mM citric acid, 200 mM Na2HPO4, pH 7.2). After washing and embedding, images were taken with a Zeiss Axiovert fluorescence microscope coupled to a CCD camera (ORCA II).

Protein fractionation (ultrafiltration)
Prior to ultrafiltration, precooled (4°C) cell culture supernatants were filtered through a 0.2 µm syringe filter to remove any cellular debris. Ultrafiltration of tumor cell supernatants with Centriprep YM-10 (10 kDa MWCO) at 1500 g (4°C) resulted in a >10 kDa retentate. This concentrated retentate was brought to the original volume with serum-free medium and thereafter filtrated through Centriprep YM-30 (30 kDa MWCO) at 1500 g (4°C), resulting in a >10–<30 kDa filtrate and a >30 kDa fraction. The filtrate was spun down with Vivaspin 20 (5000 g, 4°C) and a concentrated >20–<30 kDa fraction was obtained which was brought to the original volume with serum-free medium or PBS, 5 mM glucose, 20 µM CaCl2 buffer. All fractions were tested for biological activity concerning modulation of intercellular communication.

Reverse transcriptase–polymerase chain reaction (RT–PCR)
Total RNA was extracted using the TriFastTM reagent according to the manufacturer's protocol. Reverse transcription of 1 µg of total RNA was performed in a volume of 20 µl for 1 h at 42°C using 100 U Superscript II RNase H-Reverse Transcriptase in 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, 1 mM each dNTP and oligo(dT)12–18 (0.5 µg/20 µl). The samples were heated to 70°C for 15 min to terminate the reverse transcription reaction. Reverse transcribed cDNA (100 ng) was added to a reaction mixture containing a final concentration of 20 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM each dNTP, 0.5 µM each sense and antisense primer and 2.5 U HotStarTaq Polymerase in a final reaction volume of 50 µl. The two primer sequences used were human Cx43 (sense, 5'-CTATGTGATGCGAAAGGAAGAGAA-3'; antisense, 5'-ATCGGGGAAAT C-AAAAGGCTGTG-3') and human hypoxanthine-guanine phosphoribosyltransferase (HPRT) (sense, 5'-ATTCTTTGCTGACCTGCT GGATT-3'; antisense, 5'-CTTAGGCTTTGTATTTTGCTTTTC-3'). The mixture was heated to 95°C for 15 min. The amplification was performed for 38 (Cx43) or 34 (HPRT) sequential cycles at 94°C for 30 s, 56.5°C (Cx43) or 53°C (HPRT) for 30 s and 72°C for 60 s, followed by an incubation of 10 min at 72°C. The PCR products (727 bp for Cx43, 440 bp for HPRT) were analyzed on a 1.0% agarose gel which contained 10 µg/ml ethidium bromide in 0.5x Tris–borate/EDTA buffer. Densitometric analysis was performed using Scion Image software (Scion Corp., Frederick, MD). Densitometric data were standardized to HPRT fragment and represent x-fold increase over Cx43 mRNA level of fibroblasts incubated with conditioned medium, which was set at 1.0.

SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis
SDS–PAGE was performed according to the standard protocols published elsewhere (22), with minor modifications. Briefly, cells were lysed in 2x SDS–PAGE buffer (125 mM Tris–HCl, 4% w/v SDS, 20% w/v glycerol, 100 mM dithiothreitol, pH 6.8). After sonication, the protein amount of the samples was determined using a modified Lowry method (Bio-Rad DC). Thereafter, bromophenol blue was added (0.1% final concentration), the lysates were heated for 5 min at 95°C and the samples (10 µg total protein/lane) were applied to 10% (w/v) SDS–polyacrylamide gels. After semi-dry blotting to nitrocellulose filters (Schleicher & Schuell, Dassel, Germany), the filters were incubated with a 1:1000 dilution of primary antibody (rabbit polyclonal anti-human Cx43) and a 1:5000 dilution of anti-rabbit secondary antibody conjugated to HRP. Antigen–antibody complexes were visualized by enhanced chemiluminescence (ECL) and exposed to Hyperfilm-ECL (Amersham Biosciences). Densitometric analysis was performed using Scion Image software.

Measurement of intracellular Ca2+ concentration [Ca2+]i
Human dermal fibroblasts were grown to confluence on coverslips in 3.5 cm diameter culture dishes in phenol red-free DMEM plus supplements. Cells were washed with FCS-free DMEM and loaded with 5 µM Fura-2/acetoxymethyl ester for 30 min. After the loading period extracellular dye was removed and the coverslips were fixed in a perfusion chamber on an inverted fluorescence microscope (Zeiss, Oberkochen, Germany) and covered with conditioned medium resulting in a total bath volume of 200 µl. Cells were then alternately excited at 340 and 380 nm, respectively, for up to 30 min at a rate of 2 Hz by a monochromator, and emission was detected at 480–520 nm using a CCD camera as provided by the QuantiCell 2000® calcium imaging set-up (VisiTech, Sunderland, UK). [Ca2+]i is given as the ratio of the fluorescence measured at 340 (bound calcium) and 380 nm (free calcium) excitation wavelengths. After ~5 min, 100 µl of a 3-fold concentrated fraction of tumor cell-derived supernatant was added. At the end of each experiment 10 µl of a 100 mM ATP solution was instilled, resulting in a final concentration of 3.2 mM ATP.

Statistical analysis
In order to quantify the distribution of the number of communicating cells, box-and-whisker plots (box plot) were used which highlight and combine important parameters describing the scatter of the sample data: the median (line inside the box), 25% quartile (lower boundary of the box), 75% quartile (upper boundary of the box) and minimum and maximum values (vertical lines from the box, ‘whiskers’). In this study, box plots indicate a non-Gaussian distribution because the medians are not located in the middle of the box, the whiskers of a box are different in length and the boxes are of different sizes (inhomogeneity of the variances). Therefore, for analysis of statistical significance, non-parametric tests such as the Mann–Whitney test, Wilcoxon test and Kruskal–Wallis test were applied (23). Before the study P < 0.05, P < 0.01 and P < 0.001 were selected as the levels of significance; P > 0.05 indicated no significance.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cx43 protein in dermal fibroblasts, epidermal keratinocytes and epithelial tumor cells
The distribution of Cx43 protein within human dermal fibroblasts (Figure 1A), NHEK cells (Figure 1D) and SCC cell line SCL-1 (Figure 1G) was investigated by immunostaining. In confluent human dermal fibroblasts Cx43 proteins were localized at the cell membrane, with some protein signal detectable within the cytoplasm (Figure 1A). Figure 1B represents staining of the nuclei of the cells of Figure 1A. In contrast to confluent fibroblasts, only subconfluent NHEK cells showed Cx43 proteins located at the plasma membrane (Figure 1D). In addition, a bright fluorescence within the cytoplasm, but only close to the nuclei (Figure 1E), was detectable in normal keratinocytes, suggesting a higher amount of Cx43 protein within the cytoplasm (Figure 1D). Confluent and differentiated NHEK alter the expression pattern of their Cxs (8,9). Accordingly, Cx43 was not detected in confluent keratinocyte monolayer cultures (unpublished data). Localization of Cx43 protein at the cell membrane of fibroblasts as well as subconfluent keratinocytes suggests intercellular communication within both these non-tumorigenic cell types.



View larger version (186K):
[in this window]
[in a new window]
 
Fig. 1. Subcellular distribution of Cx43 in different cell types. (AC) HDF, (DF) NHEK and (GI) SCC SCL-1 were used for Cx43 immunofluorescence. (A) HDF showed Cx43 protein (arrowheads) at the membrane surface and some signal within the cytoplasm. (B) DAPI stained nuclei of HDF shown in (A). In contrast to confluent SCL-1 tumor cells (G), NHEK (D) showed Cx43 protein at the surface of the cell membrane (arrowheads, no cytokeratin staining performed). (D and G) High Cx43 fluorescence located around the nuclei. (E) DAPI stained nuclei of NHEK shown in (D). (H) Cytokeratin staining of the cells shown in (G). (C, F and I) Morphology of confluent HDF (C), subconfluent NHEK (F) and confluent SCL-1 cells (I). (A, B, D, E, G and H) Bar, 25 µm; (C, F and I) bar, 40 µm.

 
In comparison with confluent fibroblasts or subconfluent normal keratinocytes, in subconfluent (unpublished data) and confluent SCC SCL-1 cell cultures almost no Cx43 protein was translocated to the cell membrane. Rather, these cells showed a bright Cx43 fluorescence close to the nucleus (Figure 1G). This aberrant localization of Cx43 indicated an abrogated GJIC between the tumor cells, which has been described as a landmark of malignant transformation (5).

Furthermore, a tight network of specific CKs was detected in SCL-1 cells (Figure 1H) which did not exist in fibroblasts (Figure 3B). The antibody cocktail (Pan) used in this study recognizes, for example, CK4, CK5, CK6, CK10, CK13 and CK17, which identify the putative tissue origin of a (metastatic) carcinoma (24,25). Figure 1C, F and I show the characteristic morphology of confluent dermal fibroblasts, subconfluent epidermal keratinocytes and confluent SCL-1 tumor cells.



View larger version (161K):
[in this window]
[in a new window]
 
Fig. 3. Expression pattern of Cx43 at the membrane surface of dermal fibroblasts and squamous tumor cells in a co-culture system. (A) Confluent HDF showed Cx43 proteinn (arrowheads) at the membrane surface and some signal within the cytoplasm. (B) Cytokeratin staining and (C) DAPI stained nuclei of cells shown in (A). (D) Cx43 distribution (arrowheads) of co-cultured fibroblasts and SCL-1 tumor cells at ratios of 2:1 is independent of attendance of tumor cells compared with fibroblasts (A). (E) Cytokeratin staining of SCL-1 tumor cells in the co-culture system. (F) DAPI stained nuclei of cells shown in (D). Morphology of confluent HDF (G) and co-cultured fibroblasts (f) and SCL-1 tumor cells (t) at a ratio of 2:1 (H) used for immunofluorescence. (A–F) Bar, 25 µm; (G and H) bar, 33 µm.

 
Down-regulation of GJIC between dermal fibroblasts co-cultured with squamous tumor cell line SCL-1
In order to study GJIC between (stromal) fibroblasts during tumor–stroma interaction, dermal fibroblasts were co-cultured with SCL-1 tumor cells. Measurements of GJIC between fibroblasts by microinjection of Lucifer Yellow dye into selected fibroblasts, which were surrounded by cells of the same cell type, were performed with confluent co-cultures maintained in medium containing 10% FCS (Figure 2). Comparing the medians of the box plots, co-cultures of fibroblasts with tumor cells at different ratios (Figure 2C) resulted in a 52–70% lowering of the number of communicating fibroblasts compared with pure fibroblast monolayer cultures (Figure 2A) (P < 0.0001). The middle 50% of the values (interquartile range) indicated by the box length ranged between 10.0 and 19.0 for fibroblast monolayer cultures, while the values for the co-cultures were significantly lowered, ranging between 3.0 and 7.0 and between 4.0 and 8.0, respectively. In contrast to pure fibroblast cultures, microinjected SCL-1 tumor cells (Figure 2C) were unable to transfer dye to neighboring SCL-1 cells or fibroblasts.



View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2. GJIC capacity of dermal fibroblasts and SCL-1 tumor cells. The number of stained collateral cells adjacent to the injected cell (see micrographs A–C) was used as a measure of GJIC. Box plots show a significant down-regulation of the number of communicating fibroblasts (C) co-cultured with SCL-1 cells at ratios of 1:1 and 2:1 compared with fibroblast monolayer cultures (A). (B) GJIC between SCL-1 tumor cells was completely abrogated. The cells were maintained in DMEM + 10% FCS throughout the experiments. The upper and lower boundaries of the box are the upper and lower quartiles. The box length is the interquartile distance so the box contains the middle 50% of values (the horizontal line inside the box indicates the median). The vertical lines extend to maximum and minimum values. The values of the box plots represent the number of communicating cells of 30 microinjected cells (10 cells/dish). Similar results were obtained in two independent experiments. ***P < 0.001 versus fibroblast monolayer cultures (Kruskal–Wallis test).

 
Next, we addressed the question of whether the distribution of Cx43 protein between human dermal fibroblasts is modulated by tumor cells in the co-culture system. Fibroblast monolayer cultures served as controls and showed a typical Cx43 distribution at the cell membrane (Figure 3A). Interestingly, the Cx43 distribution at the cell membrane of the fibroblasts was not changed in the presence of SCL-1 cells, which was independent of the investigated fibroblast/tumor cell ratios (Figure 3D).

To distinguish fibroblasts and tumor cells in the co-culture system, cytokeratin staining was performed. In contrast to fibroblasts used as negative controls (Figure 3B), SCL-1 tumor cells showed a bright cytokeratin staining in the co-culture system (Figure 3E). Figure 3C and F represent DAPI stained nuclei of the cells shown in Figure 3A and D. The morphology of the confluent fibroblasts (Figure 3H) did not change in the co-culture system (Figure 3G). In evidence, the tumor cells (t) grew in clusters between the (stromal) fibroblasts (f) (Figure 3G).

Tumor cell-dependent down-regulation of homologous GJIC of fibroblasts
Data obtained from co-culture experiments suggested paracrine modulation of GJIC communication between fibroblasts via squamous cell line SCL-1. To test this hypothesis, serum-free (-FCS) and serum-containing (+FCS) supernatants of subconfluent SCL-1 monolayer cultures were harvested after 1 or 2 days and added to fibroblast monolayer cultures for 24 h. As controls, serum-free and serum-containing conditioned medium was harvested after 1 or 2 days and added to confluent fibroblasts which were subjected to gap junctional communication assays (Figure 4A). In comparison with these controls (-FCS, median = 8.5; +FCS, median = 9.0), confluent fibroblasts incubated with both supernatants of the tumor cells exhibited an almost complete loss of GJIC (-FCS, median = 0) or significant down-regulation of GJIC (+FCS, median = 5). Furthermore, the interquartile ranges (indicated by the box length) of the fibroblasts treated with supernatants of tumor cells were significantly different from the ranges of the appropriate controls treated with conditioned medium (Figure 4A). However, the use of serum-free tumor cell-derived supernatants resulted in a stronger down-regulation of homologous GJIC of fibroblasts compared with serum-containing supernatants. We hypothesize that the fetal calf serum contains a substance(s) which counteracts the observed effects and/or inhibits, at least in part, the appropriate soluble factor(s).




View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. Paracrine modulation of GJIC between fibroblasts. The number of stained collateral cells adjacent to the injected cell (see micrographs) was used as a measure of GJIC. (A) Confluent fibroblasts (HDF) were incubated with serum-free (-FCS) or serum-containing (+FCS) conditioned medium [SN(2d) HDF] and supernatants of SCL-1 cells [SN(2d) SCL-1], both harvested after 2 days. Box plots show a significant down-regulation of the number of communicating fibroblasts incubated with both SCL-1 supernatants for 24 h. (B) HDF were incubated with different FCS-free fractions (<20, >20, <30 and >30 kDa) of SCL-1 tumor cell-derived supernatants harvested after 2 days [SN(2d) SCL-1]. Box plots indicate a significant lowering of homologous GJIC of fibroblasts with the >20 kDa and <30 kDa fractions. (A and B) The values of the box plots represent the number of communicating cells of 20 microinjected cells (10 cells/dish). Experiments were performed in triplicate (A) or duplicate (B). (A) ***P < 0.001 versus controls [SN(2d) HDF, -FCS]; ***P < 0.001 versus controls [SN(2d) HDF, +FCS] (Mann–Whitney test); (B) #P > 0.05 (Mann–Whitney test).

 
Supernatants harvested after 1 day showed similar results throughout the experiments (unpublished data). Additionally, supernatants of the human epidermoid carcinoma cell line A431 [European Collection of Cell Cultures (ECACC), Salisbury, UK] and human malignant melanoma cell line A375 (ECACC) showed identical results regarding down-regulation of GJIC between fibroblasts (unpublished data). No loss of cell viability was detected after treatment of dermal fibroblasts with tumor cell-derived supernatants (unpublished data). To exclude that acidification of the cell culture supernatants (26) is responsible for the observed results, prior to incubation of fibroblasts with conditioned medium or tumor cell-derived supernatants, the pH values of the two different supernatants were tested, both ranging between 7.4 and 7.5.

To estimate the relative molecular mass(es) of the biologically active factor(s) of tumor cell supernatants, commercially available membrane filter units were used. Using this fractionation assay, the molecular masses of the assumed paracrine acting factor(s) was measured to be between 20 and 30 kDa, herein called the 20/30 fraction, showing a significant down-regulation of homologous GJIC of fibroblasts (Figure 4B). In comparison with the <20 kDa fraction (median = 6) and >30 kDa fraction (median = 7), the ‘active’ 20/30 fraction resulted in medians ranging between 1 and 2. As heating of the 20/30 fraction (5 min at 90°C) prevented down-regulation of homologous GJIC of fibroblasts, we may speculate that the 20/30 fraction contains ‘active’ protein(s).

Time course studies revealed that initiation of tumor cell-mediated down-regulation of GJIC between fibroblasts was an early event (Table I). Fibroblasts were incubated with serum-free conditioned medium harvested after 2 days [SN(2d) HDF] or serum-free supernatants of SCL-1 cells [SN(2d) SCL-1]. A significant down-regulation of GJIC was detected, starting 0.5–1 h after incubation with the supernatant of the tumor cells and persisting during the studied time period of 24 h. In experiments independent of the time course studies (Table I), fibroblasts were treated for 48 h with tumor cell-derived supernatants. The GJIC down-regulating capacity of these supernatants was identical to the data observed after 24 h (unpublished data). While the medians of the number of communicating fibroblasts used as controls ranged between 6.0 and 10.0 throughout the 24 h time interval, fibroblast monolayer cultures incubated with tumor cell supernatants showed significantly lowered medians ranging between 2.5 at 1.0 h and 0 at 24 h after incubation (Table I).


View this table:
[in this window]
[in a new window]
 
Table I. Time course analysis of paracrine modulation of GJIC between HDF

 
Reversibility of homologous GJIC or its protection by selenite supplementation
In order to investigate reversibility of tumor cell-initiated down-regulation of GJIC between confluent fibroblasts, fibroblasts were incubated for 1.5 h with SN(2d) SCL-1 (Figure 5). A significant lowering of the number of communicating fibroblasts was observed (median = 2.0) compared with fibroblast controls (median = 7.0) incubated with SN(2d) HDF. Thereafter, supernatants of tumor cells were removed and replaced either by conditioned medium or fresh serum-containing culture medium. After incubation for a further 1.5 h, both approaches resulted in a significant increase in GJIC compared with the GJIC of fibroblasts incubated only with supernatants of tumor cells (P < 0.01). The middle 50% of the values of the fibroblasts showing reversible GJIC ranged between 3.0 and 9.0, whereas the values of the fibroblasts incubated with the supernatants of the tumor cells were significantly lowered, ranging between 0 and 3.0 (Figure 5). Interestingly, the strongest effect on the increase in the number of communicating fibroblasts after removal of tumor cell supernatants was mediated by the serum-containing culture medium compared with the fibroblast-derived (conditioned) medium, suggesting an influence of a (soluble) factor(s) which may not be secreted by fibroblasts.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5. Reversibility of tumor cell-mediated GJIC between fibroblasts. Fibroblasts (HDF) were incubated with serum-free (-FCS) conditioned medium harvested after 2 days [SN(2d) HDF] or serum-free supernatants of SCL-1 cells [SN(2d) SCL-1] for 1.5 h. Furthermore, fibroblasts were incubated with serum-free supernatants of SCL-1 cells [SN(2d) SCL-1] for 1.5 h and, thereafter, with SN(2d) HDF or fresh FCS-containing DMEM for an additional 1.5 h. Box plots show that the SCL-1 tumor cell-dependent down-regulation of the number of communicating fibroblasts was reversed. The values of the box plots represent the number of communicating cells of 30 microinjected cells (10 cells/dish). Similar results were obtained in two independent experiments. **P < 0.01 versus SN(2d) SCL-1 (Friedman test).

 
Since epidemiological studies and human clinical intervention trials support a protective role of selenium against cancer development (27) and Sharov et al. (28) convincingly showed protection of gap junctional communication from peroxynitrite by selenite supplementation, we addressed the question of whether selenite supplementation protects dermal fibroblasts from tumor cell-mediated down-regulation of homologous GJIC. Therefore, confluent fibroblast monolayer cultures were incubated with a non-toxic concentration of sodium selenite (29) prior to treatment with supernatant of tumor cells or conditioned medium (Figure 6). The presence of selenite had no effect on intercellular communication in control cultures. Interestingly, tumor cell-mediated down-regulation of GJIC between fibroblasts was significantly prevented after selenite supplementation compared with selenite-deficient fibroblast cultures, which was represented by the differences in the medians and interquartile ranges (Figure 6).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. Protection of homologous GJIC by selenite supplementation. Confluent fibroblast (HDF) monolayer cultures were incubated with 0.5 µM sodium selenite for 48 h prior to treatment with supernatant of tumor cells [SN(2d) SCL-1] or conditioned medium [SN(2d) HDF] for 1 h. Box plots show that the tumor cell-dependent down-regulation of the number of communicating fibroblasts was prevented by sodium selenite. The values of the box plots represent the number of communicating cells of 20 microinjected cells (10 cells/dish) per experiment. Data are representative of two independent experiments. ***P < 0.001 (Mann–Whitney test).

 
Tumor cell-mediated down-regulation of GJIC between fibroblasts is independent of Cx43 expression and phosphorylation
To investigate the molecular mechanisms underlying the above described down-regulation of GJIC between fibroblasts, confluent fibroblasts (HDF) preincubated for 24 h either with serum-free conditioned medium or serum-free supernatant of tumor cells were subjected to RT–PCR to determine supernatant-dependent effects on the level of transcription (Figure 7A). As a positive control, fibroblasts were stimulated with 1,25-dihydroxyvitamin D3 (calcitriol) as described earlier (30). In contrast to the calcitriol-stimulated cells (lane 3), showing a 1.8-fold increase in the steady-state Cx43 mRNA level, fibroblasts incubated with serum-free supernatants of SCL-1 tumor cells (lane 2) showed no significant alteration in Cx43 mRNA level (0.9-fold) compared with fibroblasts incubated for 24 h with serum-free conditioned medium (lane 1), indicating that tumor cell-triggered GJIC down-regulation between fibroblasts seems to be independent of changes in Cx43 transcription.





View larger version (79K):
[in this window]
[in a new window]
 
Fig. 7. Effect of soluble factor(s) on Cx43 mRNA level and Cx43 phosphorylation. (A) Steady-state Cx43 and HPRT mRNA levels were detected by RT–PCR as described in Materials and methods. The ethidium bromide stained bands of 727 and 440 bp correspond to the amplification products obtained using Cx43- and HPRT-specific primers and total RNA extracted from confluent fibroblasts (HDF) incubated with supernatants of tumor cells [SN(2d) SCL-1] or conditioned medium [SN(2d) HDF] for 24 h. As a positive control, fibroblasts were stimulated for 48 h with 10 µM 1,25-dihydroxyvitamin D3 (calcitriol) prior to isolation of total RNA and RT–PCR. (B) Fibroblasts were incubated as described in (A), except that fibroblasts incubated with 100 µM TPA for 15 min were used as a positive control. After cell lysis, phosphorylation of Cx43 was analyzed by western blotting. P0, unphosphorylated form of Cx43; P1 and P2, phosphorylated Cx43. (C) Confluent fibroblasts (HDF) were incubated with fresh serum-containing medium for 1.5 h [HDF (DMEM)], with serum-free supernatants of SCL-1 cells, harvested after 1 day, for 1.5 h [SN(1d) SCL-1 (1.5 h)] or were pretreated with 10 µM U0126-containing medium for 1 h before incubation with U0126-containing SCL-1 supernatants [SN(1d) SCL-1 (1.5 h), 10 µM U0126]. Either SCL-1 supernatant was replaced by fresh serum-containing DMEM for an additional 1.5 h [SN(1d) SCL-1 (1.5 h) + DMEM (1.5 h)] or HDF were pretreated for 1 h with SCL-1 supernatant containing 1 mM sodium orthovanadate (Na3VO4) prior to replacement of this supernatant by Na3VO4-containing DMEM and incubation for a further 1.5 h [SN(1d) SCL-1 (1.5 h) + DMEM (1.5 h), 1 mM Na3VO4]. The values of the box plots represent the number of communicating cells of 30 microinjected cells (10 cells/dish). ***P < 0.001 versus SN(1d) SCL-1 (1.5 h) (Friedman test); #P > 0.05 (Mann–Whitney test). (A and B) Data are representative of three separate experiments; (C) one experiment was performed.

 
In order to check tumor cell-dependent post-translational modifications of Cx43 protein in confluent dermal fibroblasts, the fibroblasts were incubated with either the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), capable of inhibiting GJIC by phosphorylation of Cx43 (31), or with their conditioned medium and tumor cell-derived supernatants, respectively (Figure 7B). An increased phosphorylation of Cx43 was detected in fibroblasts incubated with 100 µM TPA for 15 min (lane 3), as seen from shifts in electrophoretic mobility (P2) and a slight decrease in the amount of unphosphorylated protein (P0). The electrophoretic mobility shift is also demonstrated in densitometric scans (Figure 7B, bottom). In contrast, for both fibroblasts treated with tumor cell supernatant (lane 2) and fibroblasts incubated with conditioned medium (lane 1) the phospho-Cx43 shift was prevented, indicating that tumor cell-mediated lowering in GJIC between fibroblasts may be independent of Cx43 phosphorylation.

To support this hypothesis, confluent fibroblasts were treated with a non-toxic concentration of the highly selective MEK1/2 inhibitor U0126 prior to incubation with U0126-containing tumor-cell derived supernatants (Figure 7C). U0126-containing SCL-1 supernatants (median = 1.5) and SCL-1 supernatants without MEK1/2 inhibitor (median = 0.5) led to similar significantly lowered communication rates between fibroblasts (P > 0.05) compared with the high GJIC of control fibroblasts (median = 12). Further, given that tyrosine phosphorylation was described to be an essential step in the blockade of gap junction communication (6), the involvement of tyrosine phosphatases in reversibility of tumor cell-initiated down-regulation of GJIC between fibroblasts was studied. Therefore, SCL-1 supernatants were removed and replaced by fresh serum-containing culture medium or confluent fibroblasts were pretreated for an additional 1 h with tumor-cell derived supernatants containing a non-toxic concentration of sodium orthovanadate, a broad spectrum potent inhibitor of protein tyrosine phosphatases, prior to replacement of this supernatant by orthovanadate- and serum-containing culture medium (Figure 7C). Both orthovanadate-containing and orthovanadate-free medium resulted in a significant increase in GJIC compared with the GJIC of fibroblasts incubated only with supernatants of tumor cells (P < 0.001).

Calcium influx modulates homologous GJIC of fibroblasts
To study a potential effect of a paracrine factor(s) on intracellular calcium concentration of confluent fibroblasts, 5 mM EGTA was added to SCL-1 supernatants prior to incubation of fibroblasts for 1 h. In contrast to EGTA-free SCL-1 supernatants, the number of communicating fibroblasts was significantly increased, obtaining nearly identical levels as fibroblasts incubated with conditioned medium (Figure 8A). The middle 50% of the values ranged between 6.5 and 10.0 compared with the values of EGTA-free SCL-1 supernatants, ranging between 0.5 and 3.5. In addition, incubation of HDF with the 20/30 fraction buffered in PBS, 5 mM glucose, 20 µM CaCl2 diminished the lowering of the GJIC between fibroblasts which were preincubated with the non-fluorescent intracellular Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester (5 µM, 0.5 h) (unpublished data).




View larger version (60K):
[in this window]
[in a new window]
 
Fig. 8. Increase in [Ca2+]i in confluent dermal fibroblasts on addition of tumor cell supernatants. (A) Confluent HDF incubated with both serum-free conditioned medium [SN(2d) HDF] and tumor cell-derived supernatants [SN(2d) SCL-1] plus 5 mM EGTA showed nearly identical GJIC compared with cells treated with SN(2d) SCL-1. The values of the box plots represent the number of communicating cells of 20 microinjected cells (10 cells/dish) per experiment. (B) Increase in Fura-2 fluorescence intensities ({lambda}340 nm/{lambda}380 nm ratio) indicating an increase in [Ca2+]i in human dermal fibroblasts after addition of tumor cell derived supernatants and ATP, respectively. Fibroblasts (dotted area) loaded with 5 µM Fura-2/acetoxymethyl ester for 30 min showed increased fluorescence after incubation with the 20/30 fraction of SN(2d) SCL-1 (pseudocolour image b) and ATP (c) compared with SN(2d) HDF-incubated cells (a). (A and B) Three independent experiments were performed.

 
These data suggest that changes in intracellular calcium concentrations affect GJIC between fibroblasts. In fact, the active 20/30 fraction induced rapid increases in [Ca2+]i, which, over the studied time range, did not return to the level of [Ca2+]i obtained with conditioned medium (Figure 8B). The ratio (340 nm/380 nm) of the fluorescence intensities, a rough indicator of [Ca2+]i, was <1.0 throughout the experiments, taking into account that the increase in [Ca2+]i upon treatment with supernatants of tumor cells is nonetheless too low (<60 nM) and, therefore, not sufficient for direct closure of the gap junction channels (32). A set of pseudocolour images (Figure 8B, a–c) represents the rapid Ca2+ response to tumor cell-derived supernatants (b) and ATP (c) compared with the effect of conditioned medium (a). The effect of ATP, which was shown to provoke a transient increase in [Ca2+]i (33), always exceeded the increase in fluorescence intensity induced by supernatants of tumor cells, demonstrating that the sensitivity limit of Fura-2/acetoxymethyl ester did not restrict the detection of [Ca2+]i increases.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The carcinogenic process involves the transition from a normal, GJIC-competent cell to one which is defective in GJIC, finally resulting in breakdown of intercellular communication between tumor cells during tumor promotion (5,34,35).

However, the Cx status during tumor–stroma interaction and GJIC between (stromal) fibroblasts and tumor cells or between fibroblasts or tumor cells alone have not been widely studied. The understanding of the molecular mechanisms during tumor progression is crucial for the design and effective use of novel therapeutic strategies to combat invasion and metastasis. In our studies regarding tumor–stroma interaction, we established an in vitro tumor–stroma model using the SCC cell line SCL-1 and dermal fibroblasts in co-culture. This model roughly represents the in vivo situation of SCC invasion described elsewhere (36,37).

In contrast to fibroblast monolayer cultures showing a high level of gap junction-mediated communication herein and also described earlier (38), this type of homologous cell–cell communication was abrogated in the squamous cell line SCL-1. These results perfectly fit with the hypothesis that the Cx gap junction proteins appear to act as tumor suppressors and that their tumor inhibitory effect is usually attributed to their main function of cell coupling through gap junctions (35). In that context, it was obvious that the communication with cells of another type (heterologous GJIC) may be inhibited (35), which was confirmed by co-cultures of SCL-1 with (stromal) fibroblasts. Immunostaining, RT–PCR and western blot analysis revealed that the decrease in homologous and heterologous GJIC of SCL-1 tumor cells is not due to down-regulation of Cx43 expression, but rather to the aberrant localization of Cx43 in the cytoplasm near the nucleus, as described elsewhere (35,39).

Interestingly, a significant down-regulation of homologous cell–cell communication of fibroblasts was observed in our co-culture system, indicating that tumor cells may exert a commanding influence on the surrounding stromal cells regarding GJIC. Such an influence of tumor cells on the surrounding cells was described for the expression of cell adhesion molecules and proteolytic enzymes as prerequisites for tumor malignancy (16,40). Evidence has been provided that the secretion of tumor-derived soluble factors (i.e. cytokines and growth factors) play an important role in the induction of stromal proteases (14,41). Therefore, we tested the effect of soluble factors derived from different tumorigenic cell lines on intercellular communication between fibroblasts seeded as monolayer cultures. Our data convincingly show that one or more soluble factors appears to be responsible for down-regulation of homologous GJIC of the fibroblasts. Studies are underway to identify the factor(s).

Furthermore, homologous GJIC of SCL-1 tumor cells in co-culture with dermal fibroblasts was almost completely abrogated, indicating that surrounding fibroblasts did not affect GJIC between the tumor cells. In contrast to our findings, evidence exists that the inhibition of neoplastic cell growth in co-culture with their normal counterparts is mediated via GJIC. For example, growth suppression of the oncogene-transformed C3H 10T1/2 mouse fibroblast cell line (42) or oncogene-transformed WB-F344 rat liver epithelial cells (43) was mediated by their highly communicating, non-transformed counterparts and involved an increase in GJIC between transformed and non-transformed cells. In addition, King et al. (44) reported that the ability of Cx43-positive clones of the cervical cell line HeLa to proliferate was greatly diminished via GJIC in the presence of a confluent mouse or human fibroblast monolayer culture. In that context, the question arises whether highly aggressive and metastatic squamous cell carcinoma cells or melanoma cells protect themselves from the above described (43,44) growth inhibition via down-regulation of homologous and heterologous GJIC during tumor invasion, as described in this study.

Given that the herein described homologous GJIC blockade occurred early upon treatment with the paracrine acting factor(s), post-translational modifications of the Cx43 protein seem to be responsible for the observed effects, rather than a decrease in Cx43 expression. The importance of a balance between protein phosphorylation (45) and dephosphorylation (46) initiated by growth factors (6), resulting in Cx trafficking, assembly and disassembly, as well as modulation of the open state of junctional channels, has progressively gained support over recent years. However, neither modulation of Cx43 expression or localization nor modulation of the Cx43 phosphorylation state was measured in our study, suggesting that this tumor cell-mediated down-regulation of intercellular communication between fibroblasts might be independent of protein kinases, e.g. mitogen-activated protein kinases or tyrosine kinase pp60src, which normally play a role in closure of gap junctions (6,47).

Another mechanism regulating channel gating includes intracellular levels of calcium ions (31,48). Calcium, acting as a second messenger in different signaling pathways, plays a critical role in numerous physiological and pathological processes. For example, calcium is a key regulator of keratinocyte function during epidermal differentiation (49), and coordinated cell–cell adhesion of fibroblasts via recruitment of cadherins and catenins to the actin cytoskeleton is essential for connective tissue remodeling and wound healing (50). In contrast, increasing extracellular and intracellular calcium concentrations enhanced cellular proliferation and MMP-dependent migration/invasion of oral SCC (51).

Our studies have shown that a tumor cell-derived soluble factor(s) increases intracellular calcium concentration in fibroblasts which resulted in closure of gap junctions and disruption of homologous intercellular communication of fibroblasts, as indicated by dye transfer. It is less clear how calcium regulates gap junction channel permeability. Recently, the importance of functional hemichannels (connexons) was not only described for transduction of cell survival signals (52) and ATP release (53), but also for low extracellular calcium concentrations (53). In contrast, the hemichannels are closed within a range of high extracellular calcium concentrations (1–2 mM) (53,54), which reflect the concentration in the cell culture medium. Taken together, we may speculate that functional hemichannels play no role or an unimportant one in the tumor cell-mediated down-regulation of gap junction-dependent communication between fibroblasts. However, published data suggest that calcium may act on the gap junction channel itself. Even though both the C- and N-termini of Cx proteins do not possess putative calcium-binding sites in their sequence, it was shown by a cysteine mutant (Cx46L35C) that position 35 of the first transmembrane segment of Xenopus Cx46 is responsible for direct calcium-mediated channel closure (54,55).

In addition to these findings, the calcium-binding protein calmodulin (CaM) is associated with Cxs and this may be important in calcium-dependent intercellular communication. The role of CaM in channel regulation was studied in amphibian embryonic cells by using antisense oligonucleotides to CaM mRNA. The cells injected with the antisense molecules retained the capacity for GJIC under different experimental conditions (56). Furthermore, two CaM-binding amino acid sequences in Cx32 provided evidence that CaM may function as an intracellular ligand, uncoupling Ca2+-dependent intercellular communication across gap junctions (57).

The finding in our study that a time delay exists between the tumor cell-mediated rapid increase in [Ca2+]i and down-regulation of GJIC beween fibroblasts argues in favor of calcium acting as a second messenger. However, the soluble factor(s)-mediated down-regulation of GJIC in our model of tumor invasion was observed over a rather long time period and it cannot be excluded that both calcium-dependent cellular activities and direct calcium effects are involved in that GJIC inhibition. Further studies should prove this hypothesis.

In summary, we here report a novel aspect of tumor–stroma interaction, namely that a paracrine acting factor(s) constitutively secreted by a malignant squamous cell line down-regulates homologous GJIC of fibroblasts via an increase in [Ca2+]i, which seems to be mediated by modulation of Ca2+ influx. Together with the loss of heterologous GJIC, these data suggest a mechanism by which tumor cells protect themselves from fibroblast-initiated apoptotic signaling. As invasion and metastatic spread of tumor cells continue to be the greatest barrier to curing cancer, an understanding of the cellular interaction between tumor cells and the surrounding stroma could assist us in the development of novel therapeutic strategies to combat metastasis more efficiently in the future. That micronutrients such as selenium or its metabolites might be a promising tool in that context was shown in a preliminary approach herein. Furthermore, selenium supplementation of patients with a history of basal cell or squamous carcinoma of the skin significantly reduced the overall cancer morbidity, as shown in a randomized controlled trial (58).


    Notes
 
3 To whom correspondence should be addressed at: Heinrich-Heine-University Düsseldorf, Institute for Biochemistry and Molecular Biology I, Building 22.03, Universitätsstrasse 1, D-40225 Düsseldorf, Germany. Tel: ++49 211 811 2715; Fax: ++49 211 811 3029 Email: peterbrenneisen{at}web.de Back

* These authors contributed equally to this work. Back

This article is part of the PhD thesis of D.Stuhlmann at the University DÏsseldorf. Back


    Acknowledgments
 
We are grateful to Claudia Wyrich for excellent technical assistance. H.S. is a Fellow of the National Foundation for Cancer Research (NFCR), Bethesda, MD, USA. Supported by Deutsche Forschungsgemeinschaft SPP Selenoproteine (Si 255/11-2) and SFB 575/B4.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. de Gruijl,F.R. (1999) Skin cancer and solar UV radiation. Eur. J. Cancer, 35, 2003–2009.[CrossRef][ISI][Medline]
  2. Diepgen,T.L. and Mahler,V. (2002) The epidemiology of skin cancer. Br. J. Dermatol., 146 (suppl. 61), 1–6.
  3. Yuspa,S.H. (1998) The pathogenesis of squamous cell cancer: lessons learned from studies of skin carcinogenesis. J. Dermatol. Sci., 17, 1–7.[CrossRef][ISI][Medline]
  4. Matsumura,Y. and Ananthaswamy,H.N. (2002) Molecular mechanisms of photocarcinogenesis. Front. Biosci., 7, D765–D783.[ISI][Medline]
  5. Trosko,J.E. and Ruch,R.J. (1998) Cell–cell communication in carcinogenesis. Front. Biosci., 3, D208–D236.[Medline]
  6. Hossain,M.Z. and Boynton,A.L. (2000) Regulation of Cx43 gap junctions: the gatekeeper and the password. Sci. STKE, 2000, PE1.
  7. Salomon,D., Masgrau,E., Vischer,S., Ullrich,S., Dupont,E., Sappino,P., Saurat,J.H. and Meda,P. (1994) Topography of mammalian connexins in human skin. J. Invest. Dermatol., 103, 240–247.[Abstract]
  8. Richard,G. (2000) Connexins: a connection with the skin. Exp. Dermatol., 9, 77–96.[CrossRef][ISI][Medline]
  9. Wiszniewski,L., Limat,A., Saurat,J.H., Meda,P. and Salomon,D. (2000) Differential expression of connexins during stratification of human keratinocytes. J. Invest. Dermatol., 115, 278–285.[CrossRef][ISI][Medline]
  10. Ehrlich,H.P., Gabbiani,G. and Meda,P. (2000) Cell coupling modulates the contraction of fibroblast-populated collagen lattices. J. Cell. Physiol., 184, 86–92.[CrossRef][ISI][Medline]
  11. Fitzgerald,D.J., Fusenig,N.E., Boukamp,P., Piccoli,C., Mesnil,M. and Yamasaki,H. (1994) Expression and function of connexin in normal and transformed human keratinocytes in culture. Carcinogenesis, 15, 1859–1865.[Abstract]
  12. Fusenig,N.E. and Boukamp,P. (1998) Multiple stages and genetic alterations in immortalization, malignant transformation and tumor progression of human skin keratinocytes. Mol. Carcinog., 23, 144–158.[CrossRef][ISI][Medline]
  13. Tada,J. and Hashimoto,K. (1997) Ultrastructural localization of gap junction protein connexin 43 in normal human skin, basal cell carcinoma and squamous cell carcinoma. J. Cutan. Pathol., 24, 628–635.[ISI][Medline]
  14. Mueller,M.M. and Fusenig,N.E. (2002) Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells. Differentiation, 70, 486–497.[CrossRef][ISI][Medline]
  15. Risau,W. (1997) Mechanisms of angiogenesis. Nature, 386, 671–674.[CrossRef][ISI][Medline]
  16. Edwards,D.R. and Murphy,G. (1998) Cancer. Proteases—invasion and more. Nature, 394, 527–528.[CrossRef][ISI][Medline]
  17. Zhang,W., Couldwell,W.T., Simard,M.F., Song,H., Lin,J.H. and Nedergaard,M. (1999) Direct gap junction communication between malignant glioma cells and astrocytes. Cancer Res., 59, 1994–2003.[Abstract/Free Full Text]
  18. Boukamp,P., Tilgen,W., Dzarlieva,R.T., Breitkreutz,D., Haag,D., Riehl,R.K., Bohnert,A. and Fusenig,N.E. (1982) Phenotypic and genotypic characteristics of a cell line from a squamous cell carcinoma of human skin. J. Natl Cancer Inst., 68, 415–427.[ISI][Medline]
  19. Fleischmajer,R., Perlish,J.S., Krieg,T. and Timpl,R. (1981) Variability in collagen and fibronectin synthesis by scleroderma fibroblasts in primary culture. J. Invest. Dermatol., 76, 400–403.[Abstract]
  20. Bayreuther,K., Francz,P.I., Gogol,J. and Kontermann,K. (1992) Terminal differentiation, aging, apoptosis and spontaneous transformation in fibroblast stem cell systems in vivo and in vitro. Ann. N.Y. Acad. Sci., 663, 167–179.[ISI][Medline]
  21. Glade,C.P., Seegers,B.A., Meulen,E.F., van Hooijdonk,C.A., Van Erp,P.E. and Van De Kerkhof,P.C. (1996) Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol. Arch. Dermatol. Res., 288, 203–210.[CrossRef][ISI][Medline]
  22. Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]
  23. Whitley,E. and Ball,J. (2002) Statistics review 6: nonparametric methods. Crit. Care, 6, 509–513.[CrossRef][ISI][Medline]
  24. van Dorst,E.B., van Muijen,G.N., Litvinov,S.V. and Fleuren,G.J. (1998) The limited difference between keratin patterns of squamous cell carcinomas and adenocarcinomas is explicable by both cell lineage and state of differentiation of tumour cells. J. Clin. Pathol., 51, 679–684.[Abstract]
  25. Chu,P.G. and Weiss,L.M. (2002) Expression of cytokeratin 5/6 in epithelial neoplasms: an immunohistochemical study of 509 cases. Mod. Pathol., 15, 6–10.[ISI][Medline]
  26. Ek-Vitorin,J.F., Calero,G., Morley,G.E., Coombs,W., Taffet,S.M. and Delmar,M. (1996) pH regulation of connexin43: molecular analysis of the gating particle. Biophys. J., 71, 1273–1284.[Abstract]
  27. El Bayoumy,K. (2001) The protective role of selenium on genetic damage and on cancer. Mutat. Res., 475, 123–139.[ISI][Medline]
  28. Sharov,V.S., Briviba,K. and Sies,H. (1999) Peroxynitrite diminishes gap junctional communication: protection by selenite supplementation. IUBMB Life, 48, 379–384.[ISI][Medline]
  29. Schieke,S.M., Briviba,K., Klotz,L.O. and Sies,H. (1999) Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation. FEBS Lett., 448, 301–303.[CrossRef][ISI][Medline]
  30. Clairmont,A., Tessman,D., Stock,A., Nicolai,S., Stahl,W. and Sies,H. (1996) Induction of gap junctional intercellular communication by vitamin D in human skin fibroblasts is dependent on the nuclear induction of gap junctional intercellular communication by vitamin D in human skin fibroblasts is dependent on the nuclear vitamin D receptor. Carcinogenesis, 17, 1389–1391.[Abstract]
  31. Berthoud,V.M., Rook,M.B., Traub,O., Hertzberg,E.L. and Saez,J.C. (1993) On the mechanisms of cell uncoupling induced by a tumor promoter phorbol ester in clone 9 cells, a rat liver epithelial cell line. Eur. J. Cell Biol., 62, 384–396.[ISI][Medline]
  32. Giaume,C. and Venance,L. (1998) Intercellular calcium signaling and gap junctional communication in astrocytes. Glia, 24, 50–64.[CrossRef][ISI][Medline]
  33. Koshimizu,T.A., Van Goor,F., Tomic,M., Wong,A.O., Tanoue,A., Tsujimoto,G. and Stojilkovic,S.S. (2000) Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells. Mol. Pharmacol., 58, 936–945.[Abstract/Free Full Text]
  34. Loewenstein,W.R. (1979) Junctional intercellular communication and the control of growth. Biochim. Biophys. Acta, 560, 1–65.[CrossRef][ISI][Medline]
  35. Mesnil,M. (2002) Connexins and cancer. Biol. Cell, 94, 493–500.[CrossRef][ISI][Medline]
  36. Bleuel,K., Popp,S., Fusenig,N.E., Stanbridge,E.J. and Boukamp,P. (1999) Tumor suppression in human skin carcinoma cells by chromosome 15 transfer or thrombospondin-1 overexpression through halted tumor vascularization. Proc. Natl Acad. Sci. USA, 96, 2065–2070.[Abstract/Free Full Text]
  37. Detmar,M., Velasco,P., Richard,L., Claffey,K.P., Streit,M., Riccardi,L., Skobe,M. and Brown,L.F. (2000) Expression of vascular endothelial growth factor induces an invasive phenotype in human squamous cell carcinomas. Am. J. Pathol., 156, 159–167.[Abstract/Free Full Text]
  38. Guo,H., Acevedo,P., Parsa,F.D. and Bertram,J.S. (1992) Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids. J. Invest. Dermatol., 99, 460–467.[Abstract]
  39. Yamasaki,H., Mesnil,M., Omori,Y., Mironov,N. and Krutovskikh,V. (1995) Intercellular communication and carcinogenesis. Mutat. Res., 333, 181–188.[CrossRef][ISI][Medline]
  40. Meyer,T. and Hart,I.R. (1998) Mechanisms of tumour metastasis. Eur. J. Cancer, 34, 214–221.[CrossRef][ISI][Medline]
  41. MacDougall,J.R. and Matrisian,L.M. (1995) Contributions of tumor and stromal matrix metalloproteinases to tumor progression, invasion and metastasis. Cancer Metastasis Rev., 14, 351–362.[ISI][Medline]
  42. Mordan,L.J., Martner,J.E. and Bertram,J.S. (1983) Quantitative neoplastic transformation of C3H/10T1/2 fibroblasts: dependence upon the size of the initiated cell colony at confluence. Cancer Res., 43, 4062–4067.[Abstract]
  43. Esinduy,C.B., Chang,C.C., Trosko,J.E. and Ruch,R.J. (1995) In vitro growth inhibition of neoplastically transformed cells by non-transformed cells: requirement for gap junctional intercellular communication. Carcinogenesis, 16, 915–921.[Abstract]
  44. King,T.J., Fukushima,L.H., Donlon,T.A., Hieber,A.D., Shimabukuro,K.A. and Bertram,J.S. (2000) Correlation between growth control, neoplastic potential and endogenous connexin43 expression in HeLa cell lines: implications for tumor progression. Carcinogenesis, 21, 311–315.[Abstract/Free Full Text]
  45. Cruciani,V. and Mikalsen,S.O. (2002) Connexins, gap junctional intercellular communication and kinases. Biol. Cell, 94, 433–443.[CrossRef][ISI][Medline]
  46. Herve,J.C. and Sarrouilhe,D. (2002) Modulation of junctional communication by phosphorylation: protein phosphatases, the missing link in the chain. Biol. Cell, 94, 423–432.[CrossRef][ISI][Medline]
  47. Klotz,L.O., Patak,P., Ale-Agha,N., Buchczyk,D.P., Abdelmohsen,K., Gerber,P.A., von Montfort,C. and Sies,H. (2002) 2-Methyl-1,4-naphthoquinone, vitamin K, decreases gap-junctional intercellular communication via activation of the epidermal growth factor receptor/extracellular signal-regulated kinase cascade. Cancer Res., 62, 4922–4928.[Abstract/Free Full Text]
  48. Saez,J.C., Berthoud,V.M., Moreno,A.P. and Spray,D.C. (1993) Gap junctions. Multiplicity of controls in differentiated and undifferentiated cells and possible functional implications. Adv. Second Messenger Phosphoprotein Res., 27, 163–198.[ISI][Medline]
  49. Yuspa,S.H., Kilkenny,A.E., Steinert,P.M. and Roop,D.R. (1989) Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J. Cell Biol., 109, 1207–1217.[Abstract]
  50. Ko,K.S., Arora,P.D., Bhide,V., Chen,A. and McCulloch,C.A. (2001) Cell–cell adhesion in human fibroblasts requires calcium signaling. J. Cell Sci., 114, 1155–1167.[Abstract/Free Full Text]
  51. Munshi,H.G., Wu,Y.I., Ariztia,E.V. and Stack,M.S. (2002) Calcium regulation of matrix metalloproteinase-mediated migration in oral squamous cell carcinoma cells. J. Biol. Chem., 277, 41480–41488.[Abstract/Free Full Text]
  52. Plotkin,L.I., Manolagas,S.C. and Bellido,T. (2002) Transduction of cell survival signals by connexin-43 hemichannels. J. Biol. Chem., 277, 8648–8657.[Abstract/Free Full Text]
  53. Stout,C.E., Costantin,J.L., Naus,C.C. and Charles,A.C. (2002) Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J. Biol. Chem., 277, 10482–10488.[Abstract/Free Full Text]
  54. Pfahnl,A. and Dahl,G. (1999) Gating of cx46 gap junction hemichannels by calcium and voltage. Pflugers Arch., 437, 345–353.[CrossRef][ISI][Medline]
  55. Zhang,D.Q. and McMahon,D.G. (2001) Gating of retinal horizontal cell hemi gap junction channels by voltage, Ca2+ and retinoic acid. Mol. Vis., 7, 247–252.[ISI][Medline]
  56. Peracchia,C., Wang,X., Li,L. and Peracchia,L.L. (1996) Inhibition of calmodulin expression prevents low-pH-induced gap junction uncoupling in Xenopus oocytes. Pflugers Arch., 431, 379–387.[ISI][Medline]
  57. Torok,K., Stauffer,K. and Evans,W.H. (1997) Connexin 32 of gap junctions contains two cytoplasmic calmodulin-binding domains. Biochem. J., 326, 479–483.[ISI][Medline]
  58. Clark,L.C., Combs,G.F.,Jr, Turnbull,B.W. et al. (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. J. Am. Med. Assoc., 276, 1957–1963.[Abstract]
Received May 2, 2003; revised July 14, 2003; accepted August 14, 2003.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
24/11/1737    most recent
bgg153v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Request Permissions
Google Scholar
Articles by Stuhlmann, D.
Articles by Brenneisen, P.
PubMed
PubMed Citation
Articles by Stuhlmann, D.
Articles by Brenneisen, P.