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Keywords:

  • connexin32;
  • hepatocellular carcinoma;
  • gap junction;
  • tumour progression;
  • Tet-system

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Connexins have long been believed to suppress tumour development during carcinogenesis by exerting gap junctional intercellular communication (GJIC). Although GJIC is abrogated in hepatocellular carcinoma (HCC), connexin32 (Cx32) protein tends to remain expressed in cytoplasm, but not in cell–cell contact areas; thus, it is incapable of forming gap junctions. Hypothesising that cytoplasmic Cx32 protein that has accumulated in HCC should have its proper functions, which are independent of GJIC, we established an inducible expression system of Cx32 in human HuH7 HCC cells, which were unable to support the formation of Cx32-mediated gap junctions, so that Cx32 protein could be overexpressed by doxycycline (Dox) withdrawal. Although the established clone HuH7 Tet-off Cx32 cells exhibited a 4-fold increase in Cx32 expression after Dox withdrawal, none of them were dye-coupled, and Cx32 protein was retained in the Golgi apparatus. However, the proliferation rate of the HuH7 Tet-off Cx32 cells was significantly higher in the Dox-free medium than in the Dox-supplemented one. Transwell assays also revealed that Dox withdrawal enhanced serum-stimulated motility and invasiveness into Matrigel of the HuH7 Tet-off Cx32 cells. Furthermore, when HuH7 Tet-off Cx32 cells were xenografted into the liver of SCID mice, only the mice to which no Dox was administered developed metastatic lesions, indicating that overexpression of cytoplasmic Cx32 protein induced metastasis of HuH7 cells. Our results suggest that, while Cx32-mediated GJIC suppresses the development of HCCs, cytoplasmic Cx32 protein exerts effects favourable for HCC progression, such as invasion and metastasis, once the cells have acquired a malignant phenotype. © 2007 Wiley-Liss, Inc.

Gap junction is an intercellular channel, through which water-soluble small molecules (<1 kDa) travel directly between 2 adjacent cytoplasms, and it plays pivotal roles in homeostasis of cellular society by mediating cell–cell communication.1 A gap junctional channel consists of 2 membrane-integrated hemichannels provided by each of 2 adjacent cells, and a hemichannel is a hexameric complex of connexin protein,2 which forms a connexin family composed of more than 20 members in mammals.3

A considerable number of studies have established that gap junctional intercellular communication (GJIC) suppresses tumour development, i.e., GJIC is down-regulated in almost all tumours through whatever mechanism, including no or reduced expression, aberrant localisation and aberrant phosphorylation or dephosphorylation of connexin protein.4, 5, 6 Consistently, restoration of GJIC very often reverses a malignant phenotype of transformed cells such as human HepG2 hepatoblastoma cells7 and human MCF-7 breast cancer cells.8

In the liver, normal hepatocytes express both connexin26 (Cx26) and connexin32 (Cx32), and they exhibit a high level of GJIC with neighbouring counterparts. On the other hand, GJIC is abolished in hepatocellular carcinoma (HCC),9, 10 as is the case in many other tumours. While expression of Cx26 mRNA and protein is drastically reduced in both human and rat HCCs, Cx32 protein very often continues to be expressed not in a cell–cell contact area but in cytoplasm without mutation, at least in the coding region, thus being incapable of forming gap junctions.9, 11 Further, cytoplasmic localisation of connexins has been recognised also in other tumours, including Cx32 in prostate cancers12 and Cx26 in breast cancers.13 Such an aberrant localisation of connexin proteins in tumours is generally considered to be a “loss of function” as a component of gap junction. It has, however, been reported that cytoplasmic expression of Cx26 is induced in invasive ductal carcinomas of the breast,13 and that its expression level is elevated in metastatic lesions in the lymph nodes.14 These observations raise the question whether cytoplasmic Cx32 observed in HCC exerts some other function than gap junction to the advantage of the tumour cells in terms of tumour progression, such as invasion and metastasis.

To address the question, we established an inducible expression system of Cx32 in human HuH715 and Li-716 HCC cells that were unable to support the formation of Cx32-mediated gap junction in cell–cell contact areas and examined the established clones in terms of invasiveness and metastatic ability both in vitro and in vivo. As expected, invasion and metastasis were significantly enhanced after induction of cytoplasmic Cx32 in HuH7 and Li-7 cells, suggesting that, while Cx32-mediated GJIC contributes to suppression of HCC development, aberrantly localised Cx32 should facilitate progression of developed HCCs.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cells and culture condition

Human HuH715 and Li-716 HCC cell lines were supplied by Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). Both cells and their established subclones were cultured in RPMI1640 medium supplemented with 10% Tet-system-approved foetal bovine serum (FBS) (Clontech Laboratories, Mountain View, CA), 2 mM L-glutamine, 2.4 g/l NaHCO3, 100 units/ml penicillin and 100 μg/ml streptomycin. PT-67, HeLa and its derivative cells were grown in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA), 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. All the cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 in air.

Vector construct and establishment of tetracycline-responsive Cx32 cells

The fragment containing the coding sequence of human Cx32 (GJB1) cDNA17 was amplified by polymerase chain reaction with the following set of primers: Forward, 5′-ATGAATTCAGGGAGGTGTGAACGAGG-3′; Reverse, 5′-GCGAATTCGGGAAGGAAGGTTTTGAT-3′. It was then inserted into pBluescript II SK(+) (Stratagene, La Jolla, CA) after digestion with EcoRI. After sequencing for confirmation, the insert was transferred into SalI–SphI sites of Moloney murine leukaemia virus-based vector pRevTRE (Clontech).

Tetracycline-responsive HuH7 Cx32 cells were established as follows. The regulatory vector pRev-Tet-off (Clontech) coding tTA-VP16 and the expression vector pRevTRE-Cx32 were separately transfected with Lipofectamine 2000 (Invitrogen) into the packaging cell PT-67 (Clontech), and stable transformants were selected with 400 μg/ml G418 for pRev-Tet-off transfectants or with 200 μg/ml hygromycin for pRev-Tet-off-Cx32 transfectants. HuH7 cells were first infected with the pRev-Tet-off virus-containing supernatant supplemented with 4 μg/ml of polybrene (Sigma-Aldrich, St. Louis, MO), and several parental Tet-off clones expressing a high level of tTA-VP16 protein were picked out from among 100 μg/ml G418-resistant clones. Each of these parental Tet-off clones was further transduced by the viruses carrying the construct pRevTRE-Cx32 and selected with 20 μg/ml hygromycin. Finally the most tetracycline-responsive clone (Clone 16-4) was designated as “HuH7 Tet-off Cx32 cells” in this article. To establish the control mock cells, the same parental Tet-off clone (Clone 16) that the HuH7 Tet-off Cx32 cells had originated from was transduced with the viruses carrying the empty vector pRevTRE, and the hygromycin-resistant cells were used without cloning. The HuH7 Tet-off Cx32 and the mock cells were maintained in RPMI1640 medium supplemented with 4 μg/ml doxycycline (Dox) (Sigma-Aldrich), and the medium was changed every other day.

Tetracycline-responsive Li-7 Cx32 cells were also established with the same procedure as that for HuH7 Tet-off Cx32 cells except that the transductants were selected with 400 μg/ml G418 and 200 μg/ml hygromycin.

Antibodies and employed dilutions

Anti-Cx32 polyclonal antibody (pAb) (Sigma-Aldrich) (1:500 for immunoblotting, 1:400 for immunofluorescence), Anti-Cx32 monoclonal antibody (mAb) clone CX-2C2 (Invitrogen) (1:500 for immunofluorescence, 1:200 for immunohistochemistry), Anti-VP16 pAb (Sigma-Aldrich) (1:500), Anti-E-cadherin mAb clone 36 (BD Biosciences Pharmingen, San Jose, CA) (1:3,000) and Anti-GAPDH mAb clone 6C5 (HyTest, Turku, Finland) (1:5,000), Anti-Golgi 58K protein mAb clone 58K-9 (1:50) (Sigma-Aldrich), Anti-Calreticulin pAb (1:50)18 (a kind gift from Dr. Ming Zhou, Akita University, Japan).

Immunoblotting analysis

Cells were grown in 60-mm tissue culture plates, harvested after 48 hr of induction, and lysed in CelLytic M Cell Lysis Reagent (Sigma-Aldrich). 20 μg of protein extract was loaded onto each lane of a 12% SDS-PAGE gel and separated by electrophoresis. The separated protein was then transferred to a Hybond-P membrane (GE Healthcare Bio-Sciences, Piscataway, NJ) at 1.9 mA/cm2 for 1.5 hr with a semidry transfer cell (ATTO, Tokyo, Japan). After blocking with 5% non-fat skim milk in TBS-T buffer overnight, the membrane was incubated with a primary antibody for 2 hr, then with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG antibody (GE Healthcare Bio-Sciences) at a dilution of 1:2,000 for 1 hr. The membrane was extensively washed in TBS-T buffer, and the protein–antibody complex was visualized with an ECL detection reagent (GE Healthcare Bio-Sciences).

Indirect immunofluorescence

Indirect immunofluorescence was performed as described previously.8 In brief, cells were plated on a Lab-TekII chamber slide (Nunc, Naperville, IL) at the appropriate concentration so as to be confluent after 48 hr of induction. Then the cells were washed with PBS, fixed in pure acetone for 7 min at −20°C and soaked in 0.5% blocking agent, following the protocol provided by TSA Fluorescence Systems (Perkin Elmer Life Sciences, Boston, MA). Cells were then incubated with primary antibodies for 2 hr at room temperature, followed by incubation with HRP-labelled or biotin-labelled anti-IgG (KPL, Gaithersburg, MD) for 30 min at room temperature, and finally incubated with Fluorophore Tyramid Amplification Reagent (included in the kit) or rhodamine-labelled streptavidin (KPL) for 10 min. Nuclei were stained with diamidine phenylindole dihydrochloride (KPL) at a concentration of 0.5 μg/ml. Specific signals were observed under a Nikon Microphoto-FXA microscope (Nikon, Tokyo, Japan).

Immunohistochemistry

Immunohistochemistry was performed as described previously.19 In brief, 5-μm cryosections were fixed in pure acetone for 7 min at −20°C and incubated with anti-Cx32 mAb. Specific signals were revealed with diamino-benzidine chromogen by using Envision System (DAKO, Carpinteria, CA). Nuclei were stained with Meyer's haematoxylin.

Isolation of cell surface proteins

Cell surface proteins were isolated from HuH7 Tet-off Cx32 and Li-7 Tet-off Cx32 cells by using a Pinpoint Cell Surface Protein Isolation Kit (Pierce Biotechnology, Rockford, IL). 90–95% confluent of the cells from two 75-mm flasks was biotinylated with 4.8 mg of Sulfo-NHS-SS-Biotin with gentle agitation for 30 min at 4°C. After solubilisation with lysis buffer, biotinylated protein was affinity-isolated with NeutrAvidin-agarose, eluted by 200 μl of sample buffer containing 50 mM dithiothreitol.

Scrape-loading dye-transfer assay

The assay was performed as described in el-Fouly et al.20 In brief, the confluent cells on 60-mm dishes were washed with PBS containing 1 mM CaCl2 and soaked in 2 ml of dye cocktail containing 0.2% Lucifer yellow CH (Sigma-Aldrich) and 0.05% rhodamine B isothiocyanate (RITC)-dextran (Sigma-Aldrich). Several parallel scrape lines were then made with a micropipette tip, and the cells were incubated for 5 min at 37°C. After washing with PBS, the cells dye-coupled with Lucifer yellow were observed under an Olympus IX-FLA microscope (Olympus, Tokyo, Japan). The cells positive for RITC-dextran were considered to be primarily scraped but not dye-coupled cells.

Anchorage-dependent cell proliferation assay

Cells were cultured at a density of 5 × 104 cells in 60-mm dishes in triplicate in the presence or absence of 4 μg/ml Dox. To determine the cell growth, the cells were counted every other day with a haemocytometer. Dead cells, stained by trypan blue, were excluded from the count.

Cell migration and invasion assay

Cell motility and invasiveness were assayed quantitatively with a Boyden chamber.21 For migration assay, 8-μm-pore filters of cell culture inserts (BD Biosciences Discovery Labware, Bedford, MA) were precoated with 150 ng vitronectin at 24 hr before seeding cells and installed onto the lower compartments of 6-well plates containing 4.0 ml of serum-supplemented RPMI1640. 5.0 × 105 cells resuspended in 2.0 ml of serum-free RPMI1640 containing 0.01% bovine serum albumin were seeded to each upper well. After 48 hr of incubation, cells were trypsinised and collected separately from the top of the membrane, the underside of the membrane, and the lower compartment. Cell motility was quantitated as the percentage of cells found at the underside of the membrane and the lower compartment over the total cell number.

For invasion assay, 8-μm-pore filters of cell culture inserts were precoated with 500 μg Matrigel (BD Biosciences Phrmingen), dried for 24 hr in an incubator and rehydrated by RPMI1640 for 1 hr before inoculation of cells. The assay was done according to the same protocol as that for migration assay mentioned earlier except that the cells were incubated for 72 hr.

Xenograft into mice

Male severe combined immunodeficiency (SCID) mice (C.B-17/Icr-scid/scid) were purchased from CLEA Japan (Tokyo, Japan) and maintained in a specific pathogen-free condition. The mice received humane care and were fed with autoclaved water and chow. The protocol of the animal work was approved by the Committee for Ethics of Animal Experimentation and in accordance with the Guidelines of Animal Experiments of Akita University. Orthotopic implantation of the HuH7 Tet-off Cx32 cells and the mock transductants was performed as described in Genda et al.22 The cells were harvested from subconfluent cultures, pelleted by centrifugation, washed with PBS, and resuspended in PBS at a density of 1 × 108 cells/ml. Mice were anaesthetised by intraperitoneal administration of pentobarbital (65 mg/kg body weight) and an upper-left abdominal incision was made. After the liver was carefully exposed, a 20-μl aliquot of cell suspension containing 2 × 106 cells was injected into the subserosa of the liver. Drinking water was supplemented with 2 mg/ml of Dox, given to the mice beginning 1 week before implantation of the cells, and a new bottle was provided every 3 days. The mice were euthanized with CO2 inspiration 8 weeks after implantation and autopsies were performed immediately. The removed livers were examined macroscopically for primary and metastatic tumours. The developed tumours were frozen in liquid nitrogen for subsequent immunoblotting analyses or fixed in 10% formalin for haematoxylin-eosin (HE) and elastica-Masson (EM) staining.

To examine tumourigenicity of the cells in the mice, 2 × 106 cells were injected subcutaneously (s.c.) into the backs of the athymic nude mice (CLEA). The mice were given drinking water containing 2 mg/ml Dox for 11 weeks. Size of palpable tumours was measured every other day.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Overexpression of Cx32 is induced in HuH7 Tet-off Cx32 cells by withdrawal of doxycycline

Human HuH7 hepatoma cells are a cell line derived from a well-differentiated HCC.15 A normal hepatocyte is known to express both Cx26 and Cx32.23 In contrast, our Northern blotting analysis revealed that HuH7 cells expressed not Cx26 mRNA but only Cx32 mRNA (data not shown). Cx32 protein endogenously expressed in HuH7 cells, however, does not form gap junction plaques and is retained in cytoplasm, as shown in Figure 2a: left panels. Thus, HuH7 cells present a phenotype typical of HCC with respect to the expression patterns of connexin protein, i.e., loss or reduction of expression of Cx26 and aberrant localisation of Cx32.

To evaluate the involvement of cytoplasmic Cx32 in progression of HCC without any necessity to consider clonal diversity, we generated a subclone of HuH7 cells named “HuH7 Tet-off Cx32,” as described in Material and methods, that overexpression of Cx32 could be induced by withdrawal of Dox. As shown in Figure 1a, expression of Cx32 protein declines as the amount of Dox increases. Densitometric analysis of the immunoblotting gels indicates that 8 μg/ml Dox can suppress the expression of Cx32 protein to approximately a quarter of that seen in Dox-free medium (Fig. 1b). Although no cytotoxicity of Dox against HuH7 cells was noted with a concentration of less than 20 μg/ml (data not shown), all of our in vitro experiments were performed with 4 μg/ml Dox because this was the minimal dose giving an almost maximal effect.

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Figure 1. Overexpression of Cx32 protein induced by Dox withdrawal in HuH7 Tet-off Cx32 cells. (a) HuH7 Tet-off Cx32 cells as well as HuH7 Tet-off mock cells were cultured in the media supplemented with the indicated concentrations of Dox. After 48-hr-incubation, the cells were lysed and subjected to immunoblotting to detect Cx32 protein, and GAPDH as a loading control. (b) Densitometric analysis of 3 independent immunoblottings for Cx32 protein expressed in HuH7 Tet-off Cx32 cells. Error bars represent the SD (n = 3). No error bar is indicated when the SD is too small to show.

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Cx32 protein overexpressed in HuH7 Tet-off Cx32 cells localises exclusively in cytoplasm and cannot form functional gap junctions

A small amount of Cx32 protein is detected in the cytoplasm of HuH7 Tet-off Cx32 cells by immunofluorescence when they are cultured in 4 μg/ml Dox-supplemented medium (Fig. 2a: right upper panel). In Dox-free medium, HuH7 Tet-off Cx32 cells give off strong signals in cytoplasm but not in cell–cell contact areas (Fig. 2a: right lower panel). These observations suggest that the absence of gap junction plaques from HuH7 cells and HCC should be due not to insufficient expression of Cx32 but to impaired intracellular sorting of Cx32 to the plasma membrane.

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Figure 2. (a) Indirect immunofluorescence of Cx32 protein in HuH7 Tet-off Cx32 cells as well as HuH7 Tet-off mock cells. The cells were cultured in the presence or absence of Dox for 48 hr. Scale bar, 20 μm. (b) Colocalisation of Cx32 protein with Golgi 58K protein, a Golgi marker, in HuH7 Tet-off Cx32 cells in the absence of Dox. Cx32 and Golgi 58K proteins were detected by immunofluorescence with FITC and RITC, respectively. Scale bar, 20 μm. (c) Costaining of Cx32 protein with calreticulin, a marker for the endoplasmic reticulum, in HuH7 Tet-off Cx32 cells in the absence of Dox. Cx32 protein and calreticulin were detected by immunofluorescence with FITC and RITC, respectively. Scale bar, 20 μm. (d) Immunoblotting of cell surface proteins of HuH7 Tet-off Cx32 cells. The cells were cultured in the presence or absence of Dox for 48 hr. After isolation of the cell surface protein fraction, 20 μg of cell surface proteins along with the whole cell lysates were subjected to the immunoblotting analysis to detect Cx32, E-cadherin and GAPDH.

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We further intended to specify the subcellular localisation of Cx32 protein expressed in HuH7 Tet-off Cx32 cells and costained the cells with both anti-Cx32 and anti-Golgi 58K protein antibodies after 48-hr-incubation in the Dox-free medium. Figure 2b clearly shows that the signals for Cx32 protein almost perfectly coincide with Golgi areas represented by Golgi 58K protein. On the other hand, only a small population of Cx32 protein overlaps with the signals for calreticulin, a marker for the endoplasmic reticulum (Fig. 2c). These results indicate that a great majority of Cx32 protein overexpressed in HuH7 Tet-off Cx32 cells is retained in the Golgi apparatus.

To confirm that the overexpressed Cx32 protein is not integrated into the plasma membrane, we isolated cell surface proteins from HuH7 Tet-off Cx32 cells in the presence or absence of Dox, as described in Material and methods, and examined whether Cx32 was contained in the cell surface protein fraction. As shown in Figure 2d, while Cx32 protein is overexpressed in HuH7 Tet-off Cx32 cells as a whole in the absence of Dox, no Cx32 protein is detected in the cell surface protein fractions in either the presence or the absence of Dox. In contrast, E-cadherin, an essential component of adherens junctions in hepatocytes, is successfully enriched in the cell surface protein fraction. Therefore, it has become evident that the overexpressed Cx32 protein induced by withdrawal of Dox makes no contribution to gap junctions in HuH7 Tet-off Cx32 cells.

As reported by several other groups, one type of connexin sometimes induces or suppresses expression of other types.24, 25 The Cx32 protein overexpressed in HuH7 Tet-off Cx32 cells may induce other types of connexin, which then may exert GJIC. To verify such a possibility, GJIC was measured by a scrape-loading dye-coupling assay in the presence or absence of Dox. In HuH7 Tet-off Cx32 cells as well as mock cells, cells receiving the tracer molecule Lucifer yellow coincide entirely with those receiving RITC-dextran, which cannot pass through gap junction and visualises only the primarily-scraped cells, indicating that no cells are dye-coupled in either the presence or the absence of Dox (Fig. 3). It is, thus, concluded that the overexpressed Cx32 protein is not functional as a component of gap junctions in HuH7 Tet-off Cx32 cells.

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Figure 3. Scrape-loading dye-transfer assay to measure GJIC capacity. HuH7 Tet-off Cx32 cells as well as HuH7 Tet-off mock cells were cultured in the presence or absence of Dox for 48 hr. The cells were scraped by a tip, incubated with a cocktail of Lucifer yellow CH and RITC-dextran, and observed under a fluorescence microscope after washing with PBS. Note that no dye-coupled cells with Lucifer yellow CH were observed in either the presence or absence of Dox in HuH7 Tet-off Cx32 cells. As a positive control, HeLa cells trasfected with rat Cx32 (Gjb1) gene were used.

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Overexpression of cytoplasmic Cx32 protein enhances proliferation, motility and invasiveness of HuH7 Tet-off Cx32 cells in vitro in a gap junction-independent manner

To examine the effect of the overexpressed cytoplasmic Cx32 protein on proliferation of HuH7 Tet-off Cx32 cells, an anchorage-dependent cell proliferation assay was carried out in the presence or absence of Dox. As shown in Figure 4a, HuH7 Tet-off Cx32 cells demonstrate a much higher proliferation rate in a Dox-free medium than in a Dox-supplemented one. Since HuH7 Tet-off mock cells show similar proliferation rates whether in a Dox-free or -supplemented medium, the enhanced proliferation of HuH7 Tet-off Cx32 cells in a Dox-free medium is because of the overexpression of cytoplasmic Cx32 protein.

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Figure 4. GJIC-independent effects of Cx32 protein overexpressed in HuH7 Tet-off Cx32 cells on cell proliferation, motility and invasiveness in vitro. (a) Anchorage-dependent cell proliferation assay. HuH7 Tet-off Cx32 cells and HuH7 Tet-off mock cells were cultured in the presence or absence of Dox for the indicated periods. The cells were counted every other day in triplicate dishes. Error bars represent the SD. No error bar is indicated when the SD is too small to show. Closed square, HuH7 Tet-off Cx32 Dox (−); Open square, HuH7 Tet-off Cx32 Dox (+); Closed triangle, HuH7 Tet-off mock Dox (−); Open triangle, HuH7 Tet-off mock Dox (+). (b) Transwell migration assay. The cells were seeded onto vitronectin-precoated filters of cell culture inserts and incubated for 48 hr. The cells which passed through the filter during migration toward FBS were counted and their proportion to the total cell number is indicated. Error bars represent the SD (n = 6). *p < 0.01. (c) The same experiment as (b) was performed in the presence or absence of 100 μM oleamide, a GJIC inhibitor. HeLa Cx32 cells are HeLa cells previously transfected with rat Cx32 (Gjb1) gene and overexpressing Cx32 proteins in cell–cell contact areas.26 Error bars represent the SD (n = 6). *p < 0.01. (d) Invasiveness into the matrix basement membrane. The cells were seeded onto Matrigel, which had been poured into cell culture inserts in advance. The cells that penetrated the Matrigel layer were counted and their proportion to the total cell number is indicated. Error bars represent the SD (n = 6). *p < 0.01.

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Upregulation of cell motility is one of the most important steps during tumour progression.27 We examined the effect of the overexpressed cytoplasmic Cx32 on cell motility by performing a serum-stimulated transwell migration assay, as described in Material and methods. Motility of HuH7 Tet-off Cx32 cells is significantly upregulated in a Dox-free medium compared with in a Dox-supplemented one, while Dox does not affect motility of HuH7 Tet-off mock cells (Fig. 4b). Even in a Dox-supplemented medium, HuH7 Tet-off Cx32 cells exhibit a higher motility than HuH7 Tet-off mock cells because a very low level of exogenous Cx32 protein escapes from Dox-mediated repression as a leaky expression in our HuH7 Tet-off Cx32 cells (Fig. 1a).

To exclude involvement of undetectable GJIC in the upregulation of motility, cell motility was assayed in the presence of 100 μM oleamide, a GJIC inhibitor. 100 μM oleamide is known to be sufficient to inhibit GJIC by 97% for at least 24 hr without cytotoxicity and without altering localisation of connexin proteins.28, 29 As shown in Figure 4c, the addition of oleamide does not have any effect on cell motility of either HuH7 Tet-off Cx32 cells or mock cells, confirming that GJIC is not implicated in the Cx32-mediated upregulation of motility in HuH7 Tet-off Cx32 cells. On the other hand, the Cx32-transfected HeLa cell clone,26 which exhibits a high level of GJIC and a lower motility than mock-transfected HeLa cells, regains its high motility after treatment with oleamide. Therefore, it is suggested that Cx32-mediated GJIC downregulates cell motility, which, to the contrary, cytoplasmic Cx32 protein upregulates.

We further investigated whether overexpression of cytoplasmic Cx32 could modulate invasiveness of HuH7 Tet-off Cx32 cells by evaluating the ability of HuH7 Tet-off Cx32 cells and mock cells to invade the basement membrane matrix in the presence or absence of Dox. When HuH7 Tet-off Cx32 cells are induced to overexpress cytoplasmic Cx32 protein in a Dox-free medium, they exhibit a significantly high level of invasiveness (Fig. 4d).

Taken together, our in vitro experiments showed that the Cx32 protein overexpressed in cytoplasm provided HuH7 Tet-off Cx32 cells with enhanced malignant phenotypes leading to tumour progression. This conclusion is strengthened by similar data from two other sister clones (Clones 16-5 and 16-10) of HuH7 Tet-off Cx32 cells (Fig. a, Supplemental Data). To confirm the generality of our observations, we furthermore established Tet-off Cx32 clones derived from human Li-7 HCC cells16 and obtained the results similar to those from HuH7 Tet-off Cx32 cells (Fig. b, Supplemental Data).

Overexpression of cytoplasmic Cx32 protein induces metastases of intrahepatic xenografts without enhancing tumourigenicity in SCID mice

Intrahepatic spreading is the most critical reason for liver failure caused by HCCs.30 We thus examined how HuH7 Tet-off Cx32 cells overexpressing cytoplasmic Cx32 protein behaved in vivo when they were grafted into the liver of SCID mice. Each mouse was given 2 × 106 cells of HuH7 Tet-off Cx32 cells along with mock cells into a subserosal area of the liver and was autopsied 8 weeks after the implantation to observe tumour development and metastases. For administration of Dox, potable water was supplemented with 2 mg/ml Dox throughout the experiment period.

As summarised in Table I, 5 out of 6 mice developed tumours at the implanted sites in each of the Dox (+) and the Dox (−) groups. The tumour sizes were not distinct between the Dox (+) and the Dox (−) groups (data not shown). On the other hand, macroscopic metastatic lesions were found in all of the 5 tumour-bearing mice in the Dox (−) group but in none of the mice in the Dox (+) group (Table I and Figs. 5a and 5b). This clearly indicates that the overexpressed cytoplasmic Cx32 protein can give the metastatic ability to HuH7 Tet-off Cx32 cells.

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Figure 5. Orthotopic xenografts of HuH7 Tet-off Cx32 cells into the liver of SCID mice. 2 × 106 cells were implanted into the liver of SCID mice, and the mice were autopsied 8 weeks after the implantation. (a) Gross findings of the mouse abdomen in the Dox (−) group. Blue circle, tumour developed in the liver; Yellow circle, tumour metastasised to the peritoneal wall. (b) Gross findings of the liver in the Dox (−) group. Blue circle, primary lesion; Yellow circle, metastatic lesion. (c) Microscopic findings of the primary tumour in the liver in the Dox (−) group. HE staining. Note that the tumour displays a well-differentiated morphology. (d) Micrometastasis in the liver in the Dox (−) group. EM staining. The tumour cells look paler than their normal counterparts. (e) Portal vein tumour thrombus in the liver in the Dox (−) group. EM staining. (f) Peritoneal metastasis in a mouse in the Dox (−) group. EM staining. Note that the tumour invades the abdominal muscle. (g) Expression of Cx32 proteins in a primary tumour developed in the mouse in the Dox (−) group given HuH7 Tet-off Cx32 cells. (h) Expression of Cx32 protein in a normal part of the liver of the same mouse as (g). (i) Expression of Cx32 protein in an intrahepatic meastatic tumour in the same mouse as (g). (j) Expression of Cx32 protein in a peritoneal meastatic tumour in the same mouse as (g). (k) Immunoblotting of the developed tumours for Cx32 protein. The tumours were lysed and subjected to immunoblotting analysis. Lanes 1, 2 and 3, primary tumours from 3 different mice in the Dox (+) group; Lanes 4 and 6, primary tumours from 2 different mice in the Dox (−) group; Lane 5, intrahepatic metastatic tumour from the same mouse as Lane 4; Lane 7, peritoneal metastatic tumour from the same mouse as Lane 6.

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Table I. Tumorigenicity and Metastasis of Orthotopic Xenografts in the Liver of Scid Mice
Implanted cellsDoxNo. of mice examinedNo. of mice bearing tumour (incidence)No. of micewith metastasesNo. of mice with metastases (detail)
Both intrahepatic and peritonealExclusively intrahepaticExclusively peritoneal
HuH7 Tet-off Cx32+65 (83%)0
65 (83%)5212
 HuH7 Tet-off mock+44 (100%)0
44 (100%)0

Both the primary and the metastatic lesions histologically displayed a thick trabecular pattern with pseudoglandular formation, considered to be well to moderately differentiated HCC (Fig. 5c). Micrometastases and portal vein tumour thrombi were also frequently observed in livers of the Dox (−) group given HuH7 Tet-off Cx32 cells (Figs. 5d and 5e). Besides intrahepatic metastases, peritoneal metastatic foci had developed in these mice (Figs. 5a and 5f). Immunohistochemical analyses reveal that Cx32 protein is localised in cytoplasm similarly in both primary and metastatic tumours developed in the same mice in Dox (−) group (Figs. 5g, 5i and 5j) while hepatocytes in a normal part of the host liver can form Cx32-mediated gap junction plaques in a cell–cell contact area (Fig. 5h).

As demonstrated in Figure 5k, both the primary and the metastatic tumours that developed in the Dox (−) group express a significantly higher level of Cx32 protein than those that developed in the Dox (+) group, proving that our HuH7 Tet-off Cx32 cells maintain their inducibility of Cx32 expression by Dox withdrawal in SCID mice.

Several papers have stated that Dox inhibits several members of matrix metalloproteinases.31, 32 One possible interpretation is that the absence of Dox induced the metastases of HuH7 Tet-off Cx32 cells by releasing the activity of the matrix metalloproteinases rather than by inducing overexpression of cytoplasmic Cx32. However, none of the tumours that were formed by HuH7 Tet-off mock cells could metastasize in the mice in the Dox (−) group (Table I). The possible inhibitory effect of Dox to matrix metalloproteinases is therefore negligible in our experiments.

Besides SCID mice, we xenografted our cells subcutaneously (s.c.) into the backs of athymic nude mice to evaluate the effects of the overexpressed cytoplasmic Cx32 protein on tumour growth. Three out of six mice given HuH7 Tet-off Cx32 cells in the Dox (−) group but none in the Dox (+) group developed a tumour (data not shown). Consistently with Figure 4a, it appears that cytoplasmic Cx32 protein can enhance tumourigenicity of HuH7 Tet-off Cx32 cells in nude mice.

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

While it has been established that GJIC suppresses tumour development during carcinogenesis, little is known about the roles of GJIC and/or connexins in tumour progression, including invasion and metastasis. There have so far been 2 studies describing the effects of Cx32 on metastasis in vivo. In these reports, the exogenously overexpressed Cx32 protein suppressed the metastatic ability of human Caki-1 renal cell carcinoma cells33 and human LNCaP prostatic adenocarcinoma cells12in vivo. In both cell lines, the overexpressed Cx32 protein was properly located in cell–cell contact areas and exerted a high level of GJIC, suggesting that Cx32-mediated GJIC could contribute to the suppression of metastases. In this context, translocation of Cx32 protein from the plasma membrane to cytoplasm, leading to downregulation of GJIC, is considered to be one of the causal factors for metastases, but the cytoplasmic Cx32 protein itself should not exert any function.

However, in our present study, when overexpressed in nonmetastatic human HuH7 HCC cells, the cytoplasmic Cx32 protein enhanced motility and invasiveness in vitro (Fig. 4) and induced intrahepatic and peritoneal metastases of the orthotopic xenografts implanted into mouse livers (Fig. 5 and Table I) in vivo. In many human and rat HCCs, while expression of Cx26 protein is abolished, Cx32 protein remains expressed in cytoplasm.9, 34, 35 Although such an aberrant localisation had long been thought to be responsible only for reduced or abolished GJIC, our results clearly indicate that the cytoplasmic Cx32 protein itself could facilitate progression of HCCs in a GJIC-independent manner. In genetically manipulated Cx32-deficient mice where Cx32 protein is absent from both the plasma membrane and cytoplasm, HCCs develop spontaneously,36 and the incidence of HCCs also increases in chemical hepatocarcinogenesis.36, 37 Nevertheless, there has so far been no report describing metastasis from tumours that have developed in Cx32-deficient mice. Taken together, it is most likely that, while Cx32-mediated GJIC suppresses the development of HCCs, the cytoplasmic Cx32 protein exerts effects favourable for HCC progression, such as invasion and metastasis, once the cells have acquired a malignant phenotype.

Accumulating evidences have revealed that connexin hexamers, so-called connexons, can function as pure hemichannels integrated into the plasma membrane without docking with the connexons provided by the adjacent cells.38 This has so far been the only established mechanism of GJIC-independent functions exerted by connexins. It is, however, unlikely that such hemichannels explain metastases induced by Cx32 protein overexpressed in HuH7 Tet-off Cx32 cells because Cx32 protein is unable to move to the plasma membrane in HuH7 cells (Fig. 2). Although a few examples of GJIC- and hemichannel-independent functions other than ours have been reported,39, 40, 41 their mechanism remains largely unknown.42 Further studies are required to elucidate each of GJIC-independent actions of connexins.

Although our study demonstrated that Cx32 protein that had accumulated in the cytoplasm of tumour cells exerted a GJIC-independent function, it is not clear whether a physiological amount of cytoplasmic Cx32 protein can play any significant role in cellular functions. Even in normal tissues, weak cytoplasmic signals of Cx32 protein are detectable in a few cells as revealed by immunofluorescence. On the other hand, as reviewed by Jiang and Gu,42 many of the GJIC-independent actions of connexins have been recognised only in pathological conditions. Regardless of whether or not the cytoplasmic Cx32 protein has physiological roles, it appears certain that excessive accumulation of Cx32 protein in cytoplasm is an important mechanism of disordered cell regulation leading to invasion and metastasis of HCCs. Cytoplasmic localisation of connexin proteins is not a rare phenomenon and is also found in tumours other than HCC.6 Cytoplasmic connexin proteins may therefore have the potential to modulate the progression of different tumours positively or negatively.

As shown in Figure 2b, Cx32 protein overexpressed in HuH7 Tet-off Cx32 cells is localised almost exclusively in Golgi apparatuses. Cx32 protein is transported after its synthesis from the endoplasmic reticulum to the Golgi apparatus and then assembles there together in a connexon before the trafficking to the plasma membrane.43 It is, thus, relevantly expected that the assembly of Cx32 protein may be disrupted or that the exit of Cx32-derived connexons from the Golgi apparatus may be blocked in HuH7 cells, resulting in the retention of Cx32 protein in Golgi apparatuses. In contrast, mutation of the Cx32 gene (GJB1) should not be involved in their intraGolgi retention because no mutation within the coding region has been found in human HCCs9 and because the only mutation detected in rat HCCs was a silent mutation.11 In human HepG2 hepatoblastoma cells, which express not Cx26 protein but Cx32 protein in cytoplasm, as observed in HuH7 cells and many HCCs, the exogenously expressed Cx26 protein could successfully form functional gap junctions.7 A Cx32-specific pathway of the intracellular trafficking and assembly of Cx32 protein into gap junctions should be impaired in HCCs and HCC-derived cell lines.

One of the most intriguing observations in the present study is that Cx32 protein localised in Golgi apparatuses enhanced motility and metastatic ability of HuH7 cells. How can the intraGolgi Cx32 protein exhibit such a prominent cellular function? Although everything on this issue is vague at present, we suppose 2 possible mechanisms. One possibility is that Cx32 protein excessively accumulated in Golgi apparatuses may interact with a specific unknown protein implicated in the control of cell migration or proliferation and may alter its function so as to upregulate cell motility and metastatic ability. However, no protein directly-associated with Cx32 has so far been known. Only one report describes that Cx32 protein is contained in the same complex with ZO-1 and occludin in a cell-cortical area.44 Although ZO-1 and tight junction proteins may have potential to regulate cellular function such as cell motility, they are not retained in Golgi apparatuses in either HuH7 or Li-7 cells (data not shown). Other candidates of Cx32 target proteins should be explored to elucidate the mechanisms underlying our observation. In addition, we cannot exclude another possibility that an excessive amount of Cx32 protein may disturb Golgi functions in a nonspecific manner and may lead to an enhanced motility under a specific condition such as HCC.

Since disorders of gap junctions and/or connexins are implicated in various human diseases, connexins, including Cx32 have now become new pharmacological targets.45 It has long been believed that the drugs which enhance connexin expression and GJIC should exert an anticancer effect and reduce tumour growth.46, 47 Also, in the case of benign diseases, such as cardiovascular and neurosensory diseases, many drugs targeting connexins are being designed so as to increase expression of connexins and to reinforce GJIC.45 However, it is not clear whether all the connexin molecules increased by such agents contribute to GJIC only in the cells that need a high level of GJIC. Cx32 protein overexpressed by a drug used to treat one disease may have an adverse effect on individuals carrying another disease that deteriorates due to the cytoplasmic Cx32 protein, e.g., HCC.

In conclusion, our study demonstrates that cytoplasmic accumulation of Cx32 protein, which is one of the hallmarks of HCC, is not simply a phenomenon coincident with HCC development but rather a rational event contributing to metastasis of HCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We are very grateful to Ms. Reiko Ito and Ms. Yuko Doi for their technical assistance. Q.L. is recipient of Japanese Government (MEXT) Scholarship (#022101).

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0020-7136/suppmat .

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