Influence of transfection with connexin 26 gene on malignant potential of human hepatoma cells
Akira Muramatsu,1,
Masaki Iwai,
Teruhisa Morikawa,
Saiyu Tanaka,
Takahiro Mori,
Yoshinori Harada and
Takeshi Okanoue
Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan
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Abstract
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We investigated the effect of transfection with connexin (Cx) 26 gene on the malignant potential of PLC/PRF/5 hepatoma cells, observing changes in their morphological features, alpha-fetoprotein (AFP) expression, cell proliferation and apoptosis in vitro, and their tumor growth in vivo. Fluorescence-activated cell sorting (FACS) analysis showed that 10.6% of PLC/PRF/5 hepatoma cells transfected with Cx26 cDNA expressed excessive Cx26, and the spread of lucifer yellow was wider in the colony of stable transfectants (PLC/Cx26) after its microinjection than in control. Nucleo-cytoplasmic (N/C) ratio was significantly lower in PLC/Cx26 (P < 0.0001). Cell proliferation assay showed significantly lower numbers in PLC/Cx26 on day 10 after seeding than in control (P = 0.0039), and AFP level /105cells was significantly lower in medium of PLC/Cx26 (P = 0.0039). The number of proliferating cell nuclear antigen (PCNA)-positive cells was less in PLC/Cx26 in vitro than in control (P = 0.0039), and single-stranded DNA (ssDNA)-positive cells were more abundant in the colony of PLC/Cx26 (P = 0.029). Tumor volume in SCID mice was significantly smaller in the group of PLC/Cx26 than in the control (P < 0.01) throughout the observation period, and tumor weight of PLC/Cx26 was significantly lower (P = 0.0019) week 9 after inoculation. Transfection with Cx26 cDNA inhibited dedifferentiation, suppressed cell proliferation, and apoptosis was induced. Tumor growth of PLC/Cx26 was retarded. These findings suggest that transfection with Cx26 gene into human hepatoma cells reduces their malignant potential.
Abbreviations: AFP, alpha-fetoprotein; Cx, connexin; DMEM, Dulbecco's modified Eagle's medium; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GJ, gap junction; GJIC, gap junctional intercellular communication; HBSS, Hanks' balanced salt solution; LY, lucifer yellow; PBS, phosphate buffered saline
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Introduction
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Gap junctions (GJs) are organella of cellcell contact that are composed of two hemichannels, and each hemichannel consists of six connexins (Cxs). Cx protein is composed of four transmembrane domains (M1M4), an intercellular N-terminal domain (N), two extracellular loops (E1 and E2), a cytoplasmic loop (CL) and a C-terminal domain (1,2). Small hydrophilic molecules (molecular weight: <1000 Daltons), including second messengers of adenosine 3',5'-cyclic monophosphate (cAMP), inositol 1,4,5-triphosphate (IP3), and Ca2+, can pass through gap junctional channels, so that neighboring cells can communicate. There are 16 different Cxs in vertebrate species and expression of some Cxs is specific for organ (35). In liver, Cx32 and Cx26 are expressed (68). Cx32 is the major component of GJ, being expressed in all hepatocytes of a lobule. Cx26 is the minor component in human and rat livers, and it is preferentially expressed in hepatocytes in the periportal zone (9,10). Cx26 has several specific features different from other Cxs; Cx26 has no phosphorylation sites, which have been thought to play an important role in regulating trafficking or assembly of gap junctional proteins and channel gating or turnover (10,11), and it has a limited C-terminal domain (12,13).
GJs are thought to play an important role in the control of cell proliferation and differentiation (1,2,4). In liver during ontogenesis, cell proliferation and differentiation are regulated, gap junctional channel formation is gradually increased, and gap junctional intercellular communication (GJIC) is developed (8,14). However, gap junctional channel formation is markedly decreased in hepatoma cells and GJIC is depressed (15,16), therefore induction of Cx expression in hepatoma cells may have the ability to revert them to normal proliferation and differentiation.
Hepatocellular carcinoma is responsible for
32 000 fatalities a year in Japan (17,18), and it is frequently seen in Asian and African countries. It is increasing not only in Asia but also in the United States and Europe (1921). There have been many conventional therapies for hepatocellular carcinoma (2225), but its prognosis is still poor because of its high frequency of recurrence. New therapies are required, and gene therapy has been investigated as one of the candidates for the new treatment of hepatocellular carcinomas (2629).
Cx cDNA has already been co-transfected with herpes simplex virus thymidine kinase (HSVtk) cDNA into neoplastic cells in `combination gene therapy', and the role of Cx cDNA is considered to be the augmentation of bystander effect (3032). In addition, transfection with Cx cDNA has been proposed to play an important role in reducing malignant potential of neoplastic cells or reverting them to normal biological behavior (Cx32 (33), Cx43 (34), Cx43, Cx40 and Cx26 (35)). Transfection efficiency is important in gene therapy but complete transfection into all neoplastic cells is always impossible, and transfection rate by viral or non-viral vectors is reported to be <30% except by adenovirus, therefore we used the balc system, in which a hepatoma cell line transfected with Cx cDNA is not cloned and in which expression of Cx is different in intensity among each transfected cell, to estimate the effect of its transfection on malignant potential in vitro and in vivo.
It is well known that transfection with Cx32 cDNA inhibits the growth of hepatoma cells (33), and Cx32 expression was found to have an inhibitory effect on hepatocarcinogenesis (36), but formation of GJ composed of Cx26 has not been shown to have an inhibitory effect on growth and differentiation of hepatoma cells. Cx26 has been recently reported to be a candidate for tumor suppression (35,3739), and to have a stronger anti-tumor effect than Cx43 or 40 (35,39). Both Cx32 and 43 with a deleted C-terminal domain acquired the ability to induce negative growth control as well as Cx26 (13). Formation of Cx26-positive GJ is reported to retard tumor growth of HeLa cells deficient in Cx (39), but it has not been investigated whether transfection with Cx26 cDNA has an effect on malignant potential of human hepatoma cells. Thus, we transfected Cx26 cDNA into a human hepatoma cell line, PLC/PRF/5, to examine the changes in proliferation and AFP expression comparing them with those transfected with Cx32 cDNA. A recent report revealed a new insight about the relationship between GJ formation and apoptosis (40), and apoptotic phenomena may occur in parallel with the development of GJIC. Thus, we investigated the presence of apoptotic phenomena after transfecting Cx26 cDNA into hepatoma cells. In addition, tumor growth of stable transfectants (PLC/Cx26) was also studied to assess the significance of Cx26 cDNA transfection in a new therapeutic strategy for human hepatomas.
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Materials and methods
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Cell culture
The human hepatoma cell line PLC/PRF/5 (producing AFP) was cultured at 37°C in 5% CO2/95% humidified air in DMEM (Life Technologies, Rockville, MD) containing 10% FBS (Life Technologies, Rockville, MD), 100 U/ml ampicillin and 100 µg/ml streptomycin (complete medium).
Plasmid construction and transfection
PLC/PRF/5 cells were transfected with two types of plasmids; pBEHpac18/Cx32 containing the 1480 bp coding sequence for rat Cx32 and puromycin resistant gene and pBEHpac18/Cx26 containing the 1100 bp coding sequence for rat Cx26 and puromycin resistant gene (kindly provided by Dr K.Willecke at the University of Bonn, Bonn, Germany) (41). The Cx32 and 26 gene was driven by SV40 early promoter. PLC/PRF/5 cells were seeded in 50 ml tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ) at a density of 5 x 105 cells/flask and incubated at 37°C in 5% CO2/95% humidified air. On the following day, 5 µg pBEHpac18/Cx32 and pBEHpac18/Cx26 cDNA was mixed with 30 µl polyamidoamine dendrimer, Superfect (Qiagen, Hilden, Germany) in DMEM and incubated for 10 min at room temperature (26,42). The complex was added to 1000 µl complete medium and immediately transferred to the cells that had been washed with PBS. They were incubated at 37°C in 5% CO2/95% humidified air for 3 h, followed by replacement with fresh complete medium, and the transfectants were cultured for 2 days before selection with puromycin (1.5 µg/ml puromycin dihydrochloride (Calbiochem, La Jolla, CA)). After 34 weeks, resistant clones (PLC/Cx32 and PLC/Cx26) were isolated and analyzed. Mock transfectant (control) was established in the same way with plasmid pBEHpac18 containing the puromycin resistant gene.
Cell proliferation assay in control, PLC/Cx32 and PLC/Cx26
For assessment of cell proliferation, cells were plated at a density of 1 x 104 cells per well in 12-well plates (Becton Dickinson, Franklin Lakes, NJ) in 1 ml complete medium. On day 10, the number of cells was counted using a hemocytometer after cells were harvested by mild trypsinization (exposed to 0.05% trypsin/0.02% EDTA (Life Technologies, Rockville, MD) for 5 min).
AFP level in culture medium of control, PLC/Cx32 and PLC/Cx26
Control, PLC/Cx32 or PLC/Cx26 cells were grown on 12-well plates. On day 10, AFP level in culture medium was determined by radioimmunoassay (RIA) (ng/ml/105 cells) using alpha-feto-RIA beads (Dainabot, Tokyo, Japan).
FACS analysis for Cx26 in control and PLC/Cx26
Control and PLC/Cx26 cells were grown in 150 ml tissue culture flasks. Control and PLC/Cx26 cells were collected, not fixed, incubated with an antibody to rat Cx26 in solution (dilution x100 in PBS) (43) and stained with anti-rabbit IgG (H+L)-fluorescein (Boehringer Mannheim, Mannheim, Germany). Control and PLC/Cx26 cells were resuspended in PBS, and fluorescence from 10 000 cells was analyzed by flow cytometry (FACSCalibur (Becton Dickinson, Franklin Lakes, NJ)).
Gap junctional intercellular communication (GJIC) in colonies of control and PLC/Cx26
Cells were plated at a density of 1 x 104 cells in 35 mm dishes (low density) (Becton Dickinson, Franklin Lakes, NJ). Many colonies, each of which was regarded to consist of monoclonal cells, were detected on the dishes on day 5. GJIC was assayed by transfer of the fluorescent lucifer yellow CH (5% (wt/vol) in 150 mM LiCl) (LY) (Sigma, St Louis, MO) after single-cell automatic microinjection with AIS2 (100 hPa, 0.3 s) (Carl Zeiss, Oberkochen, Germany). Microinjection was performed in each colony, and 20 colonies of control or PLC/Cx26 were used for this assay. Spread of LY was observed under a fluorescence inverted microscope 10 min after microinjection, photographed and the number of cells labeled with LY was counted.
Nucleo-cytoplasmic (N/C) ratio in control and PLC/Cx26
Cells (1 x 105) were seeded in 150 ml tissue culture flasks. On day 10, six fields were photographed randomly using 25-fold magnification. Five cells per field were selected randomly and the length and width of each nucleus and cytoplasm were measured. Size of nucleus and cytoplasm was defined as (length (mm2) x width (mm2)).
Immunohistochemistry for proliferating cell nuclear antigen (PCNA) in control and PLC/Cx26
Cells (5.26 x 103) were seeded in chamber glass slides, each of which had four chambers (20 x 10 mm) (Iwaki, Funabashi, Japan) (listed as 12-well plate used in the section `Cell proliferation assay in control, PLC/Cx32 and PLC/Cx26'). On day 10, PCNA-positive cells were detected immunohistochemically as described previously (44,45). Cells on glass slides were fixed with 70% methanol at room temperature for 2 h. They were incubated with mouse monoclonal antibody to PCNA (Dako Japan, Kyoto, Japan) in solution (dilution x100) in PBS (0.1 M, pH 7.4) at 4°C for 4 h, followed by biotinylated rabbit anti-mouse immunoglobulin (Dako Japan, Kyoto, Japan) (dilution x100) in PBS for 2 h at 4°C. They were finally incubated in peroxidase-conjugated streptavidin (Dako Japan, Kyoto, Japan) (dilution x100) and was stained with 0.06% 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Dojinkagaku, Kumamoto, Japan) in TrisHCl buffer solution (0.05 M, pH 7.6) with added H2O2. They were counter-stained with hematoxylin and examined by phase-contrast microscopy. PCNA-positive cells were photographed in peripheral and central areas of six colonies. The labeling index (LI) was expressed as the average percentage of PCNA-positive cells of 1000 in peripheral or central areas of each colony.
Immunohistochemistry for single-stranded DNA (ssDNA) in control and PLC/Cx26
Cells (5.26 x 103) were seeded in four-chamber glass slides. On day 10, apoptotic cells were detected immunohistochemically by the ssDNA method as described previously (4648). Cells on glass slides were fixed with 95% ethanol at room temperature for 5 s. These glass slides were placed in 3% H2O2/methanol solution for 5 min to quench endogenous peroxidase activity, washed with PBS (0.01 M, pH 7.4), and incubated with protein blocking agent (PBA) (Shandon, Pittsburgh, PA) for 5 min. They were incubated in a humidified chamber with rabbit primary antibody to ssDNA (Dako Japan, Kyoto, Japan) (dilution x200) for 60 min at room temperature, then washed three times in PBS (0.01 M, pH 7.4) for 5 min each, followed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody for 30 min under the same conditions. Coloration was performed with 0.06% 3,3'-diaminobenzidine tetrahydrochloride (DAB) in TrisHCl buffer solution (0.05 M, pH 7.6) with added H2O2 for 3 min at room temperature. They were counter-stained with hematoxylin and were randomly photographed. The apoptosis index (AI) was expressed as the average number of ssDNA-positive cells of 1000 in each field.
Tumorigenicity assay
Tumorigenicity assay was performed as described previously (33,34). C.B-17 scid/scid mutant (SCID) mice were purchased from CLEA (Osaka, Japan). Ten animals in each group (control and PLC/Cx26) were injected intraperitoneally with 0.2 ml acialo GM1 antibody (dilution x10 in HBSS) to reduce the activity of NK (natural killer) cells in SCID mice (49,50). The next day (day 0), they were injected with 5 x 106 cells per 0.2 ml in HBSS (Life Technologies, Rockville, MD) into the subcutaneous space of a unilateral flank. Once a week, the largest and smallest linear dimensions of all tumors (which were generally oval in shape) were measured with calipers. Tumor volumes were measured as length (mm2) x width (mm2)2 x 1/2 (51). In week 9 after subcutaneous injection, tumors were carefully removed after sacrifice and weighed.
Statistical evaluation of data
Two experiments with triplicate measurements were performed for the cell proliferation assay and determination of AFP in culture medium. Significant difference (P < 0.05) was tested using the MannWhitney U test.
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Results
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Cell proliferation assay of control, PLC/Cx32 and PLC/Cx26
To clarify whether Cx32 and 26 over-expression might reduce the proliferation of hepatoma cells, the number of control, PLC/Cx32 and PLC/Cx26 cells on day 10 after seeding was counted. Results were 25.23 ± 1.570, 7.06 ± 1.062* and 10.46 ± 0.920* (x104) (mean ± SE, n = 6, *P = 0.0039 versus control), respectively (Figure 1
). There was no significant difference in number between PLC/Cx32 and PLC/Cx26.

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Fig. 1. Number of control, PLC/Cx32 and PLC/Cx26 cells on day 10 after their seeding. The number of PLC/Cx32 and PLC/Cx26 cells was significantly (P = 0.0039) lower than that of control. However, there was no significant difference in number between PLC/Cx32 and PLC/Cx26.
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AFP level in culture medium of control, PLC/Cx32 and PLC/Cx26
To examine the influence of Cx32 and 26 over-expression on malignant potential in vitro, AFP level in culture medium of control, PLC/Cx32 and PLC/Cx26 cells was measured. It was 1801.1 ± 163.40, 31.2 ± 4.53* and 19.1 ± 2.50* (mean ± SE, n = 6, *P = 0.0039 versus control) (ng/ml/105cells), respectively (Figure 2
). There was no significant difference in AFP level between PLC/Cx32 and PLC/Cx26.

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Fig. 2. AFP level in culture medium of control, PLC/Cx32 and PLC/Cx26 cells on day 10 after seeding. AFP level (ng/ml/105cells) was significantly (P = 0.0039) lower in culture medium of PLC/Cx32 and PLC/Cx26 than that of control. However, there was no significant difference in AFP level between PLC/Cx32 and PLC/Cx26.
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Expression of Cx26 in control and PLC/Cx26
To confirm over-expression of Cx26 in PLC/Cx26, FACS analysis was performed. It showed a large group of cells expressing Cx26 weakly in control and PLC/Cx26, and there was an another group of cells overexpressing Cx26 in 10.6% (=1060 cells/total 10 000 cells) of PLC/Cx26 (Figure 3
) (arrow).

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Fig. 3. FACS analysis for Cx26 expression in control and PLC/Cx26 cells. The vertical axis indicates the counts of cells and the horizontal axis indicates the intensity of fluorescence of each cell. Large majority of control and PLC/Cx26 cells showed weak expression of Cx26, but overexpression of Cx26 was seen in 10.6% (=1060 cells/total 10 000 cells) of PLC/Cx26 (arrow). M1 + M2; number of total cells (= 10 000 cells) R4; number of cells overexpressing Cx26 (= 1060 cells) (arrow).
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GJIC in control and PLC/Cx26
To measure GJIC in control and PLC/Cx26, lucifer yellow was micro-injected. It spread only to adjacent cells or remained in the injected cells in all control colonies (Figure 4A and B
), but it spread from the injected cell into >25 neighboring cells in two of 20 PLC/Cx26 colonies (Figure 4C and D
).

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Fig. 4. Microinjection of LY into control and PLC/Cx26 cells. Phase-contrast image of control (A) and PLC/Cx26 cells (C). LY in a control colony was seen in an injected cell (B) or in a neighboring cell, but it spread widely from an injected cell in a PLC/Cx26 colony (D). *Injected cell. Arrow; fine needle. Bar = 25 µm.
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N/C ratio in control and PLC/Cx26
To observe changes in morphological features, the size of nucleus and cytoplasm was estimated and N/C ratio was examined, as a simple marker of cell differentiation. PLC/Cx26 cells were larger in size than control (Figure 5A and B
). The nuclear size in control and PLC/Cx26 was 5.00 ± 0.280 and 4.93 ± 0.477 (mean ± SE, n = 30) (mm2), respectively. There was no significant difference between them. The cytoplasmic size in control and PLC/Cx26 was 40.03 ± 4.312 and 82.80 ± 7.571* (mean ± SE, n = 30, *P < 0.0001) (mm2), and N/C ratio was 0.16 ± 0.014 and 0.07 ± 0.007* (mean ± SE, n = 30, *P < 0.0001), respectively (Figure 6
).

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Fig. 5. Morphological features of control and PLC/Cx26 cells. The cytoplasmic size in PLC/Cx26 was larger than that in control, and the nuclear size was similar in control and PLC/Cx26. (A) control colony; (B) PLC/Cx26 colony. Bar = 300 µm.
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Fig. 6. Nucleo-cytoplasmic (N/C) ratio of control and PLC/Cx26 cells on day 10 after seeding. N/C ratio of PLC/Cx26 cells was significantly (P < 0.0001) lower than that of control.
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Immunohistochemistry for PCNA in control and PLC/Cx26
To observe the influence of Cx26 over-expression on cell proliferation activity, PCNA was stained by immunoperoxidase method. Almost all of control and PLC/Cx26 cells showed PCNA-immunoreactivity in the peripheral area of each colony. PCNA-positive cells of PLC/Cx26 were lower in number in the central area than those of control (Figure 7A and B
), and there was a significant (P = 0.0039) difference in the labeling index (LI) of PCNA-positive cells between control and PLC/Cx26 (Table I
).

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Fig. 7. Immunohistochemistry for PCNA in central area of control and PLC/Cx26 colony (B). (A) There were many PCNA-positive cells in the center of control. (B) PCNA-positive cells were decreased in PLC/Cx26 colony, and they were less in number than that in control. Bar = 100 µm.
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Table I. Labeling index (LI) of PCNA-positive cells in control and PLC/Cx26 colony. LI was significantly lower in PLC/Cx26 colony than in control
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Immunohistochemistry for ssDNA in control and PLC/Cx26
To examine whether apoptosis would be induced by Cx26 overexpression or not, immunohistochemistry for ssDNA was performed. ssDNA-positive cells were scattered throughout the colonies of both control and PLC/Cx26. ssDNA-positive cells in PLC/Cx26 were more numerous than those in control (Figure 8A and B
). Apoptotic index (AI) of ssDNA-positive cells in control and PLC/Cx26 were 3.67 ± 0.045 and 22.34 ± 0.244* (mean ± SE, n = 4, P = 0.029) (Table II
). AI of ssDNA-positive cells in control was within the range of the AI of other neoplastic cells (AI = 2.39.5) (48).

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Fig. 8. Immunohistochemistry for single-stranded DNA (ssDNA) in control and PLC/Cx26 colony. (A) ssDNA-positive cells (arrow) were rarely seen in a control colony. (B) There were several positive cells (arrow) in PLC/Cx26 colony. Bar = 100 µm.
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Table II. Apoptosis index (AI) in control and PLC/Cx26 colony. AI was significantly higher in PLC/Cx26 colony than in control
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Tumor growth in control and PLC/Cx26
To examine the influence of Cx26 over-expression on tumor growth, control and PLC/Cx26 cells were injected into subcutaneous spaces in SCID mice and tumor volume was measured once a week. Tumor volume in PLC/Cx26 was significantly (P < 0.01) smaller than that in control throughout the period of observation (Figure 9A
). In addition, tumor weight was measured 9 weeks after inoculation. Tumor weight in control and PLC/Cx26 was 1.10 ± 0.126 and 0.53 ± 0.093* (mean ± SE, n = 10, *P = 0.0019) (g), respectively (Figure 9B
).

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Fig. 9. Tumor volume of control and PLC/Cx26 cells every week, and tumor weight in week 9 after inoculation. (A) Tumor volume of PLC/Cx26 cells was significantly smaller than that of control every week after inoculation. (B) Tumor weight of PLC/Cx26 cells was significantly lighter than that of control.
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Discussion
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Transfection with Cx32 cDNA was previously reported to acquire an ability for negative growth control of a hepatoma cell line (33), but it had not been determined whether Cx26 cDNA might have an inhibitory effect on tumor growth, so we investigated the influence of transfection with Cx32 and 26 cDNA on cell proliferation and AFP expression of a hepatoma cell line. The number of hepatoma cells, PLC/PRF/5, after culture was reduced by transfection with Cx26 cDNA by means of the non-viral vector as well as Cx32 (33) and AFP level was decreased in culture medium after their transfection. Our results indicated that transfection with Cx cDNA reduced not only growth rate of hepatoma cells but also their AFP production as well as that with Cx32 cDNA. Recently, it has been reported that Cx26 is also a candidate as a tumor suppressor, because transfection with Cx26 cDNA retarded the growth of cervical cancer HeLa cells deficient in expression of gap junctional proteins (39) or of choriocarcinoma Jeg-3 cells which exhibits extremely low cellcell communication (35). It was necessary to clarify whether transfection with Cx26 cDNA could affect the malignant potential of human hepatoma cells.
Our balc system, in which a hepatoma cell line is not cloned, is useful for examining the effect of transfection with Cx26 cDNA on the malignant potential of hepatoma cells, because we cannot transfect cDNA into all neoplastic cells in vivo by viral or non-viral vectors. It was important to clarify whether cell proliferation or differentiation is altered by gene transfer into a subpopulation of cells, and a mixture of transfectants with non-transfectants is necessary. Then subcutaneous inoculation of the mixture was used to examine the effect of transfection with Cx cDNA on tumor growth. We observed overexpression of Cx26 in 10.6% of PLC/Cx26 cells by FACS analysis, but we did not identify whether PLC/Cx26 cells with Cx26 overexpressed had development of GJIC or not. PLC/Cx26 cells with Cx26 overexpressed might be found to have development of GJIC in 10% of their colonies, in which LY was spread widely in comparison with control after its microinjection. Considering the effect of Cx26 gene transfer in vivo, we should apply `balc' of transfectants, in which a hepatoma cell line is not cloned, for examining the effect of Cx cDNA transfer on tumor growth or differentiation (complete transfection into all neoplastic cells is impossible in practical gene therapy).
The N/C ratio is known to be lower in differentiated neoplastic cells than in undifferentiated ones (52,53). Our transfectants with Cx26 cDNA had larger cytoplasm (34,35,39) and lower N/C ratio than that in control, and PLC/Cx26 cells acquired morphological features of differentiated neoplastic cells. AFP expression is a phenotype of hepatocellular carcinoma, and its expression is reported to be associated with loose cellcell contact, especially loss of GJIC (54). PLC/Cx26 cells had large contact area with neighboring cells and restoration of GJIC was detected in 10% of PLC/Cx26 colonies. cAMP and Ca2+ can pass through GJ (1,2,4) more abundantly in PLC/Cx26, and AFP regulatory sequence can be influenced by cAMP or Ca2+ (5557); therefore AFP was reduced to a low level in culture medium of PLC/Cx26. The `bystander effect' through GJIC might be closely related to the lowering of AFP level in the medium, and some regulatory factor might spread extracellularly through the medium to neighboring colonies and reduce AFP expression in hepatoma cells. Therefore, increase in gap junctional formation and development of GJIC may influence AFP expression in PLC/Cx26 hepatoma cells.
It was not known if transfection with Cx26 cDNA could affect proliferative activity of human hepatoma cells, thus we studied PCNA-positive cells in control and PLC/Cx26 cells by immunocytochemistry. In the peripheral areas of colonies, there was no significant difference in proliferative activity between control and PLC/Cx26 cells, but it was significantly decreased in the central areas of PLC/Cx26. Therefore, transfection with Cx26 cDNA exerts an inhibitory effect on cell proliferation in the central area inducing density-dependent inhibition of growth (58), and growth-inhibitory factors or growth-controlling signals may pass not only through GJ channels but also via the medium from the center to the surrounding area (35,39,59).
It had not been examined whether apoptosis is induced in hepatoma cells by transfection with Cx26 cDNA. ssDNA-positive cells were found to increase significantly in colonies of PLC/Cx26 cells, and apoptosis may be accelerated by transfection with Cx26 cDNA. The relationship between GJIC and apoptosis is not well understood, but apoptotic cells were reported to increase in number in the development of GJIC (40). In addition, increase of gap junctional channel formation was said to facilitate death signals (60) and it may contribute to initiating the programmed cell death (61). Tumor promoters are regarded to inhibit apoptosis (62) and to block GJIC (63,64), while tumor suppressors augment apoptosis and enhance GJIC (64). There should be a close relationship between GJIC and apoptosis, but further examination is required to clarify their relationship.
We examined the effect of transfection with Cx26 cDNA on tumor growth of hepatoma cells in vivo. Tumor volume of PLC/Cx26 in SCID mice was significantly smaller than those of control. In addition, tumor weight of PLC/Cx26 was significantly lighter week 9 after inoculation than that of control. These findings suggest that transfection with Cx26 cDNA reduced tumor growth. Transfection with Cx43 cDNA was known to reduce proliferative activity of neoplastic cells and to prolong their G1 phase (34); hence tumor growth of PLC/Cx26 may be retarded by inhibition of cell proliferation, induction of apoptosis and prolongation of cell cycle. In addition, other factors, such as angiogenesis and necrosis, should be taken into consideration for tumor growth.
Suicide gene therapy combined with transfection of Cx cDNA has been carried out to facilitate necrosis and cell death through the bystander effect (3032). Cells transfected with herpes simplex virus thymidine kinase (HSVtk) cDNA are sensitive to the toxic effect of phosphorylated gancyclovir (GCV). Toxic phosphorylated GCV passes through GJ (6567) and many neighboring tumor cells are killed by the bystander effect (6872). Transfection with Cx26 cDNA inhibited proliferation and induced apoptosis in our study. Therefore, co-transfection with Cx26 cDNA not only induces the bystander effect in gene therapy of the HSVtk/GCV system for human hepatoma cells but also reduces their malignant potential.
Transfection with Cx26 cDNA reduced the malignant potential of human hepatoma cells; therefore, it is suggested that co-transfection with Cx26 cDNA is useful for HSVtk/GCV suicide gene therapy as well as with other Cxs cDNA. Transfection with Cx26 cDNA itself should be one of the new therapeutic candidates for human hepatomas.
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Notes
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1 To whom correspondence should be addressed Email: amura{at}sun.kpu-m.ac.jp 
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Acknowledgments
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We would like to thank Dr Klaus Willecke (University of Bonn, Bonn, Germany) for pBEHpac18/Cx26 and pBEHpac18 cDNAs, Dr T.Ohkusa (Tokyo Medical and Dental University School of Medicine, Tokyo, Japan) for rabbit antibody to rat Cx26 (IgG fraction) and Dr Takafumi Ogawa (Cooperative Pathology Institute, Kobe, Japan) for assistance with pathology. We would also like to thank Dr Yoshio Sumida (Kyoto Prefectural University of Medicine, Kyoto, Japan) for his helpful advice on this study and to thank Dr M.Murozek for his revision of the English language. This investigation was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (no. 11670523).
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Received February 19, 2001;
revised November 2, 2001;
accepted November 6, 2001.