Transgenic Disruption of Gap Junctional Intercellular Communication Enhances Early but Not Late Stage Hepatocarcinogenesis in the Rat

Abstract

Much experimental evidence supports the conclusion that loss of gap junctional intercellular communication (GJIC) contributes to carcinogenesis. Transgenic rats featuring a dominant negative mutant of the connexin 32 gene under albumin promoter control (Cx32ΔTg-High and Cx32ΔTg-Low lines, respectively with high and low copy numbers of the transgene) have disrupted GJIC, as demonstrated by scrape dye-transfer assay in vivo as previous report by Asamoto et al. (2004). In the present study, we investigated the susceptibility of these transgenic rats to a single intraperitoneal administration of diethylnitrosamine (DEN), and found a significant increase in preneoplastic glutathione S-transferase placental form (GST-P) positive lesions in the livers of Cx32ΔTg-High but not Cx32ΔTg-Low rats. However, incidences of adenomas and hepatocellular carcinomas were not elevated at the end of the experiment (52 weeks). In addition, we investigated the promotional effect of phenobarbital (PB) on Cx32ΔTg-High rats pretreated with DEN and found enhanced formation of GST-P positive lesions, in contrast to the lack of promoting effects reported for Cx32 deficient mice. The results indicate that although both high and low expression of the dominant negative connexin 32 mutant gene in our rats is able to inhibit gap junctional capacity, only high expression is effective at enhancing susceptibility to early stage DEN-induced liver carcinogenesis.

Introduction

Connexin 32 (Cx32) is a major gap junction protein in the liver (Paul, 1986), forming transmembrane channels between adjacent cells which allow exchange of molecules less than about 1200 Da in mass, such as ions, second messengers, and cellular metabolites (Gilula et al., 1972; Loewenstein, 1981; Bennett et al., 1991; Yamasaki and Naus, 1996; Evans and Martin, 2002). It is reported that gap junctional intercellular communication (GJIC) plays important roles in tissue homeostasis (Loewenstein, 1981; Yamasaki and Naus, 1996), embryonic development (Guthrie and Gilula, 1989) and in carcinogenesis (Yamasaki, 1990; Trosko and Ruch, 1998). Indeed, there is a large body of evidence that disorders of GJIC have etiologic roles in tumor induction (Mesnil, 2002). Gap junctional proteins are often decreased in tumor tissue or tumor cell lines (Mesnil, 2002) and many tumor promoters, including 12-tetradecanoyl-phorbol-13-acetate (Ruch et al., 2001), phenobarbital (PB), dieldrin and DDT, inhibit GJIC (Trosko and Ruch, 1998). Chemopreventive agents such as retinoic acid, glucocorticoids, and cAMP, in contrast, often enhance GJIC (Yamasaki and Naus, 1996).

Furthermore, transfection of connexin genes into cancer cell lines may restore their communication capacity and inhibit growth in vitro (Yamasaki and Naus, 1996; Mesnil, 2002), while targeted disruption of the connexin 32 gene in mice is associated with enhanced occurrence of both spontaneous and chemically induced liver tumors (Temme et al., 1997; Moennikes et al., 1999; Evert et al., 2002). These observations have allowed connexins to be classified as tumor suppressors. It is of interest in this context that Cx32 deficient mice did not show tumor promoting effects of phenobarbital (Moennikes et al., 2000).

Transgenic mice provide good animal models for many diseases including neoplasia and are widely employed for analysis of various gene functions. For studies of chemical carcinogenesis, they are useful because of their high susceptibility to tumor induction by certain carcinogens. Cx32 knockout mice are also candidate animals for screening of carcinogens and elucidating the mechanisms of carcinogenesis (Temme et al., 1997; Moennikes et al., 1999, 2000; Evert et al., 2002).

However, rats rather than mice are more frequently used in chemical carcinogenesis studies for various reasons. For example, in the liver, a variety of enzyme-altered focal lesions have been studied in this species for their relevance to carcinoma development (Moore et al., 1981; Ito et al., 1984; Ogiso et al., 1985) and the glutathione S-transferase placental form (GST-P) has been utilized as a reliable marker for early detection of preneoplastic lesions (Sato et al., 1981, 1984; Ogiso et al., 1985; Ito et al., 1988).

We have recently established 2 lines of transgenic rats carrying a dominant negative mutant of Cx32 under control of the albumin promoter (Asamoto et al., 2004). In both strains, the normal membrane localization of endogenous Cx32 protein in the liver is disturbed and gap junction capacity measured by scrape dye-transfer assay in vivo is markedly decreased compared to the wild-type case (Asamoto et al., 2004). These animals were resistant to liver damage by hepatotoxic agents (Asamoto et al., 2004). The present study was carried out in order to investigate whether the functional disruption of GJIC affect rat liver carcinogenesis, and what role Cx32 might play in tumor progression. For these purposes, we investigated the susceptibility of our gap junction disrupted transgenic rat to diethylnitrosamine (DEN)-induced hepatocarcinogenesis, in experiments of up to 52 weeks’ duration with focusing on the effects of partial hepatectomy (PH) and PB-mediated promotion.

Materials and Methods

Animal Experiments

All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Nagoya City University Graduate School of Medical Sciences. The rats were randomly divided into groups of 3 per plastic cage with hard wood chips as bedding and maintained in an air-conditioned room at 22 ± 2°C and 55 ± 5% humidity with a 12-h light/dark cycle. Food (Oriental MF, Oriental Yeast Co., Tokyo, Japan) and tap water were available ad libitum throughout.

Establishment of Transgenic Rats

The Cx32 disrupted transgenic rats were established as described previously (Asamoto et al., 2004). Southern blotting analysis of genomic DNA from tails of the second generation of the transgenic founder rat revealed 2 sublines, one with high-copy numbers of the transgene (~50 copies) and the other with a low-copy number (~5 copies). These transgenic rats were designated as Cx32ΔTg-High and Cx32ΔTg-Low (Asamoto et al., 2004).

Production and Screening of the Transgenic Rats

DNA isolation from rat tails, and PCR-based screening assays were performed as described previously (Asamoto et al., 2000, 2001). Sequences of the PCR primers were 5′-AAC GTG GCG CAG GTG GTG TA-3′ and 5′-ATG GTG ATG GTG ATG ATG GC-3′, located on the 6xHis tag for the transgene. Southern blotting analysis for connexin 32 was also performed using the SmaI fragment of Cx32 cDNA after digestion of genomic DNA with BamHI. Heterologous transgenic males for the studies were routinely obtained by mating heterologous transgenic males and wild-type Sprague–Dawley females (Japan SLC, Inc., Shizuoka, Japan).

Animal Treatments and Identification of Preneoplastic Foci

In the first experiment, utilizing total 60 rats and medium-term bioassay system (Ito et al., 2000, 2003), male transgenic (Cx32ΔTg-High and Cx32ΔTg-Low) and wild-type rats (purchased from Japan SLC, Inc.) received a single intraperitoneal (ip) injection of 200 mg/kg body weight of diethylnitrosamine (DEN) (Tokyo Kasei Kogyo Co. Ltd, Tokyo, Japan), at the age of 6 weeks (see Table 1). They were subjected to PH at the end of the third week and all animals were sacrificed at the 8th week.

In the second experiment, total 76 male transgenic (Cx32ΔTg-High) and litter mate wild-type (Non-Tg) rats received a single ip injection of 200 mg/kg body weight of DEN at the age of 6 weeks, and were sacrificed sequentially after 0 (no treatment) and 3 days, 1, 2, 3, 8, 20, and 52 weeks (1 year) (see Table 2).

In the third experiment, male transgenic (Cx32ΔTg-High) and litter mate wild-type (Non-Tg) rats received a single ip injection of 200 mg/kg body weight of DEN at the age of 6 weeks (see Table 5). After a treatment-free interval of 2 weeks, they were randomly assigned to 2 groups of each rat strain, and maintained either on standard diet or with a 0.05% PB supplement (PB sodium salt, Wako Pure Chemicals Industries, Osaka, Japan) for 20 weeks and then sacrificed.

Immediately on sacrificing, the livers were excised, weighed, and cut into slices of 2–3 mm thickness, then fixed in ice-cold acetone or buffered formalin and routinely processed for embedding in paraffin for histological evaluation after hematoxylin and eosin staining. For demonstration of lesions, 3 μm-thick sections were cut and incubated with anti-GST-P polyclonal antibody (MBL, Nagoya, Japan) at a dilution of 1:4,000. Binding was visualized with a Vectastain Elite ABC kit (Vector Lab, Burlingame, CA) and light hematoxylin counter-staining was conducted. The average numbers and areas of GST-P positive foci >0.2 mm in diameter, and total areas of the liver section were measured with an image analyzer (IPAP; Sumica Technos, Osaka, Japan).

Immunohistochemical Staining of Cx32

Detailed methods for immunohistochemistry of Cx32 were described previously (Asamoto et al., 2004). Frozen sections were cut at 5 μm and fixed in cold acetone. A polyclonal antibody against Cx32 (kindly donated by Dr. V. Krutovskikh, IARC, Lyon, France), which recognizes the deleted part of the transgene, was used with biotin-conjugated anti-rabbit IgG and FITC-labeled streptavidin (Vector) to visualize endogenous Cx32 protein under a fluorescence microscope (Olympus AX-70, Tokyo, Japan). The polyclonal antibody applied recognizes amino acids 98 to 124 in Cx32, which are deleted in the transgene (missing amino acids 113 to 124). Therefore, the transgenes (mutated Cx32) could not be detected.

Dot Blot and Western Blot Analyses of Cx32

Dot blot protein assays were performed to clarify why signals for endogenous Cx32 were apparent in transgenic rat livers on Western blotting, but not immunofluorescence staining. In order to follow the procedures for immunofluorescence staining, 5 sections of frozen liver tissue from Non-Tg and Cx32ΔTg-High were cut at a thickness of 8 μm and collected in cooled tubes. These materials were immediately fixed in ice-cold acetone for 10 min. After washing in PBS for 10 min, the tissues were treated with suspension buffer and 2 × SDS gel-loading buffer. For the dot blotting, 3 μl of samples were simply spotted on dry nitrocellulose membranes (Hybond ECL, Amersham Bioscience, Tokyo, Japan). After drying the spots, the membranes were washed in PBS and treated with Cx32 antibody followed by anti-rabbit antibody and then the ECL plus detection system (Amersham Bioscience, Tokyo, Japan). For the Western blot analysis, the same materials as for dot blotting were used to perform conventional Western blot analysis, following methods detailed previously (Asamoto et al., 2004). Briefly, 10 μl acetone treated samples were loaded and separated on 12% SDS-polyacrylamide gels, then transferred to nitrocellulose membranes and Cx32 protein signals were detected in the same way as for the dot blot analysis.

Quantitative RT-PCR Analysis of CYP1A1 and 2E1

Cytochrome P450 (CYP) 1A1 and 2E1 are hepatic metabolic enzymes. One microgram of total RNA was converted to cDNA with avian myoblastosis virus reverse transcriptase (TaKaRa, Otsu, Japan) in 20 μl of reaction mixture and 2 μl aliquots were subjected to quantitative PCR in 20 μl reactions using FastStart DNA Master SYBR Green I and a Light Cycler apparatus (Roche Diagnostics, Mannheim, Germany). Primers for CYP1A1 and 2E1 were provided in the Rat Cytochrome P450 Competitive RT-PCR Set (TaKaRa). Primers for Cyclophilin were, 5′-TGCTGGACCAAACACAAATG-3′ and 5′-GAAGGGGAATGAGGAAAATA-3′. The fluorescence intensity of double-strand specific SYBR Green I, reflecting the amount of formed PCR-product, was monitored at the end of each elongation step and cyclophilin mRNA levels were employed to normalize the sample cDNA content.

Statistical Analysis

Statistical analysis was performed with the Fisher’s exact probability test for the incidence of liver tumors and the Student’s t-test for the remainder of the data in experiments 1 and 2, and ANOVA followed by the post hoc Bonferroni/Dunn test for the data of experiment 3 using the StatView-J 5.0 program (Berkeley, CA).

Results

Final liver/body weight ratios following DEN initiation were significantly decreased in rats undergoing partial hepatectomy (PH), regardless of the sublinein experiment 1 (Table 1), and the Tg rats at day 3 showed a lower liver/body weight ratio as compared to the Non-Tg rats in experiment 2 (Table 2).

Cx32ΔTg-High showed a high susceptibility to DEN induction of lesions, the numbers and areas of GST-P positive foci being very much greater (p ≤ 0.001) than in the other subline, with or without PH (Table 1). PH treatment significantly enhanced GST-P positive foci development, in terms of both the numbers and areas in the wild-type (p < 0.05) and Cx32ΔTg-Low (p < 0.0001) groups. In the Cx32ΔTg-High strain, this was the case for the areas (p < 0.05) but not the numbers (Table 1). In Experiment 2, Tg rats initiated with DEN demonstrated a significant increase in the numbers and areas of GST-P positive foci at 20 weeks (p < 0.05, for both) and 52 weeks (p < 0.05, p < 0.01, respectively), as compared with those of the Non-Tg group (Table 3). However, histopathological examination of the liver at each time point demonstrated no differences in DEN-induced inflammatory responses or necrotic changes between transgenic and Non-Tg (data not shown). The tumor incidences were slightly increased in the transgenic groups, but this did not reach statistical significance (Table 4).

The hepatic cytochrome P450s (CYP) are multigene family of enzymes that play a critical role in the metabolism of many drugs. CYP1A1 and 2E1 activate polycyclic aromatic hydrocarbons (PAHs) and N-nitrosamines respectively into highly carcinogenic agent (Sheweita, 2000). The mRNA expression of CYP1A1 and 2E1 after normalization to cyclophilin showed no difference between the Cx32ΔTg-High and Non-Tg rats at 0 day (no treatment) and 20 weeks in experiment 2. While CYP2E1 appeared to be increased by DEN treatment in both Cx32ΔTg-High and Non-Tg, this was not significant (data not shown). Therefore, we considered that Cx32ΔTg-High rats have normal activities of these metabolic enzymes.

Final liver/body weight ratios were significantly increased in rats undergoing PB treatment, regardless of the subline in experiment 3 (Table 5). PB treatment significantly enhanced GST-P positive foci development, in terms of both the numbers and areas in the wild-type (p < 0.05) groups. In the Cx32ΔTg-High strain, this was the case for the number (p < 0.05) but not the area (Table 5 and Figure 1).

Immunohistochemical staining of endogenous Cx32 revealed that the livers from transgenic rats lacked positive spots for gap junction plaques (Figure 2C). PB affected the normal localization of Cx32 in Non-Tg rats, causing a decrease specific to mid- and centro-lobular hepatocytes (Neveu et al., 1990).

With the dot blot analysis, strong signals were detected in Non-Tg liver, but levels were extremely low in the Cx32ΔTg-High case, in line with the results of the immunofluorescence staining (Figures 2B, 2C). However, when proteins were separated by electrophoresis in Western blotting procedures, bands for endogenous Cx32 were detected in both Non-Tg and Cx32ΔTg-High livers after acetone treatment (Figure 2A).

Discussion

Though Cx32ΔTg-High and Cx32ΔTg-Low rats both demonstrate reduced GJIC in dye transfer assays (Asamoto et al., 2004), only Cx32ΔTg-High showed higher susceptibility to DEN-induced hepatocarcinogenesis, compared with Cx32ΔTg-Low and wild-type animals in the present study, and the latter 2 showed similar responses to DEN. The findings thus suggest that high expression of dominant negative mutant Cx32 is necessary for significant influence. Furthermore, the incidences of adenomas and carcinomas in Cx32ΔTg-High strain were not significantly different from the values for the wild-type rats, so that progression from the foci to more advanced lesions was not accelerated. Therefore, a GJIC block in the rat liver in itself may not be sufficient to promote late stage hepatocarcinogenesis.

We found these Tg rats have expression of endogenous Cx32 by western blot analysis, but without visible immunofluorescence staining (Asamoto et al., 2004). Therefore, dot blot protein assays were performed to clarify this discrepancy, the results indicating that the dominant negative mutant Cx32 interacts with endogenous Cx32 and masks the functions and antigenicity for the Cx32 antibody. In our transgenic rats, the gap junction plaques could not be detected on cellular membranes by immunostaining. The results thus suggest that the 3-dimensional structure of endogenous Cx32 protein may be altered by the mutant, and the activity of channels in this Tg rat may be disrupted.

We have maintained the 2 lines of Tg rats for over 10 generations and have repeatedly checked the integrated copy numbers by Southern blot analysis, with immunofluorescence staining performed regularly. The copy numbers and results of immunofluorescence staining proved stable throughout.

Gap junctional proteins are known to be down-regulated when hepatocytes are in the S-phase of the cell cycle (Temme et al., 2000) and PH performed to induce regenerative hepatocyte proliferation enhances the development of preneoplastic lesions (Ito et al., 2003). In Cx32 deficient mice, liver regeneration after PH was found not to be disturbed by loss of the Connexin 32 gene (Temme et al., 2000) and in the present experiment, the dominant negative mutant of Cx32 did not exert any interference on PH-induced rat liver regeneration, because liver weights were not significantly different, and PH promoted GST-P positive foci formation in all groups. The results thus indicate that functional GJIC might not play a major role in mechanisms of the PH-induced enhancement of liver tumor development.

In mice with reduced GJIC after introduction of a dominant negative mutant of Cx32, established with a similar construction to that applied here, liver growth is not affected but susceptibility to DEN-induced hepato-carcinogenesis is increased (Dagli et al., 2004). Cx32 deficient mice also demonstrate high incidences of spontaneous and chemically induced liver tumors (Temme et al., 1997; Moennikes et al., 1999, 2000; Evert et al., 2002) and when transgenic and knock out mice were given a single ip injection of DEN at 2 weeks after birth, many liver adenomas and carcinomas occurred after 1 year (Temme et al., 1997; Dagli et al., 2004). Thus, there might be appreciable differences between the rat and the mouse models regarding tumorigenic responses under conditions of loss or aberrant expressions of Cx32. The fact that our rats showed a high yield of preneoplastic lesions but not adenomas or carcinomas, indicates that the reduced GJIC might be important for enhancement of early stage carcinogenesis, but other factors are involved in further progression, as observed in mice liver carcinogenesis.

It was well known that there are strain and species differences in susceptibility to liver tumor induction (Newsholme and Fish, 1994; Moennikes et al., 1999; van Ravenzwaay and Tennekes, 2002). The C57BL/6 strain, which is the background of both the Cx32 deficient and Cx32 dominant negative mutant transgenic mice, has low susceptibility with regard to spontaneous and chemical induction tumors, for example as compared with the C3H strain (Moennikes et al., 1999). The incidence of spontaneous liver tumors of C57BL/6 is 12.5% at 52 weeks of age (Temme et al., 1997) and that of DEN-induced liver tumors is 100% at 35 weeks after injection (Dagli et al., 2004). In comparison, in the present study, the tumor incidence of the Non-Tg (SD) animals was 43% at 1 year after DEN injection and the occurrence of spontaneous liver tumors in rats is well known to be much less than mice. However, progression to cancer might be possible at high incidence in the Cx32ΔTg-High strain with other treatment protocols.

Phenobarbital (PB) is a nongenotoxic carcinogen and a well-known promoter of rodent hepatocarcinogenesis both in vivo and in vitro (Kitagawa et al., 1983; Kolaja et al., 1996). In the present initiation-promotion experiment in our Tg rats, PB enhanced DEN-induced hepatocarcinogenesis. It has been reported that when PB is orally given to normal rats at a dose of 50 mg/kg, Cx32 spots in the central zone of liver lobules decreased (Ito et al., 1998). In the present study, PB enhanced the GST-P positive foci formation in both Cx32ΔTg-High and Non-Tg cases, demonstrating that the Cx32 does not play a major role in PB promotion effects on rat liver carcinogenesis. Moennikes et al. (2002) reported no promotion by PB in Cx32 deficient mice treated with DEN and therefore concluded that functional Cx32 protein is required for tumor promotion by PB in mice (Moennikes et al., 2000). However, this is clearly not the case in the rat.

In the present study, GJIC disruption by introduction of multiple copies of a transgene with deletion of 12 amino acids of the internal loop of Cx32 enhanced early stage DEN-induced in vivo hepatocarcinogenesis. This mutant has a similar deletion to that found in an X-linked Charcot-Marie-Tooth disease (CMTX) patient (Ionasescu et al., 1995) and corresponding deletion of the connexin 43 gene has been shown to exert a strong dominant negative effect in rat bladder carcinoma cells (Krutovskikh et al., 1998). Cx32-deficient mice were first established as an animal model of CMTX, and they develop late-onset progressive peripheral neuropathy with abnormalities comparable to those associated with the human disease (Anzini et al., 1997). They are highly susceptible to DEN-induced hepatocarcinogenesis (Anzini et al., 1997) although abnormalities of liver function or increased incidences of liver tumor have not been reported in CMTX patients. In summary, the present study showed high expression of mutant Cx32 to result in increased susceptibility to induction of early stage lesions by a single administration of DEN. However, in contrast to the mouse case, GJIC itself might not play major roles in the progression of the foci to adenomas and carcinomas in rat hepatocarcinogenesis. Our data thus indicate that other factors or events have more important involvement in both progression of neoplastic liver lesions and in mediation of PB promotion of DEN-initiated liver cells.

Recently we have focused on gene expression profiles with microarray analysis to elucidate mechanisms of gap junction related carcinogenesis, and obtained interesting results indicating several cytokine related signals are up-regulated in the Cx32ΔTg-High rats livers (unpublished data). Their specific roles are now under investigation. It is known that connexin proteins interact with other molecules such as tight junction and cell adhesion molecules (Giepmans, 2004), and connexin functions separate from junctions have now been proposed (Stout et al., 2004). Therefore, these issues in the Cx32 dominant negative transgenic rat models should be targeted in the future.