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[Cancer Research 60, 5087-5091, September 15, 2000]
© 2000 American Association for Cancer Research

Carcinogenesis

Lack of Phenobarbital-mediated Promotion of Hepatocarcinogenesis in Connexin32-Null Mice1

Oliver Moennikes, Albrecht Buchmann, Alessandro Romualdi, Thomas Ott, Jürgen Werringloer, Klaus Willecke and Michael Schwarz2

Institut für Toxikologie, 72074 Tübingen [O. M., A. B., J. W., M. S.], and Institut für Genetik, 53117 Bonn [A. R., T. O., K. W.], Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connexin32 (Cx32) is the major gap junction forming protein in liver. We have recently shown that hepatocarcinogenesis is strongly enhanced in mice deficient in Cx32, demonstrating that lack of functional Cx32 accelerates liver tumorigenesis. Many tumor-promoting agents, including phenobarbital, block gap junctional intercellular communication in vitro, and it has been suggested that this effect is relevant for clonal expansion of neoplastic cells in vivo. We have now tested this hypothesis by analyzing the potency of phenobarbital as a liver tumor promoter in male Cx32-wild-type (Cx32Y/+) and Cx32-null (Cx32Y/-) mice. Preneoplastic and neoplastic liver lesions were induced in 6-week-old male mice by a single injection of 90 µg/g body weight of N-nitrosodiethylamine, and groups of mice were subsequently kept on phenobarbital-containing (0.05%) or control diet for 39 weeks. Frozen liver sections were prepared, and (pre)neoplastic lesions were identified by their deficiency in glucose-6-phosphatase staining. In addition, the number and size of macroscopically visible tumors were monitored. Phenobarbital led to a ~5-fold increase in the volume fraction occupied by glucose-6-phosphatase-deficient liver lesions in Cx32Y/+ mice, whereas there was no such increase in Cx32Y/- mice. Even more pronounced differences were observed with respect to tumor response. Whereas phenobarbital clearly promoted the occurrence of numerous large hepatomas in Cx32Y/+ mice, no such effect was seen in Cx32Y/- mice. These results demonstrate, for the first time, that functional Cx32 protein is required for tumor promotion by phenobarbital.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Connexins are subunits of gap junctional channels, through which directly neighboring cells exchange low molecular weight molecules, such as ions, second messengers, and cellular metabolites. Intercellular communication mediated via gap junctions plays an important role in tissue homeostasis, embryonic development, and in cancer (for recent reviews, see Refs. 1, 2, 3, 4 ). The important role of gap junctional communication in multistage carcinogenesis is supported by several lines of evidence: Gap junctional proteins are often decreased in tumor tissue (5, 6, 7, 8, 9) , and overexpression of connexins suppresses tumorigenicity of tumor cells (10) . Moreover, targeted disruption of the Cx323 gene in mice is associated with enhanced occurrence of both spontaneous and chemically induced liver tumors (11 , 12) . The introduction of activated oncogenes into cells blocks GJIC (for review, see Ref. 4 ), and similar effects were seen if cells were exposed to tumor promoting agents in vitro. A large number of chemicals, including 12-tetradecanoyl-phorbol-13-acetate, PB, certain polyhalogenated biphenyls, and insecticides like endosulfane, chlordane, dieldrin or DDT, all known to promote carcinogenesis in skin or liver, have been tested, and many of these, albeit not all, were found to block GJIC (for reviews, see Refs. 13, 14, 15 ). Tumor promoters may affect GJIC by interference with cell signaling pathways that lead to direct blockage, e.g., by posttranslational modification of connexins or an increase in intracellular Ca2+ (for review, see Refs. 1 and 3 ). Moreover, a decrease in expression of connexins has been reported for various liver tumor promoters (7 , 9 , 16, 17, 18) .

The mechanisms by which GJIC affects the proliferation of transformed cells are not entirely understood, but two distinct types of cell-cell communication, termed homologous and heterologous communication, have to be considered (3) , the latter describing communication between transformed and surrounding normal cells. Lack of heterologous GJIC between tumor and normal cells has been observed in vivo; e.g., rat liver preneoplastic foci failed to communicate with their surrounding normal hepatocytes (8) . It has been suggested that loss of heterologous GJIC uncouples preneoplastic and neoplastic cells from growth-restraining signals of surrounding normal cells (19) . Accordingly, a clonal expansion of tumor or tumor precursor cells in solid tissues would be expected to result from inhibition of heterologous GJIC by tumor promoters, thus explaining their enhancing activity on carcinogenesis. We have now performed an initiation-promotion experiment in Cx32-wild-type and Cx32-null mice and found that the tumor promoter PB enhances DEN-induced hepatocarcinogenesis in Cx32-competent, but not in Cx32-deficient mice. This observation demonstrates the important role of the Cx32 tumor suppressor protein during the promotional phase of liver carcinogenesis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cx32-deficient mice used in the present study were generated by standard methods of targeted homologous recombination leading to a mixed genetic background of C57BL/6 and 129Sv inbred strains (20) . Cx32 heterozygous female C57BL/129Sv (Cx32+/-) mice were crossed with male C3H/He (Cx32Y/+) mice to yield male Cx32Y/+ and Cx32Y/- offspring. Tail tips were taken, and Cx32-genotyping was performed by standard PCR as recently described (12) . Two separate carcinogenesis experiments were performed. In experiment 1, mice were i.p. injected with a single dose of DEN (90 µg/g of body weight) at 6 weeks of age, while 10 µg/g body weight of the carcinogen were injected into 12–15-day-old mice in the second experiment. After a treatment-free interval of 3 weeks, DEN-treated Cx32Y/+ and Cx32Y/- mice were randomly assigned to groups, which were either kept on a standard diet or on a diet containing 0.05% PB until sacrifice. The numbers of mice in the various treatment groups are listed in Tables 1Citation 2Citation 3Citation . Mice were killed 39 weeks (experiment 1) or 25 weeks (experiment 2) after the start of PB treatment, and livers were inspected for macroscopically visible tumors. In experiment 1, where large tumors were apparent because of the longer duration of the experiment, the number and size of tumors were recorded, except for those animals where the tumor response was such that individual tumor counts were no longer possible. In both experiments, livers were frozen on blocks of dry ice and frozen sections were stained enzyme-histochemically for G6Pase activity (21) . Number and volume fraction in liver of G6Pase-deficient lesions were quantitated by means of a computer-assisted digitizer system (22) and analyzed using standard stereological techniques (23) .


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  		Table 1 Effect of PB on tumor multiplicity in Cx32-wild-type and Cx32-null mice initiated by DEN at adult age (experiment 1)a

 

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  		Table 2 Effect of PB on number and volume fraction of G6Pase-deficient lesions in mice treated with DEN at adult age (experiment 1)

 

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  		Table 3 Effect of PB on number and volume fraction of G6Pase-deficient lesions in mice treated with DEN at infancy (experiment 2)

 
Cx32 protein expression was analyzed by standard immunohistochemical protocols. In brief, frozen liver sections were fixed in ethanol (-20°C; 5 min) and air-dried, and endogenous peroxidase activities were blocked by treatment with methanol/H2O2. Sections were then incubated with polyclonal goat anti-Cx32 antibodies (SantaCruz Biotechnolgy, Santa Cruz, CA) at a 1:250 dilution, and antibody binding was visualized using horseradish peroxidaseconjugated antigoat-IgG antibodies (SantaCruz), followed by staining with 3-amino-9-ethylcarbazole/H2O2. Specimens were counterstained with hematoxylin.

For analysis of GJIC, groups (each n = 4) of adult Cx32Y/+ mice of strain B6C3F1 (Charles River) were treated with a PB (0.05%)-containing diet for 2 weeks or were kept untreated. After killing, the lateral liver lobe was immediately transferred to ice-cold buffer (Krebs-Henseleit buffer supplemented with 25 mM glucose) and used for preparation of 250-µm-thick sections, using Leica microtome VT1000S. The sections were microinjected in a microscope submerging chamber at 37°C under constant flow of oxygenated buffer (Krebs-Henseleit buffer supplemented with 25 mM fructose) after an incubation period of 60–90 min at 37°C in an artificial oxygen atmosphere (95% O2 + 5% CO2). Microinjection of the fluorescent dye Alexa Fluor488 (Molecular Probes Europe BV, Leiden, the Netherlands) was performed by iontophoresis.4 Before and after each microinjection, membrane potential was measured, and only those injections were considered where the potential was <-20 mV. Gap-junctional coupling was measured 10 min after injection with a special cross-field ocular under UV illumination with a Zeiss Axioskop FS determining the area of distribution, which was used for recalculation of the number of cells being affected.

For analysis of PB effects on liver weight and drug metabolizing enzymes, adult Cx32Y/+ and Cx32Y/- mice (three per group) were treated for 4 weeks with a PB (0.05%)-containing diet or were kept on control diet. Liver microsomes were prepared by standard procedures, and the contents of cytochrome P450 and cytochrome b5 were determined by difference spectrometry as described (24) .

Statistical analysis of data were performed using InStat for Windows NT (version 3.02) from GraphPad Software (San Diego, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1.
In this experiment, mice were given a single injection of DEN at the age of 6 weeks, and groups of mice were subsequently kept for 39 weeks on a PB-containing or control diet until sacrifice. At the end of the experiment, the tumor prevalences in Cx32Y/+ mice kept on control or PB-containing diets were 76% (13 of 17) and 94% (17 of 18), respectively, whereas the corresponding values in Cx32Y/- mice were 87% (13 of 15) and 100% (15 of 15), respectively. Macroscopically visible tumors were counted and classified according to size. In some of the animals, however, individual tumor counts were not possible because the entire liver appeared tumorigenic. The phenotype of livers from mice showing this extremely strong tumor response was therefore categorized as "polyadenomatosis." Mice with polyadenomatosis were only observed in groups receiving PB, but the frequency of occurrence differed significantly (P < 0.01) between Cx32Y/+ and Cx32Y/- mice: whereas 50% of the PB-treated Cx32Y/+ mice showed polyadenomatosis, only 7% of Cx32Y/- mice showed this extreme tumor response (Fig. 1)Citation . Because the numbers and size of individual tumors could not be quantified accurately in mice with polyadenomatosis, these animals were excluded from the tumor analyses described below. When animals were grouped according to their largest tumor observed (Fig. 1)Citation , ~50% of Cx32Y/+ control mice not treated with PB showed tumors <2 mm in diameter, whereas only ~10% had tumors of >=10 mm. By contrast, an inverse relation was found in PB-treated Cx32Y/+ mice, where ~55% of mice had tumors >=10 mm, and only ~20% had tumors <2 mm in diameter. This dramatic effect caused by PB treatment in Cx32-wild-type mice was clearly absent in mice of the Cx32-null genotype (Fig. 1)Citation . Similarly, the multiplicity of tumors stratified by size demonstrated a ~6-fold increase by PB in the number of very large tumors (>7 mm in diameter) in Cx32Y/+ mice, whereas no such increase was observed in Cx32Y/- mice (Table 1)Citation .



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  		Fig. 1. Effect of PB on tumor prevalence in Cx32-wild-type and -null mice initiated by DEN at adult age (experiment 1). Mice were categorized according to their largest liver tumor, and the percentages of mice per group represented by the indicated tumor size classes were calculated. In addition, the percentages of mice with polyadenomatosis (P) are shown; numbers of animals are indicated. {square}/-, minus PB; {blacksquare}/+, plus PB. *, significantly different from mice not treated with PB (P = 0.001; Fisher’s exact test).

 
The number and size of G6Pase-deficient neoplastic lesions in liver were subsequently quantified in 59 of 65 mice; six livers were not available for G6Pase staining. The numbers of lesions were increased by PB both in Cx32Y/+ and Cx32Y/- mice. There was, however, a highly significant ~5-fold increase in the volume fraction occupied by G6Pase-deficient tissue in livers of Cx32Y/+ mice treated with PB when compared with their counterparts not treated with the barbiturate (P = 0.0003). By contrast, no such increase was seen in Cx32Y/- mice (Table 2)Citation .

Experiment 2.
In this experiment, 12–15-day-old mice were treated with DEN followed by 25 weeks of treatment with PB-containing or control diet until sacrifice. According to literature data (Ref. 25 and references cited therein) and our own observations made in other mouse strains,5 we expected an inhibitory rather than a promotional effect of PB on hepatocarcinogenesis when using infant instead of adult mice. Because of the shorter duration of the experiment, analysis of the carcinogenic outcome in this experiment was based exclusively on the evaluation of G6Pase-deficient liver lesions. As summarized in Table 3Citation , the number of G6Pase-deficient lesions was not significantly affected by PB treatment, neither in Cx32Y/+ nor in Cx32Y/- mice. By contrast, the mean volume fraction in liver occupied by G6Pase-deficient tissue was reduced by PB in both strains, although the effect was only significant in Cx32Y/- mice (P = 0.0007).

A comparison of the effects of the Cx32 gene-knockout observed in animals not treated with PB in experiment 1 and 2 (see Tables 2Citation and 3Citation ) shows that G6Pase-deficient lesions were generally much less frequent in mice treated with DEN at adult age, resulting in an overall smaller volume fraction in liver, although a 9-fold higher carcinogen dose was used than in experiment 2, where DEN treatment was performed at infancy. This result was not unexpected because infant mice are much more susceptible to hepatocarcinogenesis, presumably because of the high rate of hepatocyte proliferation at this age (26) .

The effect of PB on Cx32 expression and GJIC in Cx32Y/+ mice was investigated as follows. Sections from livers of mice from experiment 1 and 2 were stained with anti-Cx32 antibodies. In normal liver, there was no obvious effect of the barbiturate on the expression and intracellular localization of the Cx32 protein, as demonstrated by the representative examples shown in Fig. 2ACitation . Liver tumors from Cx32Y/+ mice showed either unchanged or even slightly increased Cx32 staining (Fig. 3B)Citation , whereas the staining reaction was decreased or totally absent in others (Fig. 3A)Citation . In experiment 1, ~60% of tumors showed positive or unchanged Cx32 staining in the absence of PB treatment, whereas ~40% showed decreased or negative staining. In PB-treated Cx32Y/+ mice, however, this relation was inversed: <20% of tumors were of the positive/unchanged phenotype, whereas the majority showed decreased or negative staining. It is remarkable that these effects were not seen in experiment 2, where an unchanged/positive Cx32 immunoreaction was detected in the vast majority of tumors, both in PB-treated and untreated mice (Fig. 3C)Citation .



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  		Fig. 2. Effect of PB on Cx32 immunostaining and GJIC in normal liver tissue of Cx32Y/+ mice. A, immunohistochemical demonstration of Cx32 protein in normal liver tissue; note the presence of Cx32 containing gap-junction plaques both in control and chronically PB-treated mice. B, dye transfer in normal liver; Alexa Fluor 488 fluorescent dye was iontophoretically injected into 250-µm-thick sections from livers of control or PB-treated mice (0.05% in the diet for 2 weeks; n = 4), and GJIC was measured by the spreading of dye into the neighboring cells; top and bottom graphs, results from Cx32Y/+ and Cx32Y/- mice, respectively. Bars and error bars, means ± SD. Images were taken by use of a video camera system (Kappa, Gleichen, Germany) adapted to the microscope; magnification bar, 50 µm.

 


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  		Fig. 3. Effect of PB on Cx32 immunostaining patterns of liver tumors. A, representative example of a lacking Cx32 immunoreaction; insert, the tumor at lower magnification. B, liver tumor with positive Cx32 immunosignals; Cx32 immunostaining is similar as in normal liver; inserts, the tumor at lower magnification. A micrograph illustrating a G-6-Pase-stained serial section is also presented for better orientation. Images were taken by use of a video camera system adapted to the microscope; magnification bar, 50 µm. C, frequency distributions of tumors showing a Cx32-positive or unchanged, decreased, or negative immunoreaction in experiments 1 and 2. Numbers indicate total numbers of tumors analyzed in the respective treatment groups.

 
We then studied the effect of PB on gap junctional coupling between hepatocytes. Cx32Y/+ and Cx32Y/- mice were treated with PB-containing or control diet for 2 weeks, and liver thick-sections were prepared and microinjected with fluorescent dye Alexa Fluor 488 followed by measurement of dye spreading into adjacent cells. The results demonstrated clearly that PB treatment did not decrease GJIC in the normal liver, neither in Cx32Y/+ nor in Cx32Y/- mice (Fig. 2B)Citation .

Finally, the effect of PB on liver weight and drug metabolizing enzymes in liver was investigated in adult mice kept for 4 weeks on a PB-containing diet. The results of this short-term study are summarized in Table 4Citation . PB treatment led to a significant increase in the liver:body weight ratio both in Cx32Y/+ and in Cx32Y/- mice. Similarly, the liver microsomal contents of cytochromes P450 and b5 were significantly elevated by PB in both strains of mice, and the extent of the response to the microsomal inducer was in the same range. This indicates that the physiological response that triggers PB-mediated liver hypertrophy and microsomal enzyme changes is not affected by the connexin defect. Drug-related increases in liver weight and liver microsomal enzymes have been shown to be correlated with their tumor promotional activity (e.g., see Refs. 27 and 28 ). For example, within a series of different barbiturates, including PB, those chemicals that produced liver hypertrophy and increases in cytochrome P450b were all potent liver tumor promoters while structural analogues that failed to induce microsomal enzymes also failed to display significant promotional effects (29) . Induction of liver growth and liver promotional activity, however, do not seem to be strictly correlated but might diverge under certain conditions, as indicated by the data of the present study.


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  		Table 4 Effect of PB on liver weight and drug-metabolizing enzymes in liver

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PB has been shown to enhance hepatocarcinogenesis in mice initiated by DEN at an adult age (25 , 30 , 31) , but inhibited tumor formation when administered to mice initiated at infancy (Ref. 25 and references therein). Similar results were obtained in the present study in Cx32-wild-type mice in experiments 1 and 2, respectively. Although the suppressive effect of PB on the growth of G6Pase-deficient lesions observed in mice initiated by DEN at infancy was less dramatic than the one obtained by Lee et al. (25) and only significant in Cx32Y/- mice, it was in the range of effects seen in a comparable study with male B6C3F1 mice performed earlier (32) . The mechanisms of this paradoxical effect of the barbiturate are not entirely clear. It has been suggested (25) that PB shows opposing effects on basophilic and eosinophilic lesions in liver: in infant mice, the predominant types of lesions produced by DEN were basophilic hepatocellular adenomas, and the proliferation of these was suppressed by PB. In adult mice, however, eosinophilic lesions predominated in PB-treated mice, suggesting that the clonal outgrowth of this type of lesion was preferentially promoted by the barbiturate (25) . In accordance with these results, most of the lesions in experiment 2 were basophilic, both in control and PB-treated mice, whereas the vast majority of lesions promoted by PB in experiment 1 were of a nonbasophilic/eosinophilic phenotype (data not shown).

We have previously demonstrated that mice deficient for Cx32 show a strong increase in growth of both spontaneous and DEN-induced preneoplastic and neoplastic lesions in liver, in comparison with their wild-type counterparts. This suggests that Cx32 inhibits the proliferation of transformed hepatocytes during hepatocarcinogenesis, i.e., it acts as a tumor suppressor protein (11 , 12) . This effect is mediated most likely through cell-cell communication via gap junctions. GJIC in livers of Cx32-null mice was found to be decreased by ~80% estimated by electrophysiological measurements (33) and by direct dye injection technique into liver slices (see Fig. 2BCitation ).4 We have now found that treatment of DEN-initiated adult Cx32-wild-type mice with PB results in tumor promotion, whereas this effect is lacking in Cx32-null mice. This demonstrates that functional Cx32 is required for tumor promotion by PB and suggests that the promoting agent counteracts tumor suppression by the connexin.

A decrease in GJIC is also produced by many tumor promoters (for review, see Refs. 14 and 15 ). These results have strengthened the hypothesis that suppression of GJIC is a key mechanism by which tumor promoters enhance the clonal expansion of potential cancer cells. Unexpectedly, PB did not affect dye transfer in acute slices of wild-type mouse liver, as shown in the present study nor was there an alteration in the expression or intercellular localization of Cx32 in liver from PB-treated wild-type mice. Similarly, there was no effect of PB on GJIC in Cx32-null mice that express only Cx26. This result is at variance with findings in rat liver where PB caused inhibition of dye transfer and concomitant decrease in Cx32-positive immunostaining (16 , 34 , 35) and with results from in vitro studies with primary mouse hepatocytes where PB led to partial suppression of GJIC (36) . The reason for this discrepancy is not clear. In any case, however, normal hepatocytes are not the main target population for the tumor promoting effects of PB.

Although there was no detectable effect of PB on Cx32 expression and GJIC in normal liver from Cx32Y/+ mice, the barbiturate led to a significant increase in the frequency of tumors with decreased or negative expression of Cx32 in experiment 1, where PB promoted hepatocarcinogenesis in Cx32-wild-type mice. Similar observations have been made earlier in a comparable study with B6C3F1 mice (13) . By contrast, PB did not increase the frequency of Cx32-negative lesions in experiment 2, where no tumor promotion occurred. Potentially, there is a positive selection for tumor cells with less functional Cx32 protein because of lacking communication with their surrounding normal counterparts (heterologous GJIC). The signals exchanged between neighboring cells through GJIC for inhibition of tumor growth are presently unknown. In principle, these signals modulate cell division and/or death (apoptosis), both of which are critical for tumor development. Because Cx32-null mice were almost completely resistant to the tumor promoting activity of PB, our results clearly demonstrate that Cx32 represents an essential target for promotion of hepatocarcinogenesis by PB.

Cx32-null mice treated with the tumor initiating carcinogen DEN developed liver tumors much faster than identically treated Cx32-wild-type mice (11 , 12) . By contrast, our present data demonstrate an inverse situation for the carcinogenic response mediated by the tumor promoter PB. If this would apply to initiating and promoting hepatocarcinogens in general, the experimental system using Cx32-null and -wild-type mice would offer a very valuable toxicological tool to classify the carcinogens according to their predominant mode of action—tumor-initiating versus tumor-promoting.


   ACKNOWLEDGMENTS
 
We thank Elke Zabinski for excellent technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by the Deutsche Forschungsgemeinschaft. Also supported by a stipend of the Graduierten Kolleg Proteindomänen (to A. R.) and by the Dr. Mildred Scheel Stiftung and the Fonds of the Chemischen Industrie (to K. W.), which supported work in the Bonn laboratory. Back

2 To whom requests for reprints should be addressed, at Institut für Toxikologie, Wilhelmstrasse 56, 72074 Tübingen. Phone: 49-7071-29-77398; Fax: 49-7071-29-2273; E-mail: michael.schwarz{at}uni-tuebingen.de Back

3 The abbreviations used are: Cx32, connexin32; DEN, N-nitrosodiethylamine; G6Pase, glucose-6-phosphatase; GJIC, gap junctional intercellular communication; PB, phenobarbital. Back

4 A. Romualdi, H. Niessen, K. Willecke, and T. Ott, Quantitative analysis of gap junctional intercellular communication in precision-cut liver slices, manuscript in preparation. Back

5 K. Zuber, A. Buchmann, W. Nicklas, and M. Schwarz, unpublished observations. Back

Received 3/30/00. Accepted 7/18/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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