WY-14,643-mediated promotion of hepatocarcinogenesis in connexin32-wild-type and connexin32-null mice

Abstract

Connexin32 (Cx32) is the major gap junction forming protein in liver and lack of functional Cx32 enhances hepatocarcinogenesis. Many tumour-promoting agents block gap junctional intercellular communication, which may favour clonal expansion of neoplastic cells. We recently demonstrated that liver tumourigenesis is accelerated in Cx32-wild-type but not in Cx32-null mice by the model tumour promoter phenobarbital (PB). In the present study, male Cx32-wild-type and Cx32-null mice were treated with a single injection of 90 micro g/g body wt of N-nitrosodiethylamine (DEN) at 6 weeks of age and were subsequently kept on a diet containing the peroxisome proliferator [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid (Wy-14,643) or on control diet. Thirty-eight weeks after DEN treatment, mice were killed, frozen liver sections were prepared and (pre)-neoplastic lesions were identified by alterations in glucose-6-phosphatase (G-6-Pase) and glutamine synthetase (GS) staining. G-6-Pase-deficient lesions were generally small in size and were observed in all groups of mice. Large focal pre-neoplastic and benign neoplastic lesions, however, which demonstrated increased rather than decreased activity in G-6-Pase were exclusively present in DEN/Wy-14,643-treated mice. G-6-Pase-positive lesions were strongly promoted by Wy-14,643, both in Cx32-wild-type and Cx32-null mice without significant difference in response between mice of the two genotypes. This contrasts G-6-Pase-negative lesions and lesions over-expressing GS, which were both increased by WY-14,643 treatment in number and size in Cx32-wild-type but not in Cx32-null mice. GS-positive lesions from WY-14,643-treated mice harboured beta-catenin mutations, a hallmark of lesions selected during promotion by PB, while G-6-Pase-positive lesions, which displayed negative or diffuse GS staining, did not show beta-catenin mutations. Our results demonstrate significant differences between mouse liver lesions of differing phenotype and genotype in their response towards selection by Wy-14,643 during the promotional phase of hepatocarcinogenesis.

Effect of the tumor promoter phenobarbital on the pattern of global gene expression in liver of connexin32‐wild‐type and connexin32‐deficient mice

“…It is noteworthy, however, that the incidence of liver tumors is much higher in male as compared to female mice demonstrating the involvement of sex hormones in liver tumor development. 39 Even though the long-term effects of Cx32-deficiency on chemically induced hepatocarcinogenesis in mouse liver are rather dramatic, [5][6][7] we did not observe a concomitant strong change in the expression of a particular gene that could explain the strainspecific differential behavior. A comparable microarray analysis carried out with a conditional Cx26-deficient mouse also showed only very few genes that were moderately altered in expression in liver of the gene knockout mice (T.O., unpublished observation).…”

“…1,2 In accordance with this hypothesis, PB treatment resulted in promotion of hepatocarcinogenesis in Cx32wt mice, whereas no such effect was observed in Cx32ko mice suggesting that functional Cx32 is required for promotional activity of the barbiturate. 7 Because it is well established that PB not only affects (pre)neoplastically transformed cells but also modulates the expression of various genes within normal hepatocytes, we hypothesized that the analysis of PBmediated changes in gene expression in the normal liver tissue from Cx32wt and Cx32ko mice might give us some hint on the mechanism underlying the tumor promotional activity of the barbiturate.…”

“…1,2 In accordance with this hypothesis we showed that promotion by PB accelerates the occurrence of liver tumors in Cx32-wild-type (Cx32wt) mice whereas Cx32-null (Cx32ko) mice were almost completely resistant to promotion by the barbiturate. 7 PB is known to increase the metabolic capacity of hepatocytes by activating a variety of genes encoding drug metabolizing enzymes such as certain cytochromes P450 (CYP) and transferases. Enhanced expression of PB-inducible CYP enzymes involves activation of the constitutive androstane receptor (CAR).…”

The connection of β-catenin and phenobarbital in murine hepatocarcinogenesis: a critical discussion of Awuah et al., PLoS ONE 7(6):e39771, 2012

“…The age of mice at DEN injection is highly relevant since, in C3H mice, it has been shown that PB inhibits hepatocarcinogenesis when the initiator DEN is given to 2-week-old mice, whereas it promotes tumorigenesis after DEN injection at 6 weeks of age (Moennikes et al 2000). Comparably, PB inhibits tumor development in DENinduced (2 weeks) mice from strain B6C3F1, a crossbreed of C3H and C57BL/6 (Diwan et al 1984;Klaunig et al 1987Klaunig et al , 1988Lee et al 1998).…”

Phenobarbital Confers its Diverse Effects by Activating the Orphan Nuclear Receptor Car

“…CAR may regulate those factors through gene expressions, as well as direct protein-protein interactions. No consensus on what these factors may be has arisen yet, although c-Jun, FoxM1B, Connexin32, and Mdm2 have been suggested as candidates (Eferl et al, 2003;Kalinichenko et al, 2004;Moennikes et al, 2000, Huang et al, 2005. Although PB has been used to treat epilepsy patients for decades, liver tumors have never been associated with PB treatment, PB does not cause Drug Metabolism Reviews Downloaded from informahealthcare.com by McMaster University on 11/20/14…”