Randall J. Ruch

Gap-junction disassembly and connexin 43 dephosphorylation induced by 18β-glycyrrhetinic acid

Gap‐junction channels connect the interiors of adjacent cells and can be arranged into aggregates or plaques consisting of hundreds to thousands of channel particles. The mechanism of channel aggregation into plaques and whether plaques can disaggregate are not known. Many carcinogenic and tumor‐promoting chemicals have been identified that inhibit cell‐cell gap‐junctional coupling. Here, we provide morphological evidence that 18β‐glycyrrhetinic acid (18β‐GA), a saponin isolated from licorice root that is an inhibitor of gap‐junctional communication, caused the disassembly of gap‐junction plaques in WB‐F344 rat liver epithelial cells. This effect was dose (5–40 μM) and time dependent (1–4 h treatment). Gap‐junction channels in WB‐F344 cells are comprised of connexin 43 (Cx43), and the protein is phosphorylated to a species known as Cx43‐P2 coincident with the assembly of channels into plaques. Consistent with this, the disassembly of plaques induced by 18β‐GA was correlated with decreases in Cx43‐P2 levels and increases in nonphosphorylated Cx43. Biochemical evidence indicated that these changes in the P2 and NP forms of Cx43 represented 18β‐GA‐induced dephosphorylation of Cx43‐P2 and not its degradation or the inhibition of Cx43‐NP phosphorylation. Okadaic acid and calyculin A, which are inhibitors of type 1 and type 2A protein phosphatases, prevented the dephosphorylation of Cx43, suggesting that one or both of these phosphatases were involved in Cx43 dephosphorylation. These data indicate that 18β‐GA causes type 1 or type 2A protein phosphatase‐mediated Cx43 dephosphorylation coincident with the disassembly of gap‐junction plaques.

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB‐F344 rat liver epithelial cells strongly activated p42 and p44 ERK‐1 and ‐2, blocked gap junction‐mediated fluorescent dye‐coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis‐indolylmaleimide I; 2 μM) and ERK activation (U‐0126; 10 μM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester. J. Cell. Biochem. 83: 163–169, 2001.

Role of Inhibition of Intercellular Communication in Carcinogenesis

Subsequent to the first morphologic description of the gap junction by Revel and Karnovsky in 1967 (113), a plethora of reports has appeared describing the structure, function and pathologic changes of this plasma membrane structure. The gap junction appears to serve as a conduit for the cell-to-cell exchange of low molecular weight ions and molecules between adjacent cells (intercellular communication). A number of functions have been attributed to intercellular communication including the maintenance of normal cellular homeostasis. Another function that is clearly related to the neoplastic process is the control of cellular growth (81). Yotti, Chang, and Trosko (178) and Murray and Fitzgerald (99), working independently, reported in 1979 that tumor promoting phorbol ester compounds were capable of inhibiting gap junction-mediated intercellular communication between cells in culture. These findings resulted in the hypothesis that tumor promoters stimulate cell proliferation of initiated cells by inhibiting gap junctional intercellular communication in the initiated cells (146).

Trout hepatocyte culture: Isolation and primary culture

SummaryRainbow trout (Salmo gairdneri) hepatocytes were isolated using a two-step perfusion through the portal vein. A typical perfusion yielded 2.92×106 liver cells with a mean viability of 96.3%. Hepatocytes comprised 93.4% of the total cell isolate. Survival of hepatocytes in suspension culture was dependent on fetal bovine serum concentration and temperature of incubation. Serum concentrations of 5, 10, and 20% produced the highest survival during primary culture. Hepatocyte survival was in inverse proportion to the incubation temperature. Trout hepatocyte DNA synthesis and mitosis decreased during the culture period. Cytochromep450 activity decreased rapidly during the first 2 d of culture and then remained low but measurable during the remaining 8 d of culture. Culture temperature also influenced thep450 activity with lower temperatures producing greater activity. Morphologic changes occurred in the cells during culture. Isolated hepatocytes self-aggregated, forming strands and clumps that increased in size with time in culture. Junctional complexes between cells were evident within the aggregates. Nuclear atypia, increases in size and number of autophagic vacuoles, and the appearance of bundles of intermediate filaments also were observed with increased time in culture.

Growth inhibition in G1 and altered expression of cyclin D1 and p27kip-1after forced connexin expression in lung and liver carcinoma cells

Gap junctional intercellular communication (GJIC) and connexin expression are frequently decreased in neoplasia and may contribute to defective growth control and loss of differentiated functions. GJIC, in E9 mouse lung carcinoma cells and WB‐aB1 neoplastic rat liver epithelial cells, was elevated by forced expression of the gap junction proteins, connexin43 (Cx43) and connexin32 (Cx32), respectively. Transfection of Cx43 into E9 cells increased fluorescent dye‐coupling in the transfected clones, E9‐2 and E9‐3, to levels comparable to the nontransformed sibling cell line, E10, from which E9 cells originated. Transduction of Cx32 into WB‐aB1 cells also increased dye‐coupling in the clone, WB‐a/32‐10, to a level that was comparable to the nontransformed sibling cell line, WB‐F344. The cell cycle distribution was also affected as a result of forced connexin expression. The percentage of cells in G1‐phase increased and the percentage in S‐phase decreased in E9‐2 and WB‐a/32‐10 cells as compared to E9 and WB‐aB1 cells. Concomitantly, these cells exhibited changes in G1‐phase cell cycle regulators. E9‐2 and WB‐a/32‐10 cells expressed significantly less cyclin D1 and more p27kip‐1 protein than E9 and WB‐aB1 cells. Other growth‐related properties (expression of platelet‐derived growth factor receptor‐β, epidermal growth factor receptor, protein kinase C‐α, protein kinase A regulatory subunit‐Iα, and production of nitric oxide in response to a cocktail of pro‐inflammatory cytokines) were minimally altered or unaffected. Thus, enhancement of connexin expression and GJIC in neoplastic mouse lung and rat liver epithelial cells restored G1 growth control. This was associated with decreased expression of cyclin D1 and increased expression of p27kip‐1, but not with changes in other growth‐related functions. J. Cell. Biochem. 79:347–354, 2000.

Inhibition of lung tumor cell growth in vitro and mouse lung tumor formation by lovastatin

The HMG-CoA reductase inhibitor, lovastatin (LOV), has been reported to inhibit Ras farnesylation and the growth of Ras-transformed cells. Mouse lung tumors and human lung adenocarcinomas often have activating mutations in K-ras alleles. In the present study, we determined whether LOV inhibited the growth in vitro of mouse (C10, E9, LM1, LM2, and 82-132) and human (NCl-H125, H292, H441, H460, and H661) nor-transformed and neoplastically transformed lung epithelial cells and whether growth inhibition was related to cell transformation or K-ras activation. LOV inhibited the growth of mouse and human lung cells, but cell sensitivities were unrelated to neoplastic transformation or K-ras mutation. In addition, we evaluated whether LOV could inhibit the formation of lung adenomas induced by the tobacco-specific nitrosamine, 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in mice. LOV was administered in the diet at 0, 40, 160, or 400 ppm ad libitum to male strain A/J mice beginning 1 week after lung tumor induction with NNK (10 mumol/mouse). Mice were euthanized 6 months later. Enumeration of lung tumors revealed that LOV did not affect tumor incidence or size, but significantly reduced tumor multiplicity in a dose-related manner. These data suggest that LOV can suppress the formation of NNK-induced lung tumors, possibly at an early promotional stage. This suppression does not appear to be related to either the presence of mutated K-ras or to changes in K-ras expression.

Localization and function of the connexin 43 gap‐junction protein in normal and various oncogene‐expressing rat liver epithelial cells

Clones of rat liver epithelial cells genotypically altered by mutation or by a variety of oncogenes were analyzed by microinjection‐dye transfer, immunofluorescence confocal microscopy, and western blotting to determine at what level and to what degree these transformations disrupted gap‐junctional intercellular communication (GJIC) mediated by connexin 43 (Cx43). Compared with normal rat liver epithelial cells, cells neoplastically transformed by src, neu, ras, and myclras all displayed reduced degrees of GJIC, reduced levels of membrane‐associated Cx43 plaques, and hypophosphorylation of Cx43. Confocal analysis further demonstrated that the Cx43 protein was localized, at least in part, to the nucleus rather than to the plasma membrane in the src‐ and neu‐transformed cells, but not in the ras‐ and myclras‐transformed cells. Nuclei isolated from WB‐neu cells showed substantially higher levels of Cx43 on western blotting than did nuclei from WB‐neo control cells, supporting the idea that the nuclear‐localized immunopositive material detected by confocal microscopy was Cx43 protein. In a GJIC‐deficient mutant rat liver epithelial cell line containing normal numbers of plasma membrane‐localized Cx43 plaques that appeared to be reduced in size, the Cx43 protein was also found to be hypophosphorylated. Cells overexpressing myc, on the other hand, displayed a normal degree of GJIC, increased levels of plasma membrane‐localized Cx43 plaques, and hyperphosphorylation of the Cx43 protein. Cells expressing raf, previously shown to be GJIC competent, showed Cx43 immunostaining patterns similar to those in normal cells, whereas a cell line established from a tumor induced by injection of these raf‐expressing cells into a mouse showed a marked reduction in GJIC and plasma membrane‐associated Cx43 immunostaining. These data suggest that altered localization of the gap‐junction protein Cx43, mediated in part by changes in the phosphorylation of this protein, contributes to the disruption of GJIC in neoplastically transformed rat liver epithelial cells.

Strain and species effects on the inhibition of hepatocyte intercellular communication by liver tumor promoters

The effect of the liver tumor promoters phenobarbital (PB), 1,1-bis(4-chlorophenyl)-2,2,2-trichlorethane (DDT), and dieldrin on gap junction-mediated intercellular communication between primary cultured hepatocytes from male mice (B6C3F1), C3H, C57BL, and Balb/c strains) and male F344 rats was determined. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabelled donor hepatocytes to donor-contacting recipient hepatocytes. At non-toxic concentrations, PB (20-500 micrograms/ml) inhibited intercellular communication between B6C3F1, C3H, and Balb/c mouse hepatocytes and F344 rat hepatocytes, but not between C57BL mouse hepatocytes. DDT (1-10 micrograms/ml) inhibited intercellular communication between hepatocytes from all 4 strains of mice and the F344 rat. Dieldrin (1-10 micrograms/ml) inhibited intercellular communication between hepatocytes from the 4 strains of mice but not between rat hepatocytes. These findings showed a good correlation with the in vivo liver tumor promoting/hepatocarcinogenic actions of PB, DDT and dieldrin in the 4 mouse strains and the F344 rat strain.

Effects of tumor promoters, genotoxic carcinogens and hepatocytotoxins on mouse hepatocyte intercellular communication

Intercellular communication via gap junctions may be an important mechanism of cellular growth control. Tumor promoters can inhibit intercellular communication between cultured cells, while genotoxic carcinogens apparently lack this capability. The inhibition of intercellular communication by tumor promoters may be an essential mechanism by which tumor promotion occurs in vivo. In this study, the liver tumor promoters phenobarbital, lindane (1,2,3,4,5,6-hexachlorocyclohexane, γ-isomer), DDT (1,1-Bis[4-chlorophenyl],-2,2,2-trichloroethane), Aroclor 1254 (a polychlorinated biphenyl mixture) and dieldrin inhibited intercellular communication between male B6C3F1 mouse hepatocytes in primary culture. Intercellular communication was detected as the passage of [5-3H]uridine nucleotides from pre-labelled donor hepatocytes to non-labelled recipient heptocytes. Mouse hepatocyte intercellular communication was also inhibited by the skin tumor promoter TPA (12-0-tetradecanoyl phorbol-13-acetate), but not by the bladder tumor promoter saccharin. The genotoxic hepatocarcinogens dimethylnitrosamine, diethylnitrosamine, benzo[a]pyrene and 2-acetylaminofluorene, and the hepatocytotoxins bromobenzene, acetaminophen, carbon tetrachloride, chloroform and methotrexate had no effect on mouse hepatocyte intercellular communication at non-cytotoxic levels. These results suggest that the ability to inhibit mouse hepatocyte intercellular communication is an effect specific to tumor promoters.

Indole-3-carbinol as a chemopreventive agent in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) carcinogenesis: inhibition of PhIP–DNA adduct formation, acceleration of PhIP metabolism, and induction of cytochrome P450 in female F344 rats

The chemopreventive properties of dietary indole-3-carbinol (I3C) were evaluated by assessing its effect on DNA adduct formation and metabolism of the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and the induction of cytochromes P450 1A1 and -1A2 in female F344 rats. In experiment 1, animals on I3C diets (0, 0.02% or 0.1%, w/w) were treated by gavage with 1mg/kg/day of PhIP for 23 days. On days 2, 9, 16 and 23, their 24-hr urine was collected and unmetabolized PhIP was measured by GC/MS. On day 24, the animals were sacrificed, and DNA from pancreas, spleen, white blood cells (WBCs), lung, colon, kidney, mammary epithelial cells, caecum, heart, small intestine, liver and stomach was isolated for determination of PhIP-DNA adduct levels by (32)P-postlabelling assays. Except in the mammary gland, I3C diets significantly inhibited PhIP-DNA adduct formation in WBCs and in all organs, ranging from 34.7 to 67.7% with the 0.02% I3C diet to 68.4 to 95.3% with the 0.1% I3C diet. I3C diets also significantly decreased the concentration of urinary unmetabolized PhIP to 29.5-38.4% (0.02% I3C) and 12.8-17.8% (0.1% I3C) of values obtained with the I3C-free diet. In experiment 2, animals were either treated by intubation of I3C at 100 or 200mg/kg for 2 consecutive days or given an I3C-containing diet (0.02% or 0.1%, w/w) for 2 weeks. The expression and activity of cytochromes P450 1A1 and -1A2 were studied by Northern blots, Western blots, and in vitro enzyme determinations. Both the expression and activity of these cytochromes were induced by all of the I3C treatments. It is concluded that, in the female F344 rat, dietary I3C inhibits PhIP-DNA adduct formation and accelerates PhIP metabolism, probably through induction of cytochromes P450 1A1 and -1A2. The chemopreventive properties of I3C in PhIP-induced carcinogenesis are probably mediated through enhancement of PhIP detoxification pathways.