Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with aberrant localization of connexin proteins.
BACKGROUND
Gap junctional intercellular communication is believed to play an important role in the maintenance of tissue homeostasis, and disruption of it has been proposed to be involved in carcinogenesis. A number of tumor-promoting agents have been shown to inhibit capacity for intercellular communication in cell culture studies. Recently, we developed a simple dye-transfer technique to evaluate cell-coupling function in fresh liver slices, and we used it to show that inhibition of intercellular communication is associated with rat liver tumor progression. Using this method with analysis of gap junction protein connexin expression, we have examined whether and how different liver-specific tumor-promoting agents inhibit dye-coupling in rat liver in vivo.


METHODS
Groups of Fischer 344 rats received repeated chronic treatment of phenobarbital (PB), polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT), and clofibrate (CF) for 5 weeks. After 1, 2, and 5 weeks of treatment, intercellular communication via gap junctions was evaluated by the dye-transfer assay in liver slices taken immediately after killing the rats. In parallel, the expression of connexins (cx) 32, 26, and 43 (gap junction proteins expressed in the liver) was studied at the mRNA and protein levels.


RESULTS
All four tumor-promoting agents decreased dye-coupling in rat liver. This decrease was associated with a reduced number of gap junctions and aberrant localization of some amount of cx 32 proteins in hepatocytes; cx 32 often was observed in the cytoplasm of hepatocytes instead of at gap junctions in the plasma membrane. Western blot analysis showed only slight changes in the level of cx 32 proteins. Although cx 26 proteins at gap junctions were usually decreased by tumor promoters in rat liver, local induction of cx 26 protein expression in centrolobular groups of hepatocytes after PCB and DDT treatment was observed. The expression of cx 43 was induced in hepatocytes after PCB, DDT, and CF exposure, but this protein was also localized intracytoplasmically, suggesting no functional role. All four tested tumor-promoting agents also increased cell proliferation, as revealed by staining with an anti-Ki 67 antibody.


CONCLUSIONS
The results demonstrate that different types of liver tumor-promoting agents inhibit dye-coupling in rat liver in vivo. This inhibition may be due to aberrant localization of the major liver gap junction protein cx 32, rather than its transcriptional or translational disregulation.
Krutovskikh
V A
VA
Unit of Multistage Carcinogenesis, International Agency for Research on Cancer, Lyon, France.
Mesnil
M
M
Mazzoleni
G
G
Yamasaki
H
H
D000818:Animals; D000906:Antibodies / Q000276:immunology; D015152:Blotting, Northern; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology*; D002450:Cell Communication / Q000187:drug effects*; D017630:Connexins / Q000096:biosynthesis* / Q000276:immunology; D017629:Gap Junctions / Q000187:drug effects*; D007124:Immunoenzyme Techniques; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344
D000906:Antibodies; D002273:Carcinogens; D017630:Connexins