MULTIPLE MECHANISMS ARE RESPONSIBLE FOR ALTERED EXPRESSION OF GAP JUNCTION GENES DURING ONCOGENESIS IN RAT-LIVER

Although several abnormalities in gap junction (GJ) structure and/or f unction have been described in neoplasms, the molecular mechanisms res ponsible for many of the alterations remain unknown. The identificatio n of a family of GJ proteins, termed connexins, prompted this study of connexin32 (Cx32), connexin26 (Cx26) and connexin43 (Cx43) expression during rat hepatocarcinogenesis. Using antibody, cDNA and cRNA probes , we investigated connexin mRNA and protein expression in preneoplasti c and neoplastic rat livers. In normal liver, Cx32 is expressed in hep atocytes throughout the hepatic acinus, Cx26 is restricted to periport al hepatocytes, and Cx43 is expressed by mesothelial cells forming Gli sson's capsule. Most preneoplastic altered hepatic foci generated by d iethylnitrosamine (DEN) initiation and either phenobarbital (PB) or 2, 3,7,8-dichlorodibenzo-p-dioxin (TCDD) promotion exhibited decreased Cx 32 or increased Cx26 staining. Foci from either protocol failed to dis play Cx43 immunoreactivity. In the majority of PB-promoted foci, Cx32 immunoreactivity decreased independently of changes in mRNA abundance. Continuous thymidine labeling, following cessation of PB promotion, s howed that downregulation of Cx32 staining is reversible in foci that are promoter-dependent for growth, but irreversible in lesions that ar e promoter-independent for growth. Hepatic neoplasms from rats initiat ed with DEN and promoted with PB or TCDD also displayed modified conne xin expression. While all 24 neoplasms studied were deficient in norma l punctate Cx32 and Cx26 staining, altered cellular localization of th ese proteins was apparent in some tumors. Immunoblotting of crude tiss ue extracts revealed that neoplasms with disordered Cx32 staining show ed immunoreactive bands with altered electrophoretic mobility. These o bservations show that hepatomas may downregulate Cx32 expression throu gh changes in the primary structure of Cx32 or by post-translational m odifications. Northern blotting of total tumor mRNAs failed to demonst rate consistent changes in the abundance of Cx32, Cx26 or Cx43 transcr ipts. Some tumors expressed steady-state transcripts without observabl e immunoreactivity, indicating that some hepatomas downregulate connex in immunoreactivity independently of mRNA abundance. Increased levels of Cx43 mRNA and protein were found in several neoplasms, but immunost aining was always localized to nonparenchymal cells. Areas of bile duc t proliferation and cholangiomas displayed Cx43 staining, whereas chol angiocarcinomas were deficient in immunoreactivity. These findings sho w that alterations in the expression of connexins, by either downregul ation or differential induction, represent common modifications during hepatocarcinogenesis. Although our results imply that connexins repre sent useful markers for the boundary between tumor promotion and progr ession, preneoplastic and neoplastic rat hepatocytes fail to use a com mon mechanism to modify connexin expression.