Multiple mechanisms are responsible for altered expression of gap junction genes during oncogenesis in rat liver
Authors: Neveu Mark J. , Hully James R. , Babcock Karlee L. , Hertzberg Elliot L. , Nicholson Bruce J. , Paul David L. , Pitot Henry C. 
Published: April 2021

ABSTRACT
Although several abnormalities in gap junction (GJ) structure and/or function have been described in neoplasms, the molecular mechanisms responsible for many of the alterations remain unknown. The identifica-tion of a family of GJ proteins, termed connexins, prompted this study of connexin32 (C×32), connexin26 (C×26) and connexin43 (C×43) expression during rat hepa-tocarcinogenesis. Using antibody, cDNA and cRNA probes, we investigated connexin mRNA and protein expression in preneoplastic and neoplastic rat livers. In normal liver, C×32 is expressed in hepatocytes throughout the hepatic acinus, C×26 is restricted to periportal hepatocytes, and C×43 is expressed by mesothelial cells forming Glisson’s capsule.
Most preneoplastic altered hepatic foci generated by diethylnitrosamine (DEN) initiation and either phenobar-bital (PB) or 2,3,7,8-dichlorodibenzo-p-dioxin (TCDD) promotion exhibited decreased C×32 or increased C×26 staining. Foci from either protocol failed to display C×43 immunoreactivity. In the majority of PB-promoted foci, C×32 immunoreactivity decreased independently of changes in mRNA abundance. Continuous thymidine labeling, following cessation of PB promotion, showed that downregulation of C×32 staining is reversible in foci that are promoter-dependent for growth, but irreversible in lesions that are promoter-independent for growth.
Hepatic neoplasms from rats initiated with DEN and promoted with PB or TCDD also displayed modified connexin expression. While all 24 neoplasms studied were deficient in normal punctate C×32 and C×26 staining, altered cellular localization of these proteins was apparent in some tumors. Immunoblotting of crude tissue extracts revealed that neoplasms with disordered C×32 staining showed immunoreactive bands with altered electrophoretic mobility. These observations show that hepatomas may downregulate C×32 expression through changes in the primary structure of C×32 or by post-translational modifi-cations. Northern blotting of total tumor mRNAs failed to demonstrate consistent changes in the abundance of C×32, C×26 or C×43 transcripts. Some tumors expressed steady-state transcripts without observable immunoreactivity, indicating that some hepatomas downregulate connexin immunoreactivity independently of mRNA abundance. Increased levels of C×43 mRNA and protein were found in several neoplasms, but immunostaining was always localized to nonparenchymal cells. Areas of bile duct pro-liferation and cholangiomas displayed Cx43 staining, whereas cholangiocarcinomas were deficient in immunore-activity. These findings show that alterations in the expression of connexins, by either downregulation or dif-ferential induction, represent common modifications during hepatocarcinogenesis. Although our results imply that connexins represent useful markers for the boundary between tumor promotion and progression, preneoplastic and neoplastic rat hepatocytes fail to use a common mechanism to modify connexin expression.