INHIBITION OF GAP JUNCTIONAL INTERCELLULAR COMMUNICATION AND MALIGNANT TRANSFORMATION OF RAT-LIVER EPITHELIAL-CELLS BY NEU ONCOGENE

A retrovirus containing a neu oncogene was introduced into a Fischer F 344 rat liver epithelial cell line (WB-F344) to study the effect of th e expression of neu oncoprotein on gap junctional intercellular commun ication (GJIC), the ability to form colonies in soft agar and the abil ity to form tumors in rat liver by these cells. After viral infection, five different neu-transduced epithelial clones were randomly selecte d for further analysis. Southern blot analysis of HindIII-digested gen omic DNA hybridized with a neu-specific probe indicated that the neu o ncogene carried by the retrovirus was integrated into different chromo somal locations in the five different neu-transduced WB cell fines. Us ing the fluorescence recovery after photobleaching (FRAP) assay, we fo und that GJIC was significantly reduced in neu-transduced WB clones, c ompared with control virus-infected and parental WB cells. Western blo t analysis of connexin 43 in the neu-transduced cell lines showed alte red phosphorylation patterns compared with the normal WB-rat liver cel l line. ConfocaI image analysis of the neu-transduced cells showed tha t the connexin 43 protein, as detected by fluorescent immunostaining, was localized in the cell nucleus. The neu-transduced WB cell lines al so acquired the ability to grow in soft agar. Furthermore, cells from three of the five neu-transduced cell lines, when injected into the li ver of Fischer F344 rats through the portal vein, were highly tumorige nic (multiple focal hepatic tumors developed within 2 weeks). Cells de rived from the tumor were shown to be G-418 resistant, demonstrating t hat the tumor was derived from the injected WB-neu cells. The results of this study demonstrate that the expression of the neu oncogene is a ble to block GJIC and to induce tumorigenicity in the rat liver WB-F34 4 cell line.