Expression of gap junction genes connexin 32 and connexin 43 mRNAs and proteins,and their role in hepatocarcinogenesis

AIM: To investigate the relationship betweenhepatocarcinogenesis and the expression of connexin32(cx32), connexin43 (cx43) mRNAs and proteins in vitro.METHODS Gap junction genes cx32 and cx43 mRNA inhepatocellular carcinoma call lines HHCC, SMMC-7721 andnormal liver call line QZG were detected by in situhybridization (ISH) with digoxin-labeled cx32, and cx43cDNA probes. Expression of Cx32 and Cx43 proteins in thecell lines was revealed by indirect immuno-fluorescence andflow cytornetry (FCM).RESULTS Blue positive hybridization signals of cx32 andcx43 mRNAs detected by ISH with cx32 and cx43 cDNAprobes respectively were located in cytoplasm of cells ofHHCC, SMMC-7721 and QZG. No significant difference ofeither cx32 mRNA or cx43 mRNA was tested among HHCC,SMMC-7721 and QZG (P = 2.673, HHCC vs QZG; P =1.375, SMMC-7721 vs QZG). FCM assay showed that thepositive rates of Cx32 protein in HHCC, SMMC-7721 and QZGwere 0.7%, 1.7% and 99.0%, and the positive rates of Cx43protein in HHCC, SMMC-7721 and QZG were 7.3%, 26.5%and 99.1% respectively. Significant differences of both Cx32and Cx43 protein expression existed between hepatocellularcarcinoma cell lines and normal liver cell line ( P = 0.0069,HHCC vs QZG; P = 0.0087, SMMC-7721 vs QZG).Moreover, the fluorescent intensities of Cx32 and Cx43proteins in HHCC, SMMC-7721 were lower than that in QZG.CONCLUSION Hepatocellular carcinoma cell lines HHCCand SMMC-7721 exhibited lower positive rates andfluorescent intensities of Cx32, Cx43 proteins compared withthat of normal liver cell line QZG. lt is suggested that lowerexpression of both Cx32 and Cx43 proteins in hepatocellularcarcinoma cells could play pivotal roles in thehepatocarcinogenesis. Besides, genetic defects of cx32 andcx43 in post-translational processing should be considered.

Signal transduction of gap junctional genes,connexin32,connexin43 in human hepatocarcinogenesis

AIM: To investigate gap junctional intercellular communication (GJIC) in hepatocellular carcinoma cell lines, and signal transduction mechanism of gap junction genes connexin32(cx32),connexin43(cx43) in human hepatocarcinogenesis.METHODS: Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium [Ca2+]i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot.RESULTS: SLDT showed that ability of GJIC function was higher in QZG cell than that in HHCC and SMMC-7721 cell lines. By laser scanning confocal microscopy, concentrations of intracellular free calcium [Ca2+]i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13nmol/L) and SMMC-7721 (47.08 nmol/L) cells. Western blot suggested that only QZG cells had unphosphorylated tyrosine in Cx32 protein of 32 ku and Cx43 protein of 43ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein.CONCLUSION: The results indicated that carcinogenesis and development of human hepatocellular carcinoma related with the abnormal expression of cx genes and disorder of its signal transduction pathway, such as decrease of [Ca2+]i,post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.