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

Abstract

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. Scarped loading and dye transfer (SLDT) was employed with Lucifer Yellow (LY) to detect GJIC function in hepatocellular carcinoma cell lines HHCC, SMMC-7721 and normal control liver cell line QZG. After Fluo-3AM loading, laser scanning confocal microscope (LSCM) was used to measure concentrations of intracellular calcium (Ca(2+))i in the cells. The phosphorylation on tyrosine of connexin proteins was examined by immunoblot. 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 (Ca(2+))i was much higher in QZG cell line (108.37 nmol/L) than those in HHCC (35.13 nmol/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 43 ku; SMMC-7721 cells showed phosphorylated tyrosine Cx43 protein. 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 (Ca(2+))i, post-translation phosphorylation on tyrosine of Cx proteins which led to a dramatic disruption of GJIC.