Differential expression of S100A6 gene in human hepatocellular carcinoma

Abstract

Genes expressed differentially in hepatocellular carcinomas were screened through PCR-based subtraction and dot hybridization methods. A cDNA clone, 2C06 had been isolated and identified as human S100A6 gene. The nucleotide sequence of the S100A6 cDNA clone consists of 407 nucleotides including the ORF of 270 bases. The ORF codes for a polypeptide of 90 amino acid residues with the deduced molecular weight of about 10 KDa. Computer aided-homology analysis showed that nucleotide sequence of S100A6 shared about 30-40% homology with those of S100 family proteins. Northern blot analysis showed that S100A6 message was highly expressed with high level in the tissues of metastatic hepatocellular adenocarcinoma from colon cancer, cholangiocarcinoma and gallbladder cancer, while the expression levels in primary hepatocellular adenocarcinoma and normal liver tissues were very low. In order to elucidate the function of S100A6 protein, the ORF of S100A6 gene was reconstructed and over-expressed in E. coli cells. Antiserum and monoclonal antibody to the recombinant S100A6 protein were produced from immunized Balb/c mice. The produced monoclonal antibody KO2C12-1 did not exhibit the cross reactivity against several S100 family proteins. The site of antigen recognition was determined by western blot analysis with serially deleted S100A6 proteins. Monoclonal antibody to recombinant S100A6 protein was used to reveal the expression patterns of S100A6 protein in normal and various cancerous tissues of human origin including hepatocellular carcinoma. Immunohistochemical study demonstrated that the tissues of cholangio-carcinoma (CC), gallbladder cancer (GBC) and metastatic hepatocellular adenocarcinoma from colon cancer (McT) were positively stained in nucleus and/or cytoplasm of tumor cells. The distribution patterns of S100A6 protein varied depending on the loci of the cancerous lesions. Tumor cells located at the outskirts of the cancerous lesions were positively stained at cyt...