Expression of cytosolic ALR in HCC cells and its impact on hepatocellular growth in vitro and in vivo

Progression of HCC depends on different factors like metastasis and invasiveness. Previously, we have shown that the hepatotrophic protein Augmenter of Liver Regeneration (ALR) is highly expressed in cirrhotic livers and HCC. The aim of our study was to gain more insight in to the functional role of differential regulation of ALR in HCC and its impact on cellular growth in vitro and in vivo. Therefore the cytosolic 15 kDa isoform ALR was re-induced in hepatoma cell line HepG2 by stable transfection with a human ALR expression vector containing the 375 bp ALR cDNA under the control of the cytomegalovirus (CMV) promoter (ALR-HepG2). Quantitative RT-PCR revealed a strong induction of ALR mRNA expression in ALR-HepG2 cells, which was confirmed by matched protein levels performing western blot and immunocytochemical analysis. Interestingly, in vitro proliferations studies revealed no difference in cellular growth between wild type and ALR expressing HepG2 cells, whereas previous studies showed an increased proliferation rate after ALR application into culture medium. For analyzing the impact of ALR on tumour growth in vivo wild type and HepG2 cells stably expressing ALR were injected subcutaneously into nude mice. After three weeks body weight and tumour size were determined as well as samples for further analysis were taken. Re-expression of ALR was confirmed by RT-PCR and immunohistochemistry. Tumours originated from stably ALR expressing HCC cells compared to controls resulted in slightly lower average body weight of mice and displayed less necrotic areas, more epithelial-like cell growth as well as less polymorphisms and atypical mitotic figures. On the other hand mock transfected HepG2 derived tumours showed a more cancerous like growth with clear signs of necrosis. Based on these findings and in line with the in vitro results we assume that down regulation of 15kDa isoform of ALR expression in HCC cells supports tumorgenicity and metastatic potential of HCC cells.