Gene transfer of constitutively active caspase-3 induces apoptosis in a human hepatoma cell line.

Background: The caspase-3 gene is expressed at significantly lower levels in human hepatocellular carcinomas than in normal hepatocytes. Gene transfer technologies offer the possibility to restore caspase-3 gene expression. We explored the interest for cancer gene therapy of a constitutively active recombinant caspase-3 (RevCasp3) obtained by rearranging its subunits.

Methods: An amphotropic retroviral vector was used to express the RevCasp3 gene. HuH7 cells were infected 1 and 2 days after plating. Caspase-3 activity was measured every 24 h for the following 6 days. The level of poly (ADP-ribose) polymerase cleavage induced by caspase-3 was measured by Western blot. The percentage of apoptotic cells was estimated after Hoechst-acridine orange and TUNEL stainings.

Results: Caspase-3 activity significantly increased from days 4 to 7 after infection. Caspase-3 activity peaked on day 7, and was 5.4-fold higher in RevCasp3-transduced HuH7 cells than in control cells. Poly (ADP-ribose) polymerase cleavage was first detected 6 days after the first infection. Hoechst-acridine orange and TUNEL stainings showed that most infected HuH7 cells were apoptotic.

Conclusions: Apoptosis was selectively induced following infection of HuH7 cells with RevCasp3, demonstrating that retroviruses expressing RevCasp3 are of potential interest for the treatment of hepatocellular carcinomas and other tumour types.