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Gene transfer of constitutively active caspase‐3 induces apoptosis in a human hepatoma cell line
Author(s) -
Cam Laurence,
Boucquey Antoine,
CoulombL'hermine Aurore,
Weber Anne,
Horellou Philippe
Publication year - 2005
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.636
Subject(s) - apoptosis , acridine orange , microbiology and biotechnology , caspase 3 , biology , transfection , cell culture , caspase , poly adp ribose polymerase , tunel assay , caspase 9 , gene , polymerase , programmed cell death , biochemistry , genetics
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. Copyright © 2004 John Wiley & Sons, Ltd.