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.
Cam
Laurence
L
INSERM EMI-0020 and University Paris XI, Bat Grégory Pincus, 80 rue du Général Leclerc, 94276 Le Kremlin Bicêtre-cedex, France.
Boucquey
Antoine
A
Coulomb-L'hermine
Aurore
A
Weber
Anne
A
Horellou
Philippe
P
D017209:Apoptosis / Q000235:genetics*; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism*; D045744:Cell Line, Tumor; D024161:Collagen Type XI / Q000276:immunology; D053938:DNA Fragmentation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009876:Operon; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
C499319:COL11A2 protein, human; D024161:Collagen Type XI; D011994:Recombinant Proteins; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases