Inducible expression of chimeric recombinant caspase-3 promotes apoptosis in tumor cells

Human cervix HeLa cells were stably transfected to establish cell lines that inducibly expressed 3 types of caspase-3 constructs, respectively. These constructs involved wild-type caspase-3 (wt-casp3), recombinant caspase-3 (r-casp3) in which the order of the small and large subunits was reversed in contrast to the original protein, and chimeric recombinant (cr-casp3) in which a Pseudomonas exotoxin A (PE) -derived peptide was fused to N-terminus of r-casp3. The expression of the interest genes was detected upon induction with ponasterone. The genes of r-casp3 and cr-casp3 were demonstrated to effectively cause cell death by MTT assay and cell counting. Cells that expressed r-casp3 or cr-casp3, but not wt-casp3, underwent apoptosis in a comparable level as determined by cell cycle analysis, genomic DNA ladders, and electronic microscopy. These results prove that unlike wild-type caspase-3 which is inactive unless proteolytically processed by upstream caspase, both recombinant caspase-3s are naturally active, and the N-terminal fusion of PE translocation domain does not interfere with the natural caspase-3 activity, suggesting their applications on the construction of novel tumor therapeutics that efficiently translocate to the cytosol of tumor cells and cause cell death.