嵌合重组caspase-3诱导表达促进肿瘤细胞凋亡

通过稳定转染人宫颈癌HeLa细胞，建立了野生型caspase-3（wt-casp3），大小亚基序列颠倒的重组caspase-3 (r-casp3)，和N端融合绿脓杆菌外毒素（PE）转膜肽段的嵌合重组caspase-3 (cr-casp3)的诱导表达细胞系.蜕皮素诱导后细胞中检测到目的基因的表达，MTT检测和细胞计数结果表明，r-casp3和cr-casp3诱导表达后有效地导致HeLa细胞死亡，通过测定细胞中caspase-3活性，以及细胞周期检测、DNA梯状电泳条带检测(DNA ladder)、电镜观察等证实r-casp3和cr-casp3诱导表达后细胞发生了凋亡，且二者的促凋亡活性相当，而wt-casp3诱导表达细胞并未出现上述效应.结果表明，与野生型caspase-3活化需要上游分子的切割不同，重组caspase-3具有自发的促凋亡活性，而N端PE肽段的融合不影响这种活性，因此PE转膜结构域和重组caspase-3有望参与构建能转膜进入细胞内部，并杀伤细胞的新型肿瘤治疗分子.

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.