The Expression Of Reconstructed Human Caspase-8 Genes And Their Pro-Apoptotic Activities

Apoptosis is autonomous cell death triggered by different physiological or pathological stimuli, hi recent years, it has been firmly proved that caspase family members play essential roles in apoptosis. Caspases have been divided into three sub-families according to their different functions in apoptosis: ICE (IL-lp converting enzyme) subfamily, initiators subfamily and effectors subfamily. The initiator subfamily include caspase-2, -8, -9, and-10, the effector subfamily include caspse-3, -6 and-7.Caspase-8 belongs to the initiator caspases, which play pivotal role hi death receptor induced apoptosis and can be activated by induced proximity model. Trimetric deaths-5-Gui-Junhao. The oro-aooptotfc activities of reconstructed human casoase-8. 2002-05receptors recruit pro-caspase-8 through FADD or TRADD to form the death-inducing signaling complex (DISC), which cause three caspase-8 precursors brought in close proximity to one another and activated by autocatalytic processing.In 1997, Muzio et al demonstrated that N-prodomain truncated caspase-8 can activate caspase-3> -7 and -9 efficiently. In 1998, Srinivasula et al constructed constitutively active pro-apoptotic recombinant caspase-3 and -6 by rearrangement of their subunits. Based on the structural similarity between different caspases, we speculate that reverse caspase-8 with large subunit preceded by small subunit also can fold into active heterodimers and have constitutive activity to induce apoptosis.In our research, we cloned the catalytic domain gene of human caspase-8 by RT-PCR, and two kinds of reverse caspase-8 genes (SL and SLL) with large subunits preceded by small subunits were constructed by recombinant PCR. The reconstructed caspase-8 genes were then cloned into eukaryotic expressing vector pIRES2-EGFP. We further investigated the pro-apoptotic activities of the transiently transfected reconstructed caspase-8 as well as N-prodomain truncated caspase-8 in HeLa cells.The expression of reconstructed caspase-8 genes were first verified by Western blot and indirect immuno-fluorescence labeled assay. Using fluorescence microscope, we can see that HeLa cells transfected with SL and SLL become round and shrinked 20 h post-transfection, which differ from pIRES2-EGFP and pIRES2-EGFP-LS transected cells. The nuclei of transfected cells display the hallmarks of apoptosis by DAPI staining: chromatin condensation and clustering along the nuclei membrane. Those hallmarks were also confirmed by immunohistochemical staining assay, electronicmicroscope and cellular skeleton staining analyses. However, HeLa cells transfected-6-2002-05with pIRES2-EGFP vector grow with normal morphology, and just a few HeLa cells transfected with LS died when seen under reverse microscope. In addition, many cells transfected with SL and SLL tend to suspend in the DMEM 30 h post-transfection, which suggest their dying states.In conclusion, our results revealed that SL and SLL with large subunits preceded by small subunits can spontaneously fold into active heterodimers and promote HeLa cells apoptosis constitutively in a similar manner, while LS showed comparably poor pro-apoptotic activity even when overexpressed in HeLa cells.