Abstract
The anaphase promoting complex (APC/C) is a multi-component ubiquitin ligase complex responsible for targeting cell cycle proteins for degradation. APC/C consists of at least 12 subunits with additional cofactors. Poxvirus anaphase-promoting complex regulator (PACR) is a RING-H2 protein expressed by orf virus (ORFV), the prototype of the Parapoxvirus genus. PACR shares homology with APC/C subunit APC11, but lacks APC11’s ubiquitin ligase activity. Inclusion of PACR in APC/C, in place of APC11 has been shown to impair APC/C function causing mitotic arrest, which potentially leads to apoptosis. This investigation was the first step in attempting to make use of PACR’s unique ability to promote cell cycle arrest, by cloning PACR into an inducible expression system, Tet-One. Use of an inducible system would minimise PACR’s known toxicity to cells and allow PACR to be used as a potential therapeutic to cause cell death of murine melanoma, B16-F10 cells.
PACR with a Flag tag was successfully cloned into the Tet-One system. Transfection of this construct into human cell lines, HEK293-EBNA and HeLa, and murine melanoma, B16-F10 cells showed no detectable evidence of inducible expression of the protein, detected using western blotting. Changing inducer concentrations, transfection conditions and the timing at which these were applied also made no difference to protein expression. In contrast, when a constitutively expressing PACR-Flag construct was used, the protein was easily detected. This suggested that transfection and mode of detection (western blot) were all functioning adequately.
Use of a Tet-One control construct, in which luciferase expression is controlled by the same inducible regulator showed positive expression of luciferase in HEK293-EBNA and HeLa cells, confirming effective induction. B16-F10 cells showed no evidence of luciferase induction. HEK293-EBNA and B16-F10 cells were transfected with inducible PACR and luciferase constructs, and also constitutively expressing constructs, in an attempt to establish stable cell lines. Only HEK293-EBNA cells transfected with the constitutively expressing constructs formed visible colonies, and subsequent western blots showed expression of the target protein. B16-F10 cells transfected with the constitutively expressing constructs showed no visible colony formation or protein expression. Transfection of the inducible PACR and luciferase constructs into HEK293-EBNA and B16-F10 cells, to created stable cell lines, resulted in evidence of cell death and western blots detected no expression of the proteins when all the cells were collected together and lysed.
An inducible construct in the Tet-One system carrying another well characterised ORFV protein, VEGF-E, was also utilised. However, this too failed to generate VEGF-E when induced, as detected by western blotting. When a Tet-One GFP construct was tested, weak signals of the protein were visualised through fluorescent microscopy, but western blotting showed no detection of GFP.
A different two-plasmid induction system was also tested (Tet-On). Here PACR-Flag was cloned into the system, but again no expression of the protein was observed. However, a GFP Tet-On construct transfected into HEK293-EBNA cells allowed detection of GFP signal through both fluorescent microscopy and western blotting. Overall, these observations suggest that proteins being expressed by the Tet-One system may be present in amounts that are not readily detected by western blots. It was concluded that alternative conditional expression systems will be required to test PACR’s therapeutic potential.