Comparing the immune response to radiation-induced tumour cell death with pure apoptosis using a Doxycycline-dependent caspase 3 death switch model


Session type:

M Melis1, K Simpson1, J Honeychurch1, A Welman1, M MacFarlane2, C Dive1, T Illidge1

1Paterson Institute for Cancer Research, University of Manchester, UK, 2MRC Toxicology Centre, Leicester, UK



Recent evidence suggests that radiation (RT)-induced tumour cell death is immunogenic. However, whilst dying tumour cells may act as a source of antigen for T-cell priming, frequently treatment with RT alone is insufficient to induce robust anti-tumour activity. To further define the immunogenic potential of RT-induced cell death compared to apoptosis we have developed a doxycycline-dependent caspase-3 death switch system.


Here we describe the generation of two syngeneic murine death switch lines: B16 (melanoma) and B16 ova (transduced with a cDNA encoding the ovalbumin gene). To generate the death switch lines, cells were stably transfected with a transcriptional transactivator (rtTA2S-M2) to produce a Tet-On line and a response element containing reverse caspase 3 (revC3), under the control of an inducible promoter. In the presence of the tetracycline-analogue, doxycycline (Dox), the rtTA can bind to the inducible promoter, leading to its activation and subsequent expression of revC3, resulting in rapid and synchronous apoptosis both in vitro and in vivo.


We verified that Dox induced apoptosis using different methods including Annexin V /Propidium Iodide staining; fluorometric analysis of caspase 3; and western blotting for cleaved caspase 3 and cleaved PARP. Cell death after addition of Dox was between 60 to 80% within 24H and could be almost completely inhibited by use of the pan-caspase inhibiter Q-VD. Importantly, the revc3 clones develop with similar kinetics as wild type cells both in vitro and in vivo.


This system will allow us to explore the relationship between the amount and type of cell death and the ability to prime tumour-(ova)-specific T-cell responses in vivo; provide important clues as to what regulates immunogenicity of cell death in vivo; and eventually guide therapeutic approaches which aim to induce immune responses to dying tumour cells.