Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model.
BACKGROUND
Radiation induced transcriptional targeting is a gene therapy approach that takes advantage of the targeting abilities of radiotherapy by using radio inducible promoters to spatially and temporally limit the transgene expression. Cyclin dependent kinase inhibitor 1 (CDKN1A), also known as p21, is a crucial regulator of the cell cycle, mediating G1 phase arrest in response to a variety of stress stimuli, including DNA damaging agents like irradiation. The aim of the study was to evaluate the suitability of the p21 promoter for radiation induced transcriptional targeting with the objective to test the therapeutic effectiveness of the combined radio-gene therapy with p21 promoter driven therapeutic gene interleukin 12.


METHODS
To test the inducibility of the p21 promoter, three reporter gene experimental models with green fluorescent protein (GFP) under the control of p21 promoter were established by gene electrotransfer of plasmid DNA: stably transfected cells, stably transfected tumors, and transiently transfected muscles. Induction of reporter gene expression after irradiation was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging using fluorescence stereomicroscope in vivo. The antitumor effect of the plasmid encoding the p21 promoter driven interleukin 12 after radio-gene therapy was determined by tumor growth delay assay and by quantification of intratumoral and serum levels of interleukin 12 protein and intratumoral concentrations of interleukin 12 mRNA.


RESULTS
Using the reporter gene experimental models, p21 promoter was proven to be inducible with radiation, the induction was not dose dependent, and it could be re-induced. Furthermore radio-gene therapy with interleukin 12 under control of the p21 promoter had a good antitumor therapeutic effect with the statistically relevant tumor growth delay, which was comparable to that of the same therapy using a constitutive promoter.


CONCLUSIONS
In this study p21 promoter was proven to be a suitable candidate for radiation induced transcriptional targeting. As a proof of principle the therapeutic value was demonstrated with the radio-inducible interleukin 12 plasmid providing a synergistic antitumor effect to radiotherapy alone, which makes this approach feasible for the combined treatment with radiotherapy.
Urska
U
Kamensek
Gregor
G
Sersa
Institute of Oncology Ljubljana, Ljubljana, Slovenia. mcemazar@onko-i.si.
Maja
M
Cemazar
D000230:Adenocarcinoma / Q000628:therapy*; D000818:Animals; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics*; D008325:Mammary Neoplasms, Experimental / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009368:Neoplasm Transplantation; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000528:radiation effects; D015533:Transcriptional Activation / Q000528:radiation effects*; D014162:Transfection
D050759:Cyclin-Dependent Kinase Inhibitor p21; D049452:Green Fluorescent Proteins; D018664:Interleukin-12