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. 2013 Nov 12;12(1):136.
doi: 10.1186/1476-4598-12-136.

Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model

Urska Kamensek, Gregor Sersa, Maja Cemazar  1
Affiliations

Evaluation of p21 promoter for interleukin 12 radiation induced transcriptional targeting in a mouse tumor model

Urska Kamensek et al. Mol Cancer. .

Abstract

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.

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Figures

Figure 1
Figure 1
Flow cytometry histograms of GFP expression in (a) TS/A CMV-EGFP and (b) TS/A p21-EGFP cell line.
Figure 2
Figure 2
Induction of reporter gene expression in vitro. (a) Fluorescence intensity in TS/A cell lines stably transfected either with p21 (p21-EGFP) or constitutive promoter (CMV-EGFP) driven reporter gene GFP after 6 Gy irradiation (IR). Numbers in the bars are factors of induction of reporter gene expression or fold-induction, calculated by dividing the fluorescence obtained in the induced group by the fluorescence in the control group. The data were pooled from three independent experiments performed in 12 replicates and are presented as means + SEM; *, P < 0.05; NS, non-significant, (b) Visible and fluorescent images and surface plots of the TS/A cells stably transfected with p21 driven reporter gene GFP after 6 Gy irradiation (IR). On the surface plots fluorescence intensities are represented linearly on a rainbow scale with red being the maximum signal and black being the lowest signal.
Figure 3
Figure 3
Induction of reporter gene expression in vivo (tumors). (a) Bar chart: normalized fluorescence intensity in the stably transfected TS/A p21-EGFP tumor model with reporter gene GFP under the control of p21 promoter after 0, 2, 6 and 10 Gy irradiation, and in TS/A CMV-EGFP tumor model with reporter gene under the control of CMV promoter after 6 Gy irradiation. For all experimental groups fluorescence is expressed in arbitrary units and is normalized on the day 0 and on the appropriate controls: for the p21 promoter on the non-irradiated TS/A p21-EGFP tumors and for the CMV on the non-irradiated TS/A CMV-EGFP tumors. The data were pooled from 2 independent experiments with 4–5 animals in each experimental group and are presented as means + SEM; *, P < 0.05 vs. control; NS, non-significant. Line plot: comparison of the induction dynamics of the inducible p21 and constitutive CMV promoter after 6 Gy irradiation. (b) Images of stably transfected TS/A p21-EGFP tumors in control group and experimental group that received 6 Gy irradiation (IR). Fluorescence intensities are represented linearly on a rainbow scale with red being the maximum signal and black being the lowest signal.
Figure 4
Figure 4
Induction of reporter gene expression in vivo (muscles). (a) Bar chart: normalized fluorescence in the mouse muscles transiently transfected with the plasmid carrying reporter gene GFP under the control of p21 promoter after 6 Gy irradiation on day 1 and day 8 after gene electrotransfer (GET) of p21-EGFP plasmid. For all experimental groups fluorescence is normalized on the day 0 and is expressed in arbitrary units. The data were pooled from 2 independent experiments with 7–8 animals in each experimental group and are presented as means + SEM; *, P < 0.05 vs. control. Line plot: induction dynamics of the p21 promoter after 6 Gy irradiation on day 1 and day 8 after gene electrotransfer (GET). (b) Images of mouse legs in control group and experimental group that received 6 Gy irradiation on day 1 and day 8 after the transfection with plasmid p21-EGFP (IR 1 + 8). Fluorescence intensities are represented linearly on a rainbow scale with red being the maximum signal and black being the lowest signal.
Figure 5
Figure 5
Therapeutic effect of radioinducible IL-12 gene therapy after 6 Gy irradiation in mouse TS/A tumors. EP, electroporation; IR, irradiation; p21, intratumoral injection of the p21-mIL-12 plasmid; p21 + EP, gene electrotransfer of the p21-mIL-12 plasmid; p21 + EP + IR, radio-gene therapy with the p21-mIL-12 plasmid; pORF, intratumoral injection of the pORF-mIL-12 plasmid, pORF + EP, gene electrotransfer of the pORF-mIL-12 plasmid; etc. (a) DT, doubling time i.e. time needed for the tumor to double in size; GD, growth delay i.e. difference between the doubling time of the specific experimental group and average doubling time of the control groups); CR, complete response i.e. complete disappearance of the tumor lasting for 100 days; N, number of the animals in the individual experimental group; data are presented as mean ± SEM;*, P < 0.05 vs. control. (b) Tumor growth curves after radioinducible IL-12 gene therapy. The data were pooled from 2 independent experiments with 18–10 animals in each experimental group and are presented as means with the standard errors of the mean. (c) Serum and (d) intratumoral IL-12 protein concentrations and (e) intratumoral Il-12 mRNA levels on the 5th day after the induction with irradiation. Each column represents mean + SEM. (c) *, P < 0.05 vs. control, 6–9 animals per experimental group. (d) *, P < 0.05 vs. all experimental groups except pORF + EP, 2–3 animals per experimental group. (e) *, P < 0.05 vs. control, 2–3 animals per experimental group.
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