| BACKGROUND AND AIMCombination of irradiation therapy and gene therapy offers a promising strategy for cancer treatment, which can introduce dual cyto-killing activities derived from the radiation and the transformed gene(s), respectively. There have been several lines of evidence for the existance of irradiation-susceptible sequence(s) in cells such as E6, which acts as an enhancer and up-regulates its target gene expression. However, under the routine fractioned irrdiation dose, the expression of therapeutic gene induced by irradiation-responsive sequence is low and transient, leading to compromised therapeutic effects.The positive feedback circuit is one of important models of gene feedback circuits in which the target genes are up-regulated by themselves. Thus in this study, we will introduce the positive feedback circuit to the irradiation-gene therapy strategy and verify the effect of this modified strategy on tumor suppression. The irradiation-sensitive response element cArG[CC(A+T)6GC], i.e., E6, will be coupled with p53 response element pREs and wild type p53 gene, establishing an irradiation-controlled wt-p53 positive feedback circuit. The dynamics and the potency of up-regulating target gene wt-p53 of this feedback circuit will be investigated in vitro. Finally, the anti-tumor effects of this feedback circuit in the transplanted tumor nude mouse model will be observed to validate the effects of this circuit to up-regulate the expression of wt-p53 gene spatio-temporally. This research will explore a novel strategy of anti-tumor therapy that can not only decrease the needed irradiation dose thus to relieve the damage of irradiation to normal tissues, but also induce the persisitant expression of wt-p53 at the local tumor site, causing the apoptosis of tumor cells.METHODS1. Construction of plasmid of wt-p53 gene positive feedback circuit The CMV promoter of PCI-neo vector was replaced by six copies of synthetic cArGs (E6) element, p53 response element, or the fusioned E6 element and R4, respectivley. The wt-p53 cDNA was amplifed from the recombinant plasmid pcDNA3.1(+)-p53-EGFP by PCR. The fragment of IRES2-EGFP was prepared from plsmid IRES2-EGFP by double enzyme digestion. The two fragments were inserted into the MCS downstream of enhancer E6, R4, or E6R4 to construct pE6-p53-EGFP, pR4-p53-EGFP, or pE6R4-p53-EGFP plasmids, respectively. These plasmids were verified by both double enzyme digestion and sequencing.2. Analysis of the dynamic characteristics of positive feedback circuit regulated by irradiation:The recombinant plasmids and pcDNA3.1(+)-p53-EGFP were transfected into H1299(p53-/-) cells with liposome reagent and the cells stably expressing wt-p53 gene were selected with G418 antibiotics. The expression of wt-p53 gene in these cells was decteted with RT-PCR.1) Identification of positive feedback wt-p53 gene circuit: H1299(p53-/-) cells were infected by different dose of Ad5CMV-p53 virus, and the optimal MOI was determined by immunohistochemistry. The expression of wt-p53 protein was detcted by Western-blot assay in the pR4-53-EGFP/H1299 group (P group), Ad5CMV-p53/H1299 cell group (V group) and experimental group (V + P group).2) Identification of radiosensitive cArGs element: The level and the duration of wt-p53 protein expression were detected at different irradiation dose, or different phase with 4Gy.3) The observation of dynamic characteristics of gene circuit induced by irradiation: The expression level of EGFP was observed with microscope and FCM; The level and the duration of wt-p53 protein expression in pE6R4-p53-EGFP/H1299 cells and control groups were detected by Western-blot after irradiation.3. Analysis of anti-tumor activity of irradiation-induced wt-p53 positive circuit The cell life cycle and apoptosis caused by the irradiation-induced positive feedback gene circuit were analyzed, after the experimental group pE6R4-p53-EGFP/H1299 and the control group H1299(p53-/-), pE6-p53-EGFP/H1299, pR4-p53-EGFP/H1299, pcDNA3.1 (+)-p53-EGFP/H1299 were exposed to 4Gy radiation dose. D0 and sensitive enhancement ratio (SER) index were caculated from the irradiation dose-survival curve.4. Anti-tumor effects of irradiation-induced wt-p53 positive feedback circuit on the transplanted lung adenocarcinoma in nude mouseNude mouse lung adenocarcinoma transplant model was eastablished by subcutaneous injection of pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299, pR4-p53-EGFP/H1299 and H1299(p53-/-) cells, respectively. Tumor size in these nude mice was measured at the indicated time points post exposure to irradiation, tumor growth curve was drawn. TCD50 and SER value were calculated. At the final experimental stage, the animals were sacrificed and the tumor-inhibition rate was calculated. The morphology of the tumors was observed by HE staning and the expression of wt-p53 protein was detected by immunohistochemistry assay.RESULTS1. Construction of recombinant plasmids and transfection to H1299 cells Recombinant plasmid pE6R4-p53/EGFP, and control plasmids pE6-p53/EGFP and pR4-53/EEGFP were constructed and verified by enzymatic digestion and sequencing. After transfection of these plasmids to H1299 cells and selection with G418, wt-p53 mRNA could be amplified from the above 4 groups of transfected cells by RT-PCR method, indicating that stable p53-expressing cells were established.2. Dynamic characteristics of irradiation-induced wt-p53 positive feedback circuit The optimal multiplicity of infectin (MOI) for Ad5CMV-p53 was identified as 40 by immnohistochemistry assay. Western-blot assays showed that the p53 expression in plasmid-transfected cells did not changed during the experimental period (p>0.05) in contrast, the p53 expression in V-group and V+P group reached the peak at 72h post infection (p.i.) and thereafter decreased gradually. At 3 weeks, the p53 protein expression level in P+V group was 2.15 and 6.25 folds of that in P and V group, respectively, indicating the exsitence of the positive feedback circuit.When exposed to 0-4Gy of irradiation, pE6R4-p53-EGFP/H1299 cells of therapeutic group and pE6-p53-EGFP/H1299 cells of control group expressed increased wt-p53 protein along with the increment of irradiation doses. The summit level of wt-p53 was observed at 4Gy irradiation in therapeutic group which was 1.42 fold of that in control group, but the expression decreased significantly at 6Gy irradiation, reflecting the severe injury of cells at this dose of irradiation. Thus, 4Gy irradiation was used in following experiments in this study.At 3h post to 4Gy irradiation, wt-p53 expression could be detected in both groups. The expression level peaked at 12h post irradiation (p.i.) and at this time point, the expression in therapeutic group was 1.651 fold of that in control group. While the wt-p53 expression in control group decreased to baseline at 36h p.i., however, the cells from therapeutic group could express high level of wt-p53 even at 144h p.i., 1.86 fold of that without irradiation. FCM analyses showed that EGFP fluoresence density in therapeutic group was 3.95 and 2.5 folds of the control group at 12h and 144h p.i., respectively, indicating wt-p53 positive feedback circuit could up-regulating wt-p53 expression markedly.3. Anti-tumor effect of irradiation-induced wt-p53 positive feedback circuit in vitroAt 12h post 4Gy X-ray irradiation, FCM analysis showed that most cells in therapeutic group arrested at G0/G1 stage (75.13±1.42)%, compared with control groups (38.47±0.87)%, (62.57±0.76)%,(51.23±2.41)%, respectively. After irradiation, the cell apoptosis rate in each group was higher than that in cells without irradiation. The apoptosis rate in therapeutic group was (23.73±0.21)%, which was 5.69 folds (4.17±0.12)%, 1.91 folds (12.40±0.20)%, 1.51 folds(15.67±0.32)% and 2.57 folds(9.23±0.15)% of that in four control groups, respectively. The D0 values were 0.91Gy, 1.073 Gy and 1.413 Gy in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299, respectivley. The SER, derived from D0 values, was 2.63 and 1.34, respectively, indicating irradiation could up-regulate wt-p53 expression, regulate cell cycle and induce cell apoptosis, thus this positive feedback circuit could increase the sensitivity of lung adenocarcinoma to irradiation.4. Effects of the wt-p53 positive feedback circuit on the transplantated tumor in nude miceVarious levels of immunoreactivity for wt-p53 were found in the tissues of therapeutic group and control groups. The expression level of wt-p53 was evaluated as before described in section of materials and methods. Wt-p53 immunostaining was observed in cellular nucleus. Positive wt-p53 staining was increased greatly in H1299/pE6R4-p53-EGFP group (64.8 % ), moderately in H1299/pE6-p53-EGFP group (22.3 % ) and poorly in H1299/pR4-p53 -EGFP group(5.4%). Negative wt-p53 staining was observed in H1299 cell xenograft tumor group. The growth curves of xenograft tumors show that the tumor inhibition ratio in pE6R4-p53-EG FP/H1299/IR group is higher than that in other 3 control groups. The inhibitation radio is 86.41 % , 70.76 % , 35.53 % and 12.58 % in pE6R4-p53-EG FP/H1299+IR group, H1299/pE6-p53-EGFP+IR group, pR4-p53-EG FP/H1299+IR group and H1299+IR group, respectively. The TCD50 values were 12.1 Gy, 15.2 Gy and 19.4 Gy in pE6R4-p53-EGFP/H1299, pE6-p53-EGFP/H1299 and H1299, respectivley. The SER, derived from TCD50 values, was 1.6 and 1.28, respectively, indicating wt-p53 status were significant related to the activation of the positive feedback circuit induced by IR and the radiosensitivity of xenograft tumor in therapeautic group was enhanced significantly.CONCLUSIONSIn this study, we have constructed a recombinant pladmid containing an irradiation-induced wt-p53 positive feedback circuit, which can lead to much higher expression level of wt-p53 in transfected cells, thus it addressed the problem of current irradiation-gene therapy strategy, i.e., the weak and transient expression of target gene. Because this gene feedback circuit can obviously increase wt-p53 expression in tumor cells, it thus can cause the tumor cells to arrest at G1 stage and induce cell apoptosis, leading to the increased susceptibility of lung tumor to irradiation. This effect of the wt-p53 feedback circuit has been validated further in a transplanted tumor model in nude mice. Therefore, our results have not only improved the irradiation-gene therapy strategy but also could be a reference for other gene therapy strategy. |
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