ABSTRACT
Objective To detect specific cell killing effect of radiation combined with horseradish peroxidase (HRP)/indole-3-acetic (IAA) suicide gene therapy controlled by a novel radio-inducible and cancer-specific chimeric gene promoter in lung cancer. Methods We constructed a plasmid expressing HRP enzyme under the control of chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 CArG elements, a plasmid expressing HRP enzyme under the control of hTERT promoter carrying single CArG element, and two control plasmids, which named pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControl-luc, respectively. After radiation, the proliferation inhibition and apoptosis induction effect of each type of plasmid in lung cancer cells (A549, SPC-A1) and normal lung cells (hEL) was detected by cell counting and Annexin V-FITC staining. The change of radiosensitivity of lung cancer cells with plasmid system was also detected by clonogenic assays. Results After a single dose radiation of 6 Gy,the average proliferation inhibition rates of pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControl-luc systems were 72. 92% ,40.60% , 51.00% and 25.19% (F= 67.31 , P< 0.01) in A549 cells ,64.63%,30.02%,48.23% and 23.16% (F=64.94, P< 0.01) in SPC-A1 cells, and 20.81%,18.05%, 44.20% and 18.32% (F=52. 19,P<0.01) in normal hEL cells, respectively. The average early apoptosis rates of these four plasmid systems were 36. 63%, 22. 30%, 24. 33% and 12. 53% (F =50. 99,P <0. 01) in A549 cells, 33.73%, 17. 37%, 22. 43% and 11.20% (F = 20. 76, P < 0. 01) in SPC-A1 cells, and 13.53 %, 12. 5%, 21.93% and 12. 16% (F = 15. 08, P < 0. 01) in normal hEL cells,respectively. The sensitizing enhancement ratios of the four plasmid systems were 3.45, 2. 29, 3.05 and 1.21 in A549 cells, while 2. 68, 2. 15, 3.05 and 1.21 in SPC-A1 cells, respectively. Conclusions The new suicide gene system controlled by chimeric promoter may provide a novel therapeutic modality for lung cancer.
ABSTRACT
Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP-dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subcloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by liposome-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/PNP-CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT-PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP-CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP-CD in HepG2 cells was confirmed.This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer.
ABSTRACT
Objective:To construct and identify the chimeric mtb8.4/hIL12 eukaryotic expression plasmid.Methods:Firstly mtb8.4-linker was amplified by PCR and cloned into the single Nhe Ⅰ and Mlu Ⅰ cloning sites of pCI-neo, so pCI-neo-mtb8.4-linker(pML) plasmid was constructed.Secondly hIL-12 was amplified by PCR and cloned into the single Mlu Ⅰ and Sal Ⅰ cloning sites of pML.Finally correct pCI-neo-mtb8.4/hIL12 (pMI) plasmid was identified by PCR,RE digestion and DNA seguencing.Results:The accuracy of pMI plasmid construction was confirmed by a number of molecular biological techniques.Conclusion:The construction of mtb8.4/hIL12 chimera by linkage of M.tuberculosis mtb8.4 gene to human IL12 gene provides the possibility for investigating a new tuberculosis vaccine.