Article Figures & Data
Figures
- Fig. 1.
Construction of p2ΦC31-IFNγ and production of minicircle-IFNγ. A, restriction map of pShuttle-IFNγ. PCMV, immediate-early human cytomegalovirus enhancer/promoter; IFNγ, human γ interferon gene; poly A, bovine growth factor polyadenylation signal; Kan, kanamycin resistance gene; ori, pUC origin of DNA replication. B, flow chart of ΦC31 integrase–mediated intramolecular recombination of p2ΦC31-IFNγ. The resulting product is minicircle-IFNγ. Amp, ampicillin resistance gene; BAD, araBAD promoter; araC, araC repressor; attB, bacterial attachment site; attP, phage attachment site; attR, right hybrid sequence; I-SceIg, I-Sce I gene. C, analysis of minicircle DNA. M, 1 kb plus DNA ladder, bands of 10, 8, 5, 2, 1.6, 1, and 0.7 kb. All lanes were loaded with 0.3 μg of DNA. Lane 1, p2ΦC31-IFNγ; lane 2, p2ΦC31-IFNγ/SpeI; lane 3, minicircle-IFNγ; lane 4, minicircle-IFNγ/SpeI.
- Fig. 2.
Expression profiles of IFNγ in the supernatant of cells transfected with plasmids carrying human IFNγ expression cassette. Transfections were done with the same amount of total DNA and same molarities of IFNγ-cassette with or without stuffer DNA. A to C, columns, mean of three independent experiments, each conducted in triplicate; bars, SE. *, P < 0.05, compared with p2ΦC31-IFNγ–treated group.
- Fig. 3.
IFNγ gene transfer inhibits in vitro growth of human NPC cells. Cells were treated with minicircle-IFNγ and control plasmids for the indicated time periods. Cell viability was determined by WST assay. Data are given as relative growth rates compared with p2ΦC31-treated group. A to C, columns, mean of three independent experiments, each conducted in triplicate; bars, SE. *, P < 0.05, compared with p2ΦC31-treated group. D, r-hu-IFNγ inhibits in vitro growth of CNE-1 cell line. Cells were treated with r-hu-IFNγ at doses ranging from 10 to 40,000 IU/mL for indicated time periods. Points, mean of three independent experiments, each conducted in triplicate; bars, SE. *, P < 0.05, compared with cells treated with medium alone.
- Fig. 4.
IFNγ gene transfer exerts antiproliferative effects on NPC cells by inducing G0-G1 arrest and apoptosis. A and B, cell cycle phase distribution of minicircle-IFNγ–treated NPC cell lines. Three NPC cell lines were transfected with minicircle-IFNγ and the cell cycle phase distribution of each cell lines was analyzed at the indicated time. A, data from representative flow cytometry experiments at indicated time point after transfection. Ctrl, p2ΦC31-treated group; mc, minicircle-IFNγ treated group. B, graphical representation of the mean total population in each stage of the cell cycle. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, compared with p2ΦC31-treated group. C, graphical representation of the percentage population in pre-G1 phase from a representative experiment. D, caspase-3 activity induced by IFNγ gene transfer. Columns, mean of three independent experiments; bars, SD. *, P < 0.05, compared with p2ΦC31-treated group.
- Fig. 5.
Antitumor effect of IFNγ gene transfer on growth of NPC xenografts. A, time-dependent evolution of tumor volume in mice inoculated with the CNE-2 and C666-1 cell lines (n = 5). For CNE-2 cell–xenografted mice, mc-A versus p2ΦC31 + Lipofectamine, P < 0.05 at days 5, 9, 13, 17, and 21; mc-B versus p2ΦC31 + Lipofectamine, P < 0.05 at days 17 and 21; p2ΦC31-IFNγ versus p2ΦC31 + Lipofectamine, P < 0.05 at days 17 and 21; r-hu-IFNγ versus 0.9% NaCl, P < 0.05 at days 13, 17, and 21. For C666-1 cell–xenografted mice, mc-A versus p2ΦC31 + Lipofectamine, P < 0.05 at days 11, 16, and 21; mc-B versus p2ΦC31 + Lipofectamine, P < 0.05 at days 16 and 21; p2ΦC31-IFNγ versus p2ΦC31 + Lipofectamine, P < 0.05 at day 21; r-hu-IFNγ versus 0.9% NaCl, P < 0.05 at days 16 and 21. B, antitumor effect of IFNγ gene therapy on CNE-2 cell–xenografted and C666-1 cell–xenografted nude mice (n = 5). Mice were sacrificed after 3 weeks of treatment and tumors were resected and weighted. Columns, mean of five mice; bars SD. *, P < 0.05, compared with p2ΦC31 + Lipofectamine–treated group. C, expression profiles of IFNγ and reverse transcription-PCR analysis of IFNγ transcripts in the tumor tissue transfected with minicircle-IFNγ and p2ΦC31-IFNγ. For expression profiles of IFNγ, results are given in nanograms per 100 mg of tumor tissue. Columns, mean of three mice; bars, SD. For reverse transcription-PCR analysis of IFNγ transcripts, results from representative experiments were shown. β-Actin was used as loading control. Φ, p2ΦC31 group, 1 day after injection; b, blank control. D, effect of IFNγ gene transfer on survival (n = 10). For CNE-2 cell–xenografted mice, mc-A versus p2ΦC31-IFNγ, P < 0.0001; mc-B versus p2ΦC31-IFNγ, P < 0.02; mc-A versus r-hu-IFNγ, P < 0.0001; mc-B versus r-hu-IFNγ, P < 0.0001; p2ΦC31-IFNγ versus r-hu-IFNγ, P < 0.007. For C666-1 cell–xenografted mice, mc-A versus p2ΦC31-IFNγ, P < 0.0001; mc-B versus p2ΦC31-IFNγ, P < 0.0001; mc-A versus r-hu-IFNγ, P < 0.0001; mc-B versus r-hu-IFNγ, P < 0.0002; p2ΦC31-IFNγ versus r-hu-IFNγ, P > 0.05 (Kaplan-Meier).
Tables
- Table 1.
Treatment regimens for in vitro and in vivo transfections
(A) Treatment regimen used to transfect cells with DNA constructs with the same ratio of Lipofectamine 2000 to DNA in each case (1:1) Group Treatment per well p2ΦC31-IFNγ 11.3 kb 1 μg Minicircle-IFNγ 1.6 kb mc-A (weight:weight) 1 μg mc-B (mole:mole with stuffer DNA) 0.14 μg + 0.86 μg pSP72 mc-C (mole:mole without stuffer DNA) 0.14 μg pShuttle-IFNγ 4.6 kb 1 μg (B) Treatment regimen used for NPC-xenografted mice Group Treatment 0.9% NaCl 100 μL/d Lipofectamine* 60 μL/wk p2ΦC31 + Lipofectamine* 15 μg p2ΦC31 + 60 μL Lipofectamine/wk r-hu-IFNγ* 100 × 104 IU/kg/d p2ΦC31-IFNγ* 15 μg p2ΦC31-IFNγ + 60 μL Lipofectamine/wk mc-A* 15 μg minicircle-IFNγ + 60 μL Lipofectamine/wk mc-B* 2.1 μg minicircle-IFNγ + 12.9 μg pSP72 + 60 μL Lipofectamine/wk ↵* 0.9% NaCl solution was added to adjust the total volume to 100 μL.
Supplementary Data Wu, et al.
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Volume 12, Issue 15
