Interaction between p53 and p16 expressed by adenoviral vectors in human malignant glioma cell lines

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Object. Multiple gene replacements have been examined as a potential treatment modality for malignant gliomas. Nevertheless, no reports are available that detail the synergy, additivity, or antagonism of multiple genes. The aim of this study was to assess the interaction between p53 and p16 genes in the growth of glioma cell lines.

Methods. The human glioma cell lines U87MG and U373MG were transduced using an adenoviral vector with Ad-p53, Ad-p16, or both. Western blotting was performed to determine the expression of the protein products of the transduced p53 and p16 genes. To establish whether the combination of Ad-p53 and Ad-p16 would be beneficial, the effects of gene combinations at the median inhibitory concentration level were analyzed using the isobologram method. Annexin assays and cell cycle analyses were performed on the transduced cells. Western blotting demonstrated the expression of p53 and p16 in transduced cells. Simultaneous exposure to Ad-p53 and Ad-p16 produced additive effects in both glioma cell lines. Experimental data points in U373MG lay near the Mode I line, indicating that the vectors had a different mode of action. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis, whereas in the wild-type p53 cell lines, the overexpression of wild-type p53 resulted in a moderate degree of apoptosis and G1 arrest. Furthermore, Ad-p16 induced more marked G1 arrest than Ad-p53 in cells with wild-type p53.

Conclusions. The results show that interaction between Ad-p53 and Ad-p16 is additive, regardless of p53 gene status.

Article Information

Address reprint requests to: Byung-Kyu Cho, M.D., Ph.D., Department of Neurosurgery, Seoul National University, College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110–744, Korea. email: bkcho@snu.ac.kr.

© AANS, except where prohibited by US copyright law.

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Figures

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    Photomicrographs showing expression of β-galactosidase activity. At an MOI of 100, U87MG (left) and U373MG cells (right) show highly efficient gene transfer, with 90 to 100% of the cells stained for β-galactosidase. X-gal stain, original magnification × 100.

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    Blots showing transgene protein product expression; p53 and p16 expression by adenoviral vectors in U87MG (left) and U373MG cells (right) is confirmed by Western blotting. Actin controls confirm equal protein loading. Lane 1, sham treatment group; lane 2, cells treated with Ad-lacZ (MOI 100); lane 3, Ad-p53 (MOI 100); lane 4, Ad-p16 (MOI 100); lane 5, combined Ad-p53 and Ad-p16 (MOI 50 each).

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    Graphs showing dose—response curves for Ad-p53 in combination with Ad-p16 in U87MG (left) and U373MG cells (right). After 4 days, cell numbers were determined using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and plotted as a percentage of the control. Each point represents the mean value ± the standard error (bars).

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    Graphs depicting isobolograms of simultaneous exposure to Ad-p53 and Ad-p16 in U87MG (left) and U373MG cells (right). The data points for the combination (black squares, fixed MOI of Ad-p53; white squares, fixed MOI of Ad-p16) indicate an additive interaction. In U373MG cell lines, the experimental data points lie near the Mode I line, indicating that the vectors have different modes of action.

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    Scatterplots showing results of annexin assay of U87MG (upper) and U373MG cells (lower). (The quadrants are designated by the abbreviations UL, UR, LL, and LR.) At 72 hours, 36.1% of the U87MG cells transduced with p53 alone were positive for apoptosis and 12.2% of the cells transduced with p16 alone were positive, whereas 35.8% of the cells transduced with both p53 and p16 were positive. In U373MG cells, 42.3% of the cells transduced with p53 alone were positive for apoptosis, 18.7% transduced with p16 alone were positive, and 38.4% of the cells transduced with both p53 and p16 were positive. AV = annexin V; mock = sham-treated.

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    Graphs showing cell cycle analysis of U87MG (upper) and U373MG cells (lower). The U87MG cells transduced with p53 showed a moderate degree of G1 arrest without a significant increase in the number of cells with sub-G1 DNA content, whereas Ad-p16 induced G1 arrest. After 48 hours, U87MG cells transduced with p53 alone were 73.9% in the G1 phase and cells transduced with p16 alone were 89.3% in G1, whereas cells transduced with both p53 and p16 were 84% in G1. In U373MG cells, Ad-p53 resulted in a significant shift on the DNA profile to a sub-G1 position (53.6% in sub-G1 for Ad-p53 and 33.6% for Ad-p53 and Ad-p16). Transduction with Ad-p16 also effectively induced G1 arrest in U373MG cells (84.3% in G1 for Ad-p16 compared with 65.9% for sham treatment). G0, G1, G2, M, and S = phases of the cell cycle.

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