Overexpression of bax in human glioma cell lines

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Object. Cells that lose their ability to undergo apoptosis may promote the development of neoplasms and result in resistance to clinical treatment with DNA-damaging modalities such as radio- and chemotherapy. Four established human glioma cell lines that are resistant to apoptosis were transfected with the proapoptotic gene bax and assessed for their sensitivity to a proapoptotic stimulus.

Methods. Two cell lines had a wild-type p53 genotype (U87 and D247MG) and two had mutant p53 genotypes (U138 and U373). Constitutive overexpression of murine bax was achieved in U138 and U373 only, which resulted in an increased sensitivity of these lines to the apoptosis-inducing effect of cytosine arabinoside (ara-C). Multiple attempts to produce constitutive overexpression of bax in U87 and D247MG cells resulted in spontaneous, near-complete cell loss. Vector-only control transfections were successful in all four cell lines. Inducible overexpression of bax was achieved in the U87 cells and elevated levels of BAX were observed as early as 6 hours after gene induction. This overexpression of BAX resulted in the spontaneous induction of apoptosis in these cells.

Conclusions. Overexpression of BAX in four human glioma cell lines resulted in increased sensitivity to apoptosis. In the two lines that had a wild-type p53 genotype, overexpression of BAX produced spontaneous apoptosis. In contrast, the lines that had mutant, nonfunctional P53 did not undergo spontaneous apoptosis, but they were rendered more sensitive to the apoptosis-inducing effect of ara-C. Modulation of BAX expression may be a useful therapeutic modality for gliomas, regardless of p53 genotype.

Article Information

Address reprint requests to: Keith M. Rich, M.D., Department of Neurological Surgery, Washington University School of Medicine, 660 South Euclid Street, St. Louis, Missouri 63110.

© AANS, except where prohibited by US copyright law.

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Figures

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    Western blot showing BAX and BCL-2 expression in human glioma cell lines. Protein was isolated from 70 to 80% confluent cultures of each cell line. Equal amounts of total protein (100 µg/lane) were loaded onto 15% polyacrylamide gels and separated by SDS—polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, which were then immunoblotted for BAX or BCL-2 expression. The cell line WU37 was derived from nonneoplastic human astrocytes.

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    Western blot showing that constitutive overexpression of BAX could be achieved in two of the four human glioma cell lines. The methods used were the same as those described in the legend to Fig. 1. High levels of BAX overexpression were observed in Clones 4, 5, and 7 of the U373 cell line and Clones 1 and 5 of the U138 cell line. Lower levels of BAX expression were observed in vector (V)—only transfections of each cell line. The 2B4 is a human lymphoma cell line known to overexpress BAX.

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    Western blot showing inducible overexpression of BAX in the U87 cell line. Clones of U87 cells transfected with pMt5.neo.bax were grown in cell culture to approximately 70% confluence. Cultures were grown either in standard medium or medium with 100 µM ZnCl2. Twenty-four hours later the protein was isolated and Western blot analysis for BAX expression was performed. Relatively low levels of BAX expression were observed in native U87 cells (U) and in Clone 1 both in the absence (−) and presence (+) of Zn++. Clone 2 demonstrated marked induction of BAX after exposure to Zn++. Moderate levels of overexpression of BAX were observed in Clones 3 and 6 in the absence of Zn++, indicating the presence of a leaky promotor in these clones. Weak induction of BAX expression was observed in Clone 6. Clone 2 showed no significant leakage of promoter and strong induction of BAX in response to exposure to Zn++.

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    Western blot showing the time course of BAX overexpression following induction with ZnCl2. Eight individual plates of cells from Clone 2 of U87.pMt5.neo.bax were grown to approximately 70% confluence and then treated with 100 µM ZnCl2. Protein was isolated from each plate at the time points indicated and a Western blot analysis for BAX expression was performed. Each lane was loaded with an equal amount of protein (100 µg).

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    Photomicrographs showing apoptosis following induction of bax transcription in U87 cells. Individual cultures of U87.pMt5.neo.bax were grown to approximately 70% confluence in the absence of ZnCl2. Cultures were treated with 0 µM (A and C) or 100 µM (B and D) ZnCl2 and then assessed for apoptosis at 24 (A and B) or 48 (C and D) hours after initiation of treatment. Cells undergoing apoptosis are strongly immunofluorescent in response to TUNEL staining. Original magnification × 200.

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    Bar graph showing the percentage of U87.pMt5.neo.bax cells that underwent apoptosis following treatment with ZnCl2 (as depicted in Fig. 5A). A statistically significant increase in the number TUNEL-positive cells was observed at 48 hours after BAX induction (*p < 0.03, Student's two-tailed t-test).

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    Western blot showing BAX and BCL-2 expression in the two mutant p53 cell lines following tranfection with bax. The methods used were the same as those described in the legend to Fig. 1. B = transfection with bax; V = vector-only transfection.

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    Bar graphs showing the sensitivity of two cell lines (U373 and U138), which supported constitutive overexpression of high levels of BAX, to a proapoptotic stimulus. Untreated, these cell lines had low levels of spontaneous apoptosis (approximately ≤ 1%). Cells transfected with either pcDNA3 alone or pcDNA3.bax were treated with either a 1- to 5-µM dose of ara-C for up to 96 hours and then assessed for the presence of apoptosis. Clones that overexpressed BAX (filled columns) were more sensitive to treatment with ara-C, demonstrating higher levels of apoptosis when compared with clones transfected with pcDNA3 only (unfilled columns) (*p < 0.05; †p < 0.01, Student's two-tailed t-test).

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