Transfection of C6 glioma cells with the bax gene and increased sensitivity to treatment with cytosine arabinoside

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Object. Genes known to be involved in the regulation of apoptosis include members of the bcl-2 gene family, such as inhibitors of apoptosis (bcl-2 and bcl-xl) and promoters of apoptosis (bax). The authors investigated a potential approach for the treatment of malignant gliomas by using a gene transfection technique to manipulate the level of an intracellular protein involved in the control of apoptosis.

Methods. The authors transfected the murine bax gene, which had been cloned into a mammalian expression vector, into the C6 rat glioma cell line. Overexpression of the bax gene resulted in a decreased growth rate (average doubling time of 32.96 hours compared with 22.49 hours for untransfected C6, and 23.11 hours for clones transfected with pcDNA3 only), which may be caused, in part, by an increased rate of spontaneous apoptosis (0.77 ± 0.15% compared with 0.42 ± 0.08% for the vector-only transfected C6 cell line; p = 0.038, two-tailed Student's t-test). Treatment with 1 µM cytosine arabinoside (ara-C) resulted in significantly more cells undergoing apoptosis in the cell line overexpressing bax than in the vector-only control cell line (23.57 ± 2.6% compared with 5.3 ± 0.7% terminal deoxynucleotidyl transferase—mediated biotinylated—deoxyuridine triphosphate nick-end labeling technique—positive cells; p = 0.007). Furthermore, measurements of growth curves obtained immediately after treatment with 0.5 µM ara-C demonstrated a prolonged growth arrest of at least 6 days in the cell line overexpressing bax.

Conclusions. These results can be used collectively to argue that overexpression of bax results in increased sensitivity of C6 cells to ara-C and that increasing bax expression may be a useful strategy, in general, for increasing the sensitivity of gliomas to antineoplastic treatments.

Article Information

Address reprint requests to: Keith M. Rich, M.D., Department of Neurological Surgery, Washington University School of Medicine, 60 South Euclid Avenue, St. Louis, Missouri 63110.

© AANS, except where prohibited by US copyright law.

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Figures

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    Western blot gels showing BAX expression. A: Example of a blot used to screen BAX expression in transfected clones. Each lane was loaded with 50 µg of protein. The highest levels of BAX expression were seen in clones C6.Bax.4 and C6.Bax.7. B: Quantitative analysis of BAX expression by untransfected C6 and C6 cell lines transfected with vector only (C6.pCDNA3.10) or with the vector containing the murine bax gene (C6.Bax.7). Equivalent amounts of protein (50 µg) were loaded in each lane and the blots were probed for BAX and tubulin. Note the similar expression of tubulin in each cell line and overexpression of bax in C6.Bax.7.

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    Graph showing growth curves of C6 cells and C6 clones transfected with vector only or with vector containing the bax gene. Individual points were determined by means of a colorimetric assay. Each point represents the mean ± SD of five wells for the C6 and vector-only clones and the mean ± SD of three wells for the clones transfected with bax. Zero hour is the first time point for cell measurement, usually 24 hours after plating. Cells were examined in the exponential growth phase. By 72 hours of growth, a distinct difference in growth rates was observed between the clones transfected with bax and those transfected with vector only or untransfected.

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    Photomicrographs showing induction of apoptosis in C6 cells transfected with vector only (A and B) or with vector containing the bax gene (C and D). Cells were treated with 0.5 µM ara-C for 72 hours. They were stained using the TUNEL protocol (A and C), which reveals only nuclei of cells undergoing apoptosis, or with bis-benzamide (B and D), which stains all nuclei but allows for identification of apoptotic nuclei based on morphological criteria. Cells were viewed at × 200 magnification with the aid of a microscope equipped with fluorescent optics. Treatment of C6.pcDNA3.10 with ara-C produces only occasional TUNEL-positive cells (A) or chromatin condensation with bis-benzamide (B). Staining reveals most nuclei to be intact; in only the occasional cell is there fragmentation typical of chromatin condensation. C: In contrast, many TUNEL-positive C6.Bax.7 cells are seen after treatment with ara-C. D: The same cells as in (C) stained with bis-benzamide showing multiple nuclear fragments consistent with the frequent occurrence of chromatin condensation. Arrowheads point to examples of chromatin condensation revealed by staining with bis-benzamide (B and D).

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    Bar graph showing quantitative assessment of the prevalence of TUNEL-positive cells following treatment with 0.5 µM or 1 µM ara-C. The percentage of TUNEL-positive cells was determined for three separate high-power fields, and an average ± SD was determined. A significantly increased percentage of TUNEL-positive cells was seen in the C6.Bax.7 cells compared with the C6.pcDNA3.10 line at both doses (p < 0.008, two-tailed Student's t-test). A small but statistically significant increase in the percentage of TUNEL-positive cells was also seen in the untreated (control) C6.Bax.7 cells (p = 0.038, two-tailed Student's t-test).

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    Graph showing growth curve assay of C6.pcDNA3.10 and C6.Bax.7 cells after treatment with 0.5 µM ara-C. Each time point represents an average ± SD of three wells and is normalized to a point determined at the initiation of treatment (0 hour). Cells were treated with ara-C for 72 hours (bar under x axis), growth was assayed, and the drug was washed out and replaced with normal medium. Growth was determined at 48-hour intervals thereafter. Growth of the C6.Bax.7 cells was severely impaired over the 6 days after washout of the drug, despite regular replacement with fresh medium.

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