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. 2001 Nov 15;20(22):6306-15.
doi: 10.1093/emboj/20.22.6306.

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate

G W Makin  1 , B M Corfe, G J Griffiths, A Thistlethwaite, J A Hickman, C Dive
Affiliations

Damage-induced Bax N-terminal change, translocation to mitochondria and formation of Bax dimers/complexes occur regardless of cell fate

G W Makin et al. EMBO J. .

Abstract

Sequential steps in the activation of the pro-apoptotic protein Bax are described for cells with different sensitivity to cytotoxins. SH-EP1 and SH-SY5Y human neuroblastoma cells, derived from a single precursor cell line, differed in their sensitivity to taxol but showed the same sensitivity to cisplatin. Both drugs, in both cell lines, induced exposure of a constitutively occluded N-terminal epitope of Bax. This was reversible and occurred before the translocation of cytosolic Bax to mitochondria. The N-terminal change in Bax, its subsequent movement to mitochondria and its dimerization/complex formation were insufficient for commitment to death, occurring in the same proportion of cells that either maintained (SH-SY5Y) or lost (SH-EP1) clonogenic survival after taxol treatment. Suppression of taxol-induced apoptosis occurred upstream of cytochrome c release from mitochondria in SH-SY5Y cells. The data suggest that a further drug damage-induced event occurs after Bax dimerization/complex formation but prior to cytochrome c release. This event was absent in the taxol-resistant cells.

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Figures

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Fig. 1. Damage responses of SH-EP1 and SH-SY5Y cells to cisplatin and taxol. (A) Clonogenic survival at 10 days and (B) apoptotic response at 72 h following 1 h treatment with cisplatin or taxol. The kinetics of apoptosis after 25 µM cisplatin or 62.5 nM taxol are shown in (B) as inset graphs. SH-SY5Y, filled triangles; SH-EP1 filled squares. (C) The ratio between control or drug-treated cells as a percentage of cells in G2/M following a 1 h exposure to cisplatin (25 µM) or taxol (62.5 nM): grey bars, SH-EP1 cells; white bars, SH-SY5Y cells. The results shown in (A–C) are means of at least three independent experiments ± SEM. (D) Immunoblots for p53 protein, p21WAF1 and Bax after a 1 h exposure to cisplatin (25 µM) or taxol (62.5 nM). Representative blots of at least three independent experiments are shown.
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Fig. 2. Drug-induced exposure of the Bax N-terminus occurs regardless of cell fate. (A) (i) Antibody epitope map of Bax. (ii) Immunostaining of the Bax N-terminus with Bax N20 antibody 24 h after a 1 h exposure to cisplatin (25 µM) or taxol (62.5 nM). Bax N20 staining is seen as red fluorescence. Representative cells shown at magnification ×400. (B) The numbers of cells showing positive staining for Bax N20 at 6 and 24 h after drug treatments (means of three independent experiments ± SEM, **P <0.01 for the comparison between treated and control cells). SH-EP1, open bars; SH-SY5Y, shaded bars.
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Fig. 3. Drug-induced exposure of the N-terminus of Bax is reversible and is not blocked by caspase inhibition. (A) (i) Flow cytometric analysis of Bax 6A7 immunofluorescence (see epitope map in Figure 2) of SH-EP1 cells detached then fixed, compared with cells that have been fixed prior to detachment. IgG, histogram representing non-specific staining. These histograms are typical of at least three independent experiments. (ii) A typical cell that was detached then fixed and stained with Bax6A7 (green) (magnification ×500) is compared with Bax 6A7 and RP staining of cells that were allowed to re-attach and then examined 24 h later (magnification ×250). Nuclei were counterstained with Hoechst 33528. (B) (i) Bax N20 immunofluorescence (red) 24 h after taxol treatment (62.5 nM) in the presence or absence of Z-VAD-fmk (50 µM) (magnification ×400). (ii) Percentage of Bax N20-positive cells after taxol treatment in the presence and absence of Z-VAD-fmk. SH-EP1, grey bars; SH-SY5Y, white bars. Results represent the mean of three independent experiments ± SEM.
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Fig. 4. Translocation of Bax to mitochondria occurs regardless of cell fate. (A) Immunostaining of SH-EP1 and SH-SY5Y cells 24 h after a 1 h exposure to taxol (62.5 nM) with Bax RP (see Figure 2A), seen as red fluorescence. Nuclei are counterstained with Hoechst 33528 (blue). Magnification ×500. (B) Confocal microscopy of SH-EP1 and SH-SY5Y cells before and 24 h after a 1 h treatment with taxol (62.5 nM) or cisplatin (25 µM) stained with Bax RP (red) and cytochrome c (green). Co-localization is seen as yellow and the percentage of red fluorescence (Bax) associated with green fluorescence (cytochrome c) is shown for each image. Magnification ×500. Images are representative of at least three independent repeat experiments.
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Fig. 5. (A) The exposure of the N-terminus of Bax occurs prior to translocation to mitochondria and the release of cytochrome c. Immunostaining of SH-EP1 cells 6 h (i) and 24 h (ii and iii) after a 1 h exposure to taxol (62.5 nM) with Bax N20 (red fluorescence) and cytochrome c (green fluorescence). Co-localization is seen as yellow. Nuclei are counterstained with Hoechst 33528 (blue fluorescence). Magnification at 6 h is ×500, and at 24 h is ×1000. (B) Abrogation of taxol-induced apoptosis occurs prior to cytochrome c release from mitochondria. Immunostaining of SH-SY5Y cells 24 h after a 1 h exposure to cisplatin (25 µM) or taxol (62.5 nM) with cytochrome c (green fluorescence). Nuclei are counterstained with Hoechst 33528 (blue fluorescence). Magnification for control and taxol-treated cells, ×500; for cisplatin-treated cells, ×1000. Data are representative of at least three repeat experiments in which >100 cells were examined.
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Fig. 6. Formation of Bax dimers/complexes in the mitochondrial fraction of SH-SY5Y cells occurs regardless of cell fate. (A) At 24 h after a 1 h treatment of cells with cisplatin (25 µM) or taxol (62.5 nM), the multimerization of Bax was assessed by cross-linking the P10 fraction with DSS or BS3. Bax complexes were immunoprecipitated (using Bax 2D2) and western blotted using Bax RP. A Bax immunoreactive band at ∼46 kDa (marked with an arrow) was detected only in the presence of cross-linker, and not in the Bax null LS174T cells nor in the suspension cell line CEMC7A. Bax monomers were seen at 20 kDa (marked with an arrow) in SH-SY5Y cells but were not detectable in CEM cells despite their expression of Bax; see B(ii). Three bands noted between 50 and 70 kDa occurred in the presence or absence of the cross-linker and in the Bax null cell line (asterisks). All the data for each cross-linker are from the same gel although the middle section of the gel has been cut to exclude immunoglobulin light chain. CIT, citrate buffer alone; DMSO, dimethylsulfoxide alone. (B) The purity of SH-SY5Y P10 and S20 fractions was assessed using aldolase as a cytosolic marker and cytochrome oxidase IV (Cox IV) as a mitochondrial marker (i) and Bax was detected in the cytosolic but not the mitochondrial fractions of CEM C7A cells (ii). Results shown are typical of three independent experiments.
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