GSH depletion enhances adenoviral bax-induced apoptosis ... - Nature

Cancer Gene Therapy (2004) 11, 249–255 All rights reserved 0929-1903/04 $25.00

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GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells Tsuyoshi Honda,1 Simona Coppola,2 Lina Ghibelli,2 Song H Cho,1 Shunsuke Kagawa,3 Kevin B Spurgers,1 Shawn M Brisbay,1 Jack A Roth,3 Raymond E Meyn,4 Bingliang Fang,3 and Timothy J McDonnell1 1

Department of Molecular Pathology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA; 2Institut tor Vergata Rome, Italy; 3Department of Thoracic and Cardiovascular Surgery; and 4 Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA. The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes. Cancer Gene Therapy (2004) 11, 249–255. doi:10.1038/sj.cgt.7700684 Published online 5 March 2004 Keywords: apoptosis; bax; redox; glutathione

poptosis is a highly conserved process throughout evolution and has a major role in maintaining tissue A homeostasis. A number of genes that regulate apoptosis 1

are also conserved including members of the bcl-2 gene family. Members of this family can be divided into two functional groups: antiapoptotic genes that inhibit apoptosis and proapoptotic genes that facilitate apoptosis. Bcl2 is the prototypic member of the former group and Bax, bcl-2-associated X protein, is the prototypic member of the latter.2 Bax was first identified as a 21 kDa protein that coimmunoprecipitated with bcl-2.3 Relative levels of hetero and homodimers comprising these proteins, including bcl-2 homodimers, may function in the manner of a rheostat to regulate apoptosis. Expression of Bax may be sufficient to induce a common pathway of apoptosis. It has been demonstrated that bax directly induces cytochrome c release from mitochondria,4 suggesting that it is necessary for Bax protein to anchor to a membrane of the mitochondria to activate the apoptotic pathway. Received August 1, 2003.

Address correspondence and reprint requests to: Dr Timothy J McDonnell, MD, PhD, Department of Molecular Pathology, Box 89, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA. E-mail: [email protected]

Glutathione (GSH) is a tripeptide, g-glutamyl cysteinyl glycine, which is considered to be a key molecule in the antioxidant pathway of cells. A major role of GSH is to maintain the intracellular redox state by scavenging reactive oxygen species. GSH has also been implicated in protection against the induction of apoptosis and necrotic cell death in a variety of cell types.5,6 However, the function of GSH in apoptosis regulation is not completely understood. In this study, we determined that the H322 NSCLC cell line was significantly resistant to apoptosis induction following adenovirus bax gene transfer compared to other NSCLC cell lines. The resistance of H322 cells correlated with a significantly higher level of intracellular GSH compared to the sensitive cell lines. To gain a better understanding of the effect of GSH depletion on Bax-mediated apoptosis and to confirm if GSH depletion is a useful strategy to enhance apoptotic induction by Bax cotransfection, we performed GSH depletion in H322 and H226 cells after Bax transduction using buthionine sulfoximide (BSO), a specific inhibitor of de novo GSH synthesis. Quantification of GSH after treatment confirmed that BSO leads to a decrease in intracellular reduced GSH during the first 24 hours and resulted in a significant augmentation in Ad-baxmediated cell death. This enhanced sensitivity correlated with an increase in both mitochondrial membrane-

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associated bax protein and cytosolic cytochrome c. Together, these findings suggest that the level of intracellular GSH may be an important determinant of the sensitivity to cell death involving bax activation.

Materials and methods

Cell lines and chemicals The non-small-cell lung cancer (NSCLC) lines H1299, A549, H226, and H322 were obtained from the American Type Culture Collection. H1299, H226, and H322 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. A549 cells were maintained in HAM/F12 medium with the same supplements. Cells were incubated at 371C in a 5% CO2 incubator. BSO was purchased from Sigma Chemical Co. (St Louis, MO).

Adenovirus vectors and transfection efficiency studies The construction and properties of the Ad-GT-Bax and Ad-PGK-GV16 binary transfection system have been previously reported.7 Viral titer was determined by measurement of the optical density (OD). Final viral concentrations were made by dilution of the stock virus with phosphate-buffered saline (PBS). Adenovirus containing the LacZ gene coding for the bacterial enzyme bgalactosidase8 was used as a control vector and to measure the efficiency of infection. Cells were plated in 24-well dish at 5 104 and incubated overnight at 371C to obtain an optimal confluence. Then the cells were infected with Ad/LacZ at a range from 300 to 16,000 viral particles per ml (vp/ml). The cells were fixed with 0.5% glutaraldehyde, and stained with the b-galactosidase (bgal) substrate, x-gal (5-bromo-4-chloro-3-indolyl-b Dgalactoside; GIBCO-BRL). After 48 hours, the transduction efficiency was estimated from the percentage of cells exhibiting blue staining.

dilution of 1:200, anti-caspase-3-monoclonal antibody (PharMingen) at a dilution of 1:500, anti-DFF45-monoclonal antibody (PharMingen) at a dilution of 1:200, and anti-b-actin-monoclonal antibody (Amersham) at a dilution of 1:1000. Following incubation with the appropriate secondary antibody, detection was performed with enhanced chemiluminescence (ECL and ECL plus, Amersham).

Apoptosis assay Cells were plated in 24-well dishes at 5 104 and incubated overnight at 371C to obtain an optimal confluence. Then the cells were infected with either AdGT-Bax and Ad-PGK-GV16 or Ad-Lac-Z and Ad-PGKGV16 at the optimal vector dose for 48 hours. After fixing the cells in 4% paraformaldehyde, the cells were stained with 10 mg/ml bis-benzimide (Hoechst 33342, Sigma). The attached cells and floating cells were counted using fluorescence microscopy in 24-well dishes.10 Additionally, the floating cells were collected by cytospin for photography and imaging.

GSH assay The method developed by Hissin and Hilf11 was used to measure the GSH levels. Cells were washed once in PBS and then resuspended at 1 106/ml in cold lysis buffer consisting of 5% trichloroacetic acid, 1 mM EDTA, and 0.1 M HCl (1:1:1,v/v/v). Following lysis, nonsoluble material was spun out at 5600 r.p.m. for 20 min at 41C. A sample (0.2 ml) of the supernatant was mixed with 3.6 ml of 0.1 M phosphate/5 mM EDTA buffer (pH 8.0) and 0.2 ml of o-phthaldialdehyde (OPT) stock (0.1 mg/ml in methanol) was added. Fluorescence was read at an emission wavelength of 420 nm with excitation set at 350 nm. A standard curve was determined using known quantities of reduced GSH in order to convert fluorescence readings to GSH concentrations.

Immunoblot analysis

Detection of bax translocation to, and cytochrome c release from, mitochondria

Cells were infected with vectors when 50–70% confluent in 100-mm dishes and harvested at 48 hours. The cells were lysed for at least 1 hour at 41C in a lysis medium containing 150 M NaCl, 1% Triton X-100, 25 mM Tris (pH 7.5), and freshly added 1 mM PMSF and aprotinin (Sigma). Cell debris was pelleted by centrifugation at 14,000 r.p.m. for 5 minute at 41C. Supernatants were solublized for 5 minute at 1001C in Laemmli’s SDSPAGE sample buffer as described.9 Samples of 20 mg were resolved at 100 V in a 12.5% SDS-PAGE gel and electroblotted to nitrocellulose membranes (0.2 mm, Schielder & Schuell Inc., Keene, NH) for 2 hours at 100 V. Membranes were blocked in 5% milk PBS-T (0.1% Tween-20 in PBS) for 1 hours, then incubated with the following antibodies: anti-bcl-2-monoclonal antibody (PharMingen) at a dilution of 1:500, anti-bax-monoclonal antibody (Santa Cruz) at a dilution of 1:500, anti-bcl-Xpolyclonal antibody (Santa Cruz) at a dilution of 1:500, anti-PARP-monoclonal antibody (PharMingen) at a

H322 cells were infected with the Bax binary transfection system for 24 hours. At 3 hourrs after infection, BSO was added at 1 M concentration. At the end of the incubation, the medium containing floating cells was collected and subjected to centrifugation at 200 g. for 5 minute. After washing the cell pellet twice with cold PBS, cell pellets were resuspended in a 5 volume of hypotonic buffer (10 mM NaCl, 1.5 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail 1 tablet/10 ml). The cells were incubated on ice until they completed swelling and then dounced with the B pestle (Kontes Glass Company) until 90% of cells were lysed. A volume of 2.5 MS buffer (525 mM mannitol, 175 mM sucrose, 12.5 mM Tris-HCL pH 7.5, 2.5 mM EDTA pH 7.5, 1 mM DTT, 1 mM PMSF, protease inhibitor cocktail 1 tablet/10 ml) was quickly added to the lysate to achieve 1 MS. The mixture was subjected to centrifugation at 1300 g. The resulting supernatant was centrifuged two more times to completely remove the nuclei and unlysed

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whole cells. The supernatant was then subjected to centrifugation at 10,000 g to isolate mitochondrial membranes. The resulting pellet was lysed in loading buffer (0.0625 M Tris-HCl pH 6.8, 2% SDS, 5% b-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue) for Western blot analysis. The supernatant was subsequently subjected to centrifugation at 100,000 g for 1 hour at 41C. The resulting supernatant, the cytosolic fraction, was concentrated five- fold using a Centricon 10 concentrator (MILLIPORE). All the fractions were analyzed by Western blot analysis.

Flow cytometric analysis To quantitate the number of cells undergoing apoptosis, cells were fixed with 3.7% formaldehyde, permeabilized, and the DNA labelled with FITC-labelled terminal deoxynucleotidyl transferase (TdT) for 1 hour at 371C. Preparations were then counterstained with PI for 5 minute in the dark. The proportion of apoptotic cells was assessed using a FACSCalibur (BD Biosciences) and the data was analyzed with CELLQuest software.

Statistical analysis

Figure 1 Percentage of total cells staining positive for apoptosis. The ratio of the Ad/GT-Bax to Ad/PGK-GV16 used was 2:1 as has been reported elsewhere.7 The results for 48 hours are shown. Error bars represent SD. There is statistically a significant difference between Ad/GT-Bax plus Ad/PGK-GV16 coinfection versus Ad/LacZ plus Ad/PGK-GV16 coinfection. Po.05 for each cell line.

The results are expressed as mean7SD for the stated number of experiments. Means were compared using Student’s t-test for unpaired values. A P-value less than 0.05 was considered significant.

Results

H322 NSCLC cells are resistant to apoptosis induced by Ad-bax The vector infection efficiency and optimal vector dose was determined for each of the four NSCLC cell lines using Ad-LacZ. The optimal ratio of the Ad-GT-Bax to Ad-PGK-GV16 used was 2:1 as previously reported.7 The proportion of cells with nuclear condensation and DNA fragmentation characteristic of apoptotic cells was measured using fluorescence microscopy of Hoechst 33342stained cells. Apoptotic cells were detected in all cell lines by 48 hours after Ad-GT-Bax and Ad-PGK-GV16 (hereafter referred to as Ad-Bax) coinfection using this staining method. There was a significant difference in apoptosis induction between Ad-Bax coinfection and AdLacZ, and Ad-PGK-GV16 coinfection controls (Fig 1). Three of the four NSCLC lines (H1299, A549, and H226) had a robust apoptotic response to the Bax binary transfection system. However, the H322 cell line consistently exhibited a comparatively modest and significantly lower apoptotic response following Ad-bax gene transfer.

Resistance to Ad-bax expression in H322 cells is independent of bcl-2 and bcl-xL and correlates with reduction in processing of caspase-3, PARP, and DFF45 Bax infection, but not control virus infection, resulted in a comparable increase in Bax protein levels by 48 hours in all NSCLC cell lines studied (Fig 2). There was no

Figure 2 Comparison of Bax, bcl-2, and bcl-xL protein levels and cleavage of caspase-3, DFF45, and PARP in H1299, A549, H226, and H322 cells pre- and post Ad/Bax transfection. Protein expression following Ad/GT-Bax and Ad/PGK-GV16 coinfection was estimated at 9 hours by Western blot analysis.

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significant change in steady-state levels of bcl-xL or bcl-2 protein in Ad-bax-infected cells (Fig 2). Further, the levels of these anti-apoptotic proteins did not correlate with resistance in the H322 cell line. Western blot analysis using whole-cell extracts showed that there was processing of caspase-3, PARP, and DFF45 in Ad-bax-infected cells. The extent of PARP and DFF45 processing in the resistant H322 line was less extensive compared to the three sensitive NSCLC cell lines. Based on these results, H322 cells were examined in more detail. Floating cells, enriched for those undergoing apoptosis, and adherent cells were collected and assessed separately. Bax protein expression appeared to be equivalent in floating and adherent H322 cells (Fig 3). However, the extent of PARP and DFF45 processing was more complete in floating cells (Fig 3), indicating that processing of these proteins occurred in the apoptotic subcompartment of cells. However, the majority of the H322 cell population displayed resistance to Baxmediated apoptosis, despite the high level of Ad-bax transduction and high levels of bax expression. Additionally, the amount of bcl-2 and bcl-xL proteins was equivalent in the adherent and floating cell populations.

Quantification of GSH in NSCLC cell lines and enhancement of Ad-bax-induced apoptosis following GSH depletion by BSO The results of GSH measurements for each NSCLC line are presented in Table 1. The H322 Ad-bax-resistant cell line exhibited significantly higher steady-state levels of GSH compared to the Ad-bax-sensitive cell lines (Po.05, Po.01 and Po.05 for comparisons to H1299, A549, and H226 cells, respectively). Based on these results, it was of interest to determine whether depletion of GSH in H322 cells would sensitize them to Ad-bax-mediated apoptosis. H322 (Ad-bax resistant) and H226 (Ad-bax sensitive) cells were transfected with the Ad-Bax binary system alone or in combination with BSO to deplete intracellular GSH. Cells were harvested 24 hours after gene transfer and GSH levels were measured. As shown in Table 2, BSO alone potently lowered GSH levels in these cell lines by 24 hours. Interestingly, Ad-Bax also caused a lowering of GSH in adherent and floating H226 cells and floating H322 cells, but not in adherent H322 cells. These observations are consistent with the reported efflux of GSH during apoptosis.12 When Ad-Bax and BSO were combined, GSH levels were depleted in the adherent H322 cells. Ad-LacZ had no influence on GSH in either cell line. A morphologic assessment of apoptosis using fluorescence microscopy of Hoechst 33342-stained cells showed that BSO treatment alone exhibited negligible toxicity (Fig 4). However, a significant enhancement of Ad-baxinduced apoptosis was observed in cells treated with BSO in both cell lines tested (Fig 4). Comparable levels of apoptosis were observed in H322 and H226 cell lines after 48 hours, although the kinetics in the H322 cell line were delayed relative to H226 cells. These data suggested that GSH may be an important modulator of Bax-induced

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Figure 3 Comparison of Bax expression, cleavage of DFF45 and PARP in whole cells, floating cells and adherent H322 cells following Ad/GT-Bax and Ad/PGK-GV16 coinfection by Western blot analysis.

Table 1 GSH levels in lung cancer cell lines Cell lines H1299 A549 H226 H322

pg/cell 4.1370.71 2.0070.07 3.6970.89 6.7570.88

Data presented are the means and standard deviations of separate measurements.

apoptosis. The findings further indicate that depleting intracellular GSH could sensitize otherwise resistant NSCLC cells to cell death mediated by Bax.

Bax translocation and cytochrome c release in GSH-depleted H322 cells In order to assess whether activation events associated with Bax-induced apoptosis may be sensitive to intracel-


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Table 2 Comparison of GSH levels in H322 and H226 after BSO treatment and Ad/GT-Bax Ad/PGK-GV16 coinfection Treatment No treatment BSO AdBax adhesion AdBax floating AdBax+BSO adhesion AdBax+BSO floating Ad-LacZ

H322 pg/cell

H226 pg/cell

6.5370.40 0.9270.20 3.2970.98 0.4770.11 0.0370.02 o0.01 6.2470.30

3.8070.20 0.1370.003 0.9970.25 o0.01 0.0470.04 o0.01 3.5270.14

Data presented are the means and standard deviations of separate measurements.

lular GSH levels, subcellular fractionation and immunoblot analyses were undertaken in BSO-treated cells. Cytosolic and heavy membrane fractions were prepared using H322 whole cell extracts from cells treated with AdBax alone or in combination with BSO. In GSH-depleted H322 cells, the proportion of Bax protein associated with the mitochondrial membrane fraction was significantly increased (Fig 5). Furthermore, the proportion of cytosolic cytochrome c was also increased in Ad-Baxtreated cells in which GSH had been depleted using BSO. These findings are consistent with the enhanced apoptosis observed in H322 cells in this context.

Discussion

Adenoviral-mediated overexpression of Bax may be a new and promising strategy for treating human cancer. In previous reports from our group,7,13 as well as from others,14–16 adenoviral-mediated Bax has been shown to be an effective agent for inducing apoptosis in human lung, prostate, cervical, and ovarian cancer cells. It has also been shown that levels of intracellular GSH are frequently elevated in cancers and have been associated with therapeutic resistance.17 Four NSCLC lines were tested in this study for their sensitivity to Ad-Bax-induced apoptosis. Although Ad-Bax was generally effective at inducing apoptosis, one cell line, H322, was notably resistant, compared to the other three lines and this correlated with a higher level of GSH. These results suggest a relationship between cellular GSH level and sensitivity to Ad-Bax. However, the H1299 and H226 cell lines do have significantly more GSH than A549 cells, yet are still sensitive to Ad-bax-induced apoptosis. One possible explanation for this observation is that a threshold level of GSH, not attained by H1299, A549, or H226 cells may be required to provide resistance to apoptosis. Even so, GSH depletion can further sensitize H226 cells to Ad-bax-induced apoptosis. Therefore, the data are consistent with the interpretation that GSH is an important, but not only, determinant of sensitivity to Ad-baxinduced apoptosis. The importance of conformational changes in the bax protein and translocation of bax to the mitochondrial

Figure 4 Apoptotic index after BSO treatment, Ad/Bax, or Ad/Bax plus BSO in H226 and H332 cells. Hoechst staining was performed to detect apoptotic cells. (a) H226 cells at 24 hours. (b) H322 at 24 hours. (c) H322 at 48 hours. BSO treatment in combination with Adbax resulted in a significant enhancement of cell death compared to Ad-bax alone (Po.05 for H226 cells at 24 hours and Po.01 for H322 cells at 48 hours).

membrane following apoptotic stress has been demonstrated.18,19 Recently, Yang and Korsmeyer20 has reported that Bax is localized to the outer mitochondrial membrane by a COOH-terminal hydrophobic signal anchor when apoptosis is initiated. It is considered that a principal function of Bax is to facilitate the release of

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Figure 5 Bax translocation and cytochrome c release in H322 cells. To confirm that BSO enhanced Bax translocation to the mitochondria, we performed Western blot analysis in mitochondrial and cytosolic fractions.

molecules, including cytochrome c, from mitochondria that initiate the caspase cascade resulting in apoptosis.4 However, it has also been reported that Bax mediates cell death in the absence of an additional stimulus through a mechanism involving loss of mitochondrial membrane potential.21 Further, ectopic expression of Bax can act on the mitochondrial permeability transition (MPT) pore to induce cytochrome c release.22 Common features of these findings include the translocation of bax to the mitochondrial membrane and subsequent release of cytochrome c from the mitochondria. The translocation of Bax from the cytosol to mitochondria and its link to cytochrome c release is now established in various cell types.23,24 The cell redox state in general, and GSH in particular, are known to have modulatory effects on apoptosis. It has previously been demonstrated that lowering GSH levels sensitizes cells to apoptosis inducing stimuli such as chemotherapeutic drugs and g-irradiation.5,25–29 The potential mechanism(s) by which intracellular GSH could modulate apoptosis sensitivity remain largely undescribed. One possible mechanism involves ceramide. GSH depletion can activate the sphingomyelinase NSmase and lead to the generation of ceramide.30 Ceramide is a membrane sphingolipid that can itself induce apoptosis.31 Further, it has been shown that bcl-xL can attenuate glutathione depletion in IL-3-dependent FL5.12 cells following IL-3 deprivation.32 These findings imply an important role for GSH in the mediation of the antiapoptotic function of bcl-xL. Our observations are consistent with a protective effect of GSH in the

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inhibition of apoptosis. However, this is unlikely a consequence of bcl-xL in that no demonstrable difference in the level of bcl-xL protein between sensitive NSCLC cell lines and the resistant H322 cell line were apparent. Rather, in consideration of the findings reported herein, it seems reasonable that such sensitization may possibly be due to the facilitation of bax activation and translocation to the mitochondrial membrane. In another review, it was concluded that experimental lowering of the GSH content of cells is not sufficient by itself to induce apoptosis.33 However, we observed in the present study that H322 and H226 cells induced to undergo apoptosis following Bax induction by our cotransfection system rapidly loose reduced GSH. This is consistent with the previous report that GSH efflux occurs in apoptotic cells.34 Thus we believe that overexpressing Bax may induce GSH depletion and this effect in turn may help to drive cells to apoptosis. Taken together, these results suggest that there is an autoregulatory relationship between Bax protein localization and GSH depletion or efflux. In summary, our results suggest that the subcellular redistribution of Bax is a critical event in apoptosis induced by this binary, cotransfection system. We have shown that depletion of glutathione with BSO results in marked sensitization to the cytotoxic effects of Bax in this context. It is noteworthy that several clinical trials involving BSO have been reported.35–37 Continuous infusion of BSO was shown to deplete tumor GSH with minimal toxic effects.38 Preclinical and clinical studies also indicate that BSO treatment will also reduce GSH levels in normal tissues; however, in general it is well tolerated.39 BSO in combination with alkylating agents and platinum-based therapies may be beneficial in achieving therapeutic responses in otherwise refractory tumors. Thus, it is feasible that Bax transfection, or manipulations that induce Bax activation, in combination with agents that deplete GSH, such as BSO, could be considered as a strategy for optimizing therapeutic cell death induction.

Acknowledgments

This work was supported by NIH Grants PO1 CA78778 and RO1 CA69003. Song Cho and Kevin Spurgers were supported by NIH training Grant T32 CA67759.

References 1. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–257. 2. Bruckheimer EM, Cho SH, Sarkiss M, Herrmann J, McDonnell TJ. The Bcl-2 gene family and apoptosis. Adv Biochem Eng Biotechnol. 1998;62:75–105. 3. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell. 1993;74:609–619.


4. Jurgensmeier JM, Xie Z, Deveraux Q, Ellerby L, Bredesen D, Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA. 1998;95:4997–5002. 5. Fernandes RS, Cotter TG. Apoptosis or necrosis: intracellular levels of glutathione influence mode of cell death. Biochem Pharmacol. 1994;48:675–681. 6. Ben-Yoseph O, Boxer PA, Ross BD. Assessment of the role of the glutathione and pentose phosphate pathways in the protection of primary cerebrocortical cultures from oxidative stress. J Neurochem. 1996;66:2329–2337. 7. Kagawa S, Pearson SA, Ji L, et al. A binary adenoviral vector system for expressing high levels of the proapoptotic gene bax. Gene Ther. 2000;7:75–79. 8. Stratford-Perricaudet LD, Makeh I, Perricaudet M, Briand P. Widespread long-term gene transfer to mouse skeletal muscles and heart. J Clin Invest. 1992;90:626–630. 9. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. 10. Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell. 1995;83:493–501. 11. Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214–226. 12. Coppola S, Ghibelli L. GSH extrusion and the mitochondrial pathway of apoptotic signalling. Biochem Soc Trans. 2000;28:56–61. 13. Honda T, Kagawa S, Spurgers KB, et al. A recombinant adenovirus expressing wild-type Bax induces apoptosis in prostate cancer cells independently of their Bcl-2 status and androgen sensitivity. Cancer Biol Ther. 2002;1:163–167. 14. Tsuruta Y, Mandai M, Konishi I, et al. Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines. Eur J Cancer. 2001;37:531–541. 15. Huh WK, Gomez-Navarro J, Arafat WO, et al. Bax-induced apoptosis as a novel gene therapy approach for carcinoma of the cervix. Gynecol Oncol. 2001;83:370–377. 16. Lowe SL, Rubinchik S, Honda T, McDonnell TJ, Dong JY, Norris JS. Prostate-specific expression of Bax delivered by an adenoviral vector induces apoptosis in LNCaP prostate cancer cells. Gene Ther. 2001;8:1363–1371. 17. Chen X, Carystinos GD, Batist G. Potential for selective modulation of glutathione in cancer chemotherapy. Chem Biol Interact. 1998;111–112:263–275. 18. Gross A, Jockel J, Wei MC, Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 1998;17:3878– 3885. 19. Zhang H, Heim J, Meyhack B. Redistribution of Bax from cytosol to membranes is induced by apoptotic stimuli and is an early step in the apoptotic pathway. Biochem Biophys Res Commun. 1998;251:454–459. 20. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood. 1996;88:386–401. 21. Xiang J, Chao DT, Korsmeyer SJ. BAX-induced cell death may not require interleukin 1 beta-converting enzyme-like proteases. Proc Natl Acad Sci USA. 1996;93:14559–14563. 22. Pastorino JG, Chen ST, Tafani M, Snyder JW, Farber JL. The overexpression of Bax produces cell death upon

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

induction of the mitochondrial permeability transition. J Biol Chem. 1998;273:7770–7775. Wolter KG, Hsu YT, Smith CL, Nechushtan A, Xi XG, Youle RJ. Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol. 1997;139:1281–1292. Eskes R, Antonsson B, Osen-Sand A, et al. Bax-induced cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions. J Cell Biol. 1998;143:217–224. Mistry P, Kelland LR, Abel G, Sidhar S, Harrap KR. The relationships between glutathione, glutathione-S-transferase and cytotoxicity of platinum drugs and melphalan in eight human ovarian carcinoma cell lines. Br J Cancer. 1991;64:215–220. Timmer-Bosscha H, Timmer A, Meijer C, et al. cisDiamminedichloroplatinum(ii) resistance in vitro and in vivo in human embryonal carcinoma cells. Cancer Res. 1993;53:5707–5713. Sato N, Iwata S, Nakamura K, Hori T, Mori K, Yodoi J. Thiol-mediated redox regulation of apoptosis. Possible roles of cellular thiols other than glutathione in T cell apoptosis. J Immunol. 1995;154:3194–3203. Poppenborg H, Munstermann G, Knupfer MM, Hotfilder M, Wolff JE. C6 cells cross-resistant to cisplatin and radiation. Anticancer Res. 1997;17:2073–2077. Moritaka T, Kiura K, Ueoka H, et al. Cisplatin-resistant human small cell lung cancer cell line shows collateral sensitivity to vinca alkaloids. Anticancer Res. 1998;18:927– 933. Pettus BJ, Chalfant CE, Hannun YA. Ceramide in apoptosis: an overview and current perspectives. Biochim Biophys Acta. 2002;1585:114–125. Obeid LM, Linardic CM, Karolak LA, Hannun YA. Programmed cell death induced by ceramide. Science. 1993;259:1769–1771. Bojes HK, Datta K, Xu J, et al. Bcl-xL overexpression attenuates glutathione depletion in FL5.12 cells following interleukin-3 withdrawal. Biochem J. 1997;325(Part 2):315–319. Slater AF, Stefan C, Nobel I, van den Dobbelsteen DJ, Orrenius S. Signalling mechanisms and oxidative stress in apoptosis. Toxicol Lett. 1995;82–83:149–153. Hampton MB, Fadeel B, Orrenius S. Redox regulation of the caspases during apoptosis. Ann NY Acad Sci. 1998;854:328–335. Bailey HH, Mulcahy RT, Tutsch KD, et al. Phase I clinical trial of intravenous L-buthionine sulfoximine and melphalan: an attempt at modulation of glutathione. J Clin Oncol. 1994;12:194–205. O’Dwyer PJ, Hamilton TC, LaCreta FP, et al. Phase I trial of buthionine sulfoximine in combination with melphalan in patients with cancer. J Clin Oncol. 1996;14:249–256. Bailey HH, Ripple G, Tutsch KD, et al. Phase I study of continuous-infusion L-S,R-buthionine sulfoximine with intravenous melphalan. J Natl Cancer Inst. 1997;89:1789– 1796. Bailey HH, Ripple G, Tutsch KD, et al. Phase I study of continuous-infusion L-S,R-buthionine sulfoximine with intravenous melphalan. J Natl Cancer Inst. 1997;89:1789– 1796. Bailey HH. L-S,R-buthionine sulfoximine: historical development and clinical issues. Chem Biol Interact. 1998;111– 112:239–254.

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