Epidemiological studies have suggested that the consumption of fruits and vegetables that provide several classes of compounds, including Indole-3-carbinol (I3C), may have chemopreventive activity against breast cancer. Several in vitro and in vivo animal studies also provide convincing evidence for the anti-tumor activity of I3C, however, the molecular mechanism(s) by which I3C exerts its biological effects on breast cancer cells has not been fully elucidated. In this study, we investigated the effects of I3C in Her-2/neu over-expressing MDA-MB-435 breast cancer cells and compared these results with parental cells transfected with control vector. We focused our investigation in elucidating the molecular mechanism(s) by which I3C induces apoptosis in breast cancer cells. Our data show that I3C inhibits breast cancer cell growth in a dose dependent manner in Her-2/neu over-expressing and in normal Her-2/neu expressing cells. Induction of apoptosis was also observed in these cell lines when treated with I3C, as measured by poly (ADP-ribose) polymerase (PARP) and caspase-3 activation. In addition, we found that I3C up-regulates Bax, down-regulates Bcl-2 and, thereby, increased the ratio of Bax to Bcl-2 favoring apoptosis. These results suggest that the alteration in the expression of these genes may play an important role in mediating the biological effects of I3C. Moreover, we also show the cellular localization of Bax by confocal microscopy, which showed diffuse distribution of Bax throughout the cytoplasmic compartment in breast cancer cells in control culture. However, in I3C treated cells, Bax showed a punctate pattern of distribution that was localized in the mitochondria. From these results, we conclude that the over-expression and translocation of Bax to mitochondria causes mitochondrial depolarization and activation of caspases, which may be one of the mechanism(s) by which I3C induces apoptotic processes in I3C treated breast cancer cells. Overall, our present data provide a novel molecular mechanism(s) by which I3C elicits its biological effects on both Her-2/neu over-expressing and with normal Her-2/neu expressing breast cancer cells, suggesting that I3C could be an effective agent in inducing apoptosis in breast cancer cells.
Breast cancer is the most common cancer in women in the US, and remains the second leading cause of cancer related female deaths in the USA (Flagg et al., 2000). The present options for treating breast cancer are limited to surgery, chemotherapy, and radiation therapy or combined modality therapy. In addition, newer therapy, such as Herceptin and anti-estrogen therapy, are given to a selective group of patients with Her-2/neu and estrogen receptor positive breast tumors. There is considerable dietary and epidemiological evidence, suggesting the protective effect of fruits and vegetables (which provide isoflavones, including I3C) against cancers of the liver, colon, stomach, lung and breast (Birt et al., 1996; Freudenheim et al., 1996; Shertzer, 1984; A.I.F.C.R., 1997). The dietary compound, I3C, is an autolysis product of glucosinolate, glucobrassicin, which is found in Brassica vegetables such as cabbage, broccoli, and Brussels sprouts (Loub et al., 1975; Wattenberg and Laoub, 1978).
It has been shown that I3C possesses anti-carcinogenic effects in experimental animals and inhibits the growth of human cancer cells (Baily et al., 1987; Grubbs et al., 1995; Tiwari et al., 1994). These findings lead to significant interest in the past few years in the potential utility of I3C as a chemopreventive agent (Bradfield and Bjeldanes, 1991; Broadbent and Broadbent, 1998). However, the molecular mechanism(s) of action of I3C is not fully understood. The mechanism(s) of I3C action in human breast cancer cells could potentially lead to a novel approach for its prevention and/or treatment. One of the selective therapies available for breast cancer is anti-estrogen treatment, and this treatment is only effective on tumors that rely on estrogen for their growth. Because I3C has been shown to suppress the growth of both estrogen-dependent and estrogen-independent human breast cancer cell lines (Cover et al., 1998, 1999), a combination of these two growth suppressions may potentially provide beneficial effects for breast cancer patients. These authors have also provided molecular evidence supporting the role of I3C in the induction of G1 cell cycle arrest and inhibition of cell growth. However, it is uncertain whether I3C elicits its effects only by inhibiting cell proliferation or I3C may also induces apoptotic cell death causing ultimate anti-tumor activity.
Apoptosis is one of the vital pathways through which chemopreventive agents inhibit the growth of cancer cells. Thus, it is important to investigate whether the induction of apoptosis and alterations of apoptosis related gene expression is associated with I3C effects on breast cancer cells. The induction of apoptosis is partly mediated intracellularly by several genes, such as Bcl-2 and Bax (Chiarugi et al., 1994). Bcl-2 functions as a suppressor of apoptotic death triggered by a variety of signals (Sedlak et al., 1995), whereas a predominance of Bax over Bcl-2 promotes apoptosis upon apoptotic stimuli (Salmons et al., 1997).
Bax is a 21 KDa pro-apoptotic and death-promoting member of the Bcl-2 family of proteins localized in the mitochondria, which regulate programmed cell death (Wolter et al., 1997). Bax was identified by virtue of its association with Bcl-2 (Oltvai et al., 1993). Recently, Bax was reported to be a cytosolic protein in healthy living cells and that, upon induction of apoptosis, it translocates to the mitochondria (Gross et al., 1998; Hsu and Youle, 1997; Wolter et al., 1997). This translocation process is rapid and occurs at an early stage of apoptosis (Wolter et al., 1997). It has been reported that the intracellular movement of Bax to mitochondria, depolarizes the mitochondria and induces the release of cytochrome c, causing the adaptor Apaf-1 to activate caspase 9 and 3 thereby causing ultimate cellular demise (Desagher et al., 1999; Nomura et al., 1999). Bax mediated mitochondrial depolarization and caspase activation are also regulated by the propapoptotic members of the Bcl-2 family including Bcl-XS, Bid, and Bad (Desagher et al., 1999). On the other hand, anti-apoptotic members of the Bcl-2, such as Bcl-XL and Bcl-W, reside in the outer mitochondrial membrane and antagonize proapoptotic activity (Nomura et al., 1999). Bcl-2 prevents cytochrome c release and inhibits the activation of cysteine proteases (caspases) that initiates the apoptotic processes (Kluck et al., 1997; Kroemer, 1997). In contrast to the Bcl-2 family members, insertion of Bax family members into the mitochondriaal membrane induces the release of cytochrome c and the induction of apoptotic cell death. Thus, these Bcl-2 family members can directly influence the caspase pathway, but the role of caspase for the induction of apoptosis and translocation of Bax is complex (Nomura et al., 1999). In addition, no studies have been reported to ascertain the role of Bax in mediating I3C-induced cell death in breast cancer cells. Thus, our overall goal was to determine the molecular mechanism(s) by which I3C elicits its apoptotic effects on breast cancer cells.
We investigated the effects of I3C in breast cancer cell lines, MDA-MB-435.neo and 435.eB1 (transfected Her-2/neu cDNA), and provide molecular evidence by which I3C may induce apoptotic cell death in these cells. Our data show that I3C is an effective agent in the induction of cell growth inhibition, alterations in the expressions of cell cycle and apoptosis regulatory genes and induction of apoptosis. Most importantly, I3C-induced apoptotic processes are contributed by Bax translocation to the mitochondrial membrane.
Effects of I3C on cell growth
The effect of I3C on cell growth of MDA-MB-435 and 435 transfectants are depicted in Figure 1. The treatment of MDA-MB-435.eB1 (Figure 1, upper panel) and 435.neo (Figure 1, lower panel) breast cancer cells incubated with 30, 60 and 100 μM of I3C for 1–3 days resulted in inhibition of cell proliferation, which was dose-dependent. There appears to be more pronounced growth inhibition in 435.eB1 cells, which express more c-erbB-2 than in the control 435.neo cells. It is interesting to note that the inhibition of cell proliferation was more pronounced in control 435.neo cells at day 3 at the concentration of 100 μM of I3C. The inhibition of cell proliferation, however, could be due to the induction of apoptosis, cell cycle growth arrest and/or the inhibition of growth induced by I3C as previously described (Cover et al., 1998, 1999). Since previous studies have already documented the effect of I3C in inducing G1 cell cycle arrest and inhibition of cell growth without any direct evidence of apoptosis, we focused our subsequent investigation to determine whether I3C could induce apoptosis in these cells.
Induction of apoptosis by I3C in breast cancer cells
Apoptosis was demonstrated in all the cell lines treated with 30, 60 and 100 μM of I3C as measured by Western blot analysis of PARP. PARP cleavage analysis showed that the full size PARP 116 KDa protein was cleaved to yield an 85 KDa fragment after treatment with I3C for 48–72 h (Figure 2a) in all the cell lines tested. The induction of apoptosis was more pronounced at 48–72 h of treatment, and was directly correlated with the inhibition of cell growth. Furthermore, flow cytometric analysis with 7-amino actinomycin D staining showed an increased number of apoptotic cells with 60 or 100 μM of I3C treatment (approximately 43% apoptotic cells) compared to untreated control (6% apoptotic cells) within 24 h (Figure 2c). Similar results were also obtained with other cell lines. The fluorometric assay of caspase activity clearly showed the activation of caspase 72 h with 30, 60, and 100 μM of I3C in breast cancer cells. The activity of caspase 3 at day 3 with 100 μM of I3C was highest in 435.neo cell (Figure 2b). These three independent methods of measuring apoptosis provided convincing results showing the induction of cell growth inhibition and apoptosis in all cell lines treated with I3C. These results, however, do not provide any evidence regarding the mechanism(s) by which I3C induces apoptosis. In order to explore the mechanism(s) by which I3C induces apoptosis, we investigated the alterations in the expression of genes which are known to be involved in the apoptotic processes.
Expression of Bcl-2 and Bax
The effect of I3C on Bcl-2 and Bax expression in MDA-MB-435.eB1 and 435.neo cells were studied by Western blot analysis. The levels of Bcl-2 expression in all 435 cell lines were found to be significantly down-regulated in I3C treated cells (Figure 3a). In contrast, the expression of Bax was found to be significantly up-regulated with 60 or 100 μM of I3C treatment (Figure 3b). Optical density measurement was also conducted to obtain a quantitative value for the protein expression of Bax and Bcl-2. The ratio of Bax to Bcl-2 protein expression revealed that cells treated with 100 μM I3C showed significant induction of Bax compared to Bcl-2 at 48 h treatment (Figure 3c). We were unable to find any substantial difference in Bcl-2 and Bax expression among these cell lines, suggesting that the modulation in Bax and Bcl-2 by I3C is not dependent or influenced by c-erbB-2 expression.
Mitochondrial translocation of Bax induced by I3C treatment
Treatment of cells with 30, 60 and 100 μM of I3C for 24, 48 and 72 h resulted in a cellular redistribution of Bax as shown by immunostaining. Bax had a diffuse pattern in untreated, and punctate pattern, in I3C treated 435.eB1 cells (Figure 4). In contrast 435.neo cells display more punctate distribution of Bax at 48 or 72 h after the treatment with 60 or 100 μM of I3C (data not shown). The punctate staining of Bax was found to be colocalized with mitotracker Red-CmxROS stained mitochondria (Figure 5), suggesting mitochondrial translocation of Bax following I3C treatment. The result of these staining studies suggest that the mitochondrial translocation of Bax by I3C treatment may lead to the induction of apoptotic processes and that these results are consistent with PARP degradation and caspase activation.
Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, is well known to have a chemopreventive activity against mammary carcinogenesis in human and animals (Grubbs et al., 1995; Huber et al., 1997; Kojima et al., 1994; Niwa et al., 1994). It has been reported that I3C inhibits the growth of human MCF-7 and MDA-MB231 breast cancer cells (Niwa et al., 1994; Cover et al., 1998, 1999). In our study, I3C inhibited cell growth in a time- and dose-dependent manner in MDA-MB-435.eB1 and neo cells. These cell lines showed a significant effect with 60 or 100 μM of I3C treatment within 48 to 72 h. We also observed similar effect of I3C on G1 cell cycle arrest as shown by other investigators (Cover et al., 1998, 1999). The inhibition of cell growth observed in I3C-treated cells may be due to both cell cycle arrest and induction of apoptosis. Since previous studies have already documented the effect of I3C in inducing G1 cell cycle arrest and inhibition of cell growth without any direct evidence of apoptosis, we focused our subsequent investigation to determine whether I3C could induce apoptosis in these cells. Therefore, we used several techniques to detect the processes of apoptotic cell death in this system. The cleavage of PARP has been used as an early marker of apoptosis (Darmon et al., 1995; Li et al., 1998). We found that I3C is a potent agent in inducing apoptosis in MDA-MB-435 cells. Caspase 3 activation and PARP cleavage were observed in breast cancer cells treated with I3C for 24, 48 and 72 h. The enhancement of caspase 3 activation and cleavage of PARP could be explained by the induction of apoptosis in this system. The cleavage of PARP concomitant with the induction of apoptosis was observed in all cell lines tested, suggesting that the effect of I3C is not dependent or influenced by c-erbB2 expression. Additionally, flow cytometric analysis with 7AAD staining has been conducted to detect and quantify apoptotic cells (Philpott et al., 1996). Flow cytometric studies revealed an increase in the number of apoptotic cells when treated for 24 h with I3C in all cell lines tested. Our results clearly suggest that I3C inhibits the growth of breast cancer cells and induces apoptosis and that these effects are not influenced by c-erbB-2 expression. Our results also support the findings of other studies in other cell lines (Bradlow et al., 1997; Ge et al., 1996; Katdare et al., 1998).
Bax and Bcl-2 are members of a family of intracellular proteins that are known regulators of apoptotic processes (Oltvai et al., 1993). We hypothesized that the effect of I3C on Bax and Bcl-2 may contribute to apoptotic cell death in breast cancer. It has been reported that Bax inactivates Bcl-2 proteins, which protects cells from apoptosis and that the ratio of Bax to Bcl-2 proteins increases during apoptosis (Miyashita et al., 1994; Oltvai et al., 1993; Sedlak et al., 1995). The ratio of Bax to Bcl-2 proteins is involved in the susceptibility to apoptotic stimuli in a variety of cell lines, including leukemia, breast cancer, and lung cancer (Clarke et al., 1993; Miyashita et al., 1994; Ryan et al., 1993). In our study, a decrease in Bcl-2 expression was observed in MDA-MB-435.eB1 and neo cells treated with I3C. The expression of Bax, however, was up-regulated in MDA-MB.eB1 and neo cell after I3C treatment for 24, 48 and 72 h. Hence, the ratio of Bax to Bcl-2 was altered in favor of apoptosis. The ratio of Bax to Bcl-2 was significantly higher in neo cells after 48 and 72 h of treatment with I3C, suggesting that the over-expression of c-erbB-2 may partially protect cells against I3C-induced apoptosis. Our results corroborate the conclusions made by Salmons et al. (1997), suggesting that the ratio of Bax and Bcl-2 protein levels are important for cells undergoing apoptosis. Bax forms heterodimers with Bcl-2 and antagonizes the anti-apoptotic function of Bcl-2. Our results suggest that up-regulation of Bax and down-regulation of Bcl-2 may be one of the molecular mechanism(s) by which I3C induces apoptosis. It is important to note that Chinni et al. (Cancer Research, submitted) have found a significant down-regulation of Bcl-2 after 24, 48 and 72 h exposure of prostate cancer cells to 60 and 100 μM of I3C, but two other studies could not find any changes in the levels of Bcl-2 (Byrd et al., 1998; Parker et al., 1998). Our studies, however, show some moderate down-regulation of Bcl-2, which could be due to differences in cell lines used, but the ratio of Bax to Bcl-2 clearly provide strong evidence for the induction of apoptosis. However, further studies are needed in order to establish the role of Bax and Bcl-2 in I3C induced apoptosis in this system.
Bax is a proapototic member of the Bcl-2 family, and its translocation/or movement between the cytoplasmic and mitochondrial membrane during programmed cell death is an important biochemical event. In order to investigate the role of Bax in I3C induced apoptosis, the intracellular movement and distribution of Bax was analysed by confocal microscopy. We found that I3C induces Bax translocation to the mitochondria in breast cancer cells, which may cause mitochondrial depolarization and release of cytochrome c. These alterations, in conjunction with alteration in Bid and Bad, may contribute to the activation of caspase adapter Apaf-1 and, ultimately, activate caspase 9 and 3 during I3C induced apoptosis. In addition, Bcl-XL binds and inactivates Apaf-1, whereas proapoptotic members can displace Bcl-XL from Apaf-1, allowing Apaf-1 to activate caspase 9. Thus, these Bcl-2 family members can directly influence the caspase activation which, in turn, may activate apoptotic processes through the mitochondrial death pathway. In this study, we show that 30 and 100 μM of I3C induces translocation of Bax to the mitochondria within 72 h in eB1 cells and 60 or 100 μM of I3C at 48 or 72 h in neo cells (data not shown). Therefore, I3C not only up-regulates the gene expression of different proteins, it enhances the translocation of Bax to the mitochondria. From these results, we conclude that the translocation of Bax to the mitochondria activates the mitochondrial death pathway in I3C-induced apoptosis in breast cancer cells.
In conclusion, our results demonstrate that I3C inhibits the growth of breast cancer cells, regulates the expression of apoptosis-related genes, increases the translocation of Bax to the mitochondria and induces apoptotic cell death in breast cancer cells. These effects of I3C were found to be independent of c-erbB-2 over-expression. We have recently obtained data on the differential effects of I3C in normal breast epithelial cells compared to cancer cells derived from MCF-10A, results of which will be published elsewhere. Collectively, these effects of I3C in breast cancer cells suggest a novel effect of I3C. Our results open a new avenue and challenge the current paradigm for the prevention and/or treatment of breast cancer, however, further investigation is warranted in order to prove or disprove the usefulness of I3C for breast cancer prevention/or treatment in humans.
Materials and methods
Cell lines and culture
The human breast cancer cell lines MDA-MB-435.eB1 (transfectant cells) and control 435.neo were kindly provided by Dr Dihua Yu at the University of Texas M.D. Anderson Cancer Center. The cells were cultured in DMEM/F12 medium (Life Technologies, Inc., Rockville, MD, USA) supplemented with 10% FBS, 1% penicillin/streptomycin in a 5% CO2 atmosphere at 37%. The 435.eB transfectants were generated by transfection of the pCMVerbB-2 plasmid containing the 4.4 kb full-length human normal c-erbB-2 cDNA (Her-2/neu) and the pSV2-neo plasmid carrying the neomycin-resistance selection marker gene into MDA-MB-435 cells (Tan et al., 1997; Yu et al., 1996). 435.eB1 cells express 258-fold c-erbB-2 compared to parental MDA-MB-435 cells (Tan et al., 1997; Yu et al., 1996). The control 435.neo cell line was established by transfecting the pSV2-neo plasmid alone into MDA-MB-435 cells (Tan et al., 1997; Yu et al., 1996).
Cell growth inhibition
The MDA-MB-435.eB1 and 435.neo cells were seeded at a density of 5×104/well in a 6-well culture dish. After 24 h, the cells were treated with 30, 60 or 100 μM of I3C or DMSO (vehicle control). These concentrations were chosen based on previously published reports showing the effect of I3C on breast cancer cells (Cover et al., 1998). Cells treated with I3C or DMSO for 1–3 days were harvested by trypsinization, stained with 0.4% trypan blue and counted using a hemocytometer.
Protein extraction and Western blot analysis
The breast cancer cells (MDA-MB-435.eB1 and 435.neo) were plated and cultured in complete medium and allowed to attach for 24 h followed by the addition of 30, 60, or 100 μM of I3C and incubated for 24, 48 and 72 h. Control cells were incubated in the medium with DMSO using the same time points. After incubation, the cells were harvested by scraping the cells from culture dishes using a scraper and collected by centrifugation. Cells were re-suspended in Tris-HCl buffer, sonicated for 2×10 s and lysed using an equal volume of 4% SDS. Protein concentration was then measured using protein assay reagents (Pierce, IL, USA). Cell extracts were boiled for 10 min, and chilled on ice and subjected to 10 or 12% SDS–PAGE, and electrophoretically transferred to a nitrocellulose membrane. Each membrane was incubated with monoclonal Bcl-2 (1 : 500, Oncogene, MA, USA), Bax (1 : 5000, Biomol, PA, USA), and rabbit polyclonal β-actin (1 : 5000, Sigma, MO, USA) antibodies, washed with TTBS and incubated with secondary antibody conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Pierce, IL, USA).
Autoradiograms of Western blots were scanned with Gel Doc 1000 image scanner (Bio-Rad, CA, USA) that was linked to a Macintosh computer. The bi-dimensional optical density (O.D.) of Bcl-2, Bax, and actin proteins on the films, exposed for different period of time, were quantified and analysed with Molecular Analyst software (Bio-Rad, CA, USA). The ratio of Bax/actin, Bcl-2/actin, Bax/Bcl-2 were calculated by standardizing the ratios of each control to the unit value.
Analysis of cleavage of PARP
Cells treated with 30, 60, or 100 μM of I3C or with DMSO (as control) for 24, 48 and 72 h were lysed in lysis buffer (10 mM Tris-HCl [pH 7.1], 50 mM sodium chloride, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 μM sodium orthovandate, 2 mM iodoacetic acid, 5 μM ZnCl2, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Triton X-100). The lysates were kept on ice for 30 min and vigorously vortexed before centrifugation at 12 500 g for 20 min. Fifty μg of total proteins were resolved on 10% or 12% SDS–PAGE and then transferred to nitrocellulose membrane. The membrane was incubated with primary monoclonal anti-human PARP antibody (1 : 5000, Biomol, PA, USA), washed with TTBS and incubated with secondary antibody conjugated with peroxidase. The signal was then detected using the chemiluminescent detection system (Pierce, IL, USA).
Flow cytometry for detecting apoptosis
7-amino actinomycin D (7AAD) staining and flow cytometry were conducted to detect and quantify apoptosis (Nicholson et al., 1995). Cells treated with 60 or 100 μM I3C for 24, 48, 72 h or with DMSO for 72 h (as control) were subjected to this analysis. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a positive control for adjusting flow cytometric parameters. It is an effective uncoupler of oxidative phosphorylation in mitochondrial systems. Briefly, 7AAD (Calbiochem-Novabiochem, La Jolla, CA, USA) was dissolved in acetone and diluted in PBS to a concentration of 200 μg/ml. A total of 100 μl of 7AAD solution was added to 106 cells suspended in 1 ml of PBS and mixed well. Cells were stained for 20 min at 4°C while protected from light and pelleted by centrifugation. The cells were re-suspended in 500 μl of 1% PBS-BSA solution. Unstained fixed cells were used as negative control. Samples were analysed on a FACscan (Becton Dickinson, CA, USA) within 30 min of fixation. Data on 10 000 and 20 000 cells were acquired and processed using Lysys II software (Becton Dickinson, CA, USA). Scattergrams were generated by combining forward light scatter with 7AAD fluorescence, and regions were drawn around clear-cut populations having negative, dim, and bright fluorescence. The frequency of cells with low, medium, and high 7AAD fluorescence was assessed. The purity and enrichment of the sorted populations were then calculated.
Caspase activation assay
For caspase assays, cells were seeded at a density of 3×105 in 60×15-mm culture dishes. After 24 h, the cells were treated with 30, 60 or 100 μM of I3C or DMSO (vehicle control) for 72 h. To prepare cell extracts, cells were initially washed twice in cold PBS. Cells were thereafter treated with ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Nonindent P-40, 1 mM phenylmethylsulfonyl floride and protease inhibitor cocktail tablets, 1 tab/50 ml (Boehringer Mannheim). After two cycles of freezing and thawing, supernatants were collected after centrifugation at 2000 g for 20 min and protein concentrations were determined using protein assay reagents (Pierce, IL USA). The activation of caspases was measured essentially as described by Nicholson et al. (1995). To evaluate relative DEVDase (caspase 3-like activity), cell lysates prepared as described above were incubated with specific AMC conjugated substrate. Briefly, lysates containing 200 μg of protein were incubated with reaction buffer (20 mM Tris-HCL pH 7.5, 37 mM NaCl, 1% NP-40, 10% glycerol) containing 2 μg of specific caspase substrates Ac-DEVD-AMC (Pharmingen). The reaction mixture was incubated at 37°C for 1.5 h in the presence or absence of 50 nmol/l of specific inhibitor Ac-DEVD-CHO (Pharmingen) and caspases activity was measured in a Labsysytems Fluoroscan 2 Fluorimeter.
Flourescence staining procedure
Cells (5×104) were plated on cover slips in each well of a 6-well plate. The cells were treated with 30, 60 or 100 μM of I3C for 24, 48 and 72 h. For STS (staurosporine) treatment, the medium was supplemented with 1–3 μM staurosporine and the cells were incubated for 24 h. Cells were stained with Mitotracker Red-CmxRos (Molecular Probes, Eugene, OR, USA) using 50 ng/ml for 15 min at 37°C prior to fixation. Cells were fixed in ice cold 10% methanol for 10 min and left at 4°C until the day of staining. Cover slips were subsequently washed in PBS three times and non-specific binding sites were blocked with 0.2% BSA in PBS for 45 min. Then, PBS-BSA solution was carefully aspirated to avoid detaching the cells. Cells were incubated in PBS-0.1% saponin solution containing 1 μg/ml of Bax antibody (Trevigen, Gaithersburg, MD, USA) for 2 h. Cells were washed with PBS-0.1% saponin solution three times to remove excess unbound primary antibody. Cells were incubated with 1 μg/ml of secondary fluorescent goat anti-mouse antibody (Alexa Fluor 488, Molecular Probes, Eugene, OR, USA) in PBS-0.1% saponin with 5% serum for 1 h and washed thereafter with PBS-0.1% saponin. They were next fixed with 10% methanol and washed three times with PBS before being mounted in antifade solution (Molecular Probes, Eugene, OR, USA) on slides and the fluorescence was observed by confocal microscopy as described below.
Fluorescence images were obtained with Zeiss Laser Scanning inverted confocal microscope system 310 with a 63×/1.2 oil immersion objective. Excitation wave length/detection filter settings were as follows: Mitotracker Red-CmxRos, 585/665 nm longpass and Alexa flour 488, 495/519 nm for Bax visualization. Laser time and irradiation time was minimized to avoid photobleaching and possible photodynamic effects (Salet and Moreno, 1995). Cells were visualized in dual channel imaging where Mitotracker Red and Bax were used to compensate for effects of one channel on another.
We sincerely thank Ms Patricia Arlauskas for her editorial assistance. This project was partly funded by a pilot grant from NIEHS center grant (ES 06639) at Wayne State University, and the George Puschelberg Foundation awarded to FH Sarkar. We also thank Dr Kamiar Moin and Linda Mayernik for their invaluable assistance with the confocal microscopic imaging. Center Grants P30ES06639 from the National Institutes of Environmental Health Sciences. We would like to thank Erie Van Buren, Evano Piasentin and Dr Stephen Lerman for their assistance in the flow cytometry core facility, supported by the grant. P30CA22453 from the National Cancer Institute which supports this core facility at Wayne State University School of Medicine.