Introduction

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In the case of most chemotherapeutic agents, overexpression of bcl-2 leads to abrogation of apoptosis induction (Kamesaki et al., 1993; Miyashita and Reed, 1993; Dole et al., 1994; Teixeira et al., 1995). However, in the case of treatment of PC12 pheochromocytoma cells with reduction-dependent chemotherapeutic prodrugs, overexpression of bcl-2 potentiates apoptosis induction (Cortazzo and Schor, 1996; Tyurina et al., 1997; Schor et al., 1999a,b). It was originally hypothesized that this potentiation results from a bcl-2-induced shift in the redox potential of the cell with increased functional thiol reserves and consequent potentiation of activation of these reduction-dependent prodrugs (Cortazzo and Schor, 1996; Schor et al., 1999a). However, if overexpression of bcl-2 only increased the activation of the apoptosis-inciting drug, its protein product, Bcl-2, would be expected nonetheless to block apoptosis downstream of the action of this drug, presumably at the level of inhibition of cytochrome c release from mitochondria (Kluck et al., 1997; Yang et al., 1997). That this is not the case is demonstrated by potentiation by Bcl-2 of reduction-dependent chemotherapeutic agent-induced oxidation and externalization of membrane phosphatidylserine (Schor et al., 1999a), a late event in the apoptosis final common pathway. Furthermore, Bcl-2 prevents, rather than potentiates, apoptosis induced in MCF-7 breast cancer cells by reduction-dependent prodrugs (Schor et al., 2000). Clearly, the distal antiapoptotic effects of Bcl-2 have been thwarted in the PC12/reduction-activated prodrug system; indeed, a proapoptotic effect seems likely.

Bcl-2 is one of a family of proteins, some of which, unlike Bcl-2, are proapoptotic (for reviews, see Reed, 1994; Brady and Gil-Gomez, 1998). Previous studies have demonstrated that Bcl-2 itself can be cleaved by caspase 3 to a proapoptotic Bcl-2 fragment (Cheng et al., 1997). We have therefore examined the role of cleavage of Bcl-2 in Bcl-2-mediated potentiation of apoptosis.

	Materials and Methods

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Cells and Cell Culture. Polyclonal mock-transfected (pBabe-puro) PC12 rat pheochromocytoma cells and PC12 cells transfected with the human bcl-2 gene ligated into the retroviral vector pBabe, containing a puromycin resistance gene (bcl-2-pBabe-puro), were provided by Dale E. Bredesen (La Jolla Cancer Institute, La Jolla, CA). The ratio of Bcl-2 content of the bcl-2 transfectants relative to mock-transfected PC12 cells is 100:1 (Kane et al., 1993). Cells were maintained as adherent monolayers in Dulbecco's modified Eagle's medium made 10% in horse serum, 5% in fetal bovine serum (Atlanta Biologicals, Norcross, GA), and 1.1% in penicillin/streptomycin (Invitrogen, Carlsbad, CA). Cells were fed every 3 to 4 days; biweekly, 1 µg/ml puromycin was added to the medium. Cells were examined for Bcl-2 expression by Western blotting every 10 passages. Bcl-2 expression did not vary in either cell line over the course of these studies.

Neocarzinostatin and Cisplatin Treatment. Mock- and bcl-2-transfected PC12 and MCF-7 cells were treated with either 0.02 µM neocarzinostatin (NCS) or 10 µM cisplatin for 1 h. Subsequently, the NCS or cisplatin was washed out and fresh medium was added. Cells were then incubated for varying lengths of time as indicated. For studies of the effects of the caspase 3 inhibitor Ac-DEVD-CHO on mock- and bcl-2-transfected PC12 and MCF-7 cells, 10 µM Ac-DEVD-CHO (BD PharMingen International, San Diego, CA) was added to the cells 2 h before NCS or cisplatin treatment, and maintained in the medium thereafter.

Western Blotting Analysis of Bcl-2 and Caspase 3 proteins. At the indicated time points, PC12 and MCF-7 cells were lysed in radioimmunoprecipitation assay buffer (10 mM Tris, pH 8, 150 mM NaCl, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 4 µg/ml aprotinin, and 1 mM sodium orthovanadate). Subsequently, the protein concentrations of the lysates were estimated using the Bio-Rad protein assay (Bio-Rad, Richmond, CA) with bovine serum albumin as a standard. An aliquot of each lysate containing 500 µg of protein was loaded onto each lane and electrophoresed on a 15% SDS-polyacrylamide gel, followed by blotting on a nitrocellulose membrane (Bio-Rad). After blotting, nonspecific binding was blocked with 5% nonfat dry milk in PBS and the membrane was incubated with either anti-Bcl-2 (1:500, reactive with mouse, rat, and human Bcl-2; Santa Cruz Biotechnology) or anti-caspase 3 (1:1000; BD PharMingen) antibodies diluted in 5% nonfat dry milk in PBS at 20°C for 2 h, washed, and incubated with secondary horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Santa Cruz Biotechnology) for 1 h. The membrane was finally washed and developed with Western blotting chemiluminescence luminol reagent (Santa Cruz Biotechnology) following the manufacturer's instructions. The known molecular masses of caspase 3 and Bcl-2 and its cleavage products were confirmed by coelectrophoresis of prestained molecular mass standards (Bio-Rad) on each gel.

Determination of Adherent Cell Number. Adherent cell number was determined in control and treated cultures, as we have described previously for neuroblastoma cells (Schor, 1992; Hartsell et al., 1995, 1996; Cortazzo and Schor, 1996). Briefly, adherent cells were manually counted in each of three high-power fields from each cell culture well at each time point. Results are expressed as the mean ± S.E.M. of the three determinations. In the case of Ac-DEVD-CHO caspase 3 inhibitor treatment, 10 µM inhibitor was added 2 h before NCS treatment. The statistical significance of differences between control and treated cultures was assessed in all cases using Student's t test; P  0.05 was considered significant.

DNA Staining with Propidium Iodide. Monolayers of mock- and bcl-2-transfected PC12 cells grown on coverslips were treated with either 20 nM NCS or 10 µM cisplatin for 1 h. The NCS or cisplatin was then washed away and fresh medium was added to the cells. Twenty-four hours later, the cells were washed with PBS and fixed with cold ethanol (95%) for 5 min. After washing with PBS, the cells were treated with RNase (1 mg/ml) for 1 h at 37°C. The cells were then stained with PI (20 ng/ml) for 5 min and the coverslips were mounted on slides with Gelvatol, a solution of polyvinyl alcohol (23%; Sigma Chemical, St. Louis, MO) and glycerol [50% (v/v)] in phosphate-buffered saline. A Zeiss light microscope equipped for epifluorescent illumination was used for all observations.

Flow Cytometric Analysis of Control and NCS-Treated Cells. Apoptotic cells were quantified by flow cytometry considering 7-amino-actinomycin D (7-AAD) staining intensity to be proportional to the DNA content (Lecoeur and Gougeon, 1996). Saponin and 7-AAD were purchased from Sigma Chemical. In short, after harvesting, the cells were washed once in PBS and once in PBS/0.05% saponin, followed by addition of 4 µg of 7-AAD in 1 ml of PBS/saponin to the samples. The cells were incubated at room temperature in the dark for 30 min, and DNA histograms were obtained using a CellQuest apparatus and CellQuest software (BD Biosciences, San Jose, CA). Data on 10⁴ cells were collected. Electronic gates were set for viable and apoptotic cells with 2N-4N DNA and subnormal DNA contents, respectively, and for exclusion of debris. Percentage of apoptosis was calculated as (number of apoptotic cells/number of total cells) × 100.

Caspase 3 Colorimetric Protease Assay. At different time points, both mock- and bcl-2-transfected PC12 cells were harvested and assayed for caspase 3 activity according to the manufacturer's instructions (Medical and Biological Laboratory Co. Ltd., Naka-ku, Japan). Briefly, 50 µg of protein in cell lysate was used for the analysis. The volume of cell lysate was adjusted to 50 µl with lysis buffer, and 50 µl of 2× reaction buffer was added to each sample. Five microliters of 4 mM DEVD-pNA substrate was added, and the samples were incubated at 37°C for 1 h. The A_{405 nm} was read in a microtiter plate reader. Fold-increase in caspase 3 activity over control samples was determined by comparing these results with the A_{405 nm} of a simultaneously incubated control (i.e., vehicle- rather than NCS-treated) sample. The background reading from cell lysates and buffers was subtracted from the readings of both NCS-treated and control samples before calculating increase in caspase 3 activity.

In Vivo Studies of Effects of NCS, Cisplatin, or Vincristine on Tumor Growth from Mock- and bcl-2-Transfected PC12 Cells. Experiments involving tumor growth were performed on 5- to 7-week-old male NIH athymic mice injected subcutaneously with 10⁶ mock- or bcl-2-transfected PC12 cells into the left flank on day 0 of each study. NCS (0-5 mg/kg), cisplatin (0-25 mg/kg), or vincristine (0-1.25 mg/kg) was administered intraperitoneally on day 1 (n = 4 mice/dose). Mice were examined daily for grossly visible tumor and, once tumors appeared, they were assessed by measurement of the largest and smallest diameter of each tumor at multiple time points during the month after implantation. Tumor volume was calculated as the product of the largest diameter and the square of the smallest diameter. Mean tumor volume for the four mice in each group was calculated for each day of measurement assuming a tumor volume of zero for those mice in which a tumor was not palpable. Statistical significance of differences in mean tumor volume was determined for each day's measurements with Student's t test.

	Results

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NCS and Cisplatin Induce Apoptosis in PC12 and MCF-7 Cells. Our previous studies have demonstrated condensation, fragmentation, and margination of the nuclei (Cortazzo and Schor, 1996) and selective oxidation and externalization of membrane phosphatidylserine (Schor et al., 1999a) of mock- and bcl-2-transfected PC12 cells treated with NCS. We have further demonstrated a decrement in cell culture size in mock- and bcl-2-transfected MCF-7 cells treated with NCS (Schor et al., 2000). Sister cultures of mock- and bcl-2-transfected PC12 cells treated with NCS (0.020 µM) or cisplatin (10 µM) demonstrate qualitatively similar morphological and nuclear fragmentation features (Fig. 1, A-F). In both PC12 cells and MCF-7 cells that are either mock- or bcl-2-transfected and treated with NCS, FACS analysis shows a DNA size distribution consistent with apoptosis [Figs. 1, H, K, and M; 2, a to d; and 6D (vehicle versus NCS alone, PC12, and MCF-7 cells, respectively)].

View larger version (119K): [in this window] [in a new window]   Fig. 1.   Top, NCS and cisplatin induce apoptosis in both mock- and bcl-2-transfected PC12 cells. Both mock- and bcl-2-transfected PC12 cells were treated with either 0.02 µM NCS or 10 µM cisplatin for 1 h then washed and fed with fresh medium. After 24 h of incubation, the cells were fixed and stained with PI. A, control mock-transfected PC12 cells. B, NCS-treated mock-transfected PC12 cells. C, cisplatin-treated mock-transfected PC12 cells. D, control bcl-2-transfected PC12 cells. E, NCS-treated bcl-2-transfected PC12 cells. F, cisplatin-treated bcl-2-transfected PC12 cells. Arrows indicate apoptotic cells with condensed and fragmented nuclei. Magnification, 400×. Bottom left, flow cytometric analysis of NCS- and cisplatin-treated PC12 cells. Both mock- and bcl-2-transfected PC12 cells were treated with either 0.02 µM NCS or 10 µM cisplatin for 1 h then washed and fed with fresh medium. After 24 h of incubation, the cells were harvested and stained with 7-AAD (see Materials and Methods) and then analyzed by FACS. G, Control mock-transfected PC12 cells. H, NCS-treated mock-transfected PC12 cells. I, cisplatin-treated mock-transfected PC12 cells. J, control bcl-2-transfected PC12 cells. K, NCS-treated bcl-2-transfected PC12 cells. L, cisplatin-treated bcl-2-transfected PC12 cells. Bcl-2 potentiates NCS-induced apoptosis and attenuates cisplatin-induced apoptosis. Bottom right (M), Flow cytometric analysis of NCS- and cisplatin-treated PC12 cells. The mean ± S.D. for three independent experiments performed as described in G to L, above, is shown.

Although Bcl-2 Increases Sensitivity to NCS of PC12 Cells, It Decreases Sensitivity to NCS of MCF-7 Cells. Bcl-2 also Decreases Sensitivity to Cisplatin of PC12 Cells. Previous studies from our laboratory have demonstrated Bcl-2-mediated potentiation of apoptosis and decrease in cell culture growth in PC12 cells (Cortazzo and Schor, 1996; Schor et al., 1999a) and Bcl-2-mediated protection from decrease in cell culture growth in MCF-7 cells (Schor et al., 2000). These findings resulted in a downward shift of the EC₅₀ of NCS in PC12 cells and an upward shift of the EC₅₀ of NCS in MCF-7 cells transfected with bcl-2 relative to mock-transfected cells. Figures 1 and 2 demonstrate the effect of bcl-2 transfection of PC12 and MCF-7 cells, respectively, on apoptosis induction by NCS as determined by FACS analysis 24 h after NCS treatment. An increase in the prevalence of apoptosis is produced by bcl-2 transfection of PC12 cells. In contrast, Bcl-2 renders MCF-7 cells less sensitive to apoptosis induction by NCS. Furthermore, consistent with our previously reported studies (Cortazzo and Schor, 1996), unlike the case for NCS, Bcl-2 decreased the incidence of apoptosis in PC12 cells treated with cisplatin [Fig. 1, I, L, and M].

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Transfection with bcl-2 leads to decreased sensitivity of MCF-7 cells to NCS. In all cases, cells were treated for 1 h with NCS (0.02 µM; 37°C) and FACS analysis was performed 24 h later as described under Materials and Methods. A, control neo.1 MCF-7 cells. B, control neo.2 MCF-7 cells. C, control bcl-2.1 MCF-7 cells. D, control bcl-2.3 MCF-7 cells. a, NCS-treated neo.1 MCF-7 cells. b, NCS-treated neo.2 MCF-7 cells. c, NCS-treated bcl-2.1 MCF-7 cells. d, NCS-treated bcl-2.3 MCF-7 cells. Transfection and selection of these clonal cell lines are described under Materials and Methods. A representative set of FACS results is shown here. Figure 6D (vehicle versus NCS alone) shows the mean ± S.D. for three independent such experiments.

NCS Induces Bcl-2 Cleavage in bcl-2-Transfected PC12 Cells. Previous reports have demonstrated cleavage of Bcl-2 to its proapoptotic counterpart in genetically engineered cell systems (Cheng et al., 1997). To begin to examine the possibility that cleavage of the Bcl-2 protein plays a role in the potentiation of NCS treatment-induced apoptosis, we looked for cleavage of Bcl-2 in PC12 cells treated with NCS. For this study, bcl-2- and mock-transfected PC12 cells were subjected to NCS treatment and the cell lysate was harvested and analyzed by Western blotting. As shown in Fig. 3A, 4 h after NCS treatment, Bcl-2 was cleaved to a 23-kDa protein in the bcl-2-transfected cells. It is of particular note that cleavage of Bcl-2 is observed 12 to 20 h before the cells demonstrate light microscopic evidence of apoptosis (data not shown). The cleavage product was detected at 24 but not at 48 h after a 1-h treatment with NCS.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   A, NCS induces Bcl-2 cleavage in bcl-2-transfected PC12 cells. Mock- and bcl-2-transfected PC12 cells (polyclonal) were treated with NCS (0.02 µM; 1 h). At various time points after completion of NCS treatment, the cells were harvested and lysed for Western Blot analysis. Five hundred micrograms of protein was loaded per lane. Results of staining with an antibody for Bcl-2 (Santa Cruz Biotechnology) are shown. Mock-transfected PC12 cells, leftmost five lanes; bcl-2-transfected PC12 cells, rightmost five lanes. Note that mock-transfected PC12 cells contain 100-fold less Bcl-2 than bcl-2-transfected PC12 cells (Kane et al., 1993). The known molecular masses of the Bcl-2 band and that of its cleavage product (N34) were confirmed using simultaneously electrophoresed prestained standards (Bio-Rad). B, NCS did not induce Bcl-2 cleavage in bcl-2-transfected MCF-7 cells. Mock- and bcl-2 transfected MCF-7 cells (two independent clones each) were treated with NCS (0.02 µM; 1 h). At various time points after completion of NCS treatment, the cells were harvested and lysed for Western blot analysis. Five hundred micrograms of protein was loaded per lane. Results of staining with an antibody for Bcl-2 (Santa Cruz Biotechnology) are shown. Top, neo.1 mock-transfected cells, leftmost five lanes; bcl-2.1 bcl-2-transfected cells, rightmost five lanes. Bottom, neo.2 mock-transfected cells, leftmost five lanes; bcl-2.3 bcl-2-transfected cells, rightmost five lanes. The molecular mass of the band detected (26 kDa) was confirmed as described in the legend for A. C, Cisplatin did not induce Bcl-2 cleavage in bcl-2-transfected PC12 cells. Mock and bcl-2-transfected PC12 cells were treated with 10 µM cisplatin for 1 h. At various time points, cells were harvested for Western blot analysis for Bcl-2. Five hundred micrograms of protein was loaded per lane. Mock-transfected PC12 cells, leftmost five lanes; bcl-2-transfected PC12 cells, rightmost five lanes. The molecular mass of the band detected (26 kDa) was confirmed as described in the legend for A.

NCS Did Not Induce Bcl-2 Cleavage in bcl-2-Transfected MCF-7 Cells. To determine whether Bcl-2 cleavage after NCS treatment cosegregated only with potentiation of (as opposed to protection from) apoptosis, we examined the effects of NCS treatment on Bcl-2 in MCF-7 cells. Our previous studies demonstrated a Bcl-2-mediated shift in the NCS concentration-cell culture growth curve of MCF-7 cells (Schor et al., 2000), and Bcl-2 seemed to protect MCF-7 cells from apoptosis [Figs. 2 and 6D (vehicle versus NCS alone)]. After NCS treatment, Bcl-2 was not cleaved in either bcl-2- or mock-transfected MCF-7 cells (Fig. 3B).

Cisplatin Did Not Induce Bcl-2 Cleavage in Mock- or bcl-2-Transfected PC12 Cells. Unlike the case for NCS, bcl-2 transfection protects PC12 cells from cisplatin-induced apoptosis (Fig. 1M; Cortazzo and Schor, 1996). We therefore determined whether cisplatin induced Bcl-2 cleavage in bcl-2-transfected PC12 cells. As is shown in Fig. 3C, there was no Bcl-2 cleavage in mock- or bcl-2-transfected PC12 cells after cisplatin treatment. Again, at time points when apoptotic morphology and FACS DNA distribution were evident (Fig. 1), no cleavage of Bcl-2 could be detected.

MCF-7 Cells Do Not Express Caspase 3. Caspase 3 has been shown to cleave Bcl-2 to proapoptotic peptides in other systems (Cheng et al., 1997). In an effort to link the cleavage of Bcl-2 in PC12 cells and the lack thereof in MCF-7 cells, we measured caspase 3 expression in each of our transfectants of PC12 and MCF-7 cells. As shown in Fig. 4, caspase 3 is expressed in bcl-2- and mock-transfected PC12 cells, but not in either bcl-2- or mock-transfected MCF-7 cells. Interestingly, caspase 3 is clearly not required for apoptosis induction [Figs. 2 and 6D (vehicle versus NCS alone)] in the latter four transfected lines.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Caspase 3 expression in PC12 and MCF-7 cells. Five hundred micrograms of protein was loaded per lane. The lysate of the A431 cell line represents a known positive control. No caspase 3 was detected in any of the four different MCF-7 cell transfectants (neo.1, neo.2, bcl-2.1, bcl-2.3). In contrast, the two PC12 transfectants express caspase 3 in comparable amounts. The electrophoretic position of a simultaneously run 33-kDa molecular mass standard is indicated to the right of the blot photograph.

Caspase 3 Activity in Mock- and bcl-2-Transfected PC12 Cells after NCS Treatment. To examine caspase 3 activation over time we used the chromogenic caspase 3 substrate DEVD-pNA. As shown in Fig. 5, caspase 3 activity was elevated 30 min after NCS treatment in both mock- (Fig. 5A) and bcl-2-transfected PC12 (Fig. 5B) cells. The activity decreased to near baseline by 24 h after completion of NCS treatment.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Caspase 3 is activated in both mock- (A) and bcl-2- (B) transfected PC12 cells after NCS treatment (0.02 µM; 1 h). At the indicated time points, both mock- and bcl-2-transfected PC12 cells were harvested and assayed for caspase 3 activity by using the chromogenic substrate DEVD-pNA, according to the manufacturer's instructions (Medical and Biological Laboratory Co. Ltd.). The background reading from cell lysates and buffers was subtracted from the readings of both NCS-treated and control samples before calculating fold increase in caspase 3 activity. In both transfectants, caspase 3 activity was elevated 30 min after completion of NCS treatment, and slowly decreased to near baseline by 24 h later.

Caspase 3 Inhibitor Ac-DEVD-CHO Attenuates Sensitivity to NCS of bcl-2-Transfected PC12 cells, but Has No Effect on That of bcl-2-Transfected MCF-7 Cells. To further demonstrate the role of caspase 3 in the cleavage of Bcl-2 after NCS treatment, we pretreated bcl-2- and mock-transfected PC12 cells with the caspase 3 inhibitor Ac-DEVD-CHO (10 µM) for 2 h before NCS treatment (0.02 µM; 1 h). As shown in Fig. 6B, pretreatment with Ac-DEVD-CHO results in attenuation of the sensitivity to NCS treatment of bcl-2-transfected PC12 cells; in contrast, Ac-DEVD-CHO did not alter the sensitivity to NCS of mock-transfected PC12 cells (Fig. 6A). Note that day 2 cell counts obtained at an NCS concentration of zero did not differ significantly between control and Ac-DEVD-CHO-treated cells (P > 0.05; Student's t test). Furthermore, 2 h of pretreatment of bcl-2-transfected PC12 cells with Ac-DEVD-CHO abolished NCS-induced cleavage of Bcl-2 (Fig. 6C). The sister culture control gel (i.e., cells treated with NCS in the absence of Ac-DEVD-CHO) is shown in Fig. 3A.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Caspase 3 inhibitor Ac-DEVD-CHO increased the day 2 cell culture size after NCS treatment of bcl-2-transfected, but not mock-transfected PC12 cells. Both mock- and bcl-2-transfected PC12 cells were incubated with 10 µM Ac-DEVD-CHO for 2 h before NCS treatment (0.02 µM; 1 h). The cells were then washed and incubated with fresh medium on day 0. Cells were counted daily as we have described previously (Falcione et al., 1993; Cortazzo and Schor, 1996), and the day 2 cell counts are plotted as a reflection of cells remaining after completion of apoptosis evident by fluorescence microscopy and FACS analysis 24 h after completion of NCS treatment (day 1; Fig. 1). Plotted cell counts represent the mean counts for three high-power fields, and are expressed as a percentage of the count on day 0 ± S.E.M. Results are shown from a single representative experiment of three performed. A, mock-transfected PC12 cells. B, bcl-2-transfected PC12 cells. C, Western blot analysis of Bcl-2 cleavage in Ac-DEVD-CHO pretreated mock- and bcl-2-transfected PC12 cells. Five hundred micrograms of protein was loaded per lane. The sister culture control gel (i.e., cells treated with NCS in the absence of Ac-DEVD-CHO) is shown in Fig. 3A. D, FACS analysis performed 24 h after completion of NCS treatment of mock (two neo clones) and bcl-2 (two bcl-2 clones) transfectants of MCF-7 cells treated with NCS (0.02 µM) ± Ac-DEVD-CHO (10 or 100 µM) as described under Materials and Methods. In all cases, although NCS induces apoptosis and bcl-2 transfection significantly diminishes MCF-7 cell sensitivity to NCS-induced apoptosis (P < 0.05 for all transfectants, vehicle compared with NCS alone; p < 0.05, NCS-treated condition for each mock transfectant compared with each bcl-2 transfectant; Student's t test), Ac-DEVD-CHO has no significant effect on apoptosis induction by NCS in these cell lines.

Unlike the Case for Cisplatin Treatment, Xenografts of bcl-2-Transfected PC12 Cells Are More Susceptible to NCS Toxicity Than Are Xenografts of Mock-Transfected PC12 Cells. Nude mice (n = 4 for each transfectant at each NCS dose) were administered subcutaneous injections of mock- or bcl-2-transfected PC12 cells as described under Materials and Methods. Subcutaneous implants of bcl-2-transfected PC12 cells more readily generate gross tumors than their mock-transfected counterparts. That is, the tumors that form from them are palpable earlier and ultimately larger than those formed from implanted mock-transfected PC12 cells. Nonetheless, as is the case for in vitro treatment of bcl-2- and mock-transfected PC12 cells, implants from bcl-2-transfected cells are more susceptible to NCS (0-5 mg/kg)-induced inhibition of tumor growth rate than are implants from mock-transfected cells (Fig. 7; Table 1; tumor volume at day 32 differs as follows by Student's t test: mock-transfected cells, 3 mg/kg compared with 5 mg/kg, p < 0.025; bcl-2-transfected cells, saline compared with 1 mg/kg, P < 0.05).

View larger version (29K): [in this window] [in a new window]   Fig. 7.   NCS treatment is more effective against tumors derived from bcl-2-transfected cells than against those derived from mock-transfected cells. PC12 cells were xenografted into nude mice (n = 4/group) by subcutaneous injection of 106 cells on day 1. Saline (vehicle control), NCS, or cisplatin was administered as a single intraperitoneal dose on day 0. Tumor volume was computed at multiple time points after xenograft implantation as the product of the length (longest dimension) and square of the width (smallest dimension) of each tumor that developed. Mice in which tumors did not develop were assigned a tumor volume of zero for that day. For values for mock transfectants treated with NCS, 3 mg/kg differs from 5 mg/kg at the P < 0.025 level (Student's t test). For values for bcl-2 transfectants treated with NCS, saline differs from 1 mg/kg at the P < 0.05 level. For both mock and bcl-2 transfectants treated with cisplatin, 5 mg/kg values differ from 10 mg/kg values at the P = 0.05 level.

	Discussion

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Our previous studies have demonstrated that bcl-2 overexpression potentiates the enediyne-induced decrease in cell number in some cell lines and prevents it in others. We now show that potentiation of enediyne sensitivity of bcl-2-transfected PC12 cells is associated with enhanced incidence of apoptosis and cleavage of Bcl-2 protein. Conversely, decreased sensitivity to enediynes of bcl-2-transfected MCF-7 cells and decreased sensitivity to cisplatin of bcl-2-transfected PC12 cells are associated with protection from apoptosis and the absence of Bcl-2 cleavage. Although Bcl-2 itself is an antiapoptotic protein, it has recently been reported that the cleavage product of this protein is proapoptotic (Cheng et al., 1997; Kirsch et al., 1999). Bcl-2 cleavage has been reported to occur after Asp-34, and to result in production of a proapoptotic cleavage product lacking the N-terminal 34 amino acids of Bcl-2 (Cheng et al., 1997). This is consistent with the 23-kDa cleavage product that was detected in response to NCS treatment. Cleavage of Bcl-2 was not detected in cisplatin-treated, bcl-2-transfected PC12 cells or NCS-treated, bcl-2-transfected human MCF-7 cells.

That this Bcl-2 cleavage requires the activity of caspase 3 is suggested by two lines of evidence. NCS treatment of MCF-7 cells, which have been described previously and demonstrated herein not to express caspase 3, does not result in cleavage of Bcl-2. [The absence of caspase 3 expression in MCF-7 cells is the result of a 47-base pair deletion within exon 3 of the caspase 3 gene (Janicke et al., 1998).] Furthermore, the caspase 3-specific inhibitor Ac-DEVD-CHO blocks both the cleavage of Bcl-2, resulting from NCS treatment and the potentiation of NCS-induced apoptosis seen with bcl-2 overexpression in PC12 cells, but has no effect on MCF-7 cells. This is consistent with previous reports of the abrogation of cleavage of Bcl-2 when caspase 3 was immunodepleted from extracts of 293 cells. Conversely, immunodepletion of caspase 7 did not affect Bcl-2 cleavage in the 293 cell system, and transfection of caspase 3-deficient MCF-7 cells with the gene for caspase 3 resulted in cleavage of Bcl-2 (Kirsch et al., 1999). That caspase 7 seems not to cleave Bcl-2 is particularly interesting in light of the related structures and substrate specificities of caspases 3 and 7 and the differential substrate selectivities of the two, hypothesized to be the result of significant differences in sequence around their S4-binding sites (Sgorbissa et al., 1999; Wei et al., 2000). It is clear that caspase 3 plays a critical role in the cleavage of Bcl-2 in bcl-2-transfected PC12 cells. Although this role is likely to be a direct one, it is also possible that caspase 3 activates a downstream protease that, in turn, directly cleaves Bcl-2. The disappearance of the Bcl-2 cleavage product by 48 h after NCS treatment may represent degradation of this fragment with or without new Bcl-2 synthesis or may reflect a selection phenomenon (i.e., that those cells still alive at 48 h are those that sustained little enough Bcl-2 cleavage that they did not undergo apoptosis). Either scenario is consistent with completion of the effects of a single 1-h exposure to NCS by 48 h after treatment.

Our previous studies (Cortazzo and Schor, 1996; Schor et al., 2000) demonstrated the role of altered glutathione handling and antioxidant potential in mediating the potentiation of sensitivity to NCS by Bcl-2. These previous studies arose from the need for reductive activation of NCS and most other enediynes for their biological effects (DeGraff and Mitchell, 1985). Interestingly, caspase 3 activation depends critically on redox state. Its active site cysteine residue must be reduced for caspase activity, and the caspase 3-activating activity of the protein thioredoxin is proportional to the number of reduced cysteine residues in the thioredoxin (Baker et al., 2000). Increased reducing potential via bcl-2 overexpression may therefore provide the impetus for increased activation of both NCS and caspase 3.

It is interesting that, despite their lack of caspase 3 expression, MCF-7 cells still undergo apoptosis induced by a host of exogenous stimuli, including vitamin D (Mathiasen et al., Bcl-2 inhibition of neural death: decreased generation of reactive oxygen species. 1999), staurosporine (Janicke et al., 1998), the diazo radical initiator 2,2'-azobis(2,4'-dimethylvaleronitrile) (Schor et al., 1999b), and NCS (Schor et al., 2000). Similarly, cisplatin treatment of PC12 cells results in apoptosis, but not in cleavage of Bcl-2. Both caspase 3 and Bcl-2 cleavage are probably not involved in apoptosis in these systems; rather, activation of an alternative pathway involving caspase 7, as has been described previously for prostate carcinoma cells (Marcelli et al., 1998, 1999; Liang et al., 2001), is probable. Thus, although cleavage of Bcl-2 is clearly not required for enactment of apoptosis, it seems to potentiate this process in enediyne-treated PC12 cells.

The mechanism of apoptosis induction by NCS in MCF-7 cells may involve proapoptotic changes other than those mediated by caspase 3. Studies from our laboratory indicate that treatment of MCF-7 cells with NCS results in down-regulated expression of Bcl-2 and up-regulated expression of its proapoptotic analog, Bax (Liang et al., 2001). The resulting decrease in the Bcl-2/Bax ratio has been shown in other systems to trigger the release of cytochrome c from the mitochondria (Nakatsuka et al., 2000), and to thereby induce apoptosis (Putcha et al., 1999; Liang et al., 2000). Modulation of the Bcl-2/Bax ratio perhaps represents an alternative mechanism of potentiation of apoptosis in cells lacking caspase 3.