Drug-resistant breast carcinoma (MCF-7) cells are paradoxically sensitive to apoptosis

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Drug-Resistant Breast Carcinoma (MCF-7) Cells Are Paradoxically Sensitive to Apoptosis JACK S.K. CHEN,1 MARINA KONOPLEVA,2 MICHAEL ANDREEFF,2 ASHA S. MULTANI,3 SEN PATHAK,3 AND KAPIL MEHTA1* 1 Department of Bioimmunotherapy, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 2 Department of Blood and Marrow Transplantation, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 3 Department of Cancer Biology, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas The purpose of this study was to determine whether expression of tissue transglutaminase (TG2) and caspase-3 proteins in drug-resistant breast carcinoma MCF-7/DOX cells would render these cells selectively susceptible to apoptotic stimuli. Despite high resistance to multidrug resistance (MDR)-related drug, doxorubicin (150-fold), the MCF-7/DOX cells were extremely sensitive to apoptotic stimuli. Thus, calcium ionophore, A23187 (A23187) and the protein kinase C inhibitor staurosporine (STS) each induced rapid and time-dependent apoptosis in MCF-7/DOX cells. The apoptosis induced by either agent was accompanied by caspase-3 activation and other downstream changes that are typical of cells undergoing apoptosis. The alterations upstream of caspase-3 activation, however, such as loss in mitochondrial membrane potential (DC), release of cytochrome c, and activation of caspase-8, and caspase-9, were detected only in STS-treated cells. The A12387 failed to induce any of the caspase-3 upstream changes, implying that A23187-induced apoptosis may utilize one or more novel upstream pathways leading to the activation of caspase 3. In summary, these data demonstrate that MCF-7/DOX cells are much more sensitive to apoptotic stimuli than previously thought and that A23187-induced apoptosis may involve some novel, yet unidentified, upstream pathway that leads to the activation of caspase-3 and other downstream events. J. Cell. Physiol. 200: 223–234, 2004. ß 2004 Wiley-Liss, Inc.

The development of drug resistance poses a major problem in the treatment of cancer patients with refractory disease. When tumor cells develop resistance, they become resistant to not only the drug with which the patient has been treated but also to a variety of structurally and functionally unrelated drugs, in a phenomenon referred to as multidrug resistance (MDR). MDR is a multifactorial problem involving several genes acting either alone or in concert with other factors. Increased expression of genes, including drugtransporter genes coding for P-glycoprotein (P-gp) and MDR-related protein (MRP), and of topoisomerase II and glutathione S-transferase has been associated with the MDR phenotype (Merkel et al., 1989; Gottesman, 1993; Ling, 1997; Gottesman et al., 2002). However, modulation of proteins that are not obviously associated with drug resistance has also been observed. For example, the upregulation of protein kinase C, nuclear factor kappa B (NFkB), certain members of the integrin family of proteins, and anti-apoptotic protein Bcl2 have been frequently observed in drug-resistant tumors (Blobe et al., 1993; Green et al., 1999; Baldwin, 2001; Johnstone et al., 2002). Similarly, we and others have observed elevated expression of tissue transglutamiß 2004 WILEY-LISS, INC.

nase (TG2) in cancer cell lines selected for resistance against MDR-related drugs (Mehta, 1994; Han and Park, 1999; Chen et al., 2002; Devarajan et al., 2002a). TG2 is a member of the transglutaminase enzyme family, which catalyze calcium-dependent post-transla-

Abbreviations: [Ca2þ]i, intracellular calcium; DMSO, dimethyl sulfoxide; EGTA, ethylene glycol tetra-acetic acid; ER, endoplasmic reticulum; MDR, multidrug resistance; MTT, 3-[4,5dimethylthiazol-2-yl]-2,5-dipheyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; P-gp, P-glycoprotein; STS, staurosporine; TG2, tissue transglutaminase; DC, change in mitochondrial membrane potential. Contract grant sponsor: Department of Defense (to KM); Contract grant number: BC996884. *Correspondence to: Kapil Mehta, Department of Bioimmunotherapy, Unit 422, The University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. E-mail: [email protected] Received 10 September 2003; Accepted 6 November 2003 DOI: 10.1002/jcp.20014



tional modification of proteins by establishing isopeptide bonds. The isopeptide bonds thus formed are highly resistant to mechanical and chemical insults and confer extreme stability to the newly formed protein polymers (Chen and Mehta, 1999; Griffin et al., 2002). Some cell types, such as endothelial and smooth muscle cells, constitutively express high levels of TG2, whereas others use discrete signaling pathways to upregulate its expression (Griffin et al., 2002). In general, the expression of TG2 is markedly increased in various cell types undergoing apoptosis (Fesus et al., 1987, 1996; Melino et al., 1994; Piacentini, 1995; Autuori et al., 1998; Griffin and Verderio, 2000). TG2 stabilizes the apoptotic cells by crosslinking intracellular proteins and preventing their loss inflicting an inflammatory response (Piredda et al., 1997; Piacentini and Colizzi, 1999). Moreover, forced expression of TG2 in several cell types results in spontaneous apoptosis or renders the cells susceptible to death-inducing stimuli (Gentile et al., 1992; Melino et al., 1994; Verderio et al., 1998). Conversely, a reduction in TG2 levels by anti-sense renders the cells more resistant to apoptosis (Melino et al., 1994). These observations suggest that cells generally do not tolerate the expression of TG2 and that TG2 expression leads to apoptotic death. Similarly, caspase-3 (CPP32/Yama/apopain) is considered to play a central role in the execution of apoptosis (Cohen, 1997; Salvesen and Dixit, 1997; Liang et al., 2001). Caspase-3 is activated by different apoptotic signals. Once activated, it can cleave a variety of substrate proteins, including the TG2 and a nuclear enzyme poly(ADP-ribose) polymerase (PARP) (Tewari et al., 1995; Fabbi et al., 1999). Previous studies showed that, owing to a functional deletion in the CASP-3 gene, MCF-7 cells do not express the procapsase-3 protein (Janicke et al., 1998; Kurokawa et al., 1999). Our recent studies, suggested that the selection of caspase-3deficient wild-type MCF-7 (MCF-7/WT) cells for the drug-resistance phenotype is associated with high expression levels of caspase-3 and TG2 proteins (Devarajan et al., 2002a; Mehta et al., 2002). The expression of caspase-3 and TG2 proteins by MCF-7 cells selected for resistance against doxorubicin (MCF-7/DOX cells) was linked to the inherent property of some drug-resistant clones that are present, albeit in small numbers, in the starting tumor population (Devarajan et al., 2002a; Mehta et al., 2002). The purpose of this study was to determine whether expression of TG2 and caspase-3 in MCF-7/DOX cells would render these cells susceptible to apoptosis. We demonstrate that, despite their high degree of resistance against doxorubicin and other MDR-related drugs, MCF-7/DOX cells exhibit exquisite sensitivity towards apoptotic stimuli. Moreover, the ionophore A23187-induced apoptosis in MCF-7/DOX cells utilizes some novel upstream pathways that leads to the activation of caspase-3. MATERIALS AND METHODS Reagents and cell lines

The calcium ionophore A23187 and staurosporine (STS) were purchased from Sigma Chemical Co. (St. Louis, MO) and stored in 10 and 1 mM stock solutions, respectively, in dimethyl sulfoxide (DMSO) at 208C.

The parental (MCF-7/WT) and multidrug-resistant (MCF-7/DOX) MCF-7 cells were obtained as previously described (Mehta et al., 2002). The MCF-7/DOX cells exhibited the MDR phenotype, as revealed by their more than 200-fold resistance to doxorubicin and P-gp expression. To maintain the MDR phenotype, MCF-7/DOX cells were grown in the presence of 1 mg/ml doxorubicin (American Pharmaceutical Partners, Inc., Los Angeles, CA), however, the drug was removed from the culture 8– 10 days prior to the experiment. Caspase-3-transfected MCF-7 cells (MCF-7/Casp3) were generously provided by Dr. Ann D. Thor (Northwestern University Medical School, Evanston, IL) (Devarajan et al., 2002b). All the cell lines are maintained in RPMI 1640 culture medium, supplemented with 10% heat-inactivated fetal calf serum (FCS; Sigma), 10 mM glutamine, and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin), in a 5% CO2/95% air atmosphere at 378C. At the end of the experiment, cells were lysed in a minimal volume (100–300 ml) of the lysis buffer A (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% 2-b-mercaptoethanol, and protease inhibitors). The cell lysates were sonicated for 10 sec, and the protein contents were determined by a protein assay reagent (Bio-Rad, Hercules, CA), in accordance with the manufacturer’s instructions. To detect full-length and cleaved PARP, 2  106 cells were resuspended in 50 ml of lysis buffer B (1  sample buffer þ 6 M urea) and incubated on ice for 30 min. The samples were boiled for 3 min and then sonicated and centrifuged for 20 min at 10,000g, and the supernatant was stored at 208 C. Similarly, cytochrome c release was detected by resuspending the treated and untreated cells in 200 ml of lysis buffer C (20 mM HEPES–KOH, pH 7.0, 10 mM KCl, 1.5 mM MgCl2 1 mM EDTA, 1 mM ethylene glycol tetra-acetic acid (EGTA), 1 mM dithiothreitol [DTT], 250 mM sucrose, and protease inhibitors) on ice for 30 min. Cells were then disrupted by Dounce homogenization (30 strokes), and subsequently centrifuged in a microfuge (2,000g for 10 min). Supernatants were centrifuged again at 6,000g for 15 min, mixed with appropriate volume of sample buffer, and stored at 208C. Western blot analysis

Equal amounts of lysate protein (40 mg) were run on 8% sodium dodecyl sulfate (SDS)–polyacrylamide gels and electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ), using a semidry transfer apparatus (Hoefer Scientific Instruments, San Francisco, CA). The membrane was blocked with 5% nonfat dry milk (Bio-Rad) in 20 mM Tris-buffered saline (TBS) containing 0.01% Tween-20 (TBST) buffer and incubated overnight at 48C with one of the following primary antibodies in TBST containing 1% nonfat dry milk: 1:3,000 mouse anti-TG2 (CUB7402; Neomarkers, Fremont, CA), 1:200 mouse anti-P-gp (C219, Dako Corporation, Carpinteria, CA), 1:2,000 rabbit anti-PARP (Upstate Biotechnology, Lake Placid, NY), 1:1,000 rabbit anti-caspase-3 (Pharmingen, San Diego, CA), 1:1,000 mouse anti-caspase-8 and rabbit anti-caspase-6, -7, or -9 (Cell Signaling, Beverly, MA), 1:500 mouse anti-cytochrome c (Pharmingen), and 1:4,000 mouse anti-b-actin (Sigma). The immunoreac-



tivity was detected by the sequential incubation of the membrane with appropriate horseradish peroxidaseconjugated secondary antibody (anti-mouse or antirabbit; Amersham; prepared at 1:5,000 dilution in TBST) for 1 h at room temperature and enhanced chemluminescence reagents (Amersham). Cellular morphologic analysis

MCF-7/WT and MCF-7/DOX cells (1  106) were seeded into 75-cm2 tissue culture flasks. Two days later, the cultures were incubated with RPMI 1640 medium (supplemented with 2% FCS) alone or containing either 2 mM A23187 or 50 nM STS. At predetermined time points, cells were visualized under an inverted light microscope and photographed using Kodak Gold 400 ASA film. Total (floating and adherent) cell populations were collected and centrifuged (800g for 5 min), and the pellets were immediately processed for further experimentation. In some experiments, cells were treated with 50 mM monodansylcadaverine (MDC) overnight and then the A23187 was added. To determine the role of downstream caspases(-3, -6, and -7) in A23187-induced apoptosis, cells were treated with A23187 in the presence of IDN-1965 (N-[(1,3-dimethylindole-2-carbonyl) valinyl]-3-amino-4-oxo-5-fluoropentanoic acid; IDUN Pharmaceuticals, Inc., La Jolla, CA), a peptidomimetic fluoromethylketone that specifically inhibits downstream caspases (Grobmyer et al., 1999). To detect DNA-ladder patterns associated with apoptosis, cells were washed with PBS and lysed on ice for 15 min in Tris-HCl buffer (10 mM, pH 8.0) containing 1 mM EDTA and 0.025% Triton X-100. After being incubated for 1 h in the presence of 50 mg/ml RNAse A (Sigma) at 378C, the lysates were mixed with 2 mg/ml proteinase K (Sigma) and incubated at 378C for 1 h. The cell lysates were mixed with loading dye and analyzed on 1.8% agarose gels. The DNA was visualized by ethidium bromide staining. Apoptosis and DNA fragmentation

At the end of the treatment period, cells were washed and resuspended in PBS (0.5  106 to 1.0  106 cells/ml). Eighty-microliter aliquots were mixed with 100 ml of permeabilization buffer (0.1% Triton X-100, 0.05 N HCl, 0.15 M NaCl) and incubated on ice for 2 min. Acridine orange solution (8 mg/ml; Polysciences, Warrington, PA) was added, and then cell fluorescence was immediately determined, using a fluorescence-activated cell scan flow cytometer (Becton Dickinson, San Jose, CA) at a 488-nm excitation of a 15-mW argon laser and filter settings for green (530 nm, DNA) and red (585 nm, RNA) fluorescence. Ten thousand events were analyzed, and the percentage of sub-G1 (hypodiploid) apoptotic cells was calculated. Alterations in mitochondrial membrane potential (DC)

DC were measured by using cationic lipophilic fluorochrome, chromomethyl-X-rosamine (CMXRos; Molecular Probes, Eugene, OR). Cells (1  106) were incubated with 100 nM CMXRos in the medium for 1 h in the dark. For MCF-7/DOX cells, CMXRos staining was performed in the presence of 50 mM PSC-833 (an MDR1 blocker; Sandoz Pharmaceuticals, Hanover, NJ)

to overcome the P-gp-mediated efflux of CMXRos. After incubation with CMXRos, cells were analyzed by flow cytometry, and the percentages of cells with loss in DC were sorted and calculated. Telomeric DNA

Quntitative fluorescence in situ hybridization (QFISH) for telomeric DNA was performed using commercially available Cy-3-conjugated telomere peptide nucleic acid probe in accordance with the manufacturer’s instructions (Dako Corp.) as previously described (Multani et al., 2001). The slides were counterstained with 40 ,6-diamidino-2-phenylindole (0.1 mg/ml) and examined under a Nikon photomicroscope using a UV-2A filter (Omega Optical, Inc., Brattleboro, VT). Photomicrographs were taken with X63 oil immersion objective and quantitation of telomeric DNA was performed using the Metaview Imaging System software program (Universal Imaging Co., Westchester, PA). From each sample, at least 50 interphase nuclei were quantitated to determine the mean percentage of telomeric area compared with the total nuclear area. Statistical analysis was carried out using Student’s t-test. P values of less than 0.05 were considered to be statistically significant. RESULTS Expression of MDR and apoptosis-related proteins in MCF-7 cells

To confirm the MDR-phenotype status of the MCF-7/ DOX cells, we first checked their sensitivity to increasing doses of doxorubicin (0.01–20 mg/ml). After 2 days of treatment, more than 90% of MCF-7/WT cells succumbed to the cytotoxic effects of the drug at concentrations 1.0 mg/ml and greater. In contrast, more than 80% of MCF-7/DOX cells survived the toxic effects of doxorubicin even at a concentration as high as 20 mg/ml, as revealed by their ability to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) into insoluble formazan (data not shown). These results suggested that MCF-7/DOX cells are at least 200 times more resistant to doxorubicin than MCF-7/WT cells are. Furthermore, the MCF-7/DOX cells expressed high levels of P-gp, whereas the MCF-7/WT cells had no detectable P-gp (Fig. 1A), confirming the MDR status of the MCF-7/DOX cells. As a first step toward determining the relative responsiveness of drug-sensitive and resistant MCF-7 cells to certain apoptotic stimuli, we determined the expression of some proapoptotic (p53, bax, TG2, and caspase-3) and anti-apoptotic (Bcl-2) proteins in these cells. As previously observed (Chen et al., 2002), the MCF-7/DOX cells expressed high levels of the proapoptotic TG2 protein. Similarly, the expression of p53 and procaspase-3 proteins was much stronger in the MCF-7/ DOX cells than in the MCF-7/WT cells (Fig. 1A). Conversely, the Bcl-2 protein, which is known to inhibit apoptotic events in a cell, was abundantly expressed in MCF-7/WT cells but was deficient in the MCF-7/DOX cells (Fig. 1A). Differential response to STS-induced cell death

Although MCF-7/DOX cells are highly resistant to doxorubicin, they were equally or more sensitive to



Fig. 1. Expression of apoptosis-related proteins in MCF-7/WT and MCF-7/DOX cells and their sensitivity towards doxorubicin and staurosporine (STS)-induced cytotoxicity. A: The cell extracts from drug-sensitive (WT) and drug-resistant (DOX) MCF-7 cells were fractionated on 8% sodium dodecyl sulfate (SDS)–polyacrylamide gel, transferred onto nitrocellulose membranes, and probed with indicated antibodies, as discussed in Materials and Methods. B: The WT and DOX MCF-7 cells (3,000 cells per well) were plated in 96-well plates,

and doxorubicin (initial concentration, 110 mM) and STS (initial concentration, 1 mM) were added at 1:3 serial dilutions. After 48 h, the 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheyltetrazolium bromide (MTT) assay was employed to determine cell viability. The picture was obtained by scanning the plate optically. C: Dose-dependent cytotoxicity of STS against MCF-7/WT and MCF-7/DOX cells, as determined by MTT assay after 48 h of treatment.

STS-induced killing than the drug-sensitive MCF-7/WT cells were. Figure 1B demonstrates the differential killing effect of STS against MCF-7/WT and MCF-7/ DOX cells. Cells were plated in a 96-well plate and then treated for 48 h with increasing (1:3 dilutions) concentrations of either doxorubicin (from 0.05 to 110 mM) or STS (0.045 nM to 1 mM), and the MTT assay was performed. As shown in Figure 1B,C, MCF-7/DOX and MCF-7/WT cells exhibited similar sensitivities to STSinduced killing at lower doses (25 nM). At higher doses (50 nM), however, MCF-7/DOX cells were consistently more sensitive to STS than the MCF-7/WT cells were.

relatively more effective than STS in inflicting apoptosis in the MCF-7/DOX cells (Fig. 2A). However, under similar conditions, the MCF-7/WT cells exhibited very limited apoptosis (Fig. 2A). The STS- and A23187induced apoptosis in the MCF-7/DOX cells was timedependent, with maximum apoptosis evident at 48 h (A23187, 69  12% apoptosis; STS, 47  6.7% apoptosis; Fig. 2). Treatment of MCF-7/WT cells with A23187 also induced a time-dependent accumulation of cells in subG1 apoptotic phase, but the extent and rate of apoptotic death were significantly lower than observed in MCF-7/ DOX cells (Fig. 2B). The treatment of MCF-7/WT cells with STS, however, caused significant cell death, as revealed by MTT assay (Fig. 1B,C), but failed to cause any noticeable accumulation of cells in the sub-G1 phase, suggesting that STS treatment induces necrotic death in MCF-7/WT cells. The results of flow cytometry correlated well with morphologic observations (Fig. 3A). After they were treated with STS (50 nM) or A23187 (2 mM), most MCF7/DOX cells viewed under the phase-contrast microscope appeared to be rounded with blebs and contained fragmented nuclei (Fig. 3A), changes that are typical of cells undergoing apoptosis. In contrast, MCF-7/WT cells treated with STS under similar conditions appeared to be necrotic and aggregated in masses of dying or dead cells. A23187 treatment induced much less dramatic

Selective apoptosis in MCF-7/DOX cells

In view of the high propensity of MCF-7/DOX cells to undergo cell death and the expression of high levels of proapoptotic proteins TG2 and caspase-3 within the cells (Fig. 1A), we next determined the nature of death induced by STS in MCF-7/DOX cells. Because TG2 is a calcium-dependent enzyme and we previously demonstrated that MCF-7/DOX cells sustain TG2 expression owing to the deficient/defective intracellular calcium pools (Chen et al., 2002), we also determined the effect of A23187 on the survival of MCF-7/DOX cells. Results shown in Figure 2A demonstrate that MCF-7/DOX cells were exquisitely sensitive to STS and A23187. Both agents induced extensive apoptosis, with A23187 being



Fig. 2. A23187- and STS-induced apoptosis in MCF-7/WT and MCF7/DOX cells. A: Cells treated with or without 2 mM A23187 or 50 nM STS for 48 h were collected and analyzed for cellular DNA content by FACS. Cells exhibiting the sub-G1 level of DNA were considered apoptotic and calculated as a percentage of the total cell population. Shown are the data from a representative experiment that was

repeated at least three times with similar results. B: Cells seeded in T75 culture flasks were treated with or without 2 mM A23187 or 50 nM STS for 24 and 48 h, after which the apoptotic response was measured by determining the percentage of cells in the sub-G1 phase by FACS analysis, as described in Materials and Methods; 0 h refers to the time at which A23187 or STS was added to cells.

changes in the MCF-7/WT cells than it did in the MCF-7/ DOX cells under similar conditions (Fig. 3A). Electrophoretic analysis of the DNA contents, isolated from A23187- or STS-treated MCF-7/WT and MCF-7/DOX cells on agarose gel, further supported this contention

and revealed a classic internucleosomal DNA laddering in MCF-7/DOX cells. STS-treated MCF-7/WT cells, however, yielded a smear of DNA (Fig. 3B, lane 5), suggesting that STS-induced cell-death in MCF-7/WT cells is predominantly necrotic.

Fig. 3. A23187- and STS-induced apoptosis in MCF-7/WT and MCF7/DOX cells. A: Photomicrograph of morphologic changes induced in response to treatment of MCF-7/WT and MCF-7/DOX cells for 48 h with A23187 (2 mM) and STS (50 nM). The treated and untreated cells were observed under inverted phase-contrast microscope and photographed, as described in Materials and Methods. B: Electrophoretic

analysis of internucleosomal DNA fragmentation in WT (lanes 1, 3, and 5) and DOX (lanes 2, 4, and 6) MCF-7 cells after 48 h of treatment with A23187 (lanes 3 and 4) and STS (lanes 5 and 6). Lanes 1 and 2 represent the DNA isolated from untreated MCF-7/WT and MCF-7/ DOX cells, respectively. M, DNA size marker.



Caspase-3-induced cleavage of PARP is thought to be an important part of the cascade signaling for DNA

damage in the cells (Tewari et al., 1995). Because the parental MCF-7/WT cells lack caspase-3 as a result of a deletion mutation in the CASP-3 gene (Janicke et al., 1998), we next determined whether differences in the susceptibility of MCF-7/WT and MCF-7/DOX cells to undergo apoptosis were related to their ability to process PARP protein. Indeed, MCF-7/DOX cells have been shown to express full-length functional caspase-3 protein (Scudiero et al., 1998; Pirnia et al., 2000; Devarajan et al., 2002a; Mehta et al., 2002). The cell lysates from treated or untreated MCF-7/WT and MCF-7/DOX cells were analyzed for PARP by Western blot analysis. The results in Figure 4B (lanes 1 and 4) demonstrate the presence of a 110-kDa PARP precursor protein in both the MCF-7/WT and MCF-7/DOX cells. Treatment of the MCF-7/DOX cells with A23187 or STS consistently cleaved the PARP protein (Fig. 4B, lanes 5 and 6). In contrast, no such cleavage of PARP was observed in the MCF-7/WT cells under similar treatment conditions (Fig. 4B, lanes 2 and 3). These observations suggested that the treatment of MCF-7/DOX cells with A23187 or STS results in the activation of effector caspase-3. Because PARP can also be cleaved by other downstream caspases, caspase-6 and -7 (Salvesen and Dixit, 1997), we examined the activation status of these caspases in MCF-7 cells before and after A23187 and STS treatments. Results shown in Figure 4C demonstrated the presence of procaspase-3 protein in MCF-7/ DOX cells; procaspase-3 protein was activated in response to A23187 and STS treatments as revealed by its cleavage into 18- and 14-kDa fragments (Fig. 4C, lanes 5 and 6). Unlike caspase-3, the caspase-6 and

Fig. 4. Effect of tissue transglutaminase (TG2) inhibition on A23187and STS-induced apoptosis (A) and alterations in poly(ADP-ribose) polymerase (PARP) (B) and effector caspases (C). A: Cells were incubated with or without 50 mM MDC, a substrate inhibitor of TG2, 24 h before being treated with 2 mM A23187 or 50 nM STS. After 48 h of treatment, the cells were analyzed for apoptotic response using FACS analysis, as described in Materials and Methods. The results are the average percentages of sub-G1 cells from two independent

experiments. After the cells were incubated for 48 h with medium alone (lanes 1 and 4) or medium containing either 2 mM A23187 (lanes 2 and 5) or 50 nM STS (lanes 3 and 6), MCF-7/WT cells (lanes 1–3) and MCF-7/DOX cells (lanes 4–6) were lysed and analyzed for PARP (B) and caspase-3, -6, and -7 proteins (C) by Western blot analysis, using protein-specific antibodies, as described in Materials and Methods. One of the membranes (caspase-6) was reprobed with beta-actin antibody to ensure even loading of proteins.

Involvement of TG2 in A23187-induced apoptosis

An association between TG2 expression and apoptotic cell death has been well established (Fesus et al., 1987; Autuori et al., 1998; Fesus and Piacentini, 2002). To determine the importance of endogenous TG2 activation in A23187- and STS-induced apoptosis of MCF-7/DOX cells, we studied the effect of MDC, a competitive substrate inhibitor of TG2 (Facchiano et al., 2001). Cells were treated with 2 mM A23187 in the presence or absence of 50 mM MDC for 48 h and analyzed for apoptotic sub-G1 population, using flow cytometry. As shown in Figure 4A, approximately 75% of the MCF-7/DOX cells were apoptotic in the A23187-treated cultures. However, the presence of MDC during A23187 treatment significantly attenuated apoptosis (P < 0.003); only about 40% of cells accumulated in the sub-G1 phase. These results suggested that TG2-catalyzed protein crosslinking activity plays an essential role in the execution of A23187-induced apoptosis in MCF-7/DOX cells. Interestingly, the presence of the MDC during STS-treatment of MCF-7/DOX cells did not alter the cells’ apoptotic response. The STS alone induced about 45% apoptosis, whereas STS plus MDC induced about 40% apoptosis. Similarly, in the MCF-7/WT cells, which lack TG2 expression, MDC failed to rescue the cells from A23187-induced apoptosis (Fig. 4A). Caspase-3 and A23187-induced apoptosis in MCF-7/DOX cells



capsase-7 were present in both cell lines (Fig. 4C, lanes 1 and 4). Caspase-6 was cleaved in both the MCF-7/WT and MCF-7/DOX cells but was cleaved more efficiently in the MCF-7/DOX cells (Fig. 4C, lanes 5 and 6). This may be due to the presence and activation of caspase-3 in MCF-7/DOX cells, since caspase-6 has been shown to be a caspase-3 substrate (Salvesen and Dixit, 1997). A23187 induced activation of caspase-7 in both MCF-7/ WT and MCF-7/DOX cells (Fig. 4C, lanes 2 and 5), whereas STS activated caspase-7 only in MCF-7/DOX cells (lane 6). These data are consistent with the observation that STS did not kill MCF-7/WT cells via apoptosis (Figs. 2 and 3). Next we tested the ability of IDN 1965, a pandownstream-caspase inhibitor (Grobmyer et al., 1999), to inhibit A23187- and STS-induced apoptosis in MCF-7 cells. Cells were pretreated with 20 mM IDN 1965 for 2 h and then treated with or without 2 mM A23187 or 50 nM STS. As shown in Figure 5A, IDN 1965 remarkably attenuated A23187-induced apoptosis in the MCF-7/ DOX cells; the percentage of apoptotic cells decreased from 74% in absence to 11% in the presence of the inhibitor. Similar reversals in apoptosis levels were observed in the A23187 of IDN 1965-treated MCF-7/WT cells (from 26 to 10%) and the STS-treated MCF-7/DOX cells (from 53 to 9%). IDN 1965 had no noticeable effect on the STS-treated MCF-7/WT cells, however, further supporting the contention that STS induces necrotic rather than apoptotic death in these cells.

Because caspase 3 is differentially expressed in parental and drug-resistant MCF-7 cells and is a key mediator of PARP cleavage and DNA fragmentation in cells undergoing apoptosis, we further studied the role of caspase-3 in mediating A23187- and STS-induced apoptosis. We used MCF-7/WT cells stably transfected with procaspase-3 cDNA and assessed their apoptotic response after they were treated with A23187 and STS. Results shown in Figure 5B clearly demonstrated that the reconstitution of caspase-3 in the MCF-7/Casp3 cells significantly augmented their apoptotic response to A23187 and STS compared with the response in the nontransfected MCF-7/WT cells. In fact, the extent of apoptosis exhibited by the transfected MCF-7/Casp3 cells was comparable to that observed in the MCF-7/ DOX cells. Together, these results suggest that caspase3 plays an important role in executing A23187- and STSinduced apoptosis in MCF-7 cells. A23187-induced apoptosis was accompanied by a significant attrition of telomere in MCF-7/DOX cells (P < 0.001), the effect that was not seen in MCF-7/WT cells (Fig. 6) under similar conditions (2 mM concentration of A23187 for 48 h).

Fig. 5. Requirement of caspase-3 for A23187- and STS-induced apoptosis in MCF-7/DOX cells. A: Inhibitory effect of effector caspases on A23187- and STS-induced apoptosis in MCF-7/WT and MCF-7/ DOX cells. Cells were pretreated for 2 h with 20 mM pan-downstreamcaspase inhibitor IDN 1965 before being incubated with medium alone or medium containing either 2 mM A23187 or 50 nM STS. After 48 h of treatment, total cell populations (adherent plus floating cells) were collected and analyzed for sub-G1 distribution using the flow cytometry. Results shown are from a representative experiment of the two

experiments with similar results. B: Effect of procaspase-3 expression on A23187- and STS-induced apoptosis in MCF-7/WT cells. Caspase-3deficient (MCF-7/WT) and caspase-3 reconstituted (MCF-7/Casp3) MCF-7 cells were treated with 2 mM A23187 for 48 h and analyzed by flow cytometry for apoptotic sub-G1 cell population, as described in Materials and Methods. Caspase-3-sufficient MCF-7/DOX cells were used as controls in a parallel experiment. The results shown are from a representative experiment, performed at least three times with similar results (10% standard error).

Novel upstream pathway in A23187-induced activation of caspase-3

Three major pathways are known to induce apoptosis in cells: (a) the activation of plasma membrane death receptors, resulting in the recruitment and activation of



Fig. 6. Alterations in telomeric DNA in A23187-treated MCF-7/DOX cells. A: Increased telomere signals are evident in control MCF-7/DOX cells (plate c) compared with control MCF-7/WT cells (plate a). The loss in signal is seen 48 h after treatment of MCF-7/DOX cells with A23187 (plate d). Under identical conditions, no erosion in telomere signal was observed in MCF-7/WT cells (plate b). B: The percent telomeric area in

interphase nuclei of FISH preparations was quantitated as compared to the total nuclear areas and expressed as the mean values. The amount of telomeric DNA was significantly reduced (P < 0.001) in A23187-treated MCF-7/DOX cells whereas in A23187 treated MCF-7/ WT cells no such reduction was evident.

the initiator caspase, caspase-8 (Ashkenazi and Dixit, 1998); (b) the release of cytochrome c from mitochondria into the cytosol, cytochrome c binds to form a functional complex with APAF-1 (apoptosome) and caspase-9 and autoproteolytic cleavage and activation of caspase-9 (Green and Reed, 1998); and (c) the endoplasmic reticulum (ER)-stress-induced release and autocleavage of caspase-12 (Nakagawa et al., 2000). Activated caspase-8, -9, or -12, in turn, cleaves and activates the downstream caspase, caspase-3, which then acts on several intracellular substrate proteins, including the PARP enzyme, leading to morphologic and biochemical changes that are characteristic of cells undergoing apoptosis. To determine the nature of any upstream pathways that lead to the activation of caspase-3 and consequent apoptosis of MCF-7/DOX cells in response to STS and A23187 treatment, we first determined the alterations in mitochondrial functions. After incubation with medium alone or medium containing A23187 or STS, cells were stained with DC-sensitive probe, MitoTrackerTM (Red CMXRos) and analyzed by flow cytometry (Gilmore and Wilson, 1999). Figure 7A shows the results from a typical experiment, depicting alterations in DC of MCF7/DOX cells following treatment with A23187 or STS. The treatment of the MCF-7/DOX cells with A23187

induced a change in the DC of only 15% of cells, compared with 3% of control cells. Consistent with these results, the MCF-7/DOX cells, despite undergoing substantial apoptosis (Fig. 2), failed to show any detectable release of cytochrome c from mitochondria or cleavage and activation of caspase-9 (Fig. 7B). Treatment with STS, in contrast, induced a significant collapse in the DC of the MCF-7/DOX cells (64% cells, Fig. 7A); this collapse correlated well with the detection of cytochrome c release and activation of caspase-9 in these cells (Fig. 7B). These data clearly suggest that A23187-induced caspase-3 activation and apoptosis of MCF-7/DOX cells are independent of alterations in mitochondrial functions. Next, we determined whether death receptor-dependent, caspase-8-mediated activation of caspase-3 is involved in A23287-induced apoptosis of MCF-7/DOX cells. Ligation of death receptors, such as the tumor necrosis factor receptor-1 and the Fas receptor, leads to the activation of caspase-8. Activated caspase-8 can either cleave and activate caspase-3 directly or do so indirectly by first cleaving the Bcl-2 homology 3containing protein Bid, which causes the release of cytochrome c from the mitochondria and activation of caspase-9 (Li et al., 1998). Cell lysates from control and treated cells were evaluated by Western blot analysis for


Fig. 7. Mitochondrial membrane potential (DC) and subsequent alterations induced by A23187 and STS treatment in MCF-7/WT and MCF-7/DOX cells. A: Twenty four hours after treatment, the cells were collected and analyzed by flow cytometry for changes in DC, as described in Materials and Methods. The results shown are from a representative experiment, reproduced in three independent experiments. B: After 24 h of treatment with 2 mM A23187, cells were harvested and analyzed by Western blot analysis for cytosolic release of cytochrome c and proteolytic cleavage of procaspase-9. Lane 5 shows the extent of cytochrome c release and cleaved fragment of caspase-9 in MCF-7/DOX cells in response to STS treatment (50 nM for 24 h); , absent; þ, present.

Fig. 8. Effect of A23187 treatment on caspase-8 activation and Bid cleavage (A) and caspase-12 (B) and calpain activation (C) in MCF-7/ WT and MCF-7/DOX cells. A: After 48 h of incubation in medium alone (lanes 1 and 4) or medium containing either 2 mM A23187 (lanes 2 and 5) or 50 nM STS (lanes 3 and 6), MCF-7/WT (lanes 1–3) and MCF-7/ DOX (lanes 4–6) were harvested, and the cell lysates were analyzed


Bid cleavage. As shown in Figure 8A, no cleavage of Bid or caspase-8 was observed in MCF-7 cells in response to A23187 treatment, as suggested by the identical intensity of the Bid immunoreactive band in treated and untreated cells. This ruled out the involvement of the receptor-mediated pathway in A23187-induced apoptosis of MCF-7/DOX cells. Finally, the ER stress-induced activation of caspase12 can also lead to the activation of caspase-3 (Rao et al., 2001). To determine the involvement of this pathway in A23187-induced apoptosis of MCF-7/DOX cells, we studied caspase-12 status in treated and untreated cells. As shown in Figure 8B, treatment of MCF-7 cells with A23187 failed to induce caspase-12 activation, suggesting that this pathway is also unlikely to contribute to A23187-induced activation of caspase-3 in MCF-7/ DOX cells. Calpain, the cytosolic cysteine protease, can be activated by alterations in intracellular calcium pools and has been shown to represent an early event, upstream of the caspases, in radiation-induced apoptosis (Waterhouse et al., 1998). Moreover, activated calpain has been implicated in the cleavage of procaspase-3 (McGinnis et al., 1999; Blomgren, 2001; Varghese et al., 2001). Therefore, we determined the effect of A23187 treatment on calpain activation in the MCF-7/ DOX cells. Results shown in Figure 8C indicate that treatment of MCF-7/DOX cells with A23187 results in the activation of calpain, as revealed by the presence of its cleaved product. Under similar conditions, however, calpain was not activated in the MCF-7/WT cells, despite that these cells expressed the calpain protein at levels similar to those in the MCF-7/DOX cells (Fig. 8C). These results suggested that calpain activation may be a late event, rather than upstream of caspase-3

for caspase-8 and Bid cleavage by Western blot, as described in Materials and Methods. Similarly, cells incubated with medium alone or 2 mM A23187 for 48 h were used to analyze the activation status of caspase-12 (B) and calpain proteins (C) by Western blot. Membranes were reprobed with anti-beta actin antibody to establish even loading of proteins in each lane.



activation. Indeed, MCF-7/Casp3 cells transfected with caspase-3 and treated with A23187 revealed the presence of a cleaved calpain product (data not shown). DISCUSSION

The issue of whether drug resistance is acquired by cancer cells in response to drug treatment or results from selection of one or more inherently resistant subclones present in the starting tumor cell population remains controversial. The finding that MCF-7/WT cells lack caspase-3 expression owing to deletion of the CASP3 gene (Janicke et al., 1998) and the unexpected finding that MCF-7/DOX cells express full-length, functional procaspase-3 (Devarajan et al., 2002a) raised doubts about whether MCF-7/DOX cells are truly a derivative of parental MCF-7 cells. On the basis of DNA fingerprinting analysis and unexplained expression of fulllength caspase-3 protein, it was proposed that MCF-7/ DOX cells are not related to MCF-7/WT cells and that the MCF-7/DOX cell line was actually derived from a different donor (Pirnia et al., 2000). However, we recently demonstrated that MCF-7/DOX cells represent a subclone that is present in MCF-7/WT cells. Thus, the selection with doxorubicin and possibly other chemotherapeutic drugs leads to the specific propagation of this subclone (Mehta et al., 2002). The specific expression of procaspase-3 and TG2 (Mehta, 1994; Chen et al., 2002) in the MCF-7/DOX cells led us to believe that despite their high resistance against chemotherapeutic drugs, these cells may be sensitive to certain apoptotic stimuli. By using A23187, we demonstrated that these cells were exquisitely sensitive to apoptosis (Figs. 2 and 3). The increased sensitivity of the MCF-7/DOX cells toward A23187 seems to be related to the ability of this agent to increase the cytosolic pool of calcium; this increase in turn could lead to the activation of otherwise cryptic TG2 present in high amounts in these cells. Indeed, our recent studies provided strong evidence that the ability of MCF-7/DOX cells to sustain TG2 expression is related to their deficient/defective intracellular calcium pools (Chen et al., 2002). Conversely, transfection of MCF-7/WT cells with TG2-specific cDNA resulted in spontaneous apoptotsis of these cells (Chen and Mehta, unpublished). Moreover, other cell types (e.g., drug-resistant myeloblastic leukemia, U937 cell line and lung cancer H1299 cell line) that expressed high constitutive levels of TG2 protein exhibited higher sensitivity to A23187-induced apoptosis than did their TG2-deficient counterparts (Chen and Mehta, unpublished). Similarly, treatment of U937 cells with retinoic acid, which induces TG2 expression in these cells, rendered these cells highly sensitive to A23187-induced apoptosis (Oliverio et al., 1999). More importantly A23187-induced apoptosis of MCF-7/DOX cells was rescued to a considerable extent by the presence of MDC, a competitive inhibitor for crosslinking activity of TG2 (Fig. 4A). Under similar conditions, however, MDC failed to protect the MCF-7/ WT cells from A23187-induced apoptosis. Because reliable methods to study the in situ activity of TG2 do not exist, we could not directly substantiate the claim that A23187-induced apoptosis is a result of direct activation of TG2. Nevertheless, it is apparent that perturbation in calcium homeostasis in these cells

results in a massive cell death, probably because of the activation of endogenous TG2 and intracellular crosslinking. Although several publications have reported the involvement of TG2 in apoptosis (Fesus et al., 1987, 1996; Melino et al., 1994; Piacentini, 1995; Autuori et al., 1998; Fesus, 1998), the mechanisms that underlie this effect are far from clear. A recent report from Piacentini et al. (2002) suggested that TG2-mediated apoptosis is accompanied by the hyperpolarization of mitochondria and an increase in reactive oxygen intermediates. Similarly, Tucholski and Johnson (2002) observed that the effect of TG2 on the apoptotic process is highly dependent on the type of the stimuli and how the transamidating activity of the enzyme is affected. Thus, stimuli that induced increase in transamidating activity of TG2 significantly increased caspase-3 activation and apoptotic nuclear changes. These observations suggest that TG2 activity may facilitate apoptosis by modulating caspase-3 functions by an as yet unknown mechanism. MCF-7/DOX cells, which selectively exhibit a mighty apoptotic response against A23187 and STS, express high levels of TG2 and caspase-3 proapoptotic proteins (Fig. 1A). Accordingly, we observed the activation of caspase-3 and cleavage of its downstream substrate, PARP, in only A23187-treated MCF-7/DOX cells. In the MCF-7/WT cells, on the other hand, caspase-6 and -7 were activated under these conditions (Fig. 4C). Activation of these downstream caspases may explain how MCF-7/WT cells undergo apoptosis in response to A23187 treatment. Indeed, caspase-3 deficient MCF-7 cells have been shown to undergo apoptosis in response to various stimuli, this apoptosis was accompanied by condensation and fragmentation of nuclei and release of cytochrome c from the mitochondria (Liang et al., 2001). Reconstitution of caspase-3 in MCF-7/WT cells completely restored the apoptotic response of these cells to A23187, and this apoptotic response was almost identical to that observed in MCF-7/DOX cells under similar conditions (Fig. 5B). Moreover, the presence of a pandownstream caspase inhibitor completely rescued MCF7/DOX cells from apoptotic death, suggesting that caspase-3 plays a significant role in these events. It is likely that caspase-3 and TG2 are equally effective in driving cells into apoptosis, although they utilize independent pathways to achieve this. Indirect evidence of this notion is provided by the observation that transfection of caspase-3-deficient MCF-7/WT cells with TG2 cDNA consistently led to the spontaneous apoptosis of these cells. An interesting aspect of this study was the observation that activation of procaspase-3 was the first common step observed in A23187- and STS-induced apoptosis of MCF-7/DOX cells. The two apoptotic stimuli apparently utilize independent upstream pathways that lead to the proteolytic cleavage and activation of procaspase-3. As shown in Figures 7 and 8, STS-induced apoptosis in MCF-7/DOX cells followed a classic mitochondria-dependent pathway, as revealed by the loss of DC, cytochrome c release, and the cleavage of caspase-9 (Fig. 7). However, A23187-induced apoptosis of MCF-7/ DOX cells was not accompanied by any noticeable loss in the DC, cytochrome c release, or caspase-9 activation. Similarly, no appreciable changes in death-receptor-


mediated pathway events, such as activation of caspase8 or Bid cleavage, were observed in A23187-treated MCF-7/DOX cells. Moreover, caspase-12 that is localized on the cytoplasmic side of the ER and activated (autoprocessed) in response to ER stress stimuli (Nakagawa et al., 2000; Rao et al., 2001; Yoneda et al., 2001) failed to show any evidence of activation in MCF-7/DOX cells, despite the massive apoptosis of these cells in response to A23187 treatment (Fig. 8). A mitochondrial flavoprotein, apoptosis-inducing factor, is known to induce morphological apoptosis in a caspase-independent manner in response to certain apoptotic stimuli (Candaˆe et al., 2002a,b). In MCF-7/DOX cells, the block of A23187-induced apoptosis by the panupstream caspase inhibitor IDN 1965, excludes any significant contribution of such caspase-independent death signaling pathways. We do not know the exact mechanism that leads to the activation of caspase-3 in A23187-induced MCF-7/DOX cells. However, it is well known that an increase in cytosolic calcium levels results in the activation of some proteases, including calpain. The latter can then cleave and activate caspase-3 (McGinnis et al., 1999; Blomgren, 2001; Varghese et al., 2001). Indeed, our data provided evidence that calpain was activated in A23187treated MCF-7/DOX cells (Fig. 8C). Nevertheless, we feel that activation of calpain under these conditions is a downstream rather than upstream event for caspase-3 activation (Wood and Newcomb, 1999). In support of this contention, MCF-7/WT cells, which lack caspase-3 protein, failed to show the activation of calpain. Conversely, MCF-7/Casp3 cells reconstituted with functional caspase-3 by transfection showed the activation of calpain following A23187 treatment (data not shown). In summary, these results suggest that MCF-7/DOX cells, despite their high degree of resistance to druginduced cytotoxicity, exhibit exquisite sensitivity to undergo apoptosis. Our data support the involvement of TG2-catalyzed protein crosslinking reactions in A23187-induced apoptosis of drug resistant cells. Caspase-3 plays an equally essential role in promoting apoptosis in response to certain stimuli. The two pathways are independent and are able to induce apoptosis in the absence of each other. STS-induced activation of caspase-3 and apoptosis of MCF-7/DOX cells involved alterations in mitochondrial pathway. On the other hand, A23187-induced activation of caspase-3 in MCF-7/ DOX cells appears to be independent of death-receptorinduced caspase-8 activation, mitochondria-dependent caspase-9 activation, or ER-stress-induced caspase-12 activation. It is likely that increased cytosolic calcium induced by A23187 treatment may activate an as yet undefined protease or caspase, which in turn can cleave and activate caspase-3. Additional studies are needed to further address the exact involvement of TG2 in drugresistant tumors. In view of our observation that cancer cells selected for resistance against MDR-related drugs express high levels of TG2 protein (Mehta, 1994; Han and Park, 1999; Chen et al., 2002) and that TG2 expressing tumor cells exhibit high propensity to undergo apoptosis, TG2 could serve as an attractive target for eradicating drug-resistant tumors, which pose a major clinical challenge for successful treatment of cancer.



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