Abrogation of Mitochondrial Cytochrome c Release and Caspase-3 Activation in Acquired Multidrug Resistance*

, Acquired multidrug resistance to anti-cancer agents has been associated with overexpression of the P-glyco-protein and other members of the ATP-binding cassette superfamily. The present studies demonstrate that SCC-25 cells selected for resistance to the alkylating agent cisplatin (CDDP) overexpress the anti-apoptotic Bcl-x L protein. In contrast to parental cells, the SCC-25/ CDDP-resistant variant failed to exhibit activation of caspase-3, cleavage of protein kinase C d , and other characteristics of apoptosis in response to CDDP. Similar results were obtained when SCC-25/CDDP cells were exposed to the structurally and functionally unrelated antimetabolite 1- b - D -arabinofuranosyl-cytosine (ara-C). Other cells selected for resistance to doxorubicin or vincristine also exhibited overexpression of Bcl-x L and failed to respond to CDDP and ara-C with activation of caspase-3. The results further demonstrate that multi-drug-resistant cells exhibit a block in the release of mitochondrial cytochrome c into the cytosol and that this effect is dependent on overexpression of Bcl-x L .

Acquired multidrug resistance to chemotherapeutic agents is an obstacle to the therapy of human malignancies. The development of resistance to certain cytotoxic drugs has been associated with insensitivity to structurally and functionally unrelated classes of anticancer agents. One type of acquired multidrug resistance is attributable to overexpression of the P-glycoprotein (Pgp) (1, 2). 1 As a member of the ATP-binding cassette (ABC) superfamily of membrane transport proteins and an energy-dependent efflux pump, Pgp confers resistance to the anthracyclines, vinca alkaloids, and epipodophyllotoxins. The multidrug resistance-associated protein is another member of the ABC superfamily that when overexpressed also results in resistant phenotypes (3,4). The finding that acquired multidrug resistance occurs in the absence of ABC protein overexpression has provided support for the existence of other mechanisms (5,6).
The cellular response to the effects of diverse anticancer agents includes the induction of apoptosis (7). Whereas the precise signals responsible for the induction of apoptotic cell death remain unclear, recent studies have demonstrated that cytochrome c release from mitochondria contributes to this response (8). In the cytosol, cytochrome c associates with a complex of Apaf-1 and caspase-9 and thereby induces the activation of caspase-3 (9, 10). The induction of apoptosis is associated with caspase-3-mediated cleavage of poly(ADP-ribose) polymerase (PARP), protein kinase C ␦ (PKC␦), and other proteins (11)(12)(13). Importantly, overexpression of the anti-apoptotic Bcl-2 and Bcl-x L proteins block cytochrome c release and the activation of caspase-3 (14 -16). In addition to effects on cytochrome c release and in concert with the finding that Bcl-2 is not restricted to mitochondria (17), Bcl-2 exhibits a downstream caspase-inhibiting activity (18,19).
Treatment of cells with DNA damaging agents and staurosporine has been associated with release of cytochrome c (8,16). By contrast, cytochrome c release was not observed in tumor necrosis factor-, Fas-, or dexamethasone-induced apoptosis (20). Thus, release of cytochrome c appears to be induced by only certain forms of cellular stress. Distinct mechanisms responsible for inhibition of cytochrome c release may also be restricted to certain apoptotic stimuli. In this context, selection of cells for resistance to vincristine and doxorubicin is associated with induction of Bcl-x L , and not Bcl-2, expression (21). These findings have suggested that overexpression of Bcl-x L may contribute to inhibition of cytochrome c release in acquired multidrug resistance.
The present studies demonstrate that acquired resistance to cytotoxic drugs is associated with inhibition of cytochrome c release. The findings support a Bcl-x L -dependent mechanism that is induced during selection to cytotoxic agents and thereby confers resistance to apoptosis.

MATERIALS AND METHODS
Cell Culture and Transfection-Human SCC-25 squamous carcinoma cells were grown as monolayers in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, and 0.4 g/ml hydrocortisone. SCC-25 cells were selected for resistance to cisplatin (CDDP) (22,23). The cloning of resist-* This investigation was supported by Public Health Service Grant CA29431 awarded by the NCI, DHHS, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cytochrome c Release-Cells were washed twice with phosphatebuffered saline, and the pellet was suspended in 5 ml of ice-cold buffer A (20 mM HEPES, pH 7.5, 1.5 mM MgCl 2 , 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10 g/ml each leupeptin, aprotinin, and pepstatin A) containing 250 mM sucrose. The cells were homogenized by being run through a Dounce homogenizer 14 times with a sandpaper-polished pestle. After centrifugation for 5 min at 4°C, the supernatants were centrifuged at 105,000 ϫ g for 30 min at 4°C. The resulting supernatant was used as the soluble cytosolic fraction. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with anti-cytochrome c (Pharmingen, San Diego, CA).
Analysis of DNA Fragmentation-Cells (5 ϫ 10 6 ) were harvested, washed, and suspended in 50 l of 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, and 0.5 mg/ml proteinase K (Sigma). After incubation at 50°C for 6 h, the samples were mixed with 50 l of 10 mM EDTA (pH 8.0) containing 1% (w/v) low melting point agarose and 40% sucrose for 10 min at 70°C. The DNA was separated in 2% agarose gels. After treatment with RNase, the gels were visualized by UV illumination.
In Vitro Assay for Cytochrome c-dependent Activation of Caspase-3-S-100 cytosolic fractions from untreated cells were immunodepleted of cytochrome c by incubation with anti-cytochrome c (Pharmingen) and protein A-Sepharose beads for 2 h at 4°C (8). The immunodepletion procedure was repeated twice with centrifugation to remove the beads.  (Fig. 1A). The parental cells also responded to CDDP with internucleosomal DNA fragmentation (Fig. 1B). By contrast, there was no detectable activation of caspase-3, cleavage of PKC␦, or DNA fragmentation following exposure of SCC-25/ CDDP cells to 100 M CDDP (Fig. 1, C and D). These findings indicated that SCC-25/CDDP cells are resistant to CDDP-induced apoptosis.
Previous studies have demonstrated that cells selected for resistance to anthracyclines or vinca alkaloids overexpress the Bcl-x L protein (21). Analysis of SCC-25 cell lysates by immunoblotting with anti-Bcl-x L demonstrated that the CDDP-resistant line overexpresses this anti-apoptotic protein compared with the parental cells ( Fig. 2A). The finding that Bcl-2 is not increased in the SCC-25/CDDP cells indicates that resistance to CDDP results in the selective induction of Bcl-x L expression (data not shown). To determine whether overexpression of Bcl-x L in the SCC-25 cells contributes to the apoptosis-resistant phenotype, we stably transfected SCC-25 cells to express Bcl-x L (Fig. 2A). CDDP treatment of SCC-25/neo cells that express the empty vector was associated with activation of caspase-3 (Fig. 2B). By contrast, there was no apparent cleavage of caspase-3 to the active fragment in SCC-25/bcl-x L cells treated with CDDP (Fig. 2B). The SCC-25/bcl-x L cells also failed to respond to CDDP with cleavage of PARP or PKC␦, whereas this response was detectable in the SCC-25/neo cells (data not shown). These results suggest that overexpression of Bcl-x L in drug-resistant cells or in stable Bcl-x L transfectants blocks CDDP-induced activation of caspase-3.
To extend these findings to an agent that is structurally and mechanistically distinct from CDDP, we treated SCC-25/CDDP cells with the antimetabolite 1-␤-D-arabinofuranosylcytosine (ara-C). In contrast to CDDP, ara-C incorporates into DNA and functions as a relative chain terminator (27). Treatment of Defect in Caspase-3 Activation in Multidrug Resistance 16648 parental SCC-25 cells with ara-C was associated with activation of caspase-3 and cleavage of PARP (Fig. 3A). Similar results were obtained in the SCC-25/neo transfectants. By contrast, the SCC-25/CDDP and SCC-25/Bcl-x L cells failed to respond to ara-C with caspase-3 activation or substrate cleavage (Fig. 3A). U-937 cell lines selected for resistance to doxorubicin (U-A20) and vincristine (U-V20) overexpress both Pgp and Bcl-x L (21). Treatment of wild-type U-937, but not U-A20 or U-V20, cells with CDDP resulted in caspase-3 activation (Fig. 3B). Similar results were obtained when the U-A20 and U-V20 cells were treated with ara-C (Fig. 3C). Thus, resistance to activation of caspase-3 and apoptosis occurs in cells selected for growth in the presence of diverse agents (CDDP, doxorubicin, or vinca alkaloids).
Overexpression of Bcl-x L blocks release of mitochondrial cytochrome c to the cytosol and thereby activation of caspase-3 (16). To assess whether similar mechanisms are involved in drug-resistant cells, we assayed SCC-25 cells for increases in cytosolic cytochrome c following CDDP treatment. Whereas SCC-25 cells responded to CDDP with release of cytochrome c, CDDP had little if any effect on this response in SCC-25/CDDP or SCC-25/bcl-x L cells (Fig. 4A). Similar findings were obtained in the drug-resistant U-A20 and U-V20 cells (data not shown). These results indicate that cells selected for resistance to cytotoxic agents exhibit defects in cytochrome c release and thereby activation of caspase-3. To determine whether the SCC-25/ CDDP cells acquire other mechanisms of resistance to cytochrome c that are downstream from mitochondrial release, we prepared cell lysates and asked whether caspase-3 is activated upon addition of cytochrome c. The results demonstrate that addition of cytochrome c to SCC-25 cell lysates is associated with activation of caspase-3 (Fig. 4B). Similar findings were obtained with lysates from the SCC-25/CDDP and SCC-25/ bcl-x L cells (Fig. 4B).
The development of resistance to cytotoxic agents is often multifactorial but is for the most part selective for particular classes of drugs. To our knowledge, there are no known mechanisms that are applicable to all classes of cytotoxic agents. The present data suggest that selection for resistance to CDDP is associated with inhibition of cytochrome c release and caspase-3 activation. The finding that the CDDP-resistant cells are also unresponsive in terms of caspase-3 activation to other structurally and mechanistically distinct agents, such as ara-C, supports a phenotype that applies broadly to the response of cells to cytotoxic agents. Indeed, the U-937 cells selected for resistance to doxorubicin or vincristine failed to respond to CDDP with activation of caspase-3. Also, in contrast to wildtype U-937 cells, the U-A20 and U-V20 lines failed to respond to ionizing radiation with release of cytochrome c, caspase-3 activation, and induction of apoptosis (data not shown). Thus, resistance to apoptosis appears to be a generalized response in the selection of cells to cytotoxic agents. The SCC-25 cells selected for resistance to CDDP overexpress the Bcl-x L protein. Other studies have shown that myeloid leukemia cells selected for resistance to doxorubicin, vincristine, and taxol also overexpress Bcl-x L (21,28). These findings indicate that diverse classes of cytotoxic agents induce Bcl-x L expression in different cell types. The demonstration that overexpression of Bcl-x L reduces the cytotoxicity of CDDP, vincristine, and other agents supports an acquired mechanism by which cells are protected from apoptotic stimuli (29). Overexpression of Bcl-x L may confer resistance to apoptosis by multiple mechanisms. For example, Bcl-x L promotes cell survival by regulating the membrane potential and volume homeostasis of mitochondria (30). Other studies indicate that Bcl-x L blocks the release of cytochrome c (16). The present data confirm that induction of Bcl-x L expression in drug-resistant cells is associated with abrogation of cytochrome c release and thereby caspase-3 activation. The finding that lysates from the drugresistant cells respond to cytochrome c with activation of caspase-3 further indicates that mechanisms responsible for activating caspase-3 are otherwise intact. Nonetheless, on the basis of present data we cannot exclude the possibility that drug-resistant cells will exhibit other defects in the apoptotic pathway that are downstream to caspase-3 activation.