Tetrocarcin a Inhibits Mitochondrial Functions of Bcl-2 and Suppresses Its Anti-Apoptotic Activity

[CANCER RESEARCH 60, 1229–1235, March 1, 2000] Tetrocarcin A Inhibits Mitochondrial Functions of Bcl-2 and Suppresses Its Anti-apoptotic Activity

Takayuki Nakashima, Masayuki Miura, and Mitsunobu Hara1 Drug Discovery Research Laboratories, Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., Nagaizumi-cho, Suntou-gun, Shizuoka 411-8731 [T. N., M. H.]; and Department of Neuroanatomy, Biomedical Research Center, Osaka University Medical School, Osaka 565-0871 [M. M.], Japan

ABSTRACT of them are oligonucleotide-based, such as the use of Bcl-2 antisense RNA or hammerhead ribozyme against bcl-2 (18–21). However, Bcl-2 is an integral, intracellular membrane protein that prevents cells small molecule Bcl-2 antagonists have yet to be reported. from undergoing apoptosis in response to a variety of cell death signals. It We describe here a natural product produced by Actinomycete, negatively regulates the activation of Caspase-3, which functions as effec- 2 tor of mammalian cell death pathways. Overexpression of Bcl-2 inhibits TC-A (Fig. 1), which inhibits the anti-apoptotic functions of the the caspase activities and apoptosis. A microbial secondary metabolite, Bcl-2 family. TC-A was originally discovered as an antibiotic active Tetrocarcin A (TC-A), was identified as an inhibitor of the anti-apoptotic against Gram-positive bacteria (22–27). It also showed antitumor function of Bcl-2. Apoptosis could be induced in cell lines that overex- activity in murine experimental tumor models, such as Sarcoma 180,

pressed Bcl-2 or Bcl-XL when the cells were treated with anti-Fas anti- P388 leukemia, and B16 melanoma, although the mechanism of its body, tumor necrosis factor ␣, staurosporine, or Bax, in addition to TC-A. antitumor activity is unknown. The present study shows that TC-A TC-A showed selectivity against the pro-apoptotic Bcl-2 family members, inhibits the mitochondrial functions of Bcl-2 and, thereby, suppresses in that cells overexpressing CrmA or dominant-negative FADD could not its anti-apoptotic function in cells overexpressing Bcl-2. These results undergo apoptosis with TC-A treatment. In Bcl-2-overexpressing cell pharmacologically indicate that the mitochondrial functions regulated lines, TC-A inhibited mitochondrial functions regulated by Bcl-2, result- by Bcl-2 are crucial for the death-suppressor activity of Bcl-2 family ing in Fas-triggered mitochondrial transmembrane potential loss and cytochrome c release. Inhibition of the mitochondrial functions of Bcl-2 proteins. and, thereby, its anti-apoptotic effect could serve as useful pharmacolog- ical targets. Thus, TC-A should serve as an archetype for specific inhibitors of Bcl-2 functions. MATERIALS AND METHODS Cell Culture. HeLa/bcl-2 is a stable, bcl-2-expressing cell line derived from HeLa cells transfected with a bcl-2 expression plasmid. The details of its INTRODUCTION construction have been reported previously (28). HeLa/bcl-XL cell is a stable, bcl-X -expressing cell line derived from HeLa cells transfected with an ex- Bcl-2 is an integral membrane protein that inhibits apoptosis or L pression vector containing a cDNA encoding human bcl-XL. Bcl-XL overex- programmed cell death induced by diverse stimuli in many cell types pressing clones were isolated by selecting G418-resistant clones. 293/bcl-2 or (1–3). Both biochemical and genetic evidence indicate that Bcl-2 293/bcl-XL cells are transiently transfected 293 cells. All of the cells were prevents apoptosis at the point of activation of CED-3 family pro- grown in DMEM supplemented with 10% fetal bovine serum. teases, such as Caspase-3 (CPP32; 4–6). Recent evidence has shown Treatment of Cells with Reagents. Cells were untreated or treated with that the mitochondria play a crucial role in apoptosis by releasing cell-death-inducing stimuli including, ␣Fas (100 ng/ml) and CHX (0.2 ␮g/ml), apoptogenic factors, such as cytochrome c and apoptosis-inducing STA (1 ␮M), or TNF-␣ (10 ng/ml), and cultured in the presence of TC-A. factor, from the intermembrane space into the cytoplasm (7). Cyto- Cultivation time for TC- A treatment was 6, 24, 8, 6, and 24 h for DEVD- chrome c release activates caspases through its effects on a protein cleavage, PARP-cleavage, DiOC6(3) staining, the release of cytochrome c, and called Apaf-1 (8, 9). Anti-apoptotic Bcl-2 and Bcl-X inhibit the XTT cell viability assays, respectively. L Viability Assays. Cell viability was determined by XTT assay. Briefly, apoptosis-associated release of both cytochrome c and apoptosis- cells (1.5 ϫ 105) were plated per well, and treated with various reagents, as inducing factor, although the mechanism of these actions has re- described above. Cells were then cultured in the presence of various concen- mained elusive. trations of TC-A for 24 h and assayed for uptake of XTT as described Small molecule inhibitors of the anti-apoptotic functions of Bcl- 2, previously (29). which we would like to refer to as Bcl-2 antagonists, would be useful PARP Cleavage Assay. Cells (2 ϫ 105) were treated by the reagents reagents for understanding Bcl-2 function. In addition to aiding in described above. After 24 h of cultivation, the cells were washed with PBS and mechanistic studies, they could also be useful in the treatment of those lysed in 20 ␮l of lysis buffer [250 mM NaCl/1.0% NP40/50 mM HEPES-NaOH diseases in which Bcl-2 plays a role. Elevated expression of Bcl-2 is (pH 7.5)/protease inhibitors/1 mM DTT/1 mM EDTA]. Lysates were clarified frequently found in human cancers. Consequently, those cancer cells by centrifugation, and the protein concentration of the supernatants was de- ␮ resist apoptosis (10). In many cases, cancers, such as follicular lym- termined. Each protein sample (10 g) was subjected to SDS/PAGE (12.5% acrylamide) and transferred to nitrocellulose filters. The filters were blocked phoma and hormone-refractory prostate cancer, that overexpress and incubated with anti-PARP (Enzyme Systems Products) or anti-Bcl-2 Bcl-2 are also resistant to chemotherapeutic agents. Such cancers have (Dako) antibodies. been associated with an unfavorable prognosis (11–17). Thus, Bcl-2 DEVD-4-methyl-coumaryl-7-amide Cleavage Assay. Cells (3 ϫ 104) antagonists have the potential of treating those human malignancies were treated with reagents as described above. After6hofcultivation, the cells composed of Bcl-2-overexpressing cells. Several approaches have were lysed as described previously (28). The DEVD-specific caspase activity been pursued to inhibit the function of Bcl-2 in human cancers. Most was measured by incubating cell extracts (25 ␮l) with Acetyl-L-Aspartyl-L- Glutamyl-L-Valyl-L-Aspartic Acid ␣-(4-Methyl-Coumaryl-7-Amide) (50 ␮M, ␮ Received 5/10/99; accepted 1/7/00. Peptide Institute) in 50 l of buffer A {20 mM PIPES (pH 7.2), 100 mM NaCl, The costs of publication of this article were defrayed in part by the payment of page 5mM DTT, 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl) dimethylammino]-1- charges. This article must therefore be hereby marked advertisement in accordance with propane sulfonate}. After 120 min, the reaction was stopped by the addition of 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Drug Discovery Research Laboratories, Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., Shimo- 2 The abbreviations used are: TC-A, Tetrocarcin A; ␣Fas, anti-Fas antibody; CHX, Ј togari 1188, Nagaizumi-cho, Suntou-gun, Shizuoka 411-8731, Japan. Phone: 81-559-89- cycloheximide; PARP, poly(ADP-ribose) polymerase; DiOC6(3), 3,3 -dihexylocarbocy- 2007; Fax: 81-559-86-7430; E-mail: [email protected] nine iodide; STA, staurosporine; TNF, tumor necrosis factor. 1229

100 ␮l of 0.75 M acetic acid and placed on ice. Fluorescence at wavelength 380 to 460 nm was compared with a standard curve of 7-amino-4-methylcoumarin (AMC, Peptide Institute). Transient Transfection. Transfections were performed by using Lipo- fectamine (Life Technologies, Inc.) according to the supplier’s protocol. 293 cells were seeded in 24-well dishes and transfected with 0.5 ␮g of each

expression vector (pcDNA3-Bax, pcDNA3-Bcl-2, or pcDNA3-Bcl-XL). Twenty-four h after transfection, the cells were treated with TC-A (2 ␮M) for Fig. 1. Structure of TC-A 12 h. Cleavage of PARP was assessed as described in “Materials and Methods” above. HeLa cells were seeded in 6-well dishes and transfected with 1 ␮g of each expression vector, together with 0.1 ␮g of green fluorescent protein (GFP) expression vector (pCMX-SAH/Y145F, kindly donated by K. Umezono at

Fig. 2. Effect of TC-A on Fas-mediated apoptosis in HeLa/ bcl-2 cells. A, Western analysis of the expression level of Bcl-2 and Bcl-XL. Erk2 was used as the control for equal protein loading. B, time course of PARP cleavage in HeLa and HeLa/bcl-2. HeLa and HeLa/bcl-2 cells were treated with indicated reagents and at the indicated times; cleavage of PARP was analyzed as described in “Materials and Meth- ods.” C-F, PARP cleavage (C), DEVD cleavage (D), cell viability (E), and cell morphology (F) of HeLa/bcl-2 treated with TC-A. Cells were treated with indicated reagents and the ,ء ,analyzed as described in “Materials and Methods” [C COOH-terminal Bcl-2 cleavage product (8)]. G, time course of PARP cleavage induced by ␣Fas/CHX in HeLa cells in the presence of various concentrations of TC-A.

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normal goat serum in PBS (blocking buffer) for 10 min. They were incubated with monoclonal, antihuman Bcl-2 antibody (1:200 dilution, DAKO, Den- mark) for1hatroom temperature and washed with PBS three times. The samples were incubated with Rhodamine-labeled antimouse IgG (1:200 dilution, Jackson Laboratory) for 1 h and washed in PBS three times. The samples were mounted with PermaFlour Aqueous Mounting Medium (Immunon) and examined with an Olympus confocal laser scanning microscope (scale bar, 25 ␮m). ⌬⌿ Assessment of Mitochondrial m. Changes in the inner membrane ⌬⌿ ϫ 5 transmembrane potential ( m) were determined by incubating 3 10 cells in 40 nM DiOC6(3) for 20 min at 37°C. The cells were assayed using FACScan flow cytometry (Becton Dickinson, Mountain View, CA). In all of the cases, the cells were gated to exclude cellular debris, which physically prevents proper fluorescence-activated cell sorting detection. Subfractionation. HeLa/bcl-2 cells were washed twice with PBS, and the pellets were resuspended in ice-cold, buffer B [20 mM HEPES (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and protease inhibitors] containing 250 mM sucrose. The cells were homogenized by dounc- ing 80 times in a Dounce homogenizer (Wheaton). Nuclei and unbroken cells were separated by a centrifugation at 10,000 ϫ g for 10 min. The resulting pellet (ppt) and supernatant fractions including cytosolic cytochrome c were subjected to Western analysis as described in “Materials and Methods.”

RESULTS AND DISCUSSION Discovery of TC-A as an Inhibitor of the Anti-apoptotic Func- tions of Bcl-2. TC-A was identified by screening a library of natural products—a rich source of chemically unique, biologically active compounds—for its ability to inhibit the anti-apoptotic function(s) of Bcl-2 and, thereby, cause apoptosis in Bcl-2-overexpressing cell lines (Fig. 2A). Untransfected HeLa cells are susceptible to cell death induced by cell-death stimuli such as ␣Fas in the presence of the protein synthesis inhibitor ␣Fas/CHX or STA, both of which cause the cleavage of PARP. HeLa/bcl-2 cells are, however, resistant to apoptosis induced by those same cell-death-inducing stimuli, and no PARP-cleavage was observed (Fig. 2B). Cotreatment of the apoptosis- resistant HeLa/bcl-2 with TC-A and ␣Fas/CHX did activate caspases, as indicated by the cleavage of PARP (Fig. 2C) and tetrapeptide- substrate, Ac-DEVD-MCA (Fig. 2D). Caspase activation resulted in the loss of cell viability, and the inhibition of caspase activation by z-DEVD-fmk prevented cells from dying (Fig. 2, D and E). Cell morphological changes were consistent with the reduced viability of HeLa/bcl-2 cells treated with TC-A and ␣Fas/CHX. That is, cell rounding and detachment from the substrate were observed in HeLa/ bcl-2 treated with TC-A and ␣Fas/CHX, resembling that observed in HeLa cells treated with ␣Fas/CHX (Fig. 2F). The effect of TC-A was dose-dependent. TC-A (2.2 ␮M) caused Fig. 3. Effect of TC-A on Fas-mediated apoptosis in HeLa/bcl-XL cells. PARP cleavage (A, B) and cell viability assay (C) of HeLa/bcl-XL. Cells were treated with complete cleavage of Mr 116,000 PARP, with a significant loss of cell indicated reagents and analyzed as described in “Materials and Methods.” In B, HeLa/ ␮ ␮ viability. The small fragment of Bcl-2 that appeared with 2.2 M of bcl-XL cells were cultured in the presence of 100 M z-DEVD-fmk for 18 h and then treated with ␣Fas/CHX. TC-A may be the COOH-terminal Bcl-2 cleavage product, because

Bcl-2 is reported to be cleaved at Asp34 of the loop domain by caspase-3 (31). TC-A alone did not show any significant signs of Kyoto University, Kyoto, Japan). Expression vector for CrmA, Bcl-2, and apoptosis in HeLa/bcl-2 cells. These results indicate that TC-A seems

Bcl-XL were described previously (30). The expression vector for the domi- to abrogate the cell-death block caused by Bcl-2 overexpression, nant negative form of FADD was kindly donated by V. M. Dixit (University because the apoptotic signal due to Fas ligation led to the death of of Michigan Medical School, Ann Arbor, MI). Forty-eight h after transfection, Fas-resistant HeLa/bcl-2 cells. cells were treated with TNF-␣ (10 ng/ml) and cycloheximide (10 ␮g/ml), Fig. 2G shows the effect of TC-A on nontranfected, parental HeLa together with TC-A (2 ␮M) for 5 h. Cells were then washed with PBS and fixed cell line. Increased concentrations of TC-A did not affect the time with 4% paraformaldehyde/PBS for 10 min. GFP-positive cells were examined course of PARP-cleavage induced by ␣Fas/CHX treatment. There- with an Olympus fluorescence microscope (model IX70). Apoptotic cells were fore, without Bcl-2 overexpression, TC-A did not sensitize HeLa cells small, dense, and frequently fragmented, whereas surviving cells were flat and ␣ well attached to the dish as described previously (30). to Fas/CHX. Furthermore, TC-A did not induce apoptosis in HeLa Immunostaining of HeLa/bcl-2 Cells. HeLa/bcl-2 cells were treated with cells at the concentrations that induced Fas-dependent apoptosis in or without 2 ␮M of TC-A for 12 h, then fixed with 4% paraformaldehyde/PBS HeLa/bcl-2 cells (data not shown), thus indicating that TC-A itself had for 10 min, and permeabilized in 0.1% Triton X-100/PBS for 10 min at room no activity as a cell-death stimulus at those drug concentrations in temperature. The cells were washed 3 times with PBS and blocked with 4% HeLa cells. Taken together, these data indicate that TC-A does not 1231

Fig. 4. The effect of TC-A on TNF-␣- or STA-mediated apoptosis in HeLa/bcl-2 cells. DEVD cleavage (A, B, left), PARP cleavage (A, B, middle) and cell viability assay (A, B, the COOH-terminal Bcl-2 cleavage ,ء .right) of HeLa/bcl-2. Cells were treated with 10 ng/ml TNF-␣ and 10 ␮g/ml CHX (A)or1␮M STA (B) and were cultured in the presence of TC-A product.

cleavage of Bcl-XL, which indicated that DEVD-cleaving caspases were responsible for the proteolysis of Bcl-XL (Fig. 3B). The addition of caspase inhibitors also significantly suppressed the other activities of TC-A, for example PARP-cleavage and cell viability loss in ␣Fas-

treated HeLa/bcl-XL cells. Thus, the action of TC-A is abrogated by the inhibition of DEVD-cleaving caspase activity, which indicates that TC-A inhibits an event upstream of the activation of z-DEVD-fmk- inhibitable caspase(s). This hypothesis is consistent with the fact that anti-apoptotic Bcl-2 family members act upstream of caspase-3 (32,

33). Similar proteolysis of Bcl-XL was reported in cells induced to undergo apoptotic death after Sindbis virus infection or interleukin 3

withdrawal. Furthermore, the COOH-terminal fragment of Bcl-XL was shown to potently induce apoptosis (34). These results suggest

that the inhibition of the anti-apoptotic functions of Bcl-XL by TC-A results in the activation of a subset of caspases that are sensitive to Fig. 5. The effect of TC-A on the anti-apoptotic function of the Bcl-2 family in the Bax-mediated apoptosis. PARP cleavage in transiently transfected 293 cells. Cell were Bcl-XL, which in turn cleaves the Bcl-XL. transfected with expression vectors as indicated. Twenty-four h after transfection, cells were treated with 2 ␮M TC-A for an additional 12 h and were analyzed for PARP-cleavage as described in “Materials and Methods.” simply sensitize the cells to ␣Fas/CHX but, rather, blocks the anti- apoptosis function of Bcl-2 in HeLa/bcl-2 cells.

TC-A also Inhibits the Anti-apoptotic Function of Bcl-XL. We next tested whether TC-A inhibited the anti-apoptotic function(s) of another Bcl-2 family protein, Bcl-XL, using HeLa cells stably overexpressing Bcl-XL (HeLa/bcl-XL cells; Fig. 2A). When these cells were treated with ␣Fas/CHX, no PARP-cleavage was observed, indicating that the HeLa/bcl-XL cell line was also resistant to Fas-induced apoptosis (Fig. 3A). The addition of TC-A caused a Fas-dependent apoptosis in HeLa/bcl-XL cells, as indicated by PARP-cleavage, and an accompanying decrease in cell viability (Fig. 3, A and C). In contrast to the marginal effect of TC-A on the cleavage of Bcl-2 protein in ␣Fas/CHX-treated HeLa/bcl-2 cells, marked cleavage of the Fig. 6. The effect of TC-A on the anti-apoptotic function of CrmA and dominant negative form of FADD. Transfected expression vectors are indicated. Forty-eight h after Bcl-XL protein occurred, with a dose dependency similar to that transfection, cells were treated with TNF-␣ and cycloheximide (10 ␮g/ml) together with observed for PARP cleavage and reduced cell viability. HeLa/bcl-XL TC-A(2 ␮M) for 5 h. Apoptotic cells were counted as described in “Materials and cells treated with the caspase inhibitor z-DEVD-fmk blocked the Methods.” 1232

Similar results were obtained for STA-induced apoptosis in Rat1 cells stably transfected with bcl-2 (data not shown). Compared with the data obtained for Fas-induced apoptosis, TC-A was almost equally effective when TNF-␣/CHX or STA were used as cell-death stimuli in HeLa/bcl-2 cells. That is, the concentrations of TC-A that induced the complete cleavage of 116K PARP were 2.2–3.3 ␮M, using all three of the different apoptotic stimuli. These results suggest that TC-A inhibits a point at which independent signaling pathways to apoptosis

converge, most likely Bcl-2/Bcl-XL or common effector machinery

that can be antagonized by Bcl-2/Bcl-XL. The effect of TC-A on the anti-apoptotic function of Bcl-2 was further tested by an experimental system in which the overexpression of Bax, a pro-apoptotic homologue of Bcl-2, induced apoptosis in 293 cells (Fig. 5). The transient expression of Bax induced the activation of caspases in 293 cells, as indicated by PARP-cleavage. Coexpres-

sion of Bcl-2 or Bcl-XL suppressed Bax-induced apoptosis. The addition of TC-A to the Bax/Bcl-2 coexpressing cells at 24 h after transfection restored the PARP-cleavage induced by Bax, which indicated that TC-A inhibited the anti-apoptotic function of the Bcl-2 family in Bax-mediated apoptosis. TC-A Preferentially Inhibits the Anti-apoptotic Function of the Bcl-2 Family in Cell-Death Signaling Pathways. To gain further evidence supporting the theory that TC-A acts on Bcl-2 family members in the cell-death signaling pathway and thereby inhibits their anti-apoptotic functions, the effect(s) of TC-A on cell death suppres- sors other than the Bcl-2 family was studied. The cowpox virus CrmA is a viral serpin protein that can inhibit caspase family proteases (36). FADD mediates cell death by Fas and TNF-␣ by recruiting caspase-8 to their receptors. A dominant negative mutant of FADD (FADD-DN)

lacks the 80 NH2-terminal amino acids, which contains the death effector domain but retains the death domain. Overexpression of Fig. 7. The effect of TC-A on the localization of Bcl-2 in HeLa cells. HeLa/bcl-2 cells FADD-DN inhibits cell death initiated by Fas and TNF-␣ (37). HeLa were treated with DMSO (a)or2␮M TC-A (b) for 12 h and were then fixed and stained cells were transiently transfected with expression vectors encoding as described in “Materials and Methods.” CrmA, FADD-DN, Bcl-2, or Bcl-XL. The transfected cells were treated with TNF-␣/CHX together with TC-A, and the apoptotic cells TC-A Suppresses the Death-suppressor Activity of Bcl-2 in were measured as described in “Materials and Methods. Similar to the Apoptosis Induced by a Variety of Cell-Death Stimuli. To deter- results obtained with the HeLa cells stably transfected with bcl-2 or mine whether the effect of TC-A was restricted to Fas-induced apop- bcl-XL, the anti-apoptotic functions of Bcl-2 or Bcl-XL were inhibited tosis, we tested other apoptosis-inducing agents (Fig. 4). HeLa cells by TC-A. In contrast to this, TC-A did not show any effects on the are susceptible to cell death by TNF-␣/CHX or STA. HeLa/bcl-2 cells anti-apoptotic function(s) of CrmA and FADD-DN, as shown in Fig. are resistant to apoptosis induced by those same cell-death stimuli (28, 6. Overexpression of CrmA inhibited apoptosis induced by TNF␣, 35) but could be made sensitive by TC-A treatment. With increasing with 24% of the cells surviving, but the ratio of surviving cells remain concentrations of TC-A, the Ac-DEVD-MCA- and PARP-cleaving unchanged in the presence of TC-A. HeLa cells transfected with activities also increased, with a concomitant decrease in cell viability. FADD-DN were almost completely resistant to TNF-␣/CHX. Treat-

⌬⌿ ␣ Fig. 8. The effect of TC-A on Fas-induced m loss and cytochrome c release. A, DiOC6(3) staining of HeLa or HeLa/bcl-2 untreated or treated with Fas/CHX in the presence of various concentrations of TC-A. B, Western blot analysis of the release of cytochrome c in HeLa and HeLa/bcl-2 cells. Cells were treated with reagents as indicated and were fractionated and analyzed as described in “Materials and Methods.” 1233

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. TC-A INHIBITS MITOCHONDRIAL FUNCTIONS OF BCL-2 ment of these cells with TC-A did not affect the anti-apoptotic also be of therapeutic utility in the treatment of diseases that are function of FADD-DN. Taken together, these results confirmed that associated with the overexpression of Bcl-2 family members. TC-A does not generally inhibit anti-apoptotic effectors but preferentially inhibits the anti-apoptotic function of the Bcl-2 family in the cell-death-signaling pathways of HeLa cells. ACKNOWLEDGMENTS TC-A Does Not Affect the Subcellular Localization of Bcl-2. We thank K. Hasegawa and Y. Akasaka for technical assistance; T. J. The proper localization of Bcl-2 in intracellular membranes is re- McDonnell, E. M. Bruckheimer, M. Quinlan and S. Sharma for critical reading quired for its death-suppressor activity. Bcl-2 mutants lacking the of the manuscript; and T. Mizukami, S. Akinaga, J. Kanazawa, T. Tamaoki, M. COOH-terminal, membrane-anchoring tail are localized mainly in the Okabe, K. Inoue and H. Nakano for helpful discussions and continuous cytosolic fraction. These mutants exhibit a greatly reduced anti- support. apoptotic activity when compared with wild-type Bcl-2 (38, 39). Therefore, the inhibition of the subcellular localization of Bcl-2 could lead to the inhibition of the anti-apoptotic function of Bcl-2. To test REFERENCES whether TC-A affected the subcellular localization of Bcl-2, HeLa/ 1. Korsmeyer, S. J. Regulation of cell death. Trends Genet., J11: 101–105, 1995. bcl-2 cells treated with TC-A were incubated with anti-Bcl-2 antibody 2. Strasser, A., Huang, D. C. S., and Vaux, D. L. The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumorigenesis and processed for immunofluorescence. In the absence of TC-A, and resistance to chemotherapy. Biochim. Biophys. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. Tetrocarcin A Inhibits Mitochondrial Functions of Bcl-2 and Suppresses Its Anti-apoptotic Activity

Takayuki Nakashima, Masayuki Miura and Mitsunobu Hara

Cancer Res 2000;60:1229-1235.

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