Targeting of the c-Abl Tyrosine Kinase to Mitochondria in the Necrotic Cell Death Response to Oxidative Stress*

Shailendra KumarDagger , Ajit BhartiDagger , Neerad C. Mishra§, Deepak RainaDagger , Surender KharbandaDagger , Satya Saxena§, and Donald KufeDagger

From the Dagger  Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and the § Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87115

Received for publication, February 14, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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The ubiquitously expressed c-Abl tyrosine kinase is activated in the response of cells to genotoxic and oxidative stress. The present study demonstrates that reactive oxygen species (ROS) induce targeting of c-Abl to mitochondria. We show that ROS-induced localization of c-Abl to mitochondria is dependent on activation of protein kinase C (PKC)delta and the c-Abl kinase function. Targeting of c-Abl to mitochondria is associated with ROS-induced loss of mitochondrial transmembrane potential. The results also demonstrate that c-Abl is necessary for ROS-induced depletion of ATP and the activation of a necrosis-like cell death. These findings indicate that the c-Abl kinase targets to mitochondria in response to oxidative stress and thereby mediates mitochondrial dysfunction and cell death.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Reactive oxygen species (ROS)1 have been implicated in the regulation of both mitogenic and apoptotic signaling pathways. Mitogenic signals induced by certain growth factors and activated Ras are mediated by ROS production (1, 2). The generation of ROS through normal cellular metabolism has also been associated with damage to cellular components and the induction of apoptosis (3, 4). However, few insights are available regarding the mechanisms responsible for ROS-induced cell death. For example, ROS induce topoisomerase II-mediated cleavage of chromosomal DNA and thereby cell death (5). The p66shc adapter protein (6) and the p85 subunit of phosphatidylinositol 3-kinase (7) have also been implicated in the apoptotic response to oxidative stress. Other studies have indicated that p53-induced apoptosis is mediated by ROS (5, 8, 9) and that ROS-induced apoptosis is p53-dependent (6, 7).

The ubiquitously expressed c-Abl protein-tyrosine kinase localizes to the nucleus and cytoplasm. The nuclear form of c-Abl is activated in the cellular response to genotoxic stress (10) and contributes to the induction of apoptosis by mechanisms in part dependent on p53 and its homolog, p73 (11-15). c-Abl also functions upstream to the proapoptotic stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 MAPK pathways (10, 16-18). Other studies have demonstrated that the cytoplasmic form of c-Abl is activated in response to oxidative stress (19). ROS induce c-Abl activation by a mechanism dependent on protein kinase Cdelta (PKCdelta ) (20). Moreover, the evidence indicates that c-Abl is required for ROS-induced mitochondrial cytochrome c release and apoptosis (19). Although these findings have provided support for involvement of c-Abl in the induction of apoptosis by oxidative stress, few insights are available regarding the signals conferred by c-Abl in this response.

In the present studies, we show that treatment of cells with H2O2 induces translocation of c-Abl to mitochondria. The results demonstrate that mitochondrial targeting of c-Abl is dependent on PKCdelta and the c-Abl kinase function. We also demonstrate that c-Abl mediates ROS-induced loss of mitochondrial transmembrane potential (psi m), depletion of ATP, and a necrosis-like cell death.

    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Cell Culture-- Human U-937 myeloid leukemia cells (ATCC, Manassas, VA) were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. Wild-type c-Abl-/-, c-Abl+ mouse embryo fibroblasts (MEFs) (10, 21), MCF-7, MCF-7/c-Abl(K-R), SH-SY5Y (neuroblastoma), and 293T cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. Cells were treated with 1 mM H2O2 (Sigma) and 30 mM N-acetyl-L-cysteine (Sigma). Transient transfections were performed in the presence of calcium phosphate.

Immunofluorescence Microscopy-- Cells were plated onto poly-D-lysine-coated glass coverslips 1 day prior to H2O2 treatment (1 h) and then fixed with 3.7% formaldehyde and phosphate-buffered saline (PBS) (pH 7.4) for 10 min. Cells were washed with PBS, permeabilized with 0.2% Triton X-100 for 10 min, washed again, and incubated for 30 min in complete medium. The coverslips were then incubated with 5 µg/ml anti-c-Abl (K-12) for 1 h followed by Texas Red-goat anti-rabbit Ig (H + L) conjugate (Molecular Probes, Eugene, OR). Mitochondria were stained with 100 nM Mitotracker Green FM (Molecular Probes). Nuclei were stained with 4,6-diamino-2-phenylindole (1 µg/ml in PBS). Coverslips were mounted onto slides with 0.1 M Tris (pH 7.0) in 50% glycerol. Cells were visualized by digital confocal immunofluorescence, and images were captured with a CCD camera mounted on a Zeiss Axioplan 2 microscope. Images were deconvolved using Slidebook software (Intelligent Imaging Innovations, Inc., Denver, CO).

Isolation of Mitochondria-- Cells (3 × 106) were washed twice with PBS, homogenized in buffer A (210 mM mannitol, 70 mM sucrose, 1 mM EGTA, 5 mM HEPES, pH 7.4) and 110 µg/ml digitonin in a glass homogenizer (Pyrex no. 7727-07) and centrifuged at 5000 × g for 20 min. Pellets were resuspended in buffer A, homogenized in a small glass homogenizer (Pyrex no. 7726) and centrifuged at 2000 × g for 5 min. The supernatant was collected and centrifuged at 11,000 × g for 10 min. Mitochondrial pellets were disrupted in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 mM sodium fluoride, 10 µg/ml leupeptin and aprotinin) at 4 °C and then centrifuged at 15,000 × g for 15 min. Protein concentration was determined by the Bio-Rad protein estimation kit.

Preparation of Cell Lysates-- Whole cell lysates were prepared as described (10) and analyzed for protein concentration.

Immunoprecipitation and Immunoblot Analysis-- Soluble proteins (100 µg) were incubated with anti-c-Abl (K-12; Santa Cruz Biotechnology) for 1 h and precipitated with protein A-Sepharose for 30 min. Immunoprecipitates and lysates (5 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting with anti-c-Abl (24-11; Santa Cruz), anti-heat shock protein 60 (StressGen, Victoria BC, Canada), anti-beta -actin (Sigma), anti-proliferating cell nuclear antigen (Calbiochem), anti-platelet-derived growth factor receptor (Oncogene), and anti-PKCdelta (Santa Cruz).

Analysis of Mitochondrial Membrane Potential-- Cells were incubated with 50 ng/ml rhodamine 123 (Molecular Probes) for 15 min at 37 °C. After washing with PBS, samples were analyzed by flow cytometry using 488 nm excitation and the measurement of emission through a 575/26 (ethidium) bandpass filter.

Quantitation of ATP-- ATP levels were measured using an ATP Determination Kit (Molecular Probes).

Assessment of Apoptosis and Necrosis by Flow Cytometry-- Cells were analyzed by staining with annexin-V-fluorescein and propidium iodide (Annexin-V-FLOUS staining kit; Roche Diagnostics). Samples were analyzed by flow cytometry (BD Bioscience, San Jose, CA) using 488 nm excitation and a 515-nm bandpass filter for fluorescein detection and a >600 nm-filter for propidium iodide detection.

    RESULTS AND DISCUSSION
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To assess the effects of ROS on c-Abl, we investigated the subcellular localization of c-Abl in response to H2O2 by measuring intracellular fluorescence with a high sensitivity CCD camera and image analyzer. Examination of the distribution of fluorescence markers in control MEFs showed distinct patterns for anti-c-Abl (red signal) and a mitochondrion-selective dye (Mitotracker; green signal). By contrast exposure to H2O2 was associated with a marked change in fluorescence signals (red and green right-arrow yellow and orange) supporting translocation of c-Abl to mitochondria (Fig. 1A). To confirm targeting of c-Abl to mitochondria in response to ROS, mitochondria were isolated from MEFs treated with H2O2. Analysis of the mitochondrial fraction by immunoblotting with anti-c-Abl demonstrated an increase in c-Abl protein that was detectable at 30 min and through 3 h (Fig. 1B). Densitometric scanning of the signals demonstrated over a 5-fold increase in c-Abl protein at 0.5-1 h of H2O2 treatment (Fig. 1B). Immunoblotting for the mitochondrial HSP60 protein was used to assess loading of the lanes (Fig. 1B). Moreover, purity of the mitochondrial fraction was confirmed by reprobing the blots with antibodies against the cytoplasmic beta -actin protein, the proliferating cell nuclear antigen protein, and the cell membrane platelet-derived growth factor receptor (Fig. 1B). To estimate the amount of c-Abl protein that localizes to mitochondria, we subjected whole cell and mitochondrial lysates, each prepared from 3 × 106 cells, to immunoblotting with anti-c-Abl. Densitometric scanning of the signals (Fig. 1C) and adjustment for lysate volume indicated that mitochondrial c-Abl is ~4% of the total cellular c-Abl protein. Following treatment with H2O2, ~20% of total c-Abl localized to mitochondria (Fig. 1B). As an additional control, mitochondrial lysates were first subjected to immunoprecipitation with anti-c-Abl. Immunoblot analysis of the immunoprecipitates with anti-c-Abl showed H2O2-induced increases in levels of mitochondrial c-Abl protein (Fig. 1D). The demonstration that c-Abl levels are increased in the mitochondria of H2O2-treated human U-937 leukemia cells (Fig. 1E) and human neuroblastoma cells (data not shown) further indicated that this response occurs in diverse cell types.


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Fig. 1.   Localization of c-Abl to mitochondria in response to H2O2 treatment. A, MEFs were treated with 1 mM H2O2 for 1 h. After washing, the cells were fixed and incubated with anti-c-Abl followed by Texas Red-conjugated goat anti-rabbit IgG. Mitochondria were stained with the mitochondrial selective dye Mitotracker Green and nuclei with 4,6-diamino-2-phenylindole. The slides were visualized using a fluorescence microscope coupled to a high sensitivity CCD camera and image analyzer. Red signal, c-Abl. Green signal, Mitotracker. Yellow/orange signals, colocalization of c-Abl and Mitotracker. B, MEFs were treated with 1 mM H2O2 for the indicated times. The mitochondrial fraction (5 µg) was subjected to immunoblotting with anti-c-Abl, anti-HSP60, anti-actin, anti-proliferating cell nuclear antigen (PCNA) and anti-platelet-derived growth factor receptor (PDGF-R). C, MEFs (6 × 106) were divided into two aliquots for preparation of whole cell lysates (WCL) and mitochondrial lysates. Samples (25 µl) of the whole cell lysates (total volume 500 µl) and of the mitochondrial lysate (total volume 100 µl) were subjected to immunoblot analysis with anti-c-Abl. D, mitochondrial lysates were immunoprecipitated with anti-c-Abl and then analyzed by blotting with anti-c-Abl. E, mitochondrial lysates from U-937 myeloid leukemia cells treated with 1 mM H2O2 for the indicated times were subjected to immunoblot analysis with the indicated antibodies.

To confirm involvement of ROS in targeting of c-Abl to mitochondria, MEFs were treated with N-acetyl-L-cysteine, a scavenger of reactive oxygen intermediates and precursor of glutathione (22, 23). N-Acetyl-L-cysteine treatment inhibited H2O2-induced translocation of c-Abl to mitochondria (Fig. 2A). Also to determine whether ROS-induced activation of the c-Abl kinase function is necessary for targeting of c-Abl to mitochondria, MCF-7 cells stably expressing a kinase-inactive c-Abl(K-R) mutant at levels comparable with that of kinase-active c-Abl in MCF-7/neo cells (Fig. 2B, left panel) (11) were treated with H2O2. The finding that MCF-7/neo, but not MCF-7/c-Abl(K-R), cells respond to H2O2 with translocation of c-Abl to mitochondria supported a requirement for the c-Abl kinase function (Fig. 2B, right panel). These results indicate that ROS-induced c-Abl activation is associated with the targeting of c-Abl to mitochondria.


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Fig. 2.   Targeting of c-Abl to mitochondria is dependent on ROS-induced c-Abl activation. A, MEFs were pretreated with 30 mM N-acetyl-L-cysteine (NAC) for 1 h before adding H2O2 for an additional 1 h. Mitochondrial lysates were subjected to immunoblot analysis with anti-c-Abl, anti-HSP60, and anti-beta -actin. B, mitochondrial lysates from MCF-7 and MCF-7/c-Abl(K-R) cells treated with H2O2 were analyzed by immunoblotting with anti-c-Abl and anti-beta -actin.

The available evidence indicates that ROS activate c-Abl by a mechanism dependent on the PKCdelta kinase (19, 20). To determine whether PKCdelta contributes to mitochondrial targeting of c-Abl, MEFs were treated with the selective PKCdelta inhibitor, rottlerin (24). Although rottlerin had no effect on constitutive levels of mitochondrial c-Abl, this agent inhibited H2O2-induced c-Abl translocation (Fig. 3A). To extend the interaction of PKCdelta and c-Abl, 293 cells were cotransfected with HA-c-Abl and PKCdelta . Analysis of the mitochondrial fraction by immunoblotting with anti-HA demonstrated that targeting of HA-c-Abl to mitochondria is increased by H2O2 treatment (Fig. 3B). By contrast, cotransfection of HA-c-Abl and kinase-inactive PKCdelta was associated with less targeting of c-Abl to mitochondria and no apparent effect of H2O2 treatment (Fig. 3B). These findings provide support for the involvement of ROS-induced activation of PKCdelta in mitochondrial targeting of c-Abl.


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Fig. 3.   PKCdelta is required for targeting of c-Abl to mitochondria. A, MEFs were pretreated with 10 µM rottlerin for 0.5 h and then with H2O2 for 1 h. Mitochondrial lysates were analyzed by immunoblotting with anti-c-Abl. B, 293T cells were cotransfected to express HA-c-Abl and GFP-PKCdelta or GFP-PKCdelta (K-R). At 24 h, the cells were treated with H2O2 for 2 h. Lysates prepared from the mitochondrial fraction and intact cells were subjected to immunoblot analysis with anti-GFP and anti-HA.

Although the results indicate that c-Abl targets mitochondria in the ROS response, we hypothesized that c-Abl may directly induce mitochondrial dysfunction. To determine whether c-Abl is necessary for ROS-induced loss of psi m, wild-type and c-Abl-/- cells were treated with H2O2 and then stained with rhodamine 123. Mitochondrial transmembrane potential was substantially diminished in H2O2-treated wild-type cells (Fig. 4A). By contrast, the psi m was protected from ROS-induced loss in c-Abl-/- cells, but not in c-Abl-/- cells transfected to stably express c-Abl (c-Abl+) (Fig. 4A). Cyclosporin A prevents the reduction in psi m induced by various agents that open mitochondrial permeability transition pores (25). In this context, pretreatment of wild-type MEFs with cyclosporin A (100 µM for 1 h) abrogated the H2O2-induced change in psi m (data not shown). Both apoptosis and necrosis are associated with decreases in psi m, whereas necrosis is distinguished from apoptosis by depletion of ATP and an early loss of plasma membrane integrity (26, 27). To assess the involvement of c-Abl in ROS-induced necrosis, wild-type, c-Abl-/-, and c-Abl+ cells were treated with H2O2 and assayed for ATP levels. The results demonstrate that, whereas H2O2 treatment of wild-type and c-Abl+ cells is associated with depletion of ATP, this response was attenuated in c-Abl-/- cells (Fig. 4B). Because these findings support the involvement of c-Abl in a necrosis-like cell death, cells were stained with both annexin-V and propidium iodide to assess plasma membrane integrity. The results demonstrate that, compared with wild-type and c-Abl+ MEFs, loss of plasma membrane integrity in response to H2O2 is attenuated in c-Abl-/- cells (Fig. 4C). These findings demonstrate that ROS-induced targeting of c-Abl to mitochondria is associated with loss of mitochondrial membrane potential, ATP depletion, and necrotic cell death.


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Fig. 4.   Targeting of c-Abl to mitochondria is associated with loss of mitochondrial transmembrane potential (psi m), depletion of ATP, and necrosis-like cell death. Wild-type (solid bars), c-Abl-/- (open bars) and c-Abl+ (hatched bars) MEFs were treated with H2O2 for the indicated times. A, cells were stained with Rhodamine 123 and analyzed by flow cytometry (upper panels). Percentage (mean ± S.E.) of control psi m obtained from 3 separate experiments (lower panels). B, cells were analyzed for ATP levels. The data represent the percentage (mean ± S.E.) of control ATP levels obtained from three separate experiments. C, cells stained with annexin-V-fluorescein and propidium iodide were analyzed by flow cytometry. The data represent the percentage (mean ± S.E.) of annexin-V-positive, propidium iodide-positive cells obtained in three separate experiments.

Activation of the c-Abl kinase in the cellular response to oxidative stress is dependent on PKCdelta and associated with release of mitochondrial cytochrome c (19, 20). These findings provided support for involvement of c-Abl in the regulation of mitochondrial signaling. The present studies demonstrate that ROS target the c-Abl protein to mitochondria and that this response is dependent on the c-Abl kinase function. Moreover, in concert with the demonstration that PKCdelta is required for ROS-induced activation of c-Abl (20), we show that PKCdelta is necessary for targeting of c-Abl to mitochondria. Importantly localization of c-Abl to mitochondria is associated with loss of the mitochondrial transmembrane potential. Apoptosis and necrosis both involve the loss of mitochondrial transmembrane potential, whereas depletion of ATP is found selectively in necrosis (28). Thus, the demonstration that mitochondrial targeting of c-Abl is associated with depletion of ATP indicated that c-Abl is functional in necrosis-like cell death. In this context, wild-type, but not c-Abl-/-, MEFs also responded to oxidative stress with dysfunction of the plasma membrane. These findings and the previous demonstration that c-Abl is involved in ROS-induced cytochrome c release (19) indicate that targeting of c-Abl to mitochondria confers both proapoptotic and pronecrotic cell death signals.

    FOOTNOTES

* This work was supported by Grant CA42802 awarded by the NCI, National Institutes of Health, the Department of Health and Human Services, and by the office of Health and Biological Research, United States Department of Energy cooperative agreement DE-FC04-96AL76406.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dana-Farber Cancer Inst., 44 Binney St., Boston, MA 02115. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: donald_kufe@dfci.harvard.edu.

Published, JBC Papers in Press, February 28, 2001, DOI 10.1074/jbc.M101414200

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; PKC, protein kinase C; psi m, mitochondrial transmembrane potential; HA, hemagglutinin; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; GFP, green fluorescent protein..

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

1. Sundaresan, M., Yu, Z.-X., Ferrans, V., Irani, K., and Finkel, T. (1995) Science 270, 296-299[Abstract]
2. Irani, K., Xia, Y., Zweier, J. L., Sollott, S. J., Der, C. J., Fearon, E. R., Sundaresan, M., Finkel, T., and Goldschmidt-Clermont, P. J. (1997) Science 275, 1649-1652[Abstract/Free Full Text]
3. Croteau, D., and Bohr, V. (1997) J. Biol. Chem. 272, 25409-25412[Free Full Text]
4. Berlett, S., and Stadtman, E. (1997) J. Biol. Chem. 272, 20313-20316[Free Full Text]
5. Li, T., Chen, A., Yu, C., Mao, Y., Wang, H., and Liu, L. (1999) Genes Dev. 13, 1553-1560[Abstract/Free Full Text]
6. Migliaccio, E., Giorgio, M., Mele, S., Pelicci, G., Reboldi, P., Pandolfi, P. P., Lanfrancone, L., and Pelicci, P. G. (1999) Nature 402, 309-313[CrossRef][Medline] [Order article via Infotrieve]
7. Yin, Y., Terauchi, Y., Solomon, G., Aizawa, S., Rangarajan, P., Yazaki, Y., Kadowaki, T., and Barrett, J. (1998) Nature 391, 707-710[CrossRef][Medline] [Order article via Infotrieve]
8. Johnson, T., Yu, Z., Ferrans, V., Lowenstein, R., and Finkel, T. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11848-11852[Abstract/Free Full Text]
9. Polyak, K., Xia, Y., Zweier, J., Kinzler, K., and Vogelstein, B. (1997) Nature 389, 300-305[CrossRef][Medline] [Order article via Infotrieve]
10. Kharbanda, S., Ren, R., Pandey, P., Shafman, T. D., Feller, S. M., Weichselbaum, R. R., and Kufe, D. W. (1995) Nature 376, 785-788[CrossRef][Medline] [Order article via Infotrieve]
11. Yuan, Z. M., Huang, Y., Whang, Y., Sawyers, C., Weichselbaum, R., Kharbanda, S., and Kufe, D. (1996) Nature 382, 272-274[CrossRef][Medline] [Order article via Infotrieve]
12. Yuan, Z., Huang, Y., Fan, M.-m., Sawers, C., Kharbanda, s., and Kufe, D. (1996) J. Biol. Chem. 271, 26457-26460[Abstract/Free Full Text]
13. Yuan, Z. M., Shioya, H., Ishiko, T., Sun, X., Huang, Y., Lu, H., Kharbanda, S., Weichselbaum, R., and Kufe, D. (1999) Nature 399, 814-817[CrossRef][Medline] [Order article via Infotrieve]
14. Gong, J., Costanzo, A., Yang, H., Melino, G., Kaelin JR, W., Levrero, M., and Wang, J. Y. J. (1999) Nature 399, 806-809[CrossRef][Medline] [Order article via Infotrieve]
15. Agami, R., Blandino, G., Oren, M., and Shaul, Y. (1999) Nature 399, 809-813[CrossRef][Medline] [Order article via Infotrieve]
16. Kharbanda, S., Pandey, P., Ren, R., Feller, S., Mayer, B., Zon, L., and Kufe, D. (1995) J. Biol. Chem. 270, 30278-30281[Abstract/Free Full Text]
17. Pandey, P., Raingeaud, J., Kaneki, M., Weichselbaum, R., Davis, R., Kufe, D., and Kharbanda, S. (1996) J. Biol. Chem. 271, 23775-23779[Abstract/Free Full Text]
18. Kharbanda, S., Pandey, P., Yamauchi, T., Kumar, S., Kaneki, M., Kumar, V., Bharti, A., Yuan, Z., Ghanem, L., Rana, A., Weichselbaum, R., Johnson, G., and Kufe, D. (2000) Mol. Cell. Biol. 20, 4979-4989[Abstract/Free Full Text]
19. Sun, X., Majumder, P., Shioya, H., Wu, F., Kumar, S., Weichselbaum, R., Kharbanda, S., and Kufe, D. (2000) J. Biol. Chem. 275, 17237-17240[Abstract/Free Full Text]
20. Sun, X., Wu, F., Datta, R., Kharbanda, S., and Kufe, D. (2000) J. Biol. Chem. 275, 7470-7473[Abstract/Free Full Text]
21. Tybulewicz, V. L. J., Crawford, C. E., Jackson, P. K., Bronson, R. T., and Mulligan, R. C. (1991) Cell 65, 1153-1163[Medline] [Order article via Infotrieve]
22. Roederer, M., Staal, F. J., Raju, P. A., Ela, S. W., and Herzenberg, L. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4884-4888[Abstract]
23. Staal, F. J., Roederer, M., Herzenberg, L. A., and Herzenberg, L. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9943-9947[Abstract]
24. Gschwendt, M., Kielbassa, K., Kittstein, W., and Marks, F. (1994) FEBS Lett. 347, 773-779
25. Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S. A., Petit, P. X., Mignotte, B., and Kroemer, G. (1995) J. Exp. Med. 182, 367-377[Abstract]
26. Leist, M., Single, B., Castoldi, A. F., Kuhnle, S., and Nicotera, P. (1997) J. Exp. Med. 185, 1481-1486[Abstract/Free Full Text]
27. Nicotera, P., Leist, M., and Ferrando-May, E. (1998) Toxicology Letters 102-103, 139-142
28. Ha, H. C., and Snyder, S. H. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 13978-13982[Abstract/Free Full Text]


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