Advertisement

Comparison of Paclitaxel-, 5-Fluoro-2′-deoxyuridine-, and Epidermal Growth Factor (EGF)-induced Apoptosis

EVIDENCE FOR EGF-INDUCED ANOIKIS*
Open AccessPublished:May 28, 1999DOI:https://doi.org/10.1074/jbc.274.22.15927
      Epidermal growth factor (EGF), a hormone that stimulates proliferation of many cell types, induces apoptosis in some cell lines that overexpress the EGF receptor. To evaluate the mechanism of EGF-induced apoptosis, MDA-MB-468 breast cancer cells were examined by microscopy, flow cytometry, immunoblotting, enzyme assays, and affinity labeling after treatment with EGF, paclitaxel, or 5-fluoro-2′-deoxyuridine (5FUdR). Apoptosis induced by all three agents was accompanied by activation of caspases-3, -6, and -7, as indicated by disappearance of the corresponding zymogens from immunoblots, cleavage of substrate polypeptides in situ, and detection of active forms of these caspases in cytosol and nuclei using fluorogenic assays and affinity labeling. Further analysis indicated involvement of the cytochrome c/Apaf-1/caspase-9 pathway of caspase activation, but not the Fas/Fas ligand pathway. Interestingly, caspase activation was consistently lower after EGF treatment than after paclitaxel or 5FUdR treatment. Additional experiments revealed that the majority of cells detaching from the substratum after EGF (but not paclitaxel or 5FUdR) were morphologically normal and retained the capacity to readhere, suggesting that EGF-induced apoptosis involves cell detachment followed by anoikis. These observations not only indicate that EGF- and chemotherapy-induced apoptosis in this cell line involve the same downstream pathways but also suggest that detachment-induced apoptosis is responsible for the paradoxical antiproliferative effects of EGF.
      EGF
      The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; 5FUdR, 5-fluoro-2′-deoxyuridine; caspase, cysteine-dependent aspartate-directed protease; AFC, 7-amino-4-trifluoromethylcoumarin; DEVD-AFC, aspartylglutamylvalinylaspartyl-AFC; VEID-AFC, valinylglutamylisoleucylaspartyl-AFC; YVAD-AFC, tyrosinylvalinylalanylaspartyl-AFC; FAK, focal adhesion kinase; FasL, Fas ligand; PARP, poly(ADP-ribose) polymerase; PCD, programmed cell death; poly(HEMA), poly(2-hydroxyethyl methacrylate); Z-EK(bio)D-aomk, N-(N α-benzyloxycarbonylglutamyl-N ε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone
      1The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; 5FUdR, 5-fluoro-2′-deoxyuridine; caspase, cysteine-dependent aspartate-directed protease; AFC, 7-amino-4-trifluoromethylcoumarin; DEVD-AFC, aspartylglutamylvalinylaspartyl-AFC; VEID-AFC, valinylglutamylisoleucylaspartyl-AFC; YVAD-AFC, tyrosinylvalinylalanylaspartyl-AFC; FAK, focal adhesion kinase; FasL, Fas ligand; PARP, poly(ADP-ribose) polymerase; PCD, programmed cell death; poly(HEMA), poly(2-hydroxyethyl methacrylate); Z-EK(bio)D-aomk, N-(N α-benzyloxycarbonylglutamyl-N ε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone
      is a 6-kDa polypeptide that binds to a 170-kDa cell surface receptor (the EGFR) expressed on a wide variety of normal and neoplastic cells (reviewed in Refs.
      • Khazaie K.
      • Schirrmacher V.
      • Lichtner R.B.
      ,
      • Chrysogelos S.A.
      • Dickson R.B.
      ,
      • Rusch V.
      • Mendelsohn J.
      • Dmitrovsky E.
      ,
      • Kelloff G.J.
      • Fay J.R.
      • Steele V.E.
      • Lubet R.A.
      • Boone C.W.
      • Crowell J.A.
      • Sigman C.C.
      ). The interaction of these two molecules results in activation of the EGFR tyrosine kinase, which in turn activates the mitogen-activated protein kinase and Janus kinase signaling pathways (reviewed in Refs.
      • Khazaie K.
      • Schirrmacher V.
      • Lichtner R.B.
      ,
      • Chrysogelos S.A.
      • Dickson R.B.
      ,
      • Rusch V.
      • Mendelsohn J.
      • Dmitrovsky E.
      ,
      • Kelloff G.J.
      • Fay J.R.
      • Steele V.E.
      • Lubet R.A.
      • Boone C.W.
      • Crowell J.A.
      • Sigman C.C.
      ; see also Refs.
      • Chin Y.E.
      • Kitagawa M.
      • Kuida K.
      • Flavell R.A.
      • Fu X.
      and
      • Kato Y.
      • Tapping R.I.
      • Huang S.
      • Watson M.H.
      • Ulevitch R.J.
      • Lee J.-D.
      ). In most target cells, these events result in proliferation (
      • Khazaie K.
      • Schirrmacher V.
      • Lichtner R.B.
      ,
      • Chrysogelos S.A.
      • Dickson R.B.
      ,
      • Rusch V.
      • Mendelsohn J.
      • Dmitrovsky E.
      ,
      • Kelloff G.J.
      • Fay J.R.
      • Steele V.E.
      • Lubet R.A.
      • Boone C.W.
      • Crowell J.A.
      • Sigman C.C.
      ). In a number of cell lines, however, EGF paradoxically inhibits proliferation (
      • Filmus J.
      • Pollak M.N.
      • Cailleau R.
      • Buick R.N.
      ,
      • Lee K.
      • Tanaka M.
      • Hatanaka M.
      • Kuze F.
      ,
      • Ennis B.W.
      • Valverius E.M.
      • Bates S.E.
      • Lippman M.E.
      • Bellot F.
      • Kris R.
      • Schlessinger J.
      • Masui H.
      • Goldenberg A.
      • Mendelsohn J.
      • Dickson R.B.
      ,
      • Dong X.-F.
      • Berthois Y.
      • Martin P.M.
      ,
      • Minke J.M.H.M.
      • Schuuring E.
      • van den Berghe R.
      • Stolwijk J.A.M.
      • Roonstra J.
      • Cornelisse C.
      • Hilkens J.
      • Misdorp W.
      ,
      • Argilé A.
      • Kraft N.
      • Ootaka T.
      • Hutchinson P.
      • Atkins R.C.
      ). Studies of A431 epidermoid carcinoma cells and MDA-MB-468 breast cancer cells suggest that this inhibition of proliferation results from EGF-induced apoptosis (
      • Chin Y.E.
      • Kitagawa M.
      • Kuida K.
      • Flavell R.A.
      • Fu X.
      ,
      • Armstrong D.K.
      • Kaufmann S.H.
      • Ottaviano Y.L.
      • Furuya Y.
      • Buckley J.A.
      • Isaacs J.T.
      • Davidson J.A.
      ,
      • Schaerli P.
      • Jaggi R.
      ). Because the mechanism by which EGF induces apoptosis is not known, the physiological significance of these findings is unclear.
      Apoptosis is a morphologically and biochemically distinct form of PCD that occurs in many cell types after withdrawal of trophic stimuli (
      • Eastman A.
      ,
      • Park J.R.
      ,
      • Sachs L.
      ,
      • May W.S.
      ) or treatment with a wide variety of cytotoxic agents (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ,
      • Arends M.J.
      • Wyllie A.H.
      ). Current models separate the apoptotic process into at least two distinct phases, initiation and execution. The initiation phase involves biochemical changes that might be unique to each apoptotic stimulus (
      • Ucker D.S.
      ). In contrast, the execution phase involves a series of stereotypic morphological and biochemical changes (
      • Wyllie A.H.
      • Kerr J.F.R.
      • Currie A.R.
      ) that appear to result from the action of cysteine-dependent aspartate-directed proteases called caspases (
      • Alnemri E.S.
      • Livingston D.J.
      • Nicholson D.W.
      • Salvesen G.
      • Thornberry N.A.
      • Wong W.W.
      • Yuan J.
      ).
      In most cell types, caspases are constitutively expressed as zymogens that require proteolytic cleavage for activation (
      • Weil M.
      • Jacobson M.D.
      • Coles H.S.
      • Davies T.J.
      • Gardner R.L.
      • Raff K.D.
      • Raff M.C.
      ,
      • Martins L.M.
      • Kottke T.J.
      • Mesner P.W.
      • Basi G.S.
      • Sinha S.
      • Frigon Jr., N.
      • Tatar E.
      • Tung J.S.
      • Bryant K.
      • Takahashi A.
      • Svingen P.A.
      • Madden B.J.
      • McCormick D.J.
      • Earnshaw W.C.
      • Kaufmann S.H.
      ). Three canonical pathways of caspase activation have been identified. First, caspases can be activated by granzyme B, a major serine protease in cytotoxic lymphocyte granules (reviewed in Refs.
      • Greenberg A.H.
      and
      • Thornberry N.A.
      • Rosen A.
      • Nicholson D.W.
      ). Second, ligation of cell surface death receptors, such as Fas (the cell surface polypeptide also known as CD95 or Apo-1) or the type 1 tumor necrosis factor α receptor, results in binding of adaptor molecules, which in turn recruit procaspases-2, -8, and -10 to membrane-associated signaling complexes, leading to proximity-induced activation of at least some of these caspases (reviewed in Refs.
      • Fraser A.
      • Evan G.
      , ,
      • Nicholson D.W.
      • Thornberry N.A.
      ,
      • Wallach D.
      • Boldin M.
      • Varfolomeev E.
      • Beyaert R.
      • Vandenabeele P.
      • Fiers W.
      ,
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ). After activation, these upstream caspases appear capable of directly cleaving precursors of effector caspases (reviewed in Refs.
      • Fraser A.
      • Evan G.
      , ,
      • Nicholson D.W.
      • Thornberry N.A.
      ,
      • Wallach D.
      • Boldin M.
      • Varfolomeev E.
      • Beyaert R.
      • Vandenabeele P.
      • Fiers W.
      ,
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ). Finally, in some model systems, mitochondria are induced to release cytochromec, which interacts with the cytosolic docking protein Apaf-1, thereby facilitating binding and activation of procaspase-9 (reviewed in Refs.
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      , ,
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Green D.R.
      • Reed J.C.
      ). After activation, caspase-9 is thought to proteolytically activate caspase-3 and possibly caspase-7 (
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Li P.
      • Nijhawan D.
      • Budihardjo I.
      • Srinivasula S.M.
      • Ahmad M.
      • Alnemri E.S.
      • Wang X.
      ).
      The mechanism by which caspases are activated after treatment with chemotherapeutic agents has been the subject of considerable investigation. The Fas/FasL pathway (reviewed in Refs.
      • Fraser A.
      • Evan G.
      , ,
      • Nicholson D.W.
      • Thornberry N.A.
      ,
      • Wallach D.
      • Boldin M.
      • Varfolomeev E.
      • Beyaert R.
      • Vandenabeele P.
      • Fiers W.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      , and
      • Eischen C.
      • Leibson P.
      ) has been implicated in the initiation of apoptosis in methotrexate- or doxorubicin-treated CEM T cell leukemia cells (
      • Friesen C.
      • Herr I.
      • Krammer P.H.
      • Debatin K.-M.
      ,
      • Herr I.
      • Wilhelm D.
      • Bohler T.
      • Angel P.
      • Debatin K.M.
      ), etoposide- or teniposide-treated Jurkat T cells (
      • Kasibhatla S.
      • Brunner T.
      • Genestier L.
      • Echeverri F.
      • Mahboubi A.
      • Green D.R.
      ), bleomycin-treated HepG2 hepatoma cells (
      • Muller M.
      • Strand S.
      • Hug H.
      • Heinemann E.M.
      • Walczak H.
      • Hofmann W.J.
      • Stremmel W.
      • Krammer P.H.
      • Galle P.R.
      ), and 5-fluorouracil-treated Gc3/cl colon cancer cells (
      • Houghton J.A.
      • Harwood F.G.
      • Tillman D.M.
      ), but other studies have found no evidence for Fas/FasL interactions in drug-induced apoptosis (
      • Fuchs E.J.
      • McKenna K.A.
      • Bedi A.
      ,
      • Eischen C.M.
      • Kottke T.J.
      • Martins L.M.
      • Basi G.S.
      • Tung J.S.
      • Earnshaw W.C.
      • Leibson P.J.
      • Kaufmann S.H.
      ,
      • Villunger A.
      • Egle A.
      • Kos M.
      • Hartmann B.L.
      • Geley S.
      • Kofler R.
      • Greil R.
      ). Likewise, release of cytochrome c to the cytosol has been observed early in the process of drug-induced apoptosis in some model systems (
      • Yang J.
      • Liu X.
      • Bhalla K.
      • Kim C.N.
      • Ibrado A.M.
      • Cai J.
      • Peng T.-I.
      • Jones D.P.
      • Wang X.
      ,
      • Kharbanda S.
      • Pandey P.
      • Schofield L.
      • Israels S.
      • Roncinske R.
      • Yoshida K.
      • Bharti A.
      • Yuan Z.M.
      • Saxena S.
      • Weichselbaum R.
      • Nalin C.
      • Kufe D.
      ,
      • Kim C.N.
      • Wang X.
      • Huang Y.
      • Ibrado A.M.
      • Liu L.
      • Frang G.
      • Bhalla K.
      ,
      • Martins L.M.
      • Mesner P.W.
      • Kottke T.J.
      • Basi G.S.
      • Sinha S.
      • Tung J.S.
      • Svingen P.A.
      • Madden B.J.
      • Takahashi A.
      • McCormick D.J.
      • Earnshaw W.C.
      • Kaufmann S.H.
      ) but not others (
      • Chauhan D.
      • Pandey P.
      • Ogata A.
      • Teoh G.
      • Krett N.
      • Halgren R.
      • Rosen S.
      • Kufe D.
      • Kharbanda S.
      • Anderson K.
      ,
      • Tang D.G.
      • Li L.
      • Zhu Z.
      • Joshi B.
      ).
      Once caspase activation is initiated, the work of disassembling the cell then falls to caspases-3, -6, and -7 and the downstream activities that they activate (
      • Martins L.M.
      • Kottke T.J.
      • Mesner P.W.
      • Basi G.S.
      • Sinha S.
      • Frigon Jr., N.
      • Tatar E.
      • Tung J.S.
      • Bryant K.
      • Takahashi A.
      • Svingen P.A.
      • Madden B.J.
      • McCormick D.J.
      • Earnshaw W.C.
      • Kaufmann S.H.
      ,
      • Thornberry N.A.
      • Lazebnik Y.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ,
      • Faleiro L.
      • Kobayashi R.
      • Fearnhead H.
      • Lazebnik Y.
      ). These enzymes cleave a number of cellular polypeptides, inactivating some and producing enzymatically active forms of others (
      • Nicholson D.W.
      • Thornberry N.A.
      ,
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ). The net result of these cleavages is the disassembly of key structural components of the nucleus and cytoskeleton; inhibition of the processes of DNA repair, replication, and transcription; and activation of one or more endonucleases that irreversibly damage the genome (reviewed in Refs.
      • Thornberry N.A.
      • Lazebnik Y.
      and
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      ). Collectively, these events contribute to the biochemical and morphological changes that make up the apoptotic phenotype.
      Previous studies from our laboratories have demonstrated that MDA-MB-468 breast cancer cells undergo apoptosis after treatment with 5FUdR, paclitaxel, or EGF (
      • Armstrong D.K.
      • Kaufmann S.H.
      • Ottaviano Y.L.
      • Furuya Y.
      • Buckley J.A.
      • Isaacs J.T.
      • Davidson J.A.
      ,
      • Armstrong D.K.
      • Isaacs J.T.
      • Ottaviano Y.L.
      • Davidson N.E.
      ,
      • McCloskey D.E.
      • Kaufmann S.H.
      • Prestigiacomo L.J.
      • Davidson N.E.
      ). The caspase substrates PARP and lamin B1 were cleaved in all cases, although the cleavage was less extensive in EGF-treated cultures. The identity of the activated caspases and the mechanism of their activation were not addressed in these earlier studies. More recently, Chin et al. (
      • Chin Y.E.
      • Kitagawa M.
      • Kuida K.
      • Flavell R.A.
      • Fu X.
      ) reported that EGF treatment of MDA-MB-468 cells resulted in enhanced expression and activation of caspase-1 without any activation of caspase-3. In view of previous results indicating that caspase-1 plays a limited role in apoptosis under many circumstances (
      • Kuida K.
      • Lippke J.A.
      • Ku G.
      • Harding M.W.
      • Livingston D.J.
      • Su M.S.
      • Flavell R.A.
      ,
      • Li P.
      • Allen H.
      • Banerjee S.
      • Franklin S.
      • Herzog L.
      • Johnston C.
      • McDowell J.
      • Paskind M.
      • Rodman L.
      • Salfeld J.
      ), it was unclear whether the EGF-induced expression of caspase-1 was sufficient to explain the apoptosis observed in these cells. Accordingly, the present studies were undertaken to 1) characterize the complement of caspases activated during apoptosis induced by paclitaxel, 5FUdR, and EGF; and 2) compare the mechanisms involved in caspase activation after these treatments.

      DISCUSSION

      In the present study, we have compared the apoptotic pathways activated by paclitaxel, 5FUdR, or EGF in MDA-MB-468 breast cancer cells. Our results indicate that the vast majority of cells detaching from the tissue culture plates after treatment with paclitaxel or 5FUdR are frankly apoptotic, whereas 50–70% of the cells becoming nonadherent after EGF treatment are morphologically normal (Fig. 9,A–D). Further experiments have demonstrated that the cells becoming nonadherent after EGF treatment can reattach and survive (Fig.9 F). In contrast, cells that detach after treatment with paclitaxel or 5FUdR cannot. Despite these differences, all of the treatments appear to activate the cytochrome c/Apaf-1 ⇒ caspase-3 pathway, as indicated by the release of cytochromec to the cytosol (Fig. 7) and the appearance of active caspases-3, -6, and -7 (Figs. Figure 2, Figure 3, Figure 4, Figure 5). These observations have potentially important implications for the understanding of drug- and hormone-induced PCD in human breast cancer cells.
      Recent studies (reviewed in Refs.
      • Ruoslahti E.
      • Reed J.C.
      ,
      • Meredith J.E.
      • Schwartz M.A.
      ,
      • Khwaja A.
      • Downward J.
      ,
      • Frisch S.M.
      • Ruoslahti E.
      ) have demonstrated that cell-substratum interactions can provide survival signals for many cell types. Conversely, removal of certain cell lines from their substrata can result in apoptosis. These recent descriptions of the process termed “anoikis” lead us to propose the following sequence of events following EGF treatment: 1) EGF-treated cells adhere less tightly to the tissue culture plates; 2) a fraction of cells detach from the substratum (Figs. 1 E, 6E, and 9C) and lose integrin-mediated signaling; 3) those cells that do not reattach in a timely fashion initiate the process of anoikis, which includes release of cytochrome c to the cytosol (Fig. 7) and subsequent activation of downstream caspases (Figs. Figure 2, Figure 3, Figure 4, Figure 5).
      A number of observations support this model. First, MDA-MB-468 cells clearly undergo apoptosis when deliberately deprived of the opportunity to attach to a substratum (Fig. 9 D). Second, treatment of the tissue culture plates with poly(HEMA), which decreases the adhesion of the MDA-MB-468 cells,
      T. J. Kottke and S. H. Kaufmann, unpublished observations.
      enhances the effect of EGF (Fig. 9 E). Third, MDA-MB-468 cells that detach during EGF treatment can be rescued by replating in EGF-free medium (Fig. 9 F). Fourth, EGF-treated cells show little increase in caspase activity until they have detached, whereas paclitaxel- or 5FUdR-treated cells contain elevated levels of DEVD-AFC and VEID-AFC cleavage activity before detachment (Fig. 4). Finally, when MDA-MB-468 cells are cultured in suspension as illustrated in Fig.9 D, EGF fails to exert any pro- or anti-apoptotic effect that can be distinguished from the effect of detachment alone.3 Collectively, all of these observations support the view that EGF-induced apoptosis results from the loss of cell-substratum attachment. Although this process of anoikis has been implicated in a variety of physiological processes (
      • Ruoslahti E.
      • Reed J.C.
      ,
      • Meredith J.E.
      • Schwartz M.A.
      ,
      • Frisch S.M.
      • Ruoslahti E.
      ), the present report appears to be the first implicating anoikis in the antiproliferative effects of any hormone. Further studies are required to determine whether detachment-induced apoptosis plays a similar role in the antiproliferative effects of other hormones.
      The present studies do not address the question of how EGF treatment causes detachment. EGF is known to enhance motility of various epithelial cells (
      • Klemke R.L.
      • Yebra M.
      • Bayna E.M.
      • Cheresh D.A.
      ,
      • Nelson J.
      • Allen W.E.
      • Scott W.N.
      • Bailie J.R.
      • Walker B.
      • McFerran N.V.
      • Wilson D.J.
      ,
      • Chen P.
      • Murphy-Ullrich J.E.
      • Wells A.
      ,
      • McCawley L.J.
      • O'Brien P.
      • Hudson L.G.
      ), providing one potential explanation for the decreased adherence. A change in the affinity of integrins for their ligands (
      • Bazzoni G.
      • Hemler M.E.
      ), either in association with altered motility or independent of this process, might also contribute to the decreased adhesion. Further studies are required to examine these possibilities.
      While the present studies were in progress, Chin et al. (
      • Chin Y.E.
      • Kitagawa M.
      • Kuida K.
      • Flavell R.A.
      • Fu X.
      ) reported that EGF treatment of serum-deprived MDA-MB-468 cells resulted in enhanced procaspase-1 expression. Based in part on these results, these investigators proposed that EGF-induced apoptosis was triggered by procaspase-1 autoactivation. Our results are not compatible with this model. First, when MDA-MB-468 cells were cultured in serum-containing medium, immunoblotting failed to provide evidence that EGF treatment enhanced procaspase-1 polypeptide levels (Fig. 3 D, lanes 1, 2, and 7) even though this treatment induced apoptosis (Figs. 6 F and 9 C). Second, assays for cleavage of the caspase-1 substrate YVAD-AFC failed to demonstrate an EGF-induced increase in the low level basal activity of this enzyme (Fig. 4 C). Finally, an affinity labeling technique with nanogram sensitivity (
      • Martins L.M.
      • Kottke T.J.
      • Mesner P.W.
      • Basi G.S.
      • Sinha S.
      • Frigon Jr., N.
      • Tatar E.
      • Tung J.S.
      • Bryant K.
      • Takahashi A.
      • Svingen P.A.
      • Madden B.J.
      • McCormick D.J.
      • Earnshaw W.C.
      • Kaufmann S.H.
      ) failed to provide evidence for active caspase-1 in cytosol or nuclei of EGF-treated cells (Fig.5 A). Although these observations argue against a role for caspase-1 in EGF-induced apoptosis of the MDA-MB-468 cells, we nonetheless observed decreased levels of procaspase-1 concomitant with PCD in this cell line (Fig. 3 D). Several potential explanations could account for these observations, including the possibility that caspase-1 is exported out of the MDA-MB-468 cells upon activation (
      • Singer I.I.
      • Scott S.
      • Chin J.
      • Bayne E.K.
      • Limjuco G.
      • Weidner J.
      • Miller D.K.
      • Chapman K.
      • Kostura M.J.
      ) or the possibility that procaspase-1 has been proteolytically cleaved at one or more sites that do not result in its catalytic activation.
      In contrast to procaspase-1, cleavage of procaspases-3, -6, and -7 (Fig. 3, A–C) is clearly accompanied by their activation, as evidenced by the generation of protease species of appropriate molecular weight that react with the affinity label zEK(bio)D-aomk (Fig. 5), appearance of activities that cleave DEVD-AFC and VEID-AFC (Fig. 4, A and B), and degradation of caspase substrates to fragments that are known to reflect activity of caspases-3, -6, and/or -7 in situ (Fig. 2). In short, four separate pieces of evidence (Figs. Figure 2, Figure 3, Figure 4, Figure 5) support the view that EGF treatment is accompanied by activation of these downstream caspases. The cohort of effector caspases activated during EGF treatment is very similar to the cohort activated by paclitaxel or 5FUdR (Fig. 5), although the specific activities of these caspases in vitro(Fig. 4, A and B) and in situ (Fig. 2) are lower, presumably reflecting the lower percentage of cells that are undergoing apoptosis after EGF treatment (Figs. 6 F and9 C). These observations not only provide additional support for previous claims that different stimuli activate the same downstream effectors of PCD (
      • Eischen C.M.
      • Kottke T.J.
      • Martins L.M.
      • Basi G.S.
      • Tung J.S.
      • Earnshaw W.C.
      • Leibson P.J.
      • Kaufmann S.H.
      ,
      • Faleiro L.
      • Kobayashi R.
      • Fearnhead H.
      • Lazebnik Y.
      ,
      • Chinnaiyan A.M.
      • Orth K.
      • O'Rourke K.
      • Duan H.
      • Poirier G.G.
      • Dixit V.M.
      ,
      • Srinivasan A.
      • Foster L.M.
      • Testa M.-P.
      • Ord T.
      • Keane R.W.
      • Bredesen D.E.
      • Kayalar C.
      ) but also provide the first characterization of the caspases that are activated as cells undergo anoikis.
      Additional experiments were designed to examine, in a preliminary fashion, the potential role of various pathways in the activation of these downstream caspases. Although a number of studies have provided evidence that the Fas/FasL pathway can participate in the induction of apoptosis by chemotherapeutic agents in some Fas-expressing cells (see the Introduction), this pathway does not appear to contribute to PCD in MDA-MB-468 cells. Treatment with a blocking anti-Fas antibody failed to attenuate the effects of 5FUdR, paclitaxel, and EGF in this cell line (Fig. 6, E and F). Furthermore, ligation of Fas by exogenous antibodies did not induce PCD in MDA-MB-468 cells (Fig. 6,B and E). Although these observations are difficult to reconcile with a model in which drug treatment induces PCD by activating the Fas/FasL pathway in this cell line, they do not rule out the possibility that another death receptor (e.g. death receptor 5 (
      • Wu G.S.
      • Burns T.F.
      • McDonald E.R.
      • Jiang W.
      • Meng R.
      • Krantz I.D.
      • Kao G.
      • Gan D.D.
      • Zhou J.Y.
      • Muschel R.
      • Hamilton S.R.
      • Spinner N.B.
      • Markowitz S.
      • Wu G.
      • el-Deiry W.S.
      )) and its ligand might be involved in MDA-MB-468 cell death, nor do they rule out the possibility that the Fas/FasL pathway might play a role in chemotherapy-induced apoptosis in other cell lines. These issues require further investigation.
      Recent studies have provided evidence that a pathway involving cytochrome c, Apaf-1, and procaspase-9 can also lead to activation of downstream caspases (
      • Earnshaw W.C.
      • Martins L.M.
      • Kaufmann S.H.
      , ,
      • Srinivasula S.M.
      • Ahmad M.
      • Fernandes-Alnemri T.
      • Alnemri E.S.
      ,
      • Green D.R.
      • Reed J.C.
      ,
      • Li P.
      • Nijhawan D.
      • Budihardjo I.
      • Srinivasula S.M.
      • Ahmad M.
      • Alnemri E.S.
      • Wang X.
      ). To evaluate the possible activation of this pathway, the distribution of cytochrome cwas examined by immunoblotting. Results of these experiments (Fig. 7) revealed that cytochrome c was readily detectable in cytosol of cells that became apoptotic after treatment with EGF as well as paclitaxel or 5FUdR (Fig. 7). Because of the lack of sensitivity of currently available anti-procaspase-9 antibodies, we were unable to directly confirm that procaspase-9 was cleaved in these cells. Nonetheless, the observations in Fig. 7 are highly suggestive that the cytochrome c/Apaf-1/procaspase-9 pathway contributes to activation of the downstream caspases after treatment with any of these agents.
      Current evidence suggests that this pathway can be triggered by alterations in the expression or phosphorylation of certain Bcl-2 family members. In particular, increased levels of Bax (
      • Mandal M.
      • Adam L.
      • Mendelsohn J.
      • Kumar R.
      ,
      • Miyashita T.
      • Krajewski S.
      • Krajewska M.
      • Wang H.G.
      • Lin H.K.
      • Liebermann D.A.
      • Hoffman B.
      • Reed J.C.
      ,
      • Zhan Q.
      • Fan S.
      • Bae I.
      • Guillouf C.
      • Liebermann D.A.
      • O'Connor P.M.
      • Fornace Jr., A.J.
      ), decreased levels of Bcl-2 (
      • König A.
      • Schwartz G.K.
      • Mohammad R.M.
      • Al-Katib A.
      • Gabrilove J.L.
      ), and altered phosphorylation of Bcl-2 (
      • Haldar S.
      • Jena N.
      • Croce C.M.
      ,
      • Ibrado A.M.
      • Liu L.
      • Bhalla K.
      ,
      • Blagosklonny M.V.
      • Giannakokou P.
      • el-Deiry W.S.
      • Kingston D.G.
      • Higgs P.I.
      • Neckers L.
      • Fojo T.
      ) have been observed in various model systems prior to the onset of apoptosis. To search for similar changes in MDA-MB-468 cells, whole cell lysates were probed with antibodies that recognize six Bcl-2 family members. Consistent with previous reports (
      • Haldar S.
      • Jena N.
      • Croce C.M.
      ,
      • Ibrado A.M.
      • Liu L.
      • Bhalla K.
      ,
      • Ling Y.H.
      • Tornos C.
      • Perez-Soler R.
      ,
      • Blagosklonny M.V.
      • Giannakokou P.
      • el-Deiry W.S.
      • Kingston D.G.
      • Higgs P.I.
      • Neckers L.
      • Fojo T.
      ), paclitaxel treatment caused a transient reduction in the mobility of part of the Bcl-2 molecules present in MDA-MB-468 cells (Fig.8 A). In contrast, changes in Bcl-2 phosphorylation were not evident after treatment with 5FUdR or EGF (Fig. 8 A). Moreover, changes in phosphorylation state or levels of Bcl-xL, Mcl-1, Bak, Bax, or Bad were not discernible during treatment with EGF (Fig. 8 B), suggesting that other alterations must trigger the apoptotic cascade.
      In summary, the present studies have demonstrated that EGF treatment induces the process of anoikis in MDA-MB-468 cells. As is the case with drug-induced PCD, anoikis in these cells involves release of cytochromec from mitochondria and activation of downstream caspases, including caspases-3, -6, and -7. The changes occurring between treatment with EGF and activation of downstream caspases do not appear to involve activation of the Fas/FasL pathway or alterations in levels of commonly studied Bcl-2 family members. Although the intervening steps remain unidentified at present, EGF-treated MDA-MB-468 cells might provide a useful model for studying the process by which loss of integrin-mediated signaling results in caspase activation and cell death.

      Acknowledgments

      We thank Drs. John Reed and Guy Salvesen for antibodies to caspase-6, Bcl-xL, and Mcl-1; Dr. Guy Poirier for C-2–10 anti-PARP; Wendy Deveraux and Phyllis Svingen for technical assistance with some of the experiments; and Deb Strauss for secretarial assistance.

      REFERENCES

        • Khazaie K.
        • Schirrmacher V.
        • Lichtner R.B.
        Cancer Metastasis Rev. 1993; 12: 255-274
        • Chrysogelos S.A.
        • Dickson R.B.
        Breast Cancer Res. Treat. 1994; 29: 29-40
        • Rusch V.
        • Mendelsohn J.
        • Dmitrovsky E.
        Cytokine Growth Factor Rev. 1996; 71: 133-141
        • Kelloff G.J.
        • Fay J.R.
        • Steele V.E.
        • Lubet R.A.
        • Boone C.W.
        • Crowell J.A.
        • Sigman C.C.
        Cancer Epidemiol. Biomarkers Prev. 1996; 5: 657-666
        • Chin Y.E.
        • Kitagawa M.
        • Kuida K.
        • Flavell R.A.
        • Fu X.
        Mol. Cell. Biol. 1997; 17: 5328-5337
        • Kato Y.
        • Tapping R.I.
        • Huang S.
        • Watson M.H.
        • Ulevitch R.J.
        • Lee J.-D.
        Nature. 1998; 395: 713-716
        • Filmus J.
        • Pollak M.N.
        • Cailleau R.
        • Buick R.N.
        Biochem. Biophys. Res. Commun. 1985; 128: 898-905
        • Lee K.
        • Tanaka M.
        • Hatanaka M.
        • Kuze F.
        Exp. Cell Res. 1987; 173: 156-162
        • Ennis B.W.
        • Valverius E.M.
        • Bates S.E.
        • Lippman M.E.
        • Bellot F.
        • Kris R.
        • Schlessinger J.
        • Masui H.
        • Goldenberg A.
        • Mendelsohn J.
        • Dickson R.B.
        Mol. Endocrinol. 1989; 3: 1830-1838
        • Dong X.-F.
        • Berthois Y.
        • Martin P.M.
        Anticancer Res. 1991; 11: 737-744
        • Minke J.M.H.M.
        • Schuuring E.
        • van den Berghe R.
        • Stolwijk J.A.M.
        • Roonstra J.
        • Cornelisse C.
        • Hilkens J.
        • Misdorp W.
        Cancer Res. 1991; 51: 4028-4037
        • Argilé A.
        • Kraft N.
        • Ootaka T.
        • Hutchinson P.
        • Atkins R.C.
        Cancer Res. 1992; 52: 4356-4360
        • Armstrong D.K.
        • Kaufmann S.H.
        • Ottaviano Y.L.
        • Furuya Y.
        • Buckley J.A.
        • Isaacs J.T.
        • Davidson J.A.
        Cancer Res. 1994; 54: 5280-5283
        • Schaerli P.
        • Jaggi R.
        Cell. Mol. Life Sci. 1998; 54: 129-138
        • Eastman A.
        Semin. Cancer Biol. 1995; 6: 45-52
        • Park J.R.
        Curr. Opin. Hematol. 1996; 3: 191-196
        • Sachs L.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4742-4749
        • May W.S.
        Adv. Pharmacol. 1997; 41: 219-246
        • Wyllie A.H.
        • Kerr J.F.R.
        • Currie A.R.
        Int. Rev. Cytol. 1980; 68: 251-306
        • Arends M.J.
        • Wyllie A.H.
        Int. Rev. Exp. Pathol. 1991; 32: 223-254
        • Ucker D.S.
        Adv. Pharmacol. 1997; 41: 179-218
        • Alnemri E.S.
        • Livingston D.J.
        • Nicholson D.W.
        • Salvesen G.
        • Thornberry N.A.
        • Wong W.W.
        • Yuan J.
        Cell. 1996; 87: 171
        • Weil M.
        • Jacobson M.D.
        • Coles H.S.
        • Davies T.J.
        • Gardner R.L.
        • Raff K.D.
        • Raff M.C.
        J. Cell Biol. 1996; 133: 1053-1059
        • Martins L.M.
        • Kottke T.J.
        • Mesner P.W.
        • Basi G.S.
        • Sinha S.
        • Frigon Jr., N.
        • Tatar E.
        • Tung J.S.
        • Bryant K.
        • Takahashi A.
        • Svingen P.A.
        • Madden B.J.
        • McCormick D.J.
        • Earnshaw W.C.
        • Kaufmann S.H.
        J. Biol. Chem. 1997; 272: 7421-7430
        • Greenberg A.H.
        Adv. Exp. Med. Biol. 1996; 406: 219-228
        • Thornberry N.A.
        • Rosen A.
        • Nicholson D.W.
        Adv. Pharmacol. 1997; 41: 155-177
        • Fraser A.
        • Evan G.
        Cell. 1996; 85: 781-784
        • Nagata S.
        Cell. 1997; 88: 355-365
        • Nicholson D.W.
        • Thornberry N.A.
        Trends Biochem. Sci. 1997; 22: 299-306
        • Wallach D.
        • Boldin M.
        • Varfolomeev E.
        • Beyaert R.
        • Vandenabeele P.
        • Fiers W.
        FEBS Ltrs. 1997; 410: 96-106
        • Thornberry N.A.
        • Lazebnik Y.
        Science. 1998; 281: 1312-1316
        • Earnshaw W.C.
        • Martins L.M.
        • Kaufmann S.H.
        Annu. Rev. Biochem. 1999; 68: 383-424
        • Reed J.C.
        Cell. 1997; 91: 559-562
        • Srinivasula S.M.
        • Ahmad M.
        • Fernandes-Alnemri T.
        • Alnemri E.S.
        Mol. Cell. 1998; 1: 949-957
        • Green D.R.
        • Reed J.C.
        Science. 1998; 281: 1309-1312
        • Li P.
        • Nijhawan D.
        • Budihardjo I.
        • Srinivasula S.M.
        • Ahmad M.
        • Alnemri E.S.
        • Wang X.
        Cell. 1997; 91: 479-489
        • Eischen C.
        • Leibson P.
        Adv. Pharmacol. 1997; 41: 107-132
        • Friesen C.
        • Herr I.
        • Krammer P.H.
        • Debatin K.-M.
        Nat. Med. 1996; 2: 574-577
        • Herr I.
        • Wilhelm D.
        • Bohler T.
        • Angel P.
        • Debatin K.M.
        EMBO J. 1997; 16: 6200-6208
        • Kasibhatla S.
        • Brunner T.
        • Genestier L.
        • Echeverri F.
        • Mahboubi A.
        • Green D.R.
        Mol. Cell. 1998; 1: 543-551
        • Muller M.
        • Strand S.
        • Hug H.
        • Heinemann E.M.
        • Walczak H.
        • Hofmann W.J.
        • Stremmel W.
        • Krammer P.H.
        • Galle P.R.
        J. Clin. Invest. 1997; 99: 403-413
        • Houghton J.A.
        • Harwood F.G.
        • Tillman D.M.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8144-8149
        • Fuchs E.J.
        • McKenna K.A.
        • Bedi A.
        Cancer Res. 1997; 57: 2550-2554
        • Eischen C.M.
        • Kottke T.J.
        • Martins L.M.
        • Basi G.S.
        • Tung J.S.
        • Earnshaw W.C.
        • Leibson P.J.
        • Kaufmann S.H.
        Blood. 1997; 90: 935-943
        • Villunger A.
        • Egle A.
        • Kos M.
        • Hartmann B.L.
        • Geley S.
        • Kofler R.
        • Greil R.
        Cancer Res. 1997; 57: 3331-3334
        • Yang J.
        • Liu X.
        • Bhalla K.
        • Kim C.N.
        • Ibrado A.M.
        • Cai J.
        • Peng T.-I.
        • Jones D.P.
        • Wang X.
        Science. 1997; 275: 1129-1132
        • Kharbanda S.
        • Pandey P.
        • Schofield L.
        • Israels S.
        • Roncinske R.
        • Yoshida K.
        • Bharti A.
        • Yuan Z.M.
        • Saxena S.
        • Weichselbaum R.
        • Nalin C.
        • Kufe D.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6939-6942
        • Kim C.N.
        • Wang X.
        • Huang Y.
        • Ibrado A.M.
        • Liu L.
        • Frang G.
        • Bhalla K.
        Cancer Res. 1997; 57: 3115-3120
        • Martins L.M.
        • Mesner P.W.
        • Kottke T.J.
        • Basi G.S.
        • Sinha S.
        • Tung J.S.
        • Svingen P.A.
        • Madden B.J.
        • Takahashi A.
        • McCormick D.J.
        • Earnshaw W.C.
        • Kaufmann S.H.
        Blood. 1997; 90: 4283-4296
        • Chauhan D.
        • Pandey P.
        • Ogata A.
        • Teoh G.
        • Krett N.
        • Halgren R.
        • Rosen S.
        • Kufe D.
        • Kharbanda S.
        • Anderson K.
        J. Biol. Chem. 1997; 272: 29995-29997
        • Tang D.G.
        • Li L.
        • Zhu Z.
        • Joshi B.
        Biochem. Biophys. Res. Commun. 1998; 242: 380-384
        • Faleiro L.
        • Kobayashi R.
        • Fearnhead H.
        • Lazebnik Y.
        EMBO J. 1997; 16: 2271-2281
        • Armstrong D.K.
        • Isaacs J.T.
        • Ottaviano Y.L.
        • Davidson N.E.
        Cancer Res. 1992; 52: 3418-3424
        • McCloskey D.E.
        • Kaufmann S.H.
        • Prestigiacomo L.J.
        • Davidson N.E.
        Clin. Cancer Res. 1996; 2: 847-854
        • Kuida K.
        • Lippke J.A.
        • Ku G.
        • Harding M.W.
        • Livingston D.J.
        • Su M.S.
        • Flavell R.A.
        Science. 1995; 267: 2000-2003
        • Li P.
        • Allen H.
        • Banerjee S.
        • Franklin S.
        • Herzog L.
        • Johnston C.
        • McDowell J.
        • Paskind M.
        • Rodman L.
        • Salfeld J.
        Cell. 1995; 80: 401-411
        • Kaufmann S.H.
        J. Biol. Chem. 1989; 264: 13946-13955
        • Budihardjo I.I.
        • Walker D.
        • Svingen P.A.
        • Buckwalter C.A.
        • Desnoyers S.
        • Shah G.M.
        • Poirier G.
        • Ames M.M.
        • Kaufmann S.H.
        Clin. Cancer Res. 1998; 4: 117-130
        • Folkman J.
        • Moscona A.
        Nature. 1978; 273: 345-349
        • Smith P.K.
        • Krohn R.I.
        • Hermanson G.T.
        • Mallia A.K.
        • Gartner F.H.
        • Provenzano M.D.
        • Fujimoto E.K.
        • Goeke N.M.
        • Olson B.J.
        • Klenk D.C.
        Anal. Biochem. 1985; 150: 76-85
        • Bible K.
        • Kaufmann S.H.
        Cancer Res. 1997; 57: 3375-3380
        • Matthews D.E.
        • Farewell V.T.
        Using and Understanding Medical Statistics. S. Karger AG, Basel1988: 178-179
        • Fry D.W.
        • Kraker A.J.
        • McMichael A.
        • Ambroso L.A.
        • Nelson J.M.
        • Leopold W.R.
        • Connors R.W.
        • Bridges A.J.
        Science. 1994; 265: 1093-1095
        • Karnes W.E.
        • Weller S.G.
        • Adjei P.N.
        • Kottke T.J.
        • Glenn K.S.
        • Gores G.J.
        • Kaufmann S.H.
        Gastroenterology. 1998; 114: 930-939
        • Kaufmann S.H.
        • Desnoyers S.
        • Ottaviano Y.
        • Davidson N.E.
        • Poirier G.G.
        Cancer Res. 1993; 53: 3976-3985
        • Lazebnik Y.A.
        • Kaufmann S.H.
        • Desnoyers S.
        • Poirier G.G.
        • Earnshaw W.C.
        Nature. 1994; 371: 346-347
        • Takahashi A.
        • Alnemri E.S.
        • Lazebnik Y.A.
        • Fernandes-Alnemri T.
        • Litwack G.
        • Moir R.D.
        • Goldman R.D.
        • Poirier G.G.
        • Kaufmann S.H.
        • Earnshaw W.C.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8395-8400
        • Rao L.
        • Perez D.
        • White E.
        J. Cell Biol. 1996; 135: 1441-1455
        • Fernandes-Alnemri T.
        • Takahashi A.
        • Armstrong R.
        • Krebs J.
        • Fritz L.
        • Tomaselli K.J.
        • Wang L.
        • Yu Z.
        • Croce C.M.
        • Salveson G.
        • Earnshaw W.C.
        • Litwack G.
        • Alnemri E.S.
        Cancer Res. 1995; 55: 6045-6052
        • Yonehara S.
        • Ishii A.
        • Yonehara M.
        J. Exp. Med. 1989; 169: 1747-1756
        • Yonehara S.
        • Nishimura Y.
        • Kishil S.
        • Yonehara M.
        • Takazawa K.
        • Tamatani T.
        • Ishii A.
        Int. Immunol. 1994; 6: 1849-1856
        • Jurgensmeier J.M.
        • Xie Z.
        • Deveraux Q.
        • Ellerby L.
        • Bredesen D.
        • Reed J.C.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002
        • Gross A.
        • Jockel J.
        • Wei M.C.
        • Korsmeyer S.J.
        EMBO J. 1998; 17: 3878-3885
        • Haldar S.
        • Jena N.
        • Croce C.M.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4507-4511
        • Ibrado A.M.
        • Liu L.
        • Bhalla K.
        Cancer Res. 1997; 57: 1109-1115
        • Fang G.
        • Chang B.S.
        • Kim C.N.
        • Perkins C.
        • Thompson C.B.
        • Bhalla K.N.
        Cancer Res. 1998; 58: 3202-3208
        • Ling Y.H.
        • Tornos C.
        • Perez-Soler R.
        J. Biol. Chem. 1998; 273: 18984-18991
        • Mandal M.
        • Adam L.
        • Mendelsohn J.
        • Kumar R.
        Oncogene. 1998; 17: 999-1007
        • Ruoslahti E.
        • Reed J.C.
        Cell. 1994; 77: 477-478
        • Meredith J.E.
        • Schwartz M.A.
        Trends Cell Biol. 1997; 7: 146-150
        • Khwaja A.
        • Downward J.
        J. Cell Biol. 1997; 139: 1017-1023
        • Frisch S.M.
        • Ruoslahti E.
        Curr. Opin. Cell Biol. 1997; 9: 701-706
        • Klemke R.L.
        • Yebra M.
        • Bayna E.M.
        • Cheresh D.A.
        J. Cell Biol. 1994; 127: 859-866
        • Nelson J.
        • Allen W.E.
        • Scott W.N.
        • Bailie J.R.
        • Walker B.
        • McFerran N.V.
        • Wilson D.J.
        Cancer Res. 1995; 55: 3772-3776
        • Chen P.
        • Murphy-Ullrich J.E.
        • Wells A.
        J. Cell Biol. 1996; 134: 689-698
        • McCawley L.J.
        • O'Brien P.
        • Hudson L.G.
        Endocrinology. 1997; 138: 121-127
        • Bazzoni G.
        • Hemler M.E.
        Trends Biochem. Sci. 1998; 23: 30-34
        • Singer I.I.
        • Scott S.
        • Chin J.
        • Bayne E.K.
        • Limjuco G.
        • Weidner J.
        • Miller D.K.
        • Chapman K.
        • Kostura M.J.
        J. Exp. Med. 1995; 182: 1447-1459
        • Chinnaiyan A.M.
        • Orth K.
        • O'Rourke K.
        • Duan H.
        • Poirier G.G.
        • Dixit V.M.
        J. Biol. Chem. 1996; 271: 4573-4576
        • Srinivasan A.
        • Foster L.M.
        • Testa M.-P.
        • Ord T.
        • Keane R.W.
        • Bredesen D.E.
        • Kayalar C.
        J. Neurosci. 1996; 16: 5654-5660
        • Wu G.S.
        • Burns T.F.
        • McDonald E.R.
        • Jiang W.
        • Meng R.
        • Krantz I.D.
        • Kao G.
        • Gan D.D.
        • Zhou J.Y.
        • Muschel R.
        • Hamilton S.R.
        • Spinner N.B.
        • Markowitz S.
        • Wu G.
        • el-Deiry W.S.
        Nat. Genet. 1997; 17: 141-143
        • Miyashita T.
        • Krajewski S.
        • Krajewska M.
        • Wang H.G.
        • Lin H.K.
        • Liebermann D.A.
        • Hoffman B.
        • Reed J.C.
        Oncogene. 1994; 9: 1799-1805
        • Zhan Q.
        • Fan S.
        • Bae I.
        • Guillouf C.
        • Liebermann D.A.
        • O'Connor P.M.
        • Fornace Jr., A.J.
        Oncogene. 1994; 9: 3743-3751
        • König A.
        • Schwartz G.K.
        • Mohammad R.M.
        • Al-Katib A.
        • Gabrilove J.L.
        Blood. 1997; 90: 4307-4312
        • Blagosklonny M.V.
        • Giannakokou P.
        • el-Deiry W.S.
        • Kingston D.G.
        • Higgs P.I.
        • Neckers L.
        • Fojo T.
        Cancer Res. 1997; 57: 130-135
        • Dubrez L.
        • Savoy I.
        • Hamman A.
        • Solary E.
        EMBO J. 1996; 15: 5504-5512