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Advances in Brief |
Interdisciplinary Oncology Program, Moffitt Cancer Center, University of South Florida, Tampa, Florida 33612
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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m) and release of cyt c, thereby inhibiting the activation of the executioner caspases and apoptosis (4)
. Ectopic or endogenous Bcr-Abl expression up-regulates several antiapoptotic mechanisms, including the levels of Bcl-xL as well as the activities of NF-
B and Akt kinase (3, 4, 5, 6)
. Recent studies have shown that inhibition of Bcr-Abl TK activity by a relatively specific inhibitor, STI571, induces differentiation and apoptosis as well as causes in vitro and in vivo eradication of Bcr-Abl-positive human leukemia cells (7
, 8)
. Exposure to STI571 lowered Bcl-xL levels and Akt kinase and NFB activities as well as induced intracellular Hb and differentiation in Bcr-Abl-positive acute leukemia cells (7)
. Preclinical studies have indicated that agents that lower Bcr-Abl expression could also be highly effective against Bcr-Abl-positive leukemias (9, 10, 11, 12)
. Although arsenic trioxide (As2O3) is an active agent against acute promyelocytic leukemic cells (13)
, it was also demonstrated to lower Bcr-Abl levels and induce apoptosis of HL-60/Bcr-Abl and K562 cells (14)
. Taken together, these reports indicate that multiple strategies that lower the levels or activity of Bcr-Abl TK may have efficacy against Bcr-Abl-positive leukemias. The benzoquinone ansamycin antibiotic GA and its analogue 17-AAG bind strongly to the Hsp90 and specifically disrupt its chaperone function for several transcription factors (e.g., steroid, hormone, and retinoid receptors and hypoxia-inducible factor) and protein kinases (e.g., v-Src, Raf-1, lck, Wee 1, and cyclin-dependent kinase 4; Refs. 15, 16, 17, 18, 19, 20, 21 ). This results in their reduced stability and promotes degradation by the proteasome-based mechanism (15 , 16 , 22) . This may be biologically significant, because another ansamycin, herbimycin A, a known tyrosine kinase inhibitor, can revert the morphological phenotype of v-Src-transformed cells (20) . Previous studies have also demonstrated that herbimycin A can induce differentiation (erythroid) of K562 cells (23) . Therefore, we investigated whether GA or 17-AAG would also affect Bcr-Abl levels and its antiapoptotic effects in HL-60/Bcr-Abl and K562 cells. In the present report, we demonstrate that GA and 17-AAG decreased the association of Bcr-Abl with HSP90, resulting in its degradation through the proteasomes. Consequently, GA or 17-AAG treatment lowers Bcr-Abl levels, thereby inducing apoptosis and Hb in HL-60/Bcr-Abl and K562 cells.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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Cell Culture and Cell Growth Inhibition.
Human leukemic cells HL-60/neo, HL-60/Bcr-Abl, and K562 cells were cultured and passaged as described previously (4
, 7)
. Logarithmically growing cells were exposed to the designated concentrations of either GA or 17-AAG and/or PS341. After these treatments, cells were pelleted and washed free of the drug(s) prior to the performance of the studies described below.
Preparation of S-100 Fraction and Western Analysis of Cytosolic cyt c.
Untreated and drug-treated cells were harvested by centrifugation at 1000 x g for 10 min at 4°C. The cell pellets were washed once with ice-cold PBS and resuspended with five volumes of buffer [20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM DTT, and 0.1 mM PMSF], containing 250 mM sucrose. The cells were homogenized with a 22-gauge needle, and the homogenates were centrifuged at 100,000 x g for 30 min at 4°C (S-100 fraction; Refs. 4
and 25
). The supernatants were collected, and the protein concentrations of S-100 were determined by Bradford method (Bio-Rad, Hercules, CA). Twenty to 30 µg of the S-100 fraction were used for Western blot analysis of cyt c, as described previously (4
and 25
).
Immunoprecipitation and Immunoblotting Analyses.
After the designated treatment, cells were lysed in the lysis buffer (1% SDS, 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, and 10 µg/ml leupeptin) for 1 h, and the nuclear and cellular debris was cleared by centrifugation. Protein G-agarose beads were washed twice with RIPA 1 buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP 40, and 0.1% deoxycholate] and then incubated with Abl-specific mAb (1 in 100; Santa Cruz) at 4°C for 2 h. After washing the Protein G and the antibody mix with RIPA 1 buffer, 100 µg of total cell lysates were added and incubated overnight at 4°C. The immunoprecipitates were washed three times in RIPA 1 buffer, and proteins were eluted with the SDS sample loading buffer. Proteins were separated by SDS-PAGE as described (24
, 25) . Immunoprecipitates were examined by Western blot analysis after transfer of proteins to nitrocellulose membranes. Western blot analyses were performed using anti-Hsp90 and anti-Hsp70 antibodies (24
, 25)
.
Preparation of Detergent-soluble and -insoluble Fractions.
After the designated drug treatments, cells were lysed with TNSEV buffer [50 mM Tris-HCl (pH 7.5), 2 mM EDTA, 100 mM NaCl, 1 mM sodium orthovanadate, 1% NP40 containing 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM PMSF, 25 mM NAF, and 5 mM N-ethylmaleimide). NP40 in soluble proteins were solubilized with SDS buffer [2% SDS, 100 mM DTT, 80 mM Tris (pH 6.8), 10% glycerol]. Thirty µg of proteins from the NP40 soluble and insoluble fraction were separated on 7.5% SDS-polyacrylamide gels and analyzed by Western blotting (24
, 25
, 27)
.
Western Analyses of Proteins.
Western analyses of caspase-9, caspase-3, Bid, poly(ADP-ribose) polymerase, XIAP, cIAP, survivin, and ß-actin were performed using specific antisera or monoclonal antibodies according to protocols reported previously (24, 25, 26, 27)
. Horizontal scanning densitometry was performed on Western blots by using acquisition into Adobe Photo Shop (Apple, Inc., Cupertino, CA) and analysis by the NIH Image Program (NIH, Bethesda, MD). The expression of ß-actin was used as a control.
Apoptosis Assessment by Annexin V Staining.
After drug treatments, cells were resuspended in 100 µl of staining solution (containing Annexin V fluorescein and propidium iodide in a HEPES buffer; Annexin-V-FLUOS Staining kit; Boehringer-Mannheim, Indianapolis, IN). After incubation at room temperature for 15 min, cells were analyzed by flow cytometry (14)
. Annexin V binds to those cells that express phosphatidylserine on the outer layer of the cell membrane, and propidium iodide stains the cellular DNA of those cells with a compromised cell membrane. This allows for the discrimination of live cells (unstained with either fluorochrome) from apoptotic cells (stained only with Annexin V) and necrotic cells (stained with both Annexin V and propidium iodide; Ref. 14
).
Morphology of Apoptotic Cells.
After drug treatment, 50 x 103 cells were washed with PBS (pH 7.3) and resuspended in the same buffer. Cytospin preparations of the cell suspensions were fixed and stained with Wright stain. Cell morphology was determined by light microscopy. In all, five different fields were randomly selected for counting of at least 500 cells. The percentage of apoptotic cells was calculated for each experiment, as described previously (14
, 27)
.
Flow Cytometric Analysis of Cell Cycle Status and Apoptosis.
The flow cytometric evaluation of the cell cycle status and apoptosis was performed according to a method described previously method (27)
. The percentage of cells in the apoptotic sub-G1 as well as G1, S-phase, and G2-M phases were calculated using Multicycle software (Phoenix Flow Systems, San Diego, CA).
Assessment of Hb Levels.
HL-60/neo, HL-60/Bcr-Abl, and K562 cells were treated the designated concentrations of the drugs. Cells were then washed with PBS, and intracellular Hb levels were determined by a method described previously (3)
and expressed as µg/50 µg of cellular protein.
Akt Kinase Assay.
In untreated and STI571-treated cells, Akt kinase activity was determined by using an immunoprecipitation-kinase assay with reagents provided in a commercially available kit (New England Biolab, Beverly, MA; Ref. 7
). Briefly, cell lysates were used to immunoprecipitate Akt using a polyclonal Akt antibody. Immunoprecipitates were then incubated with GSK-3
fusion protein in the presence of ATP and kinase buffer, allowing immunoprecipitated Akt to phosphorylate GSK-3, which was analyzed by Western blotting using a phospho-GSK-3/ß (serine 219) antibody (27)
.
Statistical Analysis.
Significant differences between values obtained in a population of leukemic cells treated with different experimental conditions were determined by paired t test analyses. A one-way ANOVA was also applied to the results of the various treatment groups, and post hoc analysis was performed using the Bonferroni correction method.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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demonstrates that treatment with 5 µM GA or 17-AAG induces apoptosis of HL-60/neo > HL-60/Bcr-Abl > K562 cells (P < 0.05). Although 17-AAG induced more apoptosis of HL-60/neo than GA, the drugs had similar apoptotic effects against HL-60/Bcr-Abl and K562 cells (Fig. 1A)
. A time-dependent increase in apoptosis attributable to 5 µM GA or 17-AAG was observed in K562 cells (Fig. 1B)
. A dose-dependent (from 0.1 to 50 µM) increase in apoptosis of K562 cells was more obvious after treatment with GA than 17-AAG (Fig. 1C)
. A similar time and dose-dependent increase in apoptosis attributable to GA or 17-AAG was also observed for HL-60/Bcr-Abl cells (data not shown). As reported previously, both HL-60/Bcr-Abl and K562 cells express detectable levels of Hb (Fig. 1D)
. Treatment with 5 µM GA or 17-AAG significantly increased the intracellular levels of Hb (Fig. 1D)
and imparted a rusty color to the cell pellet of HL-60/Bcr-Abl and K562 cells (not shown).
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demonstrates that treatment with 5 µM 17-AAG induced cytosolic accumulation of cyt c in HL-60/Bcr-Abl and K562 cells, but this was less than in HL-60/neo cells (Fig. 2A)
. The levels of cyt c were controlled for protein loading (not shown). Treatment with 5 µM GA for 24 h also increased the cytosolic cyt c levels more in HL-60/neo than HL-60/Bcr-Abl and K562 cells (data not shown). Concomitantly, GA and 17-AAG also induced less processing of caspase-9 and caspase-3 in HL-60/Bcr-Abl and K562 than in HL-60/neo cells (Fig. 2A)
. As compared with HL-60/neo, for unclear reasons, HL-60/Bcr-Abl and K562 cells expressed higher levels of caspase-9. Both GA and 17-AAG treatment had no effect on Bcl-xL, Bcl-2, or Bax levels in HL-60/neo, HL-60/Bcr-Abl, or K562 cells (data not shown). Taken together, these findings indicate that GA and 17-AAG partially overcome the resistance and trigger the intrinsic "mitochondrial" pathway of apoptosis.
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demonstrates that exposure to 5 µM GA or 17-AAG reduced Bcr-Abl protein levels in HL-60/Bcr-Abl and K562 cells. As has been described previously (29)
, GA and 17-AAG also down-regulated the levels of c-Raf in Bcr-Abl-positive cells and the control HL-60/neo cells (Fig. 3A)
. The decline in c-Raf levels in all cell types and the additional decline in Bcr-Abl levels in HL-60/Bcr-Abl and K562 cells were associated with a reduction in the Akt kinase activity, which was estimated by documenting a decline in the phospho-GSK-3 levels (Fig. 3A)
. Lowering of c-Raf levels and Akt kinase activity may be promoting GA and 17-AAG-induced apoptosis of HL-60/neo cells and contributing toward the proapoptotic influence of the decline in Bcr-Abl levels in GA or 17-AAG-treated HL-60/Bcr-Abl and K562 cells (30)
. The findings that the effects of GA and 17-AAG on Raf-1 expression and Akt activity are roughly comparable in the three cell lines, despite different apoptotic responses, would appear to argue against a primary role for these genes in determining sensitivity to these agents. It is noteworthy that whereas in HL-60/Bcr-Abl and K562 cells GA and 17-AAG treatment did not significantly alter IB levels, an 
2-fold increase in IB levels was observed in HL-60/neo cells. Although we did not directly investigate it, this may also promote apoptosis because of GA or 17-AAG reducing the nuclear localization, and thereby inhibiting the transactivation and antiapoptotic effects of NF-B in HL-60/neo cells (5
, 7
, 26)
. The mechanism(s) by which GA or 17-AAG induces IB levels was not determined in the present studies.
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shows that although HL-60/neo cells have barely detectable levels, HL-60/Bcr-Abl and K562 cells display approximately 8–10-fold higher expression of Hsp70. This was further induced by treatment with 5 µM GA or 17-AAG for 24 h, as has been reported previously (15
, 16)
. In HL-60/neo but not in HL-60/Bcr-Abl or K562 cells, GA or 17-AAG also induced Hsp90 (Fig. 3B)
. We further investigated whether TK with Bcr-Abl has chaperone association with Hsp90, as well as whether treatment with GA or 17-AAG disrupts this association, as has been described for c-Raf and progesterone receptor protein (16)
. K562 cells were exposed to GA or 17-AAG for up to 6 h, an exposure interval during which Bcr-Abl levels are not affected. Cell lysates were obtained at 3 or 6 h, and Bcr-Abl was immunoprecipitated. Hsp90 or Hsp70 associated with Bcr-Abl was determined by immunoblotting with specific antibodies. Fig. 3C
shows that, as compared with untreated cells, in the cells treated with GA or 17-AAG, there is a gradual decline in the association of Bcr-Abl with Hsp90, with a concomitant increase in its binding to Hsp70 over 6 h. This loss in Bcr-Abl-associated Hsp90 and the appearance of Bcr-Abl-associated Hsp70 occurred before any detectable loss in the levels of Bcr-Abl attributable to treatment with GA or 17-AAG (data not shown). Prior to its proteasome-mediated degradation, a similar shift in chaperone association with Hsp70 has also been described for Raf and p185c-erbB-2 (16
, 17)
.
Cotreatment with a Proteasome Inhibitor Reduces GA or 17-AAG-induced Decline in Bcr-Abl and Apoptosis.
Previous studies have demonstrated that the GA-induced disruption of the binding of Hsp90 with v-src, Raf-1, and p185c-erbB-2 directs these proteins for degradation by proteasomes, which can be inhibited by proteasome inhibitors (17
, 29)
. Therefore, we investigated whether the GA- or 17-AAG-mediated decline in Bcr-Abl levels is attributable to degradation by the proteasomes. Fig. 4A
shows that cotreatment with the proteasome inhibitor PSC341 significantly inhibited the GA or 17-AAG (5 µM for 24 h)-mediated decline in Bcr-Abl and c-Raf. Fig. 4, B and C
, clearly demonstrate that Bcr-Abl and c-Raf proteins, which were protected from degradation by the proteasomes by cotreatment with PSC341, were present only in the detergent (NP40)-insoluble fraction, because they had to be extracted with the SDS buffer prior to detection. Treatment with PSC341 alone for 24 h produced an increased in c-Raf but not in Bcr-Abl levels in both detergent-soluble and -insoluble fractions (Fig. 4, B and C)
. This may be because of the differences in the half-life of the two proteins in K562 cells. Fig. 4D
demonstrates that cotreatment with a concentration of PSC341, which alone is not cytotoxic, significantly inhibited 17-AAG-induced apoptosis of K562 cells (P < 0.01). Similar observations were made with GA-induced apoptosis of K562 and HL-60/Bcr-Abl cells (data not shown).
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Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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GA is known to bind to the ATP/ADP binding pocket in the NH2-terminus region of Hsp90 (18) . This blocks the binding of ATP to Hsp90, thereby disrupting its chaperone, protein-protein association with the "client" proteins including Raf-1, v-Src, p185c-erbB-2, and mutant p53 (17 , 19 , 20) . This has been shown to result in the intracellular degradation of these proteins through the proteasomes (17 , 22 , 29) . GA-induced depletion of p185c-erbB-2, Raf, and mutant p53 has also been shown to exert an antiproliferative effect on breast cancer cells (28) . Present studies highlight a similar effect of GA-mediated inhibition of the chaperone function of Hsp90 with respect to Bcr-Abl. Our data indicate that treatment with GA or 17-AAG disrupts binding of Hsp90 with Bcr-Abl TK, while promoting its chaperone association with Hsp70 and degradation by the proteasomes. During the preparation of this report, similar findings were also reported by An et al. (37) . However, unlike their report, we have shown that cotreatment with the proteasome inhibitor PS341 not only inhibits the 17-AAG (a clinically relevant analogue of GA)-mediated decline in Bcr-Abl levels but also inhibits 17-AAG-induced apoptosis of HL-60/Bcr-Abl and K562. We also demonstrate that by lowering Bcr-Abl levels, 17-AAG and GA, similar to herbimycin A (23) , promote erythroid differentiation (Hb induction) in HL-60/Bcr-Abl and K562 cells.
Recent reports have documented several promising strategies that target either the mRNA or protein encoded by the bcr-abl fusion gene, which is pathogenetically responsible for the malignant phenotype of CML and Bcr-Abl-positive adult ALL (9, 10, 11, 12) . The relatively specific inhibitor of Bcr-Abl TK, STI-571, has been shown to produce a high rate of hematological remissions in CML, but the remissions induced in patients with the blast crisis of CML or Bcr-Abl-positive ALL have not been durable (38 , 39) . Relapses, despite continuous administration of STI-571, suggest the emergence of STI-571-resistant Bcr-Abl-positive leukemic cells. Indeed, a continuous in vitro exposure of Bcr-Abl-positive leukemic cells to STI-571 induced either bcr-abl gene amplification and mRNA and/or increased Bcr-Abl protein levels, resulting in resistance to STI-571 (40 , 41) . In a recent report, we had demonstrated that treatment with As2O3 alone reduces Bcr-Abl protein levels (14) , and a combination of As2O3 plus STI571 is more effective than either agent alone in inducing apoptosis of HL-60/Bcr-Abl and K562 cells (42) . In the present studies, our data showing that 17-AAG also lowers Bcr-Abl levels and Akt kinase activity as well as induces apoptosis create the rationale to investigate the cytotoxic effects of 17-AAG plus STI-571 in Bcr-Abl-positive leukemic cells. Collectively, the in vitro findings presented here support the in vivo studies of the combination of 17-AAG plus STI571 against Bcr-Abl-positive human leukemias that are either sensitive or resistant to STI571.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Interdisciplinary Oncology Program, Moffitt Cancer Center, 12902 Magnolia Drive, MRC 3 East, Room 3056, Tampa, FL 33612. Phone: (813) 903-6861; Fax (813) 903-6817; E-mail: bhallakn{at}moffitt.usf.edu 
2 The abbreviations used are: TK, tyrosine kinase; CML, chronic myelogenous leukemia; ALL, acute lymphocytic leukemia; cyt c, cytochrome c; NF-B, nuclear factor-B; Hb, hemoglobin; GA, geldanamycin; 17-AAG, 17-allylamino-17-demethoxygeldanamycin; Hsp, heat shock protein; PMSF, phenylmethylsulfonyl fluoride.
Received 8/23/00. Accepted 1/18/01.
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 M. Rahmani, E. Reese, Y. Dai, C. Bauer, L. B. Kramer, M. Huang, R. Jove, P. Dent, and S. Grant Cotreatment with Suberanoylanilide Hydroxamic Acid and 17-Allylamino 17-demethoxygeldanamycin Synergistically Induces Apoptosis in Bcr-Abl+ Cells Sensitive and Resistant to STI571 (Imatinib Mesylate) in Association with Down-Regulation of Bcr-Abl, Abrogation of Signal Transducer and Activator of Transcription 5 Activity, and Bax Conformational Change Mol. Pharmacol., April 1, 2005; 67(4): 1166 - 1176. [Abstract] [Full Text] [PDF]
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J. L. Grem, G. Morrison, X.-D. Guo, E. Agnew, C. H. Takimoto, R. Thomas, E. Szabo, L. Grochow, F. Grollman, J. M. Hamilton, et al. Phase I and Pharmacologic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients With Solid Tumors J. Clin. Oncol., March 20, 2005; 23(9): 1885 - 1893. [Abstract] [Full Text] [PDF]
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 K. Petersen Shay, Z. Wang, P.-x. Xing, I. F.C. McKenzie, and N. S. Magnuson Pim-1 Kinase Stability Is Regulated by Heat Shock Proteins and the Ubiquitin-Proteasome Pathway Mol. Cancer Res., March 1, 2005; 3(3): 170 - 181. [Abstract] [Full Text] [PDF]
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P. George, P. Bali, S. Annavarapu, A. Scuto, W. Fiskus, F. Guo, C. Sigua, G. Sondarva, L. Moscinski, P. Atadja, et al. Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3 Blood, February 15, 2005; 105(4): 1768 - 1776. [Abstract] [Full Text] [PDF]
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P. Bali, P. George, P. Cohen, J. Tao, F. Guo, C. Sigua, A. Vishvanath, A. Scuto, S. Annavarapu, W. Fiskus, et al. Superior Activity of the Combination of Histone Deacetylase Inhibitor LAQ824 and the FLT-3 Kinase Inhibitor PKC412 against Human Acute Myelogenous Leukemia Cells with Mutant FLT-3 Clin. Cancer Res., August 1, 2004; 10(15): 4991 - 4997. [Abstract] [Full Text] [PDF]
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P. George, P. Bali, P. Cohen, J. Tao, F. Guo, C. Sigua, A. Vishvanath, W. Fiskus, A. Scuto, S. Annavarapu, et al. Cotreatment with 17-Allylamino-Demethoxygeldanamycin and FLT-3 Kinase Inhibitor PKC412 Is Highly Effective against Human Acute Myelogenous Leukemia Cells with Mutant FLT-3 Cancer Res., May 15, 2004; 64(10): 3645 - 3652. [Abstract] [Full Text] [PDF]
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D. T. Jones, E. Addison, J. M. North, M. W. Lowdell, A. V. Hoffbrand, A. B. Mehta, K. Ganeshaguru, N. I. Folarin, and R. G. Wickremasinghe Geldanamycin and herbimycin A induce apoptotic killing of B chronic lymphocytic leukemia cells and augment the cells' sensitivity to cytotoxic drugs Blood, March 1, 2004; 103(5): 1855 - 1861. [Abstract] [Full Text] [PDF]
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S. J. H. Arlander, A. K. Eapen, B. T. Vroman, R. J. McDonald, D. O. Toft, and L. M. Karnitz Hsp90 Inhibition Depletes Chk1 and Sensitizes Tumor Cells to Replication Stress J. Biol. Chem., December 26, 2003; 278(52): 52572 - 52577. [Abstract] [Full Text] [PDF]
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M. Rahmani, C. Yu, Y. Dai, E. Reese, W. Ahmed, P. Dent, and S. Grant Coadministration of the Heat Shock Protein 90 Antagonist 17-Allylamino- 17-demethoxygeldanamycin with Suberoylanilide Hydroxamic Acid or Sodium Butyrate Synergistically Induces Apoptosis in Human Leukemia Cells Cancer Res., December 1, 2003; 63(23): 8420 - 8427. [Abstract] [Full Text] [PDF]
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R. Nimmanapalli, P. Bali, E. O'Bryan, L. Fuino, F. Guo, J. Wu, P. Houghton, and K. Bhalla Arsenic Trioxide Inhibits Translation of mRNA of bcr-abl, Resulting in Attenuation of Bcr-Abl Levels and Apoptosis of Human Leukemia Cells Cancer Res., November 15, 2003; 63(22): 7950 - 7958. [Abstract] [Full Text] [PDF]
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N. von Bubnoff, D. R. Veach, W. T. Miller, W. Li, J. Sanger, C. Peschel, W. G. Bornmann, B. Clarkson, and J. Duyster Inhibition of Wild-Type and Mutant Bcr-Abl by Pyrido-Pyrimidine-Type Small Molecule Kinase Inhibitors Cancer Res., October 1, 2003; 63(19): 6395 - 6404. [Abstract] [Full Text] [PDF]
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L. Fuino, P. Bali, S. Wittmann, S. Donapaty, F. Guo, H. Yamaguchi, H.-G. Wang, P. Atadja, and K. Bhalla Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B Mol. Cancer Ther., October 1, 2003; 2(10): 971 - 984. [Abstract] [Full Text] [PDF]
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R. Nimmanapalli, L. Fuino, P. Bali, M. Gasparetto, M. Glozak, J. Tao, L. Moscinski, C. Smith, J. Wu, R. Jove, et al. Histone Deacetylase Inhibitor LAQ824 Both Lowers Expression and Promotes Proteasomal Degradation of Bcr-Abl and Induces Apoptosis of Imatinib Mesylate-sensitive or -refractory Chronic Myelogenous Leukemia-Blast Crisis Cells Cancer Res., August 15, 2003; 63(16): 5126 - 5135. [Abstract] [Full Text] [PDF]
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 M. P. Goetz, D. O. Toft, M. M. Ames, and C. Erlichman The Hsp90 chaperone complex as a novel target for cancer therapy Ann. Onc., August 1, 2003; 14(8): 1169 - 1176. [Abstract] [Full Text] [PDF]
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R. Nimmanapalli, E. O'Bryan, D. Kuhn, H. Yamaguchi, H.-G. Wang, and K. N. Bhalla Regulation of 17-AAG--induced apoptosis: role of Bcl-2, Bcl-xL, and Bax downstream of 17-AAG--mediated down-regulation of Akt, Raf-1, and Src kinases Blood, July 1, 2003; 102(1): 269 - 275. [Abstract] [Full Text] [PDF]
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C. Yu, M. Rahmani, J. Almenara, M. Subler, G. Krystal, D. Conrad, L. Varticovski, P. Dent, and S. Grant Histone Deacetylase Inhibitors Promote STI571-mediated Apoptosis in STI571-sensitive and -resistant Bcr/Abl+ Human Myeloid Leukemia Cells Cancer Res., May 1, 2003; 63(9): 2118 - 2126. [Abstract] [Full Text] [PDF]
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 R. Villa, M. Folini, C. D. Porta, A. Valentini, M. Pennati, M. G. Daidone, and N. Zaffaroni Inhibition of telomerase activity by geldanamycin and 17-allylamino, 17-demethoxygeldanamycin in human melanoma cells Carcinogenesis, May 1, 2003; 24(5): 851 - 859. [Abstract] [Full Text] [PDF]
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E. A. Sausville Is Another Bcr-Abl Inhibitor Needed for Chronic Myelogenous Leukemia? Clin. Cancer Res., April 1, 2003; 9(4): 1233 - 1234. [Full Text] [PDF]
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O. Tikhomirov and G. Carpenter Identification of ErbB-2 Kinase Domain Motifs Required for Geldanamycin-induced Degradation Cancer Res., January 1, 2003; 63(1): 39 - 43. [Abstract] [Full Text] [PDF]
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S. Wittmann, P. Bali, S. Donapaty, R. Nimmanapalli, F. Guo, H. Yamaguchi, M. Huang, R. Jove, H. G. Wang, and K. Bhalla Flavopiridol Down-Regulates Antiapoptotic Proteins and Sensitizes Human Breast Cancer Cells to Epothilone B-induced Apoptosis Cancer Res., January 1, 2003; 63(1): 93 - 99. [Abstract] [Full Text] [PDF]
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J. V. Melo, T. P. Hughes, and J. F. Apperley Chronic Myeloid Leukemia Hematology, January 1, 2003; 2003(1): 132 - 152. [Abstract] [Full Text] [PDF]
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P. La Rosee, A. S. Corbin, E. P. Stoffregen, M. W. Deininger, and B. J. Druker Activity of the Bcr-Abl Kinase Inhibitor PD180970 against Clinically Relevant Bcr-Abl Isoforms That Cause Resistance to Imatinib Mesylate (Gleevec, STI571) Cancer Res., December 15, 2002; 62(24): 7149 - 7153. [Abstract] [Full Text] [PDF]
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R. Nimmanapalli, E. O'Bryan, M. Huang, P. Bali, P. K. Burnette, T. Loughran, J. Tepperberg, R. Jove, and K. Bhalla Molecular Characterization and Sensitivity of STI-571 (Imatinib Mesylate, Gleevec)-resistant, Bcr-Abl-positive, Human Acute Leukemia Cells to SRC Kinase Inhibitor PD180970 and 17-Allylamino-17-demethoxygeldanamycin Cancer Res., October 15, 2002; 62(20): 5761 - 5769. [Abstract] [Full Text] [PDF]
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M. E. Gorre, K. Ellwood-Yen, G. Chiosis, N. Rosen, and C. L. Sawyers BCR-ABL point mutants isolated from patients with imatinib mesylate-resistant chronic myeloid leukemia remain sensitive to inhibitors of the BCR-ABL chaperone heat shock protein 90 Blood, September 26, 2002; 100(8): 3041 - 3044. [Abstract] [Full Text] [PDF]
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C. Yu, G. Krystal, P. Dent, and S. Grant Flavopiridol Potentiates STI571-induced Mitochondrial Damage and Apoptosis in BCR-ABL-positive Human Leukemia Cells Clin. Cancer Res., September 1, 2002; 8(9): 2976 - 2984. [Abstract] [Full Text] [PDF]
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J. S. Isaacs, Y.-J. Jung, E. G. Mimnaugh, A. Martinez, F. Cuttitta, and L. M. Neckers Hsp90 Regulates a von Hippel Lindau-independent Hypoxia-inducible Factor-1alpha -degradative Pathway J. Biol. Chem., August 9, 2002; 277(33): 29936 - 29944. [Abstract] [Full Text] [PDF]
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 V. Poulaki, C. S. Mitsiades, V. Kotoula, S. Tseleni-Balafouta, A. Ashkenazi, D. A. Koutras, and N. Mitsiades Regulation of Apo2L/Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Thyroid Carcinoma Cells Am. J. Pathol., August 1, 2002; 161(2): 643 - 654. [Abstract] [Full Text] [PDF]
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N. Fujita, S. Sato, A. Ishida, and T. Tsuruo Involvement of Hsp90 in Signaling and Stability of 3-Phosphoinositide-dependent Kinase-1 J. Biol. Chem., March 15, 2002; 277(12): 10346 - 10353. [Abstract] [Full Text] [PDF]
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P. Bonvini, T. Gastaldi, B. Falini, and A. Rosolen Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK), a Novel Hsp90-Client Tyrosine Kinase: Down-Regulation of NPM-ALK Expression and Tyrosine Phosphorylation in ALK+ CD30+ Lymphoma Cells by the Hsp90 Antagonist 17-Allylamino,17-demethoxygeldanamycin Cancer Res., March 1, 2002; 62(5): 1559 - 1566. [Abstract] [Full Text] [PDF]
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C. Yu, G. Krystal, L. Varticovksi, R. McKinstry, M. Rahmani, P. Dent, and S. Grant Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells Cancer Res., January 1, 2002; 62(1): 188 - 199. [Abstract] [Full Text] [PDF]
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F. Turturro, M. D. Arnold, A. Y. Frist, and K. Pulford Model of Inhibition of the NPM-ALK Kinase Activity by Herbimycin A Clin. Cancer Res., January 1, 2002; 8(1): 240 - 245. [Abstract] [Full Text] [PDF]
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E. A. Sausville Combining Cytotoxics and 17-Allylamino, 17-Demethoxygeldanamycin: Sequence and Tumor Biology Matters : Commentary re: P. Munster et al., Modulation of Hsp90 Function by Ansamycins Sensitizes Breast Cancer Cells to Chemotherapy-induced Apoptosis in an RB- and Schedule-dependent Manner. Clin. Cancer Res., 7: 2228-2236, 2001. Clin. Cancer Res., August 1, 2001; 7(8): 2155 - 2158. [Full Text] [PDF]
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O. Tikhomirov and G. Carpenter Caspase-dependent Cleavage of ErbB-2 by Geldanamycin and Staurosporin J. Biol. Chem., August 31, 2001; 276(36): 33675 - 33680. [Abstract] [Full Text] [PDF]
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