| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Advances in Brief |
Division of Hematology/Oncology, Department of Medicine, College of Medicine [C. L., S. T. O.], Department of Molecular Biology and Biochemistry [A. F. A., C. M. W.], and Cancer Research Institute [C. M. W., S. T. O.], University of California, Irvine, California 92697, and Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, United Kingdom [J. V. M.]
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
B, glycogen synthase kinase-3ß, mTOR, and murine double minute 2 (MDM2), are actually important. Recently, other groups have used a combination of biochemistry and bioinformatics to successfully identify novel PI3K/AKT targets in a variety of cell types (3
, 4)
. We investigated whether a similar approach might identify downstream targets of the PI3K pathway in CML cells. We have identified the S6 ribosomal protein as being phosphorylated in a Bcr-Abl kinase-, PI3K-, and mTOR-dependent manner in Bcr-Abl-expressing cell lines and CML cell lines. Another mTOR substrate and important regulator of translation, 4E-BP1, is similarly regulated. The possibility of treating CML cells with a combination of imatinib and an inhibitor of mTOR, rapamycin, was also explored. |
Materials and Methods |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
Western Blot Analysis and Immunoprecipitation.
After incubations, cells were collected and washed twice with PBS and incubated on ice for 15 min in 500 µl of lysis buffer. Protein lysates were quantitated by the Bradford assay, 15 µg resolved by SDS-PAGE, and transferred to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ). The following antibodies were used for immunoblotting: c-abl (clone 8E9; PharMingen, San Diego, CA), phosphotyrosine (clone 4G10; Upstate Biotechnology, Inc., Lake Placid, NY), phospho-(Ser/Thr) AKT substrate (AKT-pSub), phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein, and 4E-BP1. All antibodies were from Cell Signaling Technology (Beverly, MA), unless otherwise stated. Immunoblotting was performed according to the manufacturers’ instructions. Immunoreactive bands were visualized by enhanced chemiluminescence (Pierce Chemical Co.) after incubation with a secondary antibody. For immunoprecipitation, K562 cells were incubated for 24 h with or without 10 µM LY. Cells were harvested under nondenaturing conditions by removing media and washing cells twice with cold PBS. Cells were then lysed with ice-cold lysis buffer [30 mM NaCl, 50 mM NaF, 10 mM ß-glycerophosphate, 20 mM HEPES (pH 7.4), 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride] for 10 min on ice, followed by centrifugation for 10 min at 4°C. Lysate was transferred to a new tube, and 20 µl of immobilized AKT-pSub antibody were used for every 200 µl of lysate for an overnight incubation at 4°C. After this, each sample was centrifuged for 1 min at 4°C. The pellet was washed five times using lysis buffer, after which 6x SDS sample buffer was added and samples were boiled for 5 min. The samples were loaded onto Bio-Rad (Hercules, CA.) 12% Tris-HCl ReadyGels to perform SDS-PAGE and analyzed by Western blotting.
Two-Dimensional Gel Electrophoresis.
Lysates of K562 cells that had been treated with or without LY (10 µM) were prepared for two-dimensional electrophoresis. Lysates were prepared with cell lysis buffer containing 30 mM NaCl, 50 mM NaF, 10 mM ß-glycerophosphate, 20 mM HEPES (pH 7.4), and 1% Triton X-100. Two-dimensional electrophoresis was performed according to the method of O’Farrell (7)
by Kendrick Labs, Inc. (Madison, WI). After slab gel electrophoresis, the gel for blotting was transferred to transfer buffer [12.5 mM Tris (pH 8.8), 86 mM glycine, and 10% methanol] and transblotted onto polyvinylidene difluoride paper overnight at 200 mA and 100 V/two gels. After transfer, polyvinylidene difluoride membranes were stained with Coomassie Blue.
Patient Samples.
Peripheral blood samples were obtained with appropriate consent and Institutional Review Board approval from CML patients at the University of California at Irvine. Mononuclear cells were obtained by centrifugation through Ficoll-Hypaque, washed twice in PBS, and cryopreserved. Western analysis was performed as described above.
Tetrazolium-based Proliferation Assays (MTS assay).
Exponentially growing cells were plated in triplicate in 96-well flat-bottomed plates. Cells (20 x 103) were plated in each well in a volume of 100 µl in RPMI with 10% FCS with imatinib (in PBS) at concentrations of 0, 1.0, 5.0, and 10.0 µM with or without rapamycin at 10 ng/ml. Because rapamycin was dissolved in alcohol, controls were also performed with cells incubated in 0.1% ethanol in media. Viability was determined at 48 h by incubating cells in 20 µl of CellTiter 96 AQueous One Solution reagent (Promega, Madison, WI) for 2 h at 37°C in the dark. An EMax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA) was used to read the absorbance at 490 nm. The absorbance of media alone without cells was subtracted from the reading of each well, and the values for the triplicates were averaged to obtain an absorbance for each condition.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
. A prominent band of Mr 28,000 to Mr 32,000 apparent molecular weight was present in both Ba/F3-Bcr-Abl-WT and K562 CML cells, which became markedly attenuated on treatment with either imatinib or LY. Thus, the phosphorylation of this protein occurs in an Abl kinase- and PI3K-dependent manner. The known specificity of the AKT-pSub antibody suggested that the target for phosphorylation was a serine or threonine within the RxRxxS/T consensus motif.
|
. Two prominent groups of proteins at Mr 30,000 (Fig. 1B
, top left, circles a and b) were detected in the lysates from untreated K562 cells. Of these two, the more basic group with a pI between 8 and 11 was no longer detectable in the immunoblot of lysates from LY-treated K562 cells, suggesting that this group represented the LY-sensitive Mr 30,000 protein (Fig. 1B
, top right, circle a).
We searched the GenPept database using the Scansite program with the AKT substrate antibody matrix, limiting the results to proteins with a molecular mass of 28–32 kDa and a pI between 8 and 11. Because the Mr 28,000 to Mr 32,000 protein was found in both murine and human cells, we further narrowed the search to only those results that included both species. This resulted in three candidate substrates, as shown in Table 1
. While we were conducting these experiments, another group also used the AKT-pSub antibody and found a similar sized Mr 30,000 protein in ES cells that was phosphorylated in a PI3K-dependent manner; this protein was identified as ribosomal protein S6 (4)
. The Scansite program predicts that the AKT-pSub antibody would recognize the phosphorylated serine found within the RxRxxS/T consensus sequence at position 236 of S6. The 236 position of S6 ribosomal protein is known to be phosphorylated by p70 S6 kinase in a PI3K- and rapamycin-dependent fashion (8)
. The detection of S6 using an antibody designed to recognize phosphorylated AKT substrates is likely caused by very similar linear substrate recognition motifs shared between these two families of kinases.
|
was S6, we used the pS6(Ser235/236) antibody, which recognizes only the Ser235/236-phosphorylated form of S6 to probe the two-dimensional electrophoresis. The pS6(Ser235/236) antibody detected the same proteins in circles a and b in untreated K562 cells; in LY-treated cells, no phosphoS6 was observed in circle a, and some residual protein was observed in circle b (Fig. 1B
, bottom). These results strongly suggested that the Mr 30,000 protein detected by the AKT-pSub antibody was S6. S6 ribosomal protein is phosphorylated sequentially at Ser235, Ser236, Ser240, and Ser244 (9)
. As described previously, the pS6(Ser236/235) antibody recognizes two forms of phosphorylated S6: a higher mobility band corresponding to phosphorylation at Ser235/236 only, and a lower mobility band corresponding to phosphorylation at all four sites (10)
. It is, therefore, likely that the proteins in circles a and b correspond to phosphorylated and hyperphosphorylated S6, respectively, or that the protein species in circle b is a distinct postranslational modification of S6. An alternative explanation is that the species observed in circle b is entirely distinct from S6. However, because subsequent experiments demonstrate that all AKT-pSub immunoreactive species detected in our Western blots disappear in the presence of rapamycin (see below), this band must necessarily be a downstream substrate of a kinase regulated by mTOR.
To confirm that phosphorylated S6 was indeed the Mr 30,000 protein recognized by the AKT-pSub antibody, we performed immunoprecipitation with this antibody, followed by SDS-PAGE and Western blot analysis for phosphorylated S6, using lysates from untreated and LY-treated K562 cells (Fig. 1C)
. As expected, the pS6(Ser235/236) antibody detected a Mr 30,000 band from untreated cells and a significantly less prominent band from LY-treated cells.
Phosphorylation of S6 at Ser235/236 is performed by two related kinases, S6K1 and S6K2, both of which are, in turn, activated (phosphorylated) by mTOR. This predicted that treatment of cells with the mTOR inhibitor rapamycin should block S6 phosphorylation. As expected, phosphorylation of the Mr 30,000 protein was sensitive to rapamycin in both murine and human CML cells in immunoblots with either phospho-S6 or the AKT-pSub antibody (Fig. 2A)
. The levels of total S6 were not affected by imatinib or rapamycin treatment, indicating that the differences in S6 phosphorylation were not because of differences in total S6 (Fig. 2A)
. Interestingly, in K562 cells, despite complete inhibition of Bcr-Abl kinase activity by imatinib (as measured by Bcr-Abl autophosphorylation on tyrosines), there was some residual S6 phosphorylation. This indicated that K562 cells have both Abl kinase-dependent and -independent mechanisms for S6 phosphorylation.
|
.
4E-BP1, a negative regulator of cap-dependent mRNA translation, is also phosphorylated by mTOR, but unlike S6 is inactivated by this modification (9)
. We, therefore, tested whether 4E-BP1 phosphorylation also occurred in CML cells in a Bcr-Abl kinase- and mTOR-dependent manner. We used an antibody to total 4E-BP1 that is able to detect unphosphorylated, phosphorylated, and hyperphosphorylated forms of 4E-BP1 as bands of decreasing mobility. We found that 4E-BP1 was constitutively phosphorylated in parental BaF/3 cells as well as in Ba/F3-Bcr-Abl-WT cells (Fig. 2C)
. However, a decrease in 4E-BP1 phosphorylation, after imatinib treatment, was only seen in Bcr-Abl-expressing cells, indicating that 4E-BP1 phosphorylation is dependent on Abl kinase activity. In parental Ba/F3 cells, which are IL-3 dependent, 4E-BP1 phosphorylation is likely a result of IL-3-mediated signaling (9)
. Furthermore, in Baf-BCR-ABL-R cells, increasing concentrations of imatinib resulted in a progressive shift to less phosphorylated 4E-BP1 forms. In contrast, imatinib did not affect Ba/F3-Bcr-Abl-T315I cells at all doses. In all cells, rapamycin resulted in an increase in unphosphorylated 4E-BP1 (Fig. 2C)
.
To determine whether S6 and 4E-BP1 are similarly regulated in primary CML cells, we performed Western analysis on peripheral blood mononuclear cells from a patient (CML-1) with cytogenetically confirmed CML. In this patient, we find that 4E-BP1 phosphorylation is inhibited by imatinib to the same extent as treatment with rapamycin and that phosphorylated S6 is at least, in part, dependent on Bcr-Abl-kinase activity (Fig. 2D)
.
These data demonstrate that two regulators of translation, ribosomal protein S6 and 4E-BP1, are constitutively phosphorylated in CML cells and that phosphorylation is dependent on Bcr-Abl and mTOR signaling. These results add mTOR to the list of AKT substrates activated in CML and suggest that targeting this intermediate in Bcr-Abl-expressing leukemias might be therapeutically useful. Because mTOR inhibitors are already in Phase I/II clinical trials for a number of cancers, these findings prompted us to determine whether targeting the mTOR pathway with the inhibitor rapamycin might inhibit growth of CML cells (11) .
The mTOR Inhibitor Rapamycin Enhances Imatinib-mediated Cell Killing.
In initial experiments, we found that rapamycin alone had a minimal or no effect on the viability of either parental Ba/F3 or Ba/F3-Bcr-Abl-WT cells (Fig. 3A)
. Because imatinib induces apoptosis in CML cells, we asked whether rapamycin could improve the efficacy of imatinib-mediated cell killing. We found that the combination of rapamycin and imatinib enhanced the killing of Ba/F3-Bcr-Abl-WT cells but had no effect on parental Ba/F3 cells (Fig. 3, B and C)
. Accordingly, we wondered whether the combination would be able to overcome imatinib resistance in CML cells. Three forms of cellular resistance to imatinib have been described in patients: (a) amplification of the wild-type Bcr-Abl gene, resulting in overexpression of the oncoprotein; (b) resistance-conferring mutations in the Abl-kinase domain (12)
; and (c) LYN kinase overexpression (13)
. We tested the ability of the combination to overcome two of these forms of resistance by treating imatinib-resistant Ba/F3 cells overexpressing wild-type Bcr-Abl (Baf-BCR-ABL-R) and Ba/F3 cells expressing Bcr-Abl containing the T315I mutation (Ba/F3-Bcr-Abl-T315I) with rapamycin and imatinib. We found that the addition of rapamycin was able to overcome imatinib resistance in Baf-BCR-ABL-R cells at doses of imatinib as low as 1 µM. Baf-BCR-ABL-R cells are completely resistant to imatinib at this concentration and, in fact, are maintained at 1 µM imatinib (14)
. In contrast, Ba/F3-Bcr-Abl-T315I cells were completely resistant to imatinib and rapamycin (Fig. 3C)
.
|
. |
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
The present study demonstrates that the mTOR pathway is constitutively activated by Bcr-Abl in CML cells and that two of its known substrates, ribosomal protein S6 and 4E-BP1, are constitutively phosphorylated in a Bcr-Abl-dependent manner. mTOR acts as a nutrient and mitogen sensor to positively regulate translation and ribosome biogenesis (17) . On activation by mitogens and amino acids, mTOR phosphorylates two key translational regulators: S6K1 and 4E-BP1. S6K1 activation leads to the phosphorylation of the ribosomal protein S6. S6 is important in the translation of mRNAs bearing a 5' terminal oligopolypyrimidine tract (9) , such as ribosomal proteins, elongation factors, and growth factors (8) . 4E-BP1, in contrast, is a negative regulator of translation. It is phosphorylated in response to growth signals; on phosphorylation, 4E-BP1 dissociates from eIF4E, the translation initiation factor that binds the 5' methyl cap structure on mRNAs, allowing translation to proceed. eIF4E function is particularly important for a subset of genes that are poorly translated in resting cells but are recruited to ribosomes after a proliferative signal (9) . The differing translation efficiencies for these mRNAs seem to be a result of a highly structured 5' untranslated region, or the presence of multiple open reading frames, both of which inhibit efficient scanning (9) .
Examples of genes with long, highly structured 5' untranslated regions that are translationally regulated by mTOR include c-myc and cyclin D1 (18 , 19) . Of note, both of these genes have been implicated in the pathogenesis of CML and are essential for Bcr-Abl-mediated transformation (20 , 21) . Our finding that both ribosomal protein S6 and 4E-BP1 are phosphorylated in a Bcr-Abl kinase-dependent manner in CML cells suggests that deregulated Abl kinase activity leads to activation of critical genes such as c-myc and cyclin D1 by altering their translational control. Post-transcriptional mechanisms mediating Bcr-Abl leukemogenesis have been reviewed recently (22) , although the main focus was on the ability of Bcr-Abl to increase the stability of RNA binding proteins involved in regulating translation. Whereas mechanisms and signaling pathways mediating the effect of Bcr-Abl on protein stability remain ill-defined, PI3K signaling has been implicated (23) . Thus, Bcr-Abl may signal through PI3K to affect target gene expression post-transcriptionally, both at the level of translational efficiency and protein stability.
How might rapamycin enhance imatinib-mediated killing of CML cells? The combination of imatinib and rapamycin had minimal effect on viability in Ba/F3-Bcr-Abl-T315I cells but was effective in increased killing of Baf-BCR-ABL-R cells (Fig. 3C)
. This suggests that the combination of imatinib and rapamycin acts to inhibit signals distal to Bcr-Abl and mTOR below a critical threshold required for viability. A model to explain the results is that there are two parallel Bcr-Abl-dependent pathways transmitting survival signals: one that acts via mTOR, and another that is independent of mTOR (Fig. 3E)
. These two pathways then converge on an integrator of survival signals that transmits such signals only when a threshold level is reached. Thus, inhibition of mTOR alone would have no effect on survival, if the survival threshold is achieved by mTOR-independent signaling. Conversely, inhibition of Bcr-Abl kinase by imatinib, by affecting both pathways, would lower the signals below the survival threshold, resulting in death. In situations when imatinib is able to partially reduce mTOR-independent signaling, mTOR inhibition could then further reduce the integrated signal below the threshold for survival. In addition, our observation that IL-3 is able to rescue the effects of rapamycin indicates that intermediates in the mTOR-independent pathway might include Jak2 and c-myc. This is suggested by the work of Xie et al. (16)
, who have shown that Jak2 is a key mediator of the effects of Bcr-Abl on the IL-3 receptor pathway and is associated with increased c-myc expression. The differential effect of the combined treatment on the viability of Ba/F3-Bcr-Abl-WT, Baf-BCR-ABL-R, and Ba/F3-Bcr-Abl-T315I cells is reminiscent of the findings of Hoover et al. (24)
, using the combination of the farnesyl transferase inhibitor SCH66336 and imatinib. These investigators invoked a similar model to explain their findings (24)
. After the initial report of the T315I kinase domain mutation (which confers a high level of imatinib resistance) in patients with imatinib resistance, several other mutations have been described that confer a moderate degree of imatinib resistance (12)
. If the threshold model is true, then the combination of rapamycin (or SCH66336) and imatinib may be able to overcome resistance in these patients as well.
In summary, phosphospecific motif antibodies and newly available Web-based bioinformatic programs facilitate the proteomic identification of novel kinase substrates, and can lead to the elucidation of signaling pathways in human malignancies that may be clinically exploitable.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by the Cancer Research Institute, the University of California at Irvine (to S. T. O. and C. M. W.), the Oxnard Foundation (to S. T. O.), and NIH Grant R01-AI050606 (to C. M. W.). 
2 To whom requests for reprints should be addressed, at Division of Hematology/Oncology, Department of Medicine, College of Medicine, 839 Medical Sciences Court, 116 Sprague Hall, Irvine, CA 92697. Phone: (949) 824-2808; Fax: (949) 824-4023; E-mail: ongt{at}uci.edu
3 The abbreviations used are: CML, chronic myelogenous leukemia; PI3K, phosphoinositide 3-kinase; IL-3, interleukin 3; mTOR, mammalian target of rapamycin.
4 Internet address: http://scansite.mit.edu.
Received 5/ 1/03. Revised 6/28/03. Accepted 7/14/03.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  E. Katsoulidis, A. Sassano, B. Majchrzak-Kita, N. Carayol, P. Yoon, A. Jordan, B. J. Druker, E. N. Fish, and L. C. Platanias Suppression of Interferon (IFN)-inducible Genes and IFN-mediated Functional Responses in BCR-ABL-expressing Cells J. Biol. Chem., April 18, 2008; 283(16): 10793 - 10803. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Carayol, E. Katsoulidis, A. Sassano, J. K. Altman, B. J. Druker, and L. C. Platanias Suppression of Programmed Cell Death 4 (PDCD4) Protein Expression by BCR-ABL-regulated Engagement of the mTOR/p70 S6 Kinase Pathway J. Biol. Chem., March 28, 2008; 283(13): 8601 - 8610. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. F. Arechiga, B. D. Bell, S. Leverrier, B. M. Weist, M. Porter, Z. Wu, Y. Kanno, S. J. Ramos, S. T. Ong, R. Siegel, et al. A Fas-Associated Death Domain Protein/Caspase-8-Signaling Axis Promotes S-Phase Entry and Maintains S6 Kinase Activity in T Cells Responding to IL-2 J. Immunol., October 15, 2007; 179(8): 5291 - 5300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Perrotti and P. Neviani From mRNA Metabolism to Cancer Therapy: Chronic Myelogenous Leukemia Shows the Way Clin. Cancer Res., March 15, 2007; 13(6): 1638 - 1642. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yoshida, F. Yoshida, D. E. Sears, S. M. Hart, D. Ikebe, A. Muto, S. Basu, K. Igarashi, and J. V. Melo Bcr-Abl signaling through the PI-3/S6 kinase pathway inhibits nuclear translocation of the transcription factor Bach2, which represses the antiapoptotic factor heme oxygenase-1 Blood, February 1, 2007; 109(3): 1211 - 1219. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Yoon, N. Giafis, J. Smith, H. Mears, E. Katsoulidis, A. Sassano, J. Altman, A. J. Redig, M. S. Tallman, and L. C. Platanias Activation of mammalian target of rapamycin and the p70 S6 kinase by arsenic trioxide in BCR-ABL-expressing cells. Mol. Cancer Ther., November 1, 2006; 5(11): 2815 - 2823. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ikezoe, C. Nishioka, T. Tasaka, Y. Yang, N. Komatsu, K. Togitani, H. P. Koeffler, and H. Taguchi The antitumor effects of sunitinib (formerly SU11248) against a variety of human hematologic malignancies: enhancement of growth inhibition via inhibition of mammalian target of rapamycin signaling. Mol. Cancer Ther., October 1, 2006; 5(10): 2522 - 2530. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Boissel, D. Rea, V. Tieng, N. Dulphy, M. Brun, J.-M. Cayuela, P. Rousselot, R. Tamouza, P. Le Bouteiller, F.-X. Mahon, et al. BCR/ABL oncogene directly controls MHC class I chain-related molecule A expression in chronic myelogenous leukemia. J. Immunol., April 15, 2006; 176(8): 5108 - 5116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Notari, P. Neviani, R. Santhanam, B. W. Blaser, J.-S. Chang, A. Galietta, A. E. Willis, D. C. Roy, M. A. Caligiuri, G. Marcucci, et al. A MAPK/HNRPK pathway controls BCR/ABL oncogenic potential by regulating MYC mRNA translation Blood, March 15, 2006; 107(6): 2507 - 2516. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Parmar, J. Smith, A. Sassano, S. Uddin, E. Katsoulidis, B. Majchrzak, S. Kambhampati, E. A. Eklund, M. S. Tallman, E. N. Fish, et al. Differential regulation of the p70 S6 kinase pathway by interferon {alpha} (IFN{alpha}) and imatinib mesylate (STI571) in chronic myelogenous leukemia cells Blood, October 1, 2005; 106(7): 2436 - 2443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. S. Hammerman, C. J. Fox, M. J. Birnbaum, and C. B. Thompson Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival Blood, June 1, 2005; 105(11): 4477 - 4483. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Gu, L. Santiago, and B. S. Mitchell Synergy between imatinib and mycophenolic acid in inducing apoptosis in cell lines expressing Bcr-Abl Blood, April 15, 2005; 105(8): 3270 - 3277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Deininger, E. Buchdunger, and B. J. Druker The development of imatinib as a therapeutic agent for chronic myeloid leukemia Blood, April 1, 2005; 105(7): 2640 - 2653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Kharas and D. A. Fruman ABL Oncogenes and Phosphoinositide 3-Kinase: Mechanism of Activation and Downstream Effectors Cancer Res., March 15, 2005; 65(6): 2047 - 2053. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Recher, O. Beyne-Rauzy, C. Demur, G. Chicanne, C. Dos Santos, V. M.-D. Mas, D. Benzaquen, G. Laurent, F. Huguet, and B. Payrastre Antileukemic activity of rapamycin in acute myeloid leukemia Blood, March 15, 2005; 105(6): 2527 - 2534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. P. Shah Loss of Response to Imatinib: Mechanisms and Management Hematology, January 1, 2005; 2005(1): 183 - 187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Mohi, C. Boulton, T.-L. Gu, D. W. Sternberg, D. Neuberg, J. D. Griffin, D. G. Gilliland, and B. G. Neel Combination of rapamycin and protein tyrosine kinase (PTK) inhibitors for the treatment of leukemias caused by oncogenic PTKs PNAS, March 2, 2004; 101(9): 3130 - 3135. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), phosphorylated (ß), and hyperphosphorylated (
) forms of 4E-BP1, as bands of decreasing mobility. Bottom, Baf-BCR-ABL-R and Ba/F3-Bcr-Abl-T315I cells were treated with increasing doses of imatinib in the presence or absence of rapamycin. D, cell lysates of untreated peripheral blood mononuclear cells from a patient with CML, CML-1, or those treated with 10 µM imatinib or 10 ng/ml rapamycin were subjected to Western analysis with anti-4E-BP1, anti-pS6(Ser235/236), and anti-Abl.