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Cell, Tumor, and Stem Cell Biology |
1 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India and 2 Department of Pathology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma
Requests for reprints: Kumaravel Somasundaram, Department of Microbiology and Cell Biology, Indian Institute of Science, Sir C.V. Raman Road, Bangalore, Karnataka, India 560012. Phone: 91-80-22932973; Fax: 91-80-23602697; E-mail: skumar{at}mcbl.iisc.ernet.in.
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Introduction |
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The four mammalian Notch genes (Notch1-4), which encode 300-kDa single-pass transmembrane receptors, are evolutionarily conserved with an important role in cell fate determination and differentiation. Notch activation is initiated by interactions with ligands of the Delta and Serrate/Jagged families and results in cleavage and release of the intracellular region of the Notch receptor by proteolytic cleavage and nuclear translocation (3). The released COOH-terminal fragment of Notch translocates to the nucleus, in which it interacts with the transcription factor CBF1 (RBPjk) to transactivate target genes, including HES1 (4).
Constitutively active intracellular forms of Notch have been shown to have oncogenic activity in T-cell acute lymphoblastic leukemia (T-ALL; ref. 5), mouse mammary tumors (6), and transformed kidney epithelial cells in cooperation with adenovirus oncogene E1A (7). Furthermore, Notch1 has been shown to inhibit p53-mediated apoptosis in immortalized epithelial cells (8), T-cell receptor–induced apoptosis in mature cells (9), and dexamethasone-mediated apoptosis in thymocytes (10) and is antiapoptotic in T cells (11). However, the mechanism is not clearly understood. Here, we show that activated Notch1 signaling confers chemoresistance in a wild-type (WT) p53-dependent manner. Notch1 inhibited p53 through PI3K-Akt/protein kinase B (PKB)-mammalian target of rapamycin (mTOR)-eukaryotic initiation factor 4E (eIF4E) pathway, and the inhibitors of this pathway reversed the chemoresistance. Collectively, these results suggest that cancers with activated Notch1 signaling are chemoresistant and provide basis for the reversal of chemoresistance.
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Materials and Methods |
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ABluc were described before (12–16). Ad-LacZ and Ad-p53 lack both E1A and E1B but carry ß-galactosidase and p53, respectively (17). Adenovirus expressing NIC-1 (Ad-NIC-1), which lacks both E1A and E1B but carries NIC-1, was constructed using the method described previously (15). NIC-1 was cloned as a XhoI/XbaI fragment into pAdTrack-CMV to create pAdTrack-CMV-NIC-1. HepG2, HT1080, PA1, LNCaP, A375, and MCF7/c3 cell lines of different tissue origin carrying WT p53, SW480 and U373MG carrying mutated p53, and H1299 with p53 deletion were grown in DMEM with 10% FCS. H460, a lung carcinoma cell line carrying WT p53, was grown in RPMI 1640 supplemented with 10% FCS. T-ALL cell lines Jurkat and MOLT4 were grown in RPMI 1640 along with 10% FCS. LY294002, wortmannin, PD98059, and rapamycin (Cell Signaling Technology, Danvers, MA) were used at a final concentration of 50 µmol/L, 1 µmol/L, 50 µmol/L, and 100 nmol/L, respectively. Transfection and reporter assays. Cells were plated in 35-mm dishes and transfected using Escort III transfection reagent (Sigma, St. Louis, MO) with indicated plasmids. After 24 hours of transfection, cell lysates were prepared using reporter lysis buffer (Promega, Madison, WI), and luciferase assay was carried out using luciferase assay reagent (Promega). Luciferase activity was measured by using either luminometer (Supplementary Fig. S5A) or scintillation counter (Figs. 2A, 3A, 4A, and 5A; Supplementary Fig. S5B). Chloramphenicol acetyltransferase assay was done as described previously (12).
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Western blot analysis, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blot analysis was done as described previously (18). The details of the antibodies used are given in Supplementary Data. Fluorescence-activated cell sorting (FACS) analysis was done as described (18). The antibodies used were anti-bromodeoxyuridine (1 µg Ab-3, NA61; Calbiochem, La Jolla, CA) and FITC-conjugated goat anti-mouse fluorescein conjugate (2 µg DC13L; Calbiochem). The percentage of cells undergoing DNA synthesis (%S) and the percentage of cells containing <2N amount of DNA (%A) were calculated and shown. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was done as described previously (18). The viability of control untreated cells was considered as 100%. All MTT experiments were done at least thrice, and representative data are shown.
Electrophoretic mobility shift assay. The mouse anti-human p53 monoclonal antibody pAb421 (Ab1; Oncogene Science, Uniondale, NY) was used to activate sequence-specific DNA binding by p53 in electrophoretic mobility shift assays (EMSA) as described previously (12). Briefly, a p53-binding site from p21 promoter was used as the probe (5'-CAGGAACATGTCCCAACATGTTGAGC-3'), and the following reaction conditions were used: 10% glycerol, 20 mmol/L HEPES (pH 7.5), 25 mmol/L KCl, 2 mmol/L DTT, 2 mmol/L MgCl2, 0.2% NP40, 1 µg poly(deoxyinosinic-deoxycytidylic acid), 0.3 µg pAb421, and 2 µg nuclear extracts. Nuclear extracts were prepared as described previously (12).
Generation of stable cell lines. Stable cell lines expressing different constructs of mTOR and eIF4E were generated by transfecting respective plasmids into H460 cells and selecting for either G418 (700 µg/mL; Calbiochem) or puromycin (1 µg/mL; Sigma) resistance. Individual colonies were cloned and screened for the expression of proteins. Purified clones were used for all experiments.
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Notch1 affects the post-translational modifications and nuclear localization of p53. As our results suggest that Notch1-mediated protection against chemotherapy is p53 dependent and we have shown previously that p53-mediated apoptosis is inhibited by Notch1, we then determined the mechanism of inhibition of p53 by Notch1. NIC-1 inhibited transcription very efficiently from a p53-specific reporter, PG13-Luc, by p53 (Fig. 2A, compare lane 2 with lane 4 ). However, we found that Notch1 inhibition of p53 does not involve activation domain of p53 (Supplementary Fig. S2A and B), although both Notch1 and p53 use p300 as coactivators (12, 21, 22).
Next, we analyzed the effect of Notch1 on sequence-specific DNA binding, nuclear localization, and post-translational modifications of p53. p53 expressed from Ad-p53 bound to DNA 1.55-fold less efficiently if the nuclear extract was made from cells preinfected with Ad-NIC-1 (Fig. 2B, top, compare lane 2 with lane 4). To measure the nuclear p53 in NIC-1-overexpressing cells, we monitored the levels of p53 in nuclear extract made from Ad-NIC-1-preinfected cells. Prior expression of NIC-1 by Ad-NIC-1, infection resulted in 1.28-fold decrease of nuclear p53 in Ad-p53-infected cells (Fig. 2B, middle, compare lane 2 with lane 4). Similar results were obtained in cells where p53 is induced by chemotherapy treatment (Supplementary Fig. S2E). We then analyzed the levels of phosphorylation status of p53 as a measure of active p53. The levels of phosphorylation at Ser15, Ser20, and Ser392 of p53 expressed from Ad-p53 significantly reduced in NIC-1-overexpressing cells (Fig. 2C, compare lanes 4-6 with lanes 10-12). These results suggest that NIC-1 abrogates p53 by inhibiting its sequence-specific DNA binding, which may involve both reduced nuclear localization and reduced activating phosphorylations of p53. In good agreement with the above results, H460 cells preinfected with Ad-NIC-1 are significantly resistant to cytotoxicity induced by Ad-p53 (Supplementary Fig. S2D). Further, we found that the protective effect of NIC-1 overexpression against cytotoxicity induced by Ad-p53 infection is correlated with reduced apoptosis by p53 in the presence of NIC-1 as seen by decreased poly(ADP-ribose) polymerase (PARP) cleavage (Fig. 2C, compare lanes 4-6 with lanes 10-12) and decreased sub-G1 cells (Fig. 2D). Whereas Ad-p53 infection of Ad-LacZ-preinfected cells resulted in efficient PARP cleavage by 48 hours (
33% of PARP is cleaved), Ad-p53 infection of Ad-NIC-1-preinfected cells failed to induce PARP cleavage by 48 hours (Fig. 2C, compare lane 6 with lane 12). Similarly, the percentage apoptotic cells as measured by cells with <2N DNA content in Ad-p53-infected cells reduced from 31.60% to 9.95% after 48 hours if the cells were preinfected with Ad-NIC-1. By 72 hours of Ad-p53 infection, the percentage apoptotic cells further reduced from 61.32% to 11.51% in Ad-NIC-1-preinfected cells. Thus, these results suggest that NIC-1 protects cells against p53-induced cytotoxicity by inhibiting the ability of p53 to activate transcription and consequently apoptosis.
Notch1 confers chemoresistance through PI3K-Akt/PKB-mTOR pathway. Because Notch1 inhibits p53 through PI3K (8), which signals cell survival through Akt/PKB (23), we investigated the role of PI3K-Akt/PKB pathway in Notch1-mediated inhibition of p53 functions and consequently survival signaling. We found that PI3K inhibitors wortmannin and LY294002, but not MEK1 inhibitor PD98059, abrogated Notch1 inhibition of p53-mediated transcription very efficiently (Fig. 3A, compare lane 4 with lanes 5-7 ). These results correlated well with the inability of NIC-1 to protect cells against cytotoxicity induced by chemotherapeutic drugs Adriamycin, cisplatin, etoposide, and Taxol (Fig. 3B) and Ad-p53 infection (Supplementary Fig. S3A) in the presence of PI3K inhibitors wortmannin and LY294002. This loss of protective effect by NIC-1 in the presence of PI3K inhibitors is due to the efficient apoptosis induction by p53 (Fig. 3C). Further, Ad-NIC-1-infected cells showed dramatically increased phosphorylated form of (Ser473) Akt in the presence and absence of induced p53 (Fig. 3D, compare lanes 1-3 with lanes 7-9 and lanes 4-6 with lanes 10-12, respectively), which was inhibited by wortmannin (Supplementary Fig. S3B, compare lanes 1-3 with lanes 4-6 and lanes 7-9 with lanes 10-12, respectively). These results confirm the activation of PI3K in NIC-1-overexpressing cells and suggest a vital role for PI3K-Akt/PKB pathway in Notch1-mediated survival signaling.
As the Akt/PKB signals cell survival mainly through mTOR (23), we next analyzed the role of mTOR in Notch1-induced survival signaling, although other targets, such as forkhead in rhabdomyosarcoma (FKHR) and glycogen synthase kinase-3ß (GSK3ß), in addition to mTOR were found to be activated (Fig. 3D, compare lanes 1-3 with lanes 7-9 and lanes 4-6 with lanes 10-12). Indeed, we found mTOR inhibitor rapamycin abrogated NIC-1 inhibition of p53-mediated transcription (Fig. 3A, compare lane 4 with lane 8) and protection against cytotoxicity induced by chemotherapy (Fig. 3B) and p53 (Supplementary Fig. S3A) with similar efficiency to that of PI3K inhibitors. This loss of protective effect by NIC-1 in the presence of rapamycin is due to the efficient apoptosis induction by p53 (Fig. 3C). The percentage apoptotic cells measured as cells possessing <2N amount of DNA on Ad-p53 infection in Ad-NIC-1-preinfected cells increased from 7.37% to 43.41% by the addition of rapamycin. Further, Ad-NIC-1-infected cells showed dramatically increased phosphorylated form of two mTOR targets p70 S6 kinase 1 (S6K1; Thr389) and 4E-binding protein 1 (4E-BP1; Ser65) in the presence and absence of induced p53 (Fig. 3D, compare lanes 1-3 with lanes 7-9 and lanes 1-3 with lanes 10-12), which was inhibited by rapamycin (Supplementary Fig. S3C, compare lanes 1-3 with lanes 4-6 and lanes 7-9 with lanes 10-12). Moreover, the phosphorylated S6K1 activated its downstream target S6 (Ser235/236) as seen by the appearance of phosphorylated S6 only in the absence of rapamycin (Supplementary Fig. S3C, compare lanes 1-3 with lanes 4-6 and lanes 7-9 with lanes 10-12). Taken together, these results suggest that mTOR is the key downstream target of Akt/PKB in Notch-mediated survival signaling.
Essential role of mTOR in Notch1-mediated survival signaling. To further confirm the importance of mTOR and its functions required in survival signaling by Notch1, we used mTOR rr and its derivatives (Supplementary Fig. S4), which are defective for its functions in our assays. These experiments were carried out in the presence of rapamycin to inactivate endogenous mTOR WT; hence, one can study the effect of ectopically expressed mutants of mTOR. In rapamycin-treated cells, Notch1 failed to inhibit p53-mediated transcription in the presence of exogenously expressed mTOR WT but not mTOR rr and mTOR rr del 10 (Fig. 4A, compare lane 5 with lanes 6 and 7 ). Further, NIC-1 failed to inhibit p53-mediated transcription in the presence of mTOR rr del 91 as well as mTOR SIDA (Fig. 4A, compare lane 5 with lanes 8 and 9). These results suggest that the first HEAT repeat (region of mTOR between amino acids 11 and 91) and the kinase function are essential for Notch1 to inhibit p53-mediated transcription. To determine whether results obtained in promoter assays can be extended to chemosensitivity, we made stable cell clones of H460 cells expressing mTOR WT, mTOR rr, mTOR rr del 10, mTOR rr del 91, or mTOR SIDA. These stable clones were tested for their defects in mTOR functions in the presence and absence of rapamycin by measuring the phosphorylated levels of S6K1 and 4E-BP1. On rapamycin treatment, Ad-NIC-1 infection failed to phosphorylate S6K1 and 4E-BP1 in mTOR WT stable cells but not in mTOR rr and mTOR rr del 10 stable cells (Fig. 4D, lanes 4-6, 10-12, and 16-18, respectively). Further, Ad-NIC-1 failed to phosphorylate S6K1 and 4E-BP1 in mTOR rr del 91 and mTOR SIDA stable cells (Fig. 4D, lanes 22-24 and 28-30, respectively). As expected, Ad-NIC-1 infection resulted in phosphorylation of S6K1 and 4E-BP1 in all stable clones in the absence of rapamycin (Fig. 4D, lanes 1-3, 7-9, 13-15, 19-21, and 25-27). We then used these stable cell lines to study the requirement of mTOR functions in Notch1-mediated survival signaling. On rapamycin treatment, NIC-1 failed to protect mTOR WT stable cells but not mTOR rr and mTOR rr del 10 stable cells from Adriamycin-induced cytotoxicity (Fig. 4B). Further, NIC-1 failed to protect mTOR rr del 91 and mTOR SIDA stable cells against Adriamycin-induced cytotoxicity in the presence of rapamycin (Fig. 4B). As expected, in all stable cell lines, Ad-NIC-1 was able to protect them against Adriamycin-induced cytotoxicity in the absence of rapamycin (Fig. 4B). Similar results were obtained against cytotoxicity induced by Ad-p53 as well. On rapamycin treatment, NIC-1 failed to inhibit Ad-p53-induced apoptosis in mTOR WT stable cells but not in mTOR rr and mTOR rr del 10 stable cells (Fig. 4C). Further, Notch1 failed to inhibit Ad-p53-induced apoptosis in mTOR rr del 91 and mTOR SIDA stable cells in the presence of rapamycin (Fig. 4C). As expected, in all stable cell lines, Ad-NIC-1 was able to inhibit p53-induced apoptosis in the absence of rapamycin (Fig. 4C). These results further prove that an activated, functional mTOR is essential for Notch1-mediated inhibition of p53 functions and survival signaling.
eIF4E is the major downstream target of mTOR in Notch1 inhibition of p53-induced cytotoxicity. eIF4E, the downstream target of 4E-BP1, has transforming and antiapoptotic activities in vitro and overexpressed in many tumors (13, 24, 25). eIF4E has also been shown to signal mTOR-dependent drug resistance in vivo (23). Therefore, we determined the direct effect of ectopic expression of eIF4E on p53 functions. We found that eIF4E inhibited the p53-mediated transcription (Fig. 5A, compare lane 3 with lane 4 ) and is not abrogated by rapamycin (Fig. 5A, lane 6). Further, to confirm these results, we tested the ability of Ad-p53 to inhibit H460 cells stably expressing eIF4E. Three independent stable clones of H460 (1, 2, and 3) expressed increased amounts eIF4E in comparison with a vector-stable clone (H460/Puro; Fig. 5B, compare lane 1 with lanes 2-4). Ad-p53 inhibited efficiently the growth of H460/Puro cells but not H460/eIF4E#1 cells (Fig. 5C). The inability of p53 to induce cytotoxicity in eIF4E-overexpressing cells correlated well with lack of apoptosis induction by Ad-p53 in H460/eIF4E#1 cells (Fig. 5D). Based on these results that the ectopic expression of eIF4E can block transcription, cytotoxicity, and apoptosis induced by p53, we conclude that eIF4E can recapitulate Notch1 action in inhibition of p53-mediated cytotoxicity.
Cancers with aberrant Notch1 signaling are chemoresistant, which can be reversed by PI3K or mTOR inhibitors. The results described thus far suggest that Notch1 overexpression results in chemoresistance due to inhibition of p53 through PI3K-Akt/PKB-mTOR-dependent signaling. To determine whether tumors, which arise due to aberrant Notch1 signaling, are chemoresistant due to similar reasons, we analyzed the effect of PI3K and mTOR inhibitors on chemosensitivity of cells derived from such tumors. Aberrant Notch signaling is suspected to play a role in the development of human breast cancer (26–28). The breast cancer cell line MCF7 has been shown to have activated Notch signaling (29). As expected, MCF7/c3, a caspase-3-stable derivative of MCF7, activated Notch-responsive reporter HES-1-luc but not HES-1-ABluc, which carries mutated RBPjk binding site (Supplementary Fig. S5A, compare lane 1 with lanes 2 and 3). We found that treatment of MCF7/c3 cells with wortmannin, LY294002, or rapamycin resulted in activation of PG13-Luc, a p53-specific reporter (Supplementary Fig. S5B, compare lane 1 with lanes 2-4). We then tested whether the increased p53-mediated transcription by PI3K or mTOR inhibitor treatment in MCF7/c3 cells results in increased chemosensitivity and apoptosis induction on chemotherapy. MCF7/c3 cells pretreated with PI3K or mTOR inhibitor were more sensitive (1.8- to 3.4-fold) to chemotherapeutic drugs Adriamycin, cisplatin, etoposide, and Taxol (Fig. 6A
) and underwent apoptosis severalfold (4.03, 4.92, and 4.70-fold, respectively) higher on treatment with Adriamycin (Fig. 6B). Neither PI3K inhibitors nor mTOR inhibitor treatment alone inhibited the growth of or induced apoptosis to significant levels in MCF7/c3 in comparison with untreated control population (Fig. 6A and B).
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Discussion |
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Although the transforming and antiapoptotic functions of Notch1 requires PI3K (8, 11), the exact signaling downstream of PI3K is not known. In addition to the fact that we show PI3K is essential for Notch1-mediated chemoresistance, we provide conclusive evidence that further downstream signaling occurs via Akt/PKB-mTOR pathway. Akt/PKB, an important mediator of PI3K signaling, phosphorylates multiple downstream effectors (23). Although our results show that many of these pathways are activated in NIC-1-overexpressing cells (e.g., GSK3ß, FKHR, and mTOR are activated), it is the mTOR pathway that seems to play a vital role as a downstream target of Akt/PKB in Notch1-mediated survival signaling. Firstly, the mTOR inhibitor rapamycin is able to reverse as efficiently as PI3K inhibitors the Notch1-mediated inhibition of p53 functions as well as chemoresistance. In good correlation, in cells stably transfected with mTOR rr, rapamycin failed to inhibit survival signaling by Notch1. Secondly, overexpression of eIF4E, a downstream target of mTOR, is able to protect the cells from p53-mediated cytotoxicity as efficiently as Notch1.
Although our results undoubtedly show that PI3K seems to play a major role in Notch1-mediated chemoresistance, how exactly Notch1 activates PI3K is less clear. NIC-1 has been shown to require the Src family protein tyrosine kinase p56lck for Notch1-mediated activation of Akt-PKB, and it forms a Notch1-p56lck-PI3K complex (11). On the contrary, activated Notch1 is believed to work in the nucleus by activating transcription as a heterodimer along with RBPjk. Recent research indicates that Notch proteins can also signal via an alternative intracellular pathway, which requires the cytoplasmic protein Deltex (33, 34). We do not know whether Deltex participates in Notch1-mediated survival signaling. Efforts are under progress to dissect the requirement of RBPjk and Deltex as well as the ability of Notch1 to activate transcription in survival signaling by Notch1.
Additional important conclusions we make from this study are that (a) eIF4E seems to be the major mediator of mTOR survival signaling in Notch1-mediated chemoresistance and (b) Notch1 overexpression confers chemoresistance only in WT p53-containing cells. mTOR normally regulates translation in response to nutrients and growth factors by phosphorylating key components of the protein synthesis machinery, including S6K1 and 4E-BP1 proteins (35). Phosphorylation of 4E-BP1 in turn releases the translation initiation factor eIF4E to stimulate cap-dependent translation. eIF4E has been shown to have transforming and antiapoptotic activities in vitro (13, 23) and is overexpressed in some tumor types (25). Our results indicate that eIF4E by itself is able to inhibit p53-mediated transcription and cytotoxicity to similar extent that of Notch1. This indicates that the Notch1-mediated survival signaling may actually be mediated by eIF4E through Akt/PKB-mTOR signaling. The other important conclusion from this study is that Notch1 confers chemoresistance only in WT p53-harboring cells. Recently, it was reported that mTOR inhibitor RAD001 sensitizes the cells to cisplatin by inhibiting p53-induced p21 expression (36). If inhibition of p53-induced p21 expression by mTOR inhibitor is the primary mechanism by which sensitization to chemotherapy occurs, one would expect a complete rescue by ectopically expressed p21 from chemotherapy-induced cytotoxicity. However, we found previous expression of p21 through an adenovirus rescued the cells from Adriamycin-induced cytotoxicity only partly (data not shown), suggesting the existence of other mechanisms. In fact, it has been shown that the translation of survivin, a member of inhibitor of apoptosis protein family is reduced in rapamycin-treated Jurkat cells (37). Moreover, our results show that transcription activation by chemotherapy-induced endogenous p53 is completely shut off in cells possessing activated mTOR signaling. We also found that p53 failed to induce the levels of its transcriptional targets, such as p21WAF1/CIP1, Gadd45
, PUMA, and Noxa, in cells overexpressing NIC-1.3 Thus, based on our results presented, we conclude that although mTOR plays a key role in conferring chemoresistance in cells possessing activated Notch1 signaling the actual mechanism would involve essentially preferential translation of multiple but specific mRNA species perhaps encoding antiapoptotic factors.
This study also provides, in principle, a method for combination therapy for cancers with aberrant Notch1 signaling, which would include conventional chemotherapy and either PI3K or mTOR inhibitor. We show here that chemoresistance, which arises due to apoptotic defects, could be reversed as seen by the fact that both PI3K inhibitors and mTOR inhibitor reversed the chemoresistance of Notch1-overexpressing tumor-derived cells. Although the activation of mTOR pathway has been reported in tumors arising due to aberrant Akt signaling (23), we are showing for the first time similar finding in cancer cells having ectopically expressed activated Notch1 and cells derived from Notch1-overexpressing tumors; hence, a combination treatment with rapamycin and conventional chemotherapeutic agents could be considered for Notch-overexpressing tumors. Yet, another key finding with clinical relevance from this study is that Notch1-mediated chemoresistance is WT p53 dependent. It is tempting to say that the chemoresistance seen in other tumors with activated PI3K-Akt signaling may also be WT p53 dependent. The implication of this observation is that combination chemotherapy with PI3K or mTOR inhibitor may be effective only in tumors carrying WT p53. Thus, our results provide a critical factor to be considered that p53 status perhaps is an important determinant in combination therapeutic strategies, which use mTOR inhibitors and chemotherapy.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Tom Kadesch for pCMV NeoPoly2-TAN 1 cyt (contains Notch1 amino acids 1,758-2,556 and is referred to as pCMV Neo/NIC-1 in this article), HES-1-luc, and HES-1-ABluc; Prof. Nahum Sonenberg (McGill University, Montreal, Quebec, Canada) for eIF4E expression vector; Dr. Robert Abraham (Burnham Institute, La Jolla, CA) for mTOR WT construct; Dr. Peter J. Houghton (St. Jude Children's Research Hospital, Memphis, TN) for providing mTOR rr, mTOR rr del 10, mTOR rr del 91, and mTOR SIDA constructs; and Dr. Omana Joy for technical assistance in FACS experiments; Indian Council for Medicine Research (Center for Advanced Studies in Molecular Medicine), Department of Biotechnology (Program Support and Genomics Program), Department of Science and Technology (Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions), and UGC (Special Assistance) for infrastructural support.
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3 S.K. Mungamuri and K. Somasundaram, unpublished data. 
Received 10/24/05. Revised 1/ 5/06. Accepted 2/27/06.
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