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[Cancer Research 65, 7800-7808, September 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Activation of Mammalian Target of Rapamycin in Transformed B Lymphocytes Is Nutrient Dependent but Independent of Akt, Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase, Insulin Growth Factor-I, and Serum

Pawel Wlodarski1,4, Monika Kasprzycka1, Xiaobin Liu1, Michal Marzec1, Erle S. Robertson2, Artur Slupianek3 and Mariusz A. Wasik1

Departments of 1 Pathology and Laboratory Medicine and 2 Microbiology, University of Pennsylvania; 3 College of Science and Technology, Temple University, Philadelphia, Pennsylvania; and 4 Department of Histology and Embryology, Medical University of Warsaw, Warsaw, Poland

Requests for reprints: Mariusz A. Wasik, Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, 7.106 Founders, Philadelphia, PA 19104. Phone: 215-662-3467; Fax: 215-662-7529; E-mail: wasik{at}mail.med.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Addendum
 References
 
The study examines the preponderance and mechanism of mammalian target of rapamycin (mTOR) activation in three distinct types of transformed B lymphocytes that differ in expression of the EBV genome. All three types [EBV-immortalized cells that express a broad spectrum of the virus-encoded genes (type III latency; EBV+/III), EBV-positive cells that express only a subset of the EBV-encoded genes (EBV+/I), and EBV-negative, germinal center–derived cells (EBV–)] universally displayed activation of the mTOR signaling pathway. However, only the EBV+/III transformed B cells displayed also activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that is considered to be the key activator of mTOR and of the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK pathway that coactivates one of the immediate targets of mTOR, p70 S6K1. Activation of the PI3K/Akt and MEK/ERK, but not of the mTOR pathway, was inhibited by serum withdrawal and restored by insulin growth factor-I. In contrast, activation of mTOR, but not PI3K/Akt and MEK/ERK, was sensitive to nutrient depletion. Both direct Akt (Akt inhibitors I-III) and a PI3K inhibitor (wortmannin at 1 nmol/L) suppressed Akt phosphorylation without significantly affecting mTOR activation. Furthermore, rapamycin, a potent and specific mTOR inhibitor, suppressed profoundly proliferation of cells from all three types of transformed B cells. U0126, a MEK inhibitor, had a moderate antiproliferative effect only on the EBV+/III cells. These results indicate that mTOR kinase activation is mediated in the transformed B cells by the mechanism(s) independent of the PI3K/Akt signaling pathway. They also suggest that inhibition of mTOR signaling might be effective in therapy of the large spectrum of B-cell lymphomas.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Addendum
 References
 
B-cell lymphomas represent a rather heterogeneous group of malignant disorders (1). Differential expression of the EBV genome is one of the distinguishing features (1–3). Whereas most do not show any evidence of harboring the virus, some, such as a subset of post-transplant lymphoproliferative disorders (PTLD), AIDS-related lymphomas, and Hodgkin lymphoma, express the broad spectrum of EBV-encoded RNAs and proteins (latency type III; EBV+/III) or only a few of these molecules (latency type I; EBV+/I) as typically seen in Burkitt-type lymphomas.

Cell signaling by a highly conserved serine/threonine kinase mammalian target of rapamycin (mTOR) has been shown to play a critical role in protein synthesis and enlargement and proliferation of normal and malignant cells (4, 5). However, the exact mechanisms of mTOR activation are still poorly understood. Nutrients, on one hand, and cytokines and growth factors, such as interleukin-2, insulin, and tumor necrosis factor–related proteins, on the other hand, have been shown to be involved in mTOR activation (6–9). The cytokines and growth factors seem to act via phosphatidylinositol 3-kinase (PI3K)/Akt [protein kinase B (PKB)] signaling pathway that activates mTOR indirectly through modulating the TSC1/TSC2 complex (10–14). In turn, mTOR signals via activating of at least two signaling pathways by directly phosphorylating p70 S6K1 kinase and 4eBP1 protein (4, 5). mTOR signaling, and hence phosphorylation and activation of p70 S6K1 and 4eBP1, is effectively blocked by rapamycin and its derivatives that seem to be absolutely specific for mTOR (4, 5).

Our previous studies have shown that PTLD-type, EBV+/III transformed B cells are highly sensitive to inhibition of mTOR (15, 16). However, the mechanism of the mTOR activation in such cells as well as the role of mTOR in other B-cell lymphoproliferative disorders remained undefined. Here, we report that mTOR signaling pathway is activated and required for cell proliferation not only in the EBV+/III but also in the EBV+/I and EBV– B lymphocytes. Noteworthy, this mTOR activation does not require activation of Akt and is also mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK), insulin growth factor (IGF), and serum independent. It is, however, nutrient dependent. Based on these findings, we postulate that mTOR activation occurs in the transformed B lymphocytes by a novel, currently undefined mechanism independent of the PI3K/Akt pathway. Furthermore, mTOR activation is frequent in transformed B cells regardless of their EBV status and therefore may represent an attractive direct therapeutic target in a variety of B-cell lymphomas.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Addendum
 References
 
Cell lines. The EBV-negative, diffuse large B-cell lymphoma, germinal center–derived cell lines (LY 18, LY 1pn, and Val) were a kind gift of L. Pasqualucci (Columbia University, New York, NY). The EBV+/I, Burkitt lymphoma–derived B-cell lines (Raji, Ramos, and Daudi) were from American Type Culture Collection (Manassas, VA). EBV+/III lymphoblastoid B-cell lines (MM, HH, NW, and RB) were established in our laboratory by the EBV-mediated immortalization of peripheral blood B lymphocytes (15, 16). All cell lines were cultured in RPMI (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Cellgro, Herndon, VA), unless indicated otherwise.

Cell signaling inhibitors. LY294002, U0126, and rapamycin were purchased from Cell Signaling Technology (Beverly, MA). Wortmannin and Akt inhibitors I to III were obtained from Calbiochem (San Diego, CA). All inhibitors were reconstituted in DMSO to the suitable stock concentration and diluted shortly before the experiments to the final concentrations depicted in the figures.

Serum starvation and serum stimulation. In the serum depletion experiments, the exponentially growing cells cultured in complete medium were centrifuged, washed twice with serum-free medium, and incubated overnight in RPMI supplemented with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO). Shortly before stimulation, the cells were centrifuged and resuspended in serum-containing medium for 15 to 20 minutes, centrifuged, washed in PBS, and lysed. When kinase inhibitors were used, serum-starved cells were treated with the indicated concentration of the specified inhibitors for 30 minutes before serum was added.

Nutrient starvation. The exponentially growing cells were washed twice with PBS and resuspended in PBS or RPMI (both without any additives) for 20 hours (long starvation) or 2 hours (short starvation). In the short starvation experiment, the cells were subsequently stimulated with IGF-I (Sigma) for 10 minutes. In both kinds of experiments, cells were washed very briefly in PBS before they were lysed for the Western blot analysis.

Western blots. The cells were washed briefly in PBS, centrifuged, and lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA] supplemented with 0.5 mmol/L phenylmethylsulfonyl fluoride, phosphatase inhibitor cocktails I and II (Sigma), and protease inhibitor cocktail (Roche, Nutley, NJ) according to the manufacturer's specifications. For normalization of the gel loading, the protein extracts were assayed with Lowry method [Bio-Rad (Hercules, CA) Dc protein assay]. Typically, 20 to 30 µg of the protein per lane were loaded. All phosphospecific antibodies were from Cell Signaling Technology. Antibodies for Akt isoforms were from Upstate (Charlottesville, VA).

Immunoprecipitation. Lysates were prepared as for the Western blots. The protein concentration was adjusted with RIPA buffer to 1 mg/mL; typically, 0.5 mg of the protein was used for immunoprecipitation. Five micrograms of antibody coupled to protein A-Sepharose beads were used for each overnight immunoprecipitation. The beads were washed four times in RIPA buffer with the protease and phosphatase inhibitors. Next, the beads were centrifuged, decanted, and boiled in 2x SDS sample buffer. The samples were loaded into SDS-PAGE gels for Western blot analysis. Protein extracts from the transformed B-cell lines were incubated with anti-Akt1 (mouse monoclonal) and anti-Akt3 (rabbit) antibodies; the resulting immunoprecipitates were run on SDS-PAGE gels using new membrane each time. Phospho-Akt-specific antibody (S473) and the Akt1 antibody were detected with the secondary anti-mouse horseradish peroxidase (HRP) antibody. The Akt3 antibody was detected with protein A-HRP.

Cell proliferation assays. These were done by detection of bromodeoxyuridine (BrdUrd) incorporation using the commercially available kit Cell Proliferation ELISA (Roche). In brief, the cells were seeded in the 96-well plates (Corning, Acton, MA) at 1 x 104 cells per well in RPMI supplemented with 10% FBS and cultured either alone or in the presence of the inhibitors for 20 hours and exposed to 10 mmol/L BrdUrd for additional 4 hours. Whereas rapamycin and U0126 were added once at the culture initiation, wortmannin was given at 2- to 3-hour intervals. After the plate centrifugation (10 minutes at 300 x g), supernatant removal, and plate drying, the cells were fixed and the DNA was denaturated by adding 200 mL FixDenat reagent. The incorporated BrdUrd was detected by a specific antibody conjugated with HRP. The immune complexes were detected colorimetrically by the HRP substrate conversion at the 450 nm using an ELISA reader.

Apoptotic cell death assay. Cell apoptosis was detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining using ApoAlert DNA Fragmentation Assay kit (BD Bioscience, San Jose, CA) according to manufacturer's protocol. In brief, 3 x 106 cells were collected, washed twice in PBS, and fixed with 1% formaldehyde/PBS. The cells were washed in PBS and permeabilized with 70% ice-cold ethanol for at least 2 hours. After washing, cells were incubated in terminal deoxynucleotidyl transferase incubation buffer for 1 hour at 37°C. Reaction was stopped by adding 20 mmol/L EDTA and cells were washed twice in 0.1% Triton X-100/BSA/PBS. Finally, the samples were resuspended in 0.5 mL propidium iodide/RNase/PBS and analyzed by flow cytometry (FACSort BD) using CellQuest PRO software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Addendum
 References
 
EBV-positive and EBV-negative transformed B lymphocytes display activation of mammalian target of rapamycin. The noted potent antiproliferative effect of an mTOR inhibitor in several different EBV+/III lymphoblastoid B-cell line populations (15, 16) indicated that mTOR is activated in the EBV-transformed B lymphocytes. To analyze in detail mTOR signaling in such cells, we evaluated in four lymphoblastoid B-cell lines activation status of mTOR and its two immediate downstream targets p70 S6K1 and 4eBP1. In addition, to determine if mTOR is activated also in other types of transformed B cells, we examined several cell lines derived from Burkitt lymphoma and diffuse large B-cell lymphoma/germinal center cells that are EBV+/I and EBV–, respectively.

As shown in Fig. 1A, all three types of the transformed B cells, EBV+/III, EBV+/I, and EBV–, displayed universal, seemingly perpetual activation of mTOR as determined by phosphorylation of the kinase itself and of p70 S6K1 and 4eBP1 that are phosphorylated by mTOR at the T389 and S65 residues, respectively (4, 5). Although the EBV+/III lines are all of the lymphoblastoid type, similar results (data not shown) were obtained also with an EBV+/III cell line called PTLD-1 that was derived directly from tissue section of a pulmonary PTLD described by us before (16). The apparent single exception was the noted lack of 4eBP1 phosphorylation in the Burkitt lymphoma–derived Raji cell line; this was due, however, to the lack of expression by Raji cells of the 4eBP1 protein (data not shown). Furthermore, the T389 p70 S6K1 phosphorylation tended to be uniformly strong in the EBV+/III cells but more heterogeneous in the other two types of transformed B cells. Treatment of the selected transformed B-cell lines with the mTOR inhibitor rapamycin inhibited phosphorylation of both p70 S6K1 at T389 and 4eBP1 at S65 (Fig. 1B), confirming the role of mTOR as their kinase and indicating exquisite sensitivity of these cells to the drug used at a low dose of 10 nmol/L. To examine the drug effect also on the cell function, we determined the effect of rapamycin on cell proliferation using seven cell lines from the EBV+/III lymphoblastoid B-cell, EBV+/I Burkitt lymphoma, and EBV– germinal center groups. As shown in Fig. 1C, all cell lines have shown dramatic inhibition of proliferation in response to the same low dose of the drug.



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  		Figure 1. Constant mTOR activation in transformed B lymphocytes regardless of their EBV genome expression status. A, expression of members of the mTOR signaling pathway in the activated state. Western blot was done with antibodies specific for the examined proteins phosphorylated at the indicated residues. Detection of actin expression served as the control of protein loading. EBV+/III lymphoblastoid B-cell line; EBV-transformed B cells that express broad spectrum of the EBV-encoded proteins and RNAs, EBV+/I Burkitt lymphoma; Burkitt lymphoma–derived cells that express selected EBV molecules, EBV-germinal center; germinal center–derived B-cell lymphoma cells that are EBV–. B, effect of mTOR inhibitor rapamycin on activation of the mTOR signaling pathway. MM (EBV+/III) and Ramos (EBV+/I) cells were incubated for 1 hour with 10 nmol/L rapamycin. Their protein lysates were examined for activation of the mTOR pathway with the listed phosphospecific antibodies. C, rapamycin-mediated inhibition of cell proliferation. The listed cell populations were cultured for 20 hours in medium containing 10 nmol/L rapamycin or solvent alone. Incorporation of BrdUrd added for additional 4 hours into the rapamycin-treated cells (filled columns) is presented as the percentage of the incorporation of the control counterparts (open columns).

 
Phosphatidylinositol 3-kinase/Akt pathway is activated in the EBV+/III lymphoblastoid B-cell but not in EBV+/I Burkitt lymphoma and EBV– germinal center cells. Because Akt has been reported to be responsible for mTOR activation in other cell types (11–14), we examined next activation status of this serine/threonine kinase in the transformed B cells. As shown in Fig. 2A, the EBV+/III lymphoblastoid B-cell line cells showed almost uniformly strong activation of Akt as determined by its phosphorylation at two different residues, T308 and S473. In addition, these cells showed phosphorylation of the known Akt target GSK3ß at S9. None of the EBV+/III cell lines showed loss of PTEN protein that has been shown to lead to the permanent Akt activation in some tumors (17). Surprisingly, almost none of the seven EBV+/I and EBV– cell lines tested displayed Akt T308 or S473 phosphorylation. The only exception was the LY 1pn cell line cell line in which the noted Akt activation was associated with loss of PTEN. In accordance with the lack of Akt activation, all other lines showed variably diminished GSK3ß-S9 phosphorylation; the noted residual phosphorylation could be due to the activity of different serine kinase(s) that targets the same site (18, 19). In turn, TSC2 phosphorylation on T1462 (Fig. 2B) was present with some differences in intensity in all the cell lines examined (but not normal peripheral blood lymphocytes; data not shown), suggesting the existence in transformed B lymphocytes of its Akt-independent regulation, possibly by at least one the recently described mechanisms (20–24).



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  		Figure 2. Differential activation status of Akt/PKB in different types of transformed B cells. A, expression of the activated Akt and functionally related proteins detected with the phosphospecific and control antibodies. B, expression of phosphorylated and total TSC2. C, expression of the Akt isoforms as determined by the isoform-specific antibodies. D, activation status of the Akt1 and Akt3 isoforms expressed by the EBV+/III cells. Immunoprecipitation of Akt1 and Akt3 was followed by Western blot with phospho-Akt antibody.

 
Three different isoforms of Akt protein, Akt1, Akt2, and Akt3, encoded by three separate genes have been found in mammalian cells (25, 26). As shown in Fig. 2C, the EBV+/III cells expressed Akt1 and Akt3; only traces of Akt2 were detected in one of the four lymphoblastoid B-cell lines examined. In turn, the EBV+/I Burkitt lymphoma and EBV– germinal center cells expressed only the Akt1 isoform with an exception of one germinal center line, LY 18, which expressed also Akt3. Because only the EBV+/III lymphoblastoid B cells, but none of the other two groups, displayed Akt phosphorylation (Fig. 2A), it seemed interesting to determine if phosphorylation of Akt is restricted in the EBV+/III cells to the differentially expressed Akt3. However, immunoprecipitation of the expressed isoforms (Akt1 and Akt3) followed by immunoblotting with the antibody specific for Akt phospho-serine showed that both these Akt isoforms are phosphorylated in the EBV+/III cells (Fig. 2D). The same pattern was also seen when Akt phosphorylation at the T308 site was examined (data not shown).

Mitogen-activated protein kinase signaling pathways are activated in EBV+/III but not the other two types of transformed B cells. Among pathways activated in B cells immortalized by EBV, MAPK signaling is believed to be critical for survival (27). Furthermore, recent findings (28) indicate that the member of the MAPK pathways, ERK1/2, phosphorylates p70 S6K1 albeit on the different residues (T421/S424) than mTOR and, as a result, augments p70 S6K1 activity. As shown in Fig. 3A, the general pattern of MAPK activation in transformed B lymphocytes closely resembles the one of Akt. Specifically, there is a significant phosphorylation of ERK1/2 in all four EBV+/III cell line populations tested and no such activation in the EBV+/I and EBV– cells (Fig. 3A). Two other MAPKs [stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) at T183/Y185 and p38 MAPK at T180/Y182] were also, respectively, selectively and preferentially phosphorylated in the EBV+/III cells. Treatment of the cells with the U0126 inhibitor of MEK had a marked inhibitory effect on phosphorylation of ERK1/2 and its target S6K1 T421/S424, SAPK/JNK, and, to a lesser degree, p38 MAPK (Fig. 3B). To determine if the activated MAPK plays a functional role in the EBV+/III cells, we examined an effect of the U0126 inhibitor on their proliferation. As shown in Fig. 3C, U0126 diminished BrdUrd incorporation into these cells by as much as 50% to 60% but had essentially no effect on proliferation of the EBV+/I and EBV– B cells.



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  		Figure 3. Differential activation status of MAPKs in the transformed B-cell types. A, activation status of MAPK signaling proteins (ERK1/2, SAPK/JNK, and p38 MAPK) as determined by phosphorylation of the proteins and of p70 S6K1 at the T421/S424 site targeted by ERK1/2 (23). B, effect of mTOR inhibitor (rapamycin at 10 nmol/L) and MEK1/2 inhibitor (U0126 at 2 µmol/L) on MAPK, mTOR, and Akt activation in the EBV+/III B cells. The MM cells were exposed to the inhibitors or their solvent for 1 hour before the protein extraction. C, U0126-mediated inhibition of cell proliferation. The listed cell populations were cultured in medium containing either 2 µmol/L of the MEK1/2 inhibitor or its solvent for 20 hours followed by a 4-hour pulse with BrdUrd. The BrdUrd incorporation by the U0126-treated cells (filled columns) is presented as the percentage of the incorporation of the control counterparts (open columns).

 
Activation of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathways but not of the mammalian target of rapamycin pathway is serum dependent. Although Akt has been identified as the mTOR activator (11, 12, 14), the presence of mTOR pathway activation in the EBV+/I and EBV– cells in the absence of PI3K/Akt activation and, in addition, MEK/ERK activation indicated that mTOR activation is mediated in these two types of transformed B cells by an Akt- and MEK-independent mechanism. Furthermore, it suggested that activation of mTOR in the EBV+/III B cells might also be independent of Akt. To determine if serum growth factors may play a role in activation of any of these pathways, we cultured overnight in the serum-free medium the cell lines representative of the three types of transformed B cells. As shown in Fig. 4A, the prolonged serum depletion resulted in the essentially complete inhibition in the EBV+/III cells of Akt activation. In contrast, mTOR activation status remained unaffected in this as well as the other two types of transformed B cells. Similar to Akt, activation of MEK pathway was markedly diminished in the EBV+/III cells. These findings strongly suggested that, similar to the EBV+/I and EBV– B cells, activation of mTOR in the EBV+/III cells is Akt and, less surprisingly, MEK independent.



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  		Figure 4. Serum and IGF-I dependence of Akt and ERK1/2 activation in the EBV+/III B lymphocytes. A, effect of serum withdrawal and reconstitution on activation status of PI3K/Akt, mTOR, and MEK/ERK signaling pathways. The listed cell populations that represent the three types of the transformed B cells were incubated for 20 hours in RPMI supplemented with 1% BSA. The cells were subsequently stimulated with 10% FBS (+) for 20 minutes; the unstimulated cells (–) served as controls. B, effect of stimulation with IGF-I on serum-deprived EBV+/III B cells. The listed EBV+/III cell populations were incubated for 20 hours in RPMI supplemented with 1% BSA. The cells were subsequently stimulated for 20 minutes with 10 ng/mL IGF-I or 10% FBS; the unstimulated cells served as a control.

 
We asked next which of the serum factors might be responsible for the Akt and ERK activation. Because IGF-I has been shown previously to activate both PI3K/Akt (29–31) and MAPK (29, 32–34) signaling pathways in various cell types, we examined if it is capable of activating these pathways in the serum-depleted EBV+/III B cells. As shown in Fig. 4B, IGF-I used at the physiologic dose of 10 ng/mL, indeed, induced phosphorylation of Akt, c-Raf, and ERK1/2. As can also be seen, the degree of the IGF-I-mediated phosphorylation was somewhat lower compared with the effect of the whole serum. IGF-I could not match the serum-mediated phosphorylation level, even if used at 50, 200, and 1,000 ng/mL (data not shown), indicating that other serum growth factors contribute to activation of the PI3K/Akt and MAPK pathways in the EBV+/III-transformed B cells. Similarly to serum, IGF-I had essentially no effect on the TOR activation.

Activation of the mammalian target of rapamycin pathway but not of the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathways is nutrient dependent. Because the dependence of mTOR activation on nutrients has been well documented (6), we tested next whether nutrient deprivation would affect mTOR and, where relevant, the other two signaling pathways in the three types of transformed B cells. To accomplish this aim, we cultured the transformed B-cell lines for 2 hours in the serum- and nutrient-free PBS and, as a control, serum-free RPMI. As shown in Fig. 5A, this relatively brief serum depletion did not manage to affect Akt and ERK1/2 activation; additional lack of nutrients had also no effect on these pathways. In striking contrast, depletion of nutrients resulted in greatly diminished to completely abrogated mTOR activation in all three types of the transformed B-cell lines. These results indicate that the nutrient supply–dependent regulation of mTOR signaling remains intact in the transformed B cells.



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  		Figure 5. Nutrient dependence of mTOR activation in the transformed B cells. A, effect of short-term serum and serum and nutrient deprivation on activation of the PI3K/Akt, mTOR, and MEK/ERK pathways. The listed cell populations that represent the three types of transformed B cells were incubated for 2 hours in serum-free RPMI and serum- and nutrient-free PBS. B, effect of stimulation with IGF-I on serum-starved cells. Long-term (20 hours) serum-starved transformed B cells were incubated short-term (2 hours) in PBS or RPMI and briefly (20 minutes) stimulated with IGF-I. The IGF-I unstimulated cells served as a control.

 
To determine if IGF-I could possibly activate mTOR in the absence of nutrients, we stimulated with the factor selected transformed B-cell lines that were precultured overnight in either the serum- and nutrient-free PBS or serum-free RPMI. Whereas activation of Akt and ERK1/2 that became abrogated by the prolonged serum deprivation could be restored by IGF-I not only in RPMI but also in PBS, mTOR activation was seen only in RPMI with the IGF-I stimulation having little, if any, additional effect (Fig. 5B). This indicates that mTOR activation in the transformed B cells is strictly nutrient dependent. The differential effect of IGF-I on the Akt and ERK1/2 versus mTOR provides additional argument for the Akt-independent mTOR activation in the EBV+/III B cells.

Selective inhibition of the phosphatidylinositol 3-kinase/Akt pathway does not affect mammalian target of rapamycin signaling in the transformed EBV+/III B cells. To obtain direct evidence that mTOR is, indeed, activated in the EBV+/III B cells independently of Akt, we did a series of experiments aimed at blocking the Akt activity. Whereas despite numerous attempts the genetic approaches with a dominant-negative Akt mutant and siRNA have proven very difficult due to the extremely poor transfectability of the EBV+/III B cells (data not shown), the pharmacologic approaches with small molecule inhibitors were overall much more informative with some, but not all, of the inhibitors examined. We begun with LY294002, a well-known inhibitor of PI3K that has been widely used to study Akt activation (35–37). As shown in Fig. 6A, the drug when applied at 10 and 50 µmol/L but not at 1 µmol/L, indeed, profoundly inhibited activation of Akt and mTOR signaling in the EBV+/III transformed B cells. However, it also inhibited in a similar fashion mTOR signaling in the EBV+/I cells that do not display Akt activation. This indicates that, besides acting on PI3K, LY294002 inhibits another serine kinase involved in mTOR activation, most likely mTOR itself (38). Faced with this lack of LY294002 specificity, we employed next the recently described, direct inhibitor of Akt (39), designated Akt inhibitor I, which acts by competitively binding the Akt pleckstrin homology domain of the kinase. As shown in Fig. 6B, the inhibitor suppressed Akt phosphorylation at the 10 to 40 µmol/L range in the dose-dependent manner, yet it exerted no effect on mTOR signaling in the cells with and without activated Akt. This observation supports the conclusion that mTOR in the EBV+/III transformed B cells is activated independently of Akt. Noteworthy, activation of ERK1/2 was also inhibited in these cells at the similar drug doses, indicating that the inhibitors of this class are not strictly specific for Akt.



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  		Figure 6. Differential effect of Akt inhibition on Akt, mTOR, and ERK1/2 activation in the EBV+/III B cells. A, inhibitory effect on protein phosphorylation of the dual specificity PI3K/mTOR inhibitor LY294002. EBV+/III (HH) and EBV+/I (Ramos) cells were treated for 1 hour with LY294002 applied at the indicated doses. Cells treated with rapamycin (at 10 nmol/L) served as a control. B, inhibitory effect of the direct Akt inhibitor I, a phosphoinositol analogue. HH and Ramos cells were treated for 1 hour with the inhibitor used at 0, 10, and 40 µmol/L. C, inhibitory effect of the PI3K inhibitor wortmannin. The cells were treated for 1 hour with the indicated doses of the inhibitor. Cells treated with rapamycin (at 10 nmol/L) served as a control. D, effect of low-dose wortmannin on proliferation of the transformed B cells. The listed cell populations seeded at 105 cells/mL were exposed for 4 hours to 1 nmol/L wortmannin (filled columns) or its solvent (open columns) given twice at the 0- and 2-hour time points. E, effect of high-dose wortmannin on proliferation of the transformed B cells. The listed cell populations were exposed for 10 hours to wortmannin at the indicated doses or its solvent given at the 3-hour intervals. F, effect of wortmannin on apoptotic cell death of the transformed B cells. The listed cell populations were exposed for 10 hours to wortmannin at the indicated doses or its solvent given at the 3-hour intervals.

 
Lastly, we evaluated another well-known PI3K inhibitor, wortmannin (40–42). In contrast to LY294002 and the direct Akt inhibitor, wortmannin is effective at the low nanomolar doses, which suggests that it may be more specific than the other two inhibitors. As shown in Fig. 6C, Akt activation was totally inhibited in the EBV+/III B cells with as little as 1 nmol/L wortmannin. Activation of mTOR remained essentially unaffected by 1 nmol/L and only mildly affected by 10 nmol/L of the drug; in contrast, 100 nmol/L was able to apparently completely inhibit mTOR activation. Notably, this inhibitory effect on mTOR occurred in both phospho-Akt-positive and phospho-Akt-negative B cells, suggesting a possible direct mTOR inhibition. Importantly, the marked differential in the drug efficacy noted in the EBV+/III cells further indicates the lack of functional relationship between the Akt and mTOR activation in these B cells. This conclusion is further strengthened by the lack of significant effect of wortmannin used at the low dose of 1 nmol/L on proliferation of the transformed B cells (Fig. 6D). Only when used at higher, PI3K less specific doses, wortmannin was able to markedly inhibit proliferation of the transformed B cells (Fig. 6E) in the dose-dependent manner. Because Akt is well known to protect cells from apoptotic death, we examined next if wortmannin would enhance apoptosis of the transformed B lymphocytes. As shown in Fig. 6F, the compound mildly to moderately increased the apoptotic cell rate in the EBV+/III cells that display activation of the PI3K/Akt pathway. Noteworthy, the noted proapoptotic effect was achieved at the low, seemingly PI3K-specific (1 nmol/L) dose of the compound; the 10- and 100-fold higher concentrations of the compound added little to the apoptotic cell rate. These findings indicate that the PI3K/Akt signaling pathway contributes to survival of the EBV+/III cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Addendum
 References
 
Here, we report that three types of transformed B lymphocytes universally display continuous activation of the mTOR signaling pathway regardless of their EBV genome expression status. Although Akt kinase has been identified as a key activator of mTOR in various cell types (11, 12, 14), our data clearly show that in the transformed B cells mTOR activation is Akt independent. Accordingly, as shown in Fig. 1A, only EBV+/III B cells display concomitant activation of Akt and mTOR; none of the EBV+/I and only one of the EBV– B-cell populations examined shows evidence of Akt activation. Akt activity in the EBV+/III cells is induced by IGF-I and serum; mTOR activity is IGF-I and serum independent in all three types of the transformed B lymphocytes (Fig. 4). Conversely, mTOR activation is strictly dependent on availability of nutrients; Akt can be activated by IGF-I even in their absence (Fig. 5). Finally, suppression of Akt activity by a direct inhibitor (Fig. 6B) or by the appropriately dosed PI3K inhibitor wortmannin had no effect on the mTOR-mediated signaling as determined by phosphorylation status of the mTOR targets (Fig. 6C) and cell proliferation (Fig. 6D). Results from other investigators also support the conclusion that Akt may not be critical for mTOR function in some cellular systems. Accordingly, stimulation of thyroid epithelial cells with thyroid-stimulating hormone had no effect on Akt phosphorylation but significantly induced S6K1 phosphorylation (43). Furthermore, although Akt activation leads to mTOR phosphorylation on the S2448 residue (44), the functional relevance of this phosphorylation is not clear (12). Finally, very recent report indicates that Akt involvement is not required for the proper mTOR function during Drosophila development (45).

The presence of PI3K/Akt and MEK/ERK pathway activation in the EBV+/III but not the EBV/I and EBV– B cells suggests that the EBV-encoded proteins expressed specifically in the type III latency are responsible for activation of these pathways. Accordingly, cell transfection with LMP2A has been shown before to activate the PI3K/Akt pathway (46–48). Interestingly, our finding that in the EBV+/III B cells serum withdrawal silences the PI3K/Akt and MEK/ERK signaling (Fig. 4) suggests that LMP2A might act in such cells through promoting their sensitivity to IGF-I and/or similar stimuli.

Our experiments depicted in Fig. 6A warrant caution in using LY294002 as a PI3K inhibitor (35–37). This reagent, indeed, profoundly inhibits PI3K activity as determined by Akt phosphorylation (Fig. 6A) when used at the high doses of 10 to 50 µmol/L. However, at the same dose range, it inhibits also mTOR activity in cells with no evidence of PI3K/Akt activation (Fig. 6A). This mTOR inhibitory effect is most likely due to the direct inhibition by LY294002 of mTOR itself because the compound has been shown to inhibit the in vitro kinase activity of purified mTOR (38). Therefore, the effects of LY294002 on cell signaling and biological function seem to stem from inhibition of not only PI3K but also mTOR and possibly other serine/threonine kinases. In contrast, wortmannin, effective in the low nanomolar dose range, is a more specific PI3K inhibitor because PI3K/Akt-independent inhibition of mTOR activity required ~100-fold higher doses of the drug (Fig. 6C). Therefore, it should be considered more reliable in differentiating between the effects of PI3K and mTOR. This marked difference in target specificity between wortmannin and LY294002 provides the best explanation for the difference in results reported by Brennan et al. (49) and seen by us (Fig. 6D) in regard to inhibition of cell proliferation of transformed B cells. Whereas exposure of cells to 1 nmol/L wortmannin had no detectable effect on their proliferation (Fig. 6D), exposure to 5 to 10 µmol/L Ly294002 inhibited cell growth (49). Based on our current (Fig. 1C) and previously published observations (15, 16) that showed sensitivity of the transformed B cells to growth-suppressive effect of mTOR inhibition, it is likely that the suppressive effect of LY294002 stemmed from its inhibitory effect on mTOR (ref. 38; Fig. 6A) rather than on PI3K.

Considering the well-documented oncogenicity of the continuously activated Akt, the recently described direct inhibitors of this serine kinase (39) invoke a considerable interest. By virtue of being phosphoinositol analogues, these agents occupy pleckstrin homology domain of Akt and, consequently, block interaction of the kinase with its partners, including the immediate activator phosphoinositide-dependent protein kinase 1 (PDK1). As shown in Fig. 6B, the leading representative compound from this group was, indeed, able to inhibit Akt activity. However, its effect was not very striking; even the high (40 µmol/L) dose resulted in only partial inhibition of Akt phosphorylation. Furthermore, the compound is not strictly specific for Akt because it inhibited also activation of ERK1/2 (Fig. 6B) at the same doses. Two other, closely related derivatives of the compound (see Materials and Methods for the drug characteristics) yielded similar results (data not shown). Noteworthy, a structurally similar drug named perifosine has recently been identified as a direct Akt inhibitor (50, 51). Beside Akt, this drug inhibited MAPK, JNK, and PDK1 (50). The relative lack of specificity of this class of drugs may not be surprising considering the large number of proteins that contain pleckstrin homology domain (51). Notably, perifosine seems to have a rather good therapeutic index in patients. It may, therefore, prove to be an effective anticancer drug with the caveat that its mode of action in the given tumor type could combine inhibition of several key oncogenic proteins, some of which may remain currently poorly characterized or unknown.

The Akt independence of mTOR activation in the transformed B lymphocytes shown here poses the question of the mechanism of mTOR activation in these cells. The mTOR activation is clearly not mediated by serum growth factors, including IGF-I (Fig. 4). It is also independent of the MEK/ERK signaling pathway because MEK inhibitor U0126 had no effect on phosphorylation of the mTOR-targeted residues in p70 S6K1 and 4eBP1 (Fig. 3). It seems important in this regard that mTOR activity was virtually shutoff in the transformed B cells by even a brief depletion of nutrients (Fig. 5). This finding is in agreement with the previous studies where amino acids, particularly arginine and leucine, have been identified as critical for mTOR signaling (52, 53). Our observation that deoxy-D-glucose that competitively inhibits glucose activity had no effect on the signaling (data not shown) supports the key role of amino acids in mTOR activation. However, it is likely that amino acids are required but not sufficient per se to fully activate mTOR. Therefore, other members of the cell signaling network alternative to Akt are likely to be involved. Recent studies suggest that activation of mTOR may occur by phosphatidic acid through phospholipase D1 (PLD1) independently of PI3K/Akt (54). However, when we inhibited PLD1 with 0.3% 1-butanol (54), we could not convincingly show a specific mTOR inhibition in our experimental system (data not shown). Recent studies indicate that mTOR may be indirectly activated by several other signaling pathways. Accordingly, phorbol esters and Ras isoforms that poorly activate Akt activate mTOR primarily through inhibition of TSC2 by involving p90 ribosomal S6 kinase (20) and protein kinase C/MAPK signaling (21). Furthermore, mTOR activity can be inhibited in the Akt-independent, TSC2-dependent manner by LKB1 (22)/AMP-activated kinase (23) signaling pathways and REDD1 (24). Finally, fibroblast growth factor-9 induced in endometrial stromal cells mTOR activation in the phospholipase C{gamma}– and calcium-dependent, PI3K- and Akt-independent manner (55). Our finding that, similar to mTOR and its downstream targets, TCS2 displays continuous phosphorylation regardless of the activation status of Akt (Fig. 2B) suggests that TSC2 may be implicated in the transformed B cells in mTOR activation. Which, if any, of the above mechanisms that control TSC2 function independently of Akt (20–24) activates mTOR in the transformed B cells remains to be elucidated.

Our findings have also potentially important implications for treatment of lymphomas and other malignancies. Several pharmacologic grade mTOR inhibitors are now available. Beside rapamycin, three rapamycin analogues (RAD001, CCI-779, and AP23573) have been introduced. Whereas they differ in some characteristics (e.g., pharmacokinetics and availability in an oral form), their basic function of inhibiting mTOR with high specificity and low toxicity seems to be essentially the same. We have found previously that rapamycin and the related mTOR inhibitors hold promise of becoming effective therapeutic agents in various types of T-cell lymphoma (56), and particularly in EBV+/III B-cell lymphomas (15, 16). An independent conformation of the inhibitory effect of rapamycin on the EBV+/III B cells was recently published (57). Furthermore, a few patients with B-cell transplant–related lymphoproliferative disorders were successfully treated with a combination of an antibody against a pan-B-cell marker CD20 and rapamycin (58). Contribution of rapamycin to the positive outcome has not, however, been fully determined in those studies. Noteworthy, our findings presented in this report suggest that mTOR inhibitors may be equally effective against B-cell lymphomas that are either EBV+/I or EBV– (Fig. 1A and C). In a preliminary immunohistochemical analysis of a reactive lymph node using an anti-phospho-4eBP1 antibody (data not shown), we detected staining of large subset of germinal center cells and of scattered interfollicular cells with essentially no staining of cells within mantle zone. Considering that germinal centers and, sometimes interfollicular zones, are typically the areas of cell activation, growth, and proliferation, these results suggest potentially higher sensitivity to mTOR inhibitors of the more aggressive (intermediate and high-grade) lymphomas that are more dependent on protein synthesis than of the indolent (low-grade) lymphomas comprised mostly of quiescent cells.

It could be argued that rationally designed combination of an mTOR inhibitor with other signal transduction inhibitors might have an even more pronounced anticancer effect. As shown in Fig. 3, MEK1/2 inhibitor U0126 effectively inhibits proliferation in the EBV+/III B-cell. However, we could not show any benefit of combining the MEK1/2 and mTOR inhibitors, with the latter clearly having the more profound effect on cell proliferation (data not shown). Potential combination of a mTOR inhibitor with an inhibitor of PI3K/Akt signaling also deserves consideration. Accordingly, loss of PTEN and the related aberrant activation of PI3K/Akt render the affected cells particularly sensitive to inhibitors of TOR (59, 60). However, the current lack of pharmacologic grade PI3K inhibitors and the relative nonspecificity of the direct Akt inhibitors as discussed above may impede development of such combinations.

In summary, we show that transformed B lymphocytes display continuous activation of mTOR serine/threonine kinase regardless of their EBV genome expression status. This mTOR activation is independent of PI3K/Akt and MEK/ERK signaling pathways as well as of serum growth factors, including IGF-I. It is, however, dependent on the availability of nutrients. The study also suggests that mTOR represents an attractive therapeutic target in a large spectrum of B-cell lymphomas.


   Addendum
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Addendum
References
 
The Burkitt lymphoma-derived cell line Ramos seems to be EBV– rather than EBV+/I. This apparent difference does not impact on the conclusions of the study.


   Acknowledgments
 
Grant support: National Cancer Institute grants R01-CA89194 and R01-CA96856.

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.

Received 11/22/04. Revised 6/ 6/05. Accepted 6/17/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Addendum
 References
 

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