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Cell Growth & Differentiation Vol. 13, 285-296, July 2002
© 2002 American Association for Cancer Research

The Inducible Expression of the Tumor Suppressor Gene PTEN Promotes Apoptosis and Decreases Cell Size by Inhibiting the PI3K/Akt Pathway in Jurkat T Cells1

Zheng Xu, David Stokoe, Lawrence P. Kane and Arthur Weiss2

Departments of Medicine and of Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, California 94143-0795 [Z. X., L. P. K., A. W.], and Cancer Research Institute, University of California, San Francisco, California 94115 [D. S.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we characterize the function of the tumor suppressor gene PTEN in Jurkat T cells. We established stable clones of Jurkat T cells that inducibly express either wild-type or phosphatase-inactive PTEN. We show here that PTEN potently inhibited the growth and reduced the size of Jurkat cells. The growth-suppressive effect of PTEN was associated with its ability to induce apoptotic cell death with little or no effect on cell cycle. PTEN also rendered Jurkat cells more susceptible to apoptosis induced by various stimuli. Furthermore, PTEN expression led to a reduction in the level of 3'-phosphorylated phospholipids and thus altered the activity and localization of Akt. Finally, coexpression of constitutively active Akt reversed the effects caused by PTEN. In summary, our results suggest that PTEN suppresses cell growth, promotes apoptosis, and decreases cell size by negatively regulating the phosphoinositide 3-kinase/Akt pathway in Jurkat T cells.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Proper control of survival and proliferation in hematopoietic cells is critical to maintain peripheral homeostasis and prevent the onset of tumors and autoimmune diseases. Inactivation of tumor suppressor genes is one way in which cancer cells circumvent normal growth control. Recently, PTEN,3 originally identified in 1997 as a candidate tumor suppressor gene (1, 2, 3) , has been implicated in control of the immune system. PTEN is one of the most common targets of mutation in multiple human cancers, including melanoma, meningioma, and glial, prostate, endometrial, renal, and small cell lung tumors (4) . Germ-line mutations in PTEN are also recognized as the cause of three autosomal dominant inherited cancer predisposition syndromes: Cowden disease, Lhermitte-Duclos disease, and Bannayan-Zonana syndrome (4) . Knockout and gene transfer studies have further confirmed the critical role of PTEN in tumor suppression. Three groups generated PTEN-deficient mice (5, 6, 7) . Homozygous disruption of the PTEN gene in all three lines results in early embryonic lethality. Heterozygous mice are viable; however, as they age, the mice display hyperplastic-dysplastic features and a high incidence of spontaneous tumors of various histological origins, including an increased frequency of T-cell lymphomas/leukemias; moreover, lymph node hyperplasia is frequently observed in PTEN heterozygotes, with consequent disruption of lymphoid architecture (5, 6, 7) . Therefore, PTEN is critical for the survival and proliferation of lymphocytes, particularly in T cells. Indeed, PTEN+/– mice develop a lethal autoimmune syndrome, supporting the notion that PTEN is an essential repressor of autoimmunity (8) . More recently, it has been shown that T cell-specific loss of PTEN leads to defects in central and peripheral tolerance in mice (9) . Finally, transient overexpression of PTEN causes growth suppression in PTEN-deficient glioblastoma, prostate, melanoma, and breast cancer cell lines (10, 11, 12, 13, 14, 15) . Taken together, these findings demonstrate that PTEN is required for normal development and that loss of PTEN function contributes to carcinogenesis and autoimmune diseases.

The cDNA sequence and crystal structure of PTEN provide intriguing clues as to how the protein might act as a tumor suppressor. PTEN contains a protein tyrosine phosphatase domain, the functional importance of which is underscored by the fact that most mutations in PTEN detected in primary tumors and in cell lines are confined to this domain (16 , 17) . The unusual structural features in the protein tyrosine phosphatase domain allow PTEN to act on lipid as well as protein substrates (17) . One protein substrate identified thus far is the focal adhesion kinase (18) . By regulating focal adhesion kinase phosphorylation in response to integrin ligation, PTEN could modulate various aspects of cell adhesion and migration, consistent with the frequent loss of the gene observed in late-stage metastatic tumors. However, it appears that only the lipid phosphatase activity is indispensable for its tumor-suppressive effect (14) . PTEN specifically cleaves, in vitro and in vivo, the D3 phosphate of PtdIns(3,4)P2 and PtdIns(3,4,5)P3, two major phospholipid products of PI3K (19 , 20) . The role of the PI3K pathway in cell proliferation and survival is well documented. Accumulation of phospholipids at the membrane after PI3K activation allows the recruitment of proteins containing a PH domain, which binds those lipids. One of these proteins is the proto-oncogene serine/threonine kinase Akt. Upon membrane recruitment, Akt is activated by phosphorylation and subsequently transmits survival signals. The putative targets of Akt include caspase 9, BAD, glycogen synthase kinase 3, and transcription factors of the forkhead family, each of which has been shown to play a role in preventing apoptosis (21, 22, 23) . Recent studies have confirmed the key role of some of the Akt targets in mediating the effect of PTEN (24 , 25) . Therefore, by keeping the level of D3 phospholipids low, PTEN negatively regulates an important set of cellular processes, such as proliferation and survival.

The PI3K pathway participates in T-cell growth and function. The activation of PI3K and Akt protects T cells from Fas-mediated apoptosis in T-cell lines as well as in transgenic mice expressing an active form of Akt under the control of a T cell-specific promoter (26 , 27) . Likewise, T cells from PTEN heterozygous mice show reduced activation-induced cell death and increased proliferation upon stimulation. Fas-mediated apoptosis is impaired, and PI3K inhibitors can restore Fas responsiveness in cells from those mice (8) . On the basis of these findings, it was hypothesized that PTEN antagonized the effects of PI3K in T cells by reducing the levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3. To further characterize the function of PTEN in T cells, we chose the Jurkat leukemic line because it is used as a model system to study T-cell function, and it does not express endogenous PTEN (28) . We have generated Jurkat clones that stably express PTEN in an inducible manner. We report here that the restoration of PTEN protein to a physiological level suppressed Jurkat growth and reduced cell size. The growth suppression of PTEN was reversible upon withdrawal of the inducer. PTEN did not have a significant influence on cell cycle; instead, it induced a low level of apoptotic cell death in Jurkat T cells. PTEN also increased the sensitivity of Jurkat cells to apoptotic stimuli. Furthermore, PTEN expression altered the level of phospholipids and the activity and localization of Akt. Finally, the PI3K inhibitor LY294002 had a similar effect on growth, viability, size, and coexpression of constitutively active Akt efficiently countered the action of PTEN. Taken together, we conclude that PTEN suppresses cell growth, promotes apoptosis, and decreases cell size by negatively regulating the PI3K/Akt pathway in Jurkat T cells.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
PTEN Can Be Inducibly Expressed in Jurkat T Cells.
We first examined the expression of PTEN in our E6-1 clone of Jurkat T cells. We failed to detect PTEN protein by Western blot analysis using a panel of anti-PTEN antibodies. The results from two of these antibodies are shown in Fig. 1ACitation . Two positive controls, the EGF-stimulated A431 cells and a human T cell line Hut78, express endogenous PTEN. To assess the effects of PTEN in Jurkat T cells, we generated Jurkat stable clones that express the WT or mutant PTEN (G129R) under the control of the tetracycline-inducible system (Tet-on). G129R is a tumor-derived mutation that is defective in both the protein and lipid phosphatase activity (11 , 13) . Our stable clones are under the tight control of DOX, a tetracycline analogue. As shown in Fig. 1BCitation , without DOX induction no protein could be detected in any of our clones. Upon addition of DOX, equal amounts of either WT or G129R protein were induced in two representative clones (Fig. 1B)Citation . The level of PTEN could be finely controlled by the concentration of DOX (Fig. 1C)Citation . As low as 0.2 µg/ml DOX was sufficient to induce PTEN expression. PTEN protein appeared 6 h after adding DOX and reached a steady state after 24 h (Fig. 1D)Citation . The level of PTEN remained constant up to 6 days in the presence of DOX (data not shown). The two representative clones exhibited similar time-dependent and concentration-dependent induction by DOX. The tight control of PTEN expression was seen in other clones as well (data not shown). To study the function of PTEN at a physiological level, 1 µg/ml DOX treatment for at least 24 h was used in subsequent studies because the level of PTEN induced under this condition is equivalent to that of the endogenous PTEN in Hut78 T cells (Fig. 1, B–D)Citation . The experiments hereafter were carried out in two clones of each type, and the results were essentially the same.


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Fig. 1. PTEN is inducibly expressed in stable clones of Jurkat. A, PTEN is not expressed in Jurkat cells. Whole cell lysates from Jurkat, EGF-stimulated A431, and Hut78 were probed with two anti-PTEN antibodies. The same blot was then reprobed with an anti-tubulin antibody to control for the loading. B, PTEN is inducibly expressed in stable clones under the control of DOX. Representative clones were cultured in the absence or presence of 1 µg/ml DOX for 24 h. A blot of whole cell lysates was first probed with an anti-PTEN antibody and then with an anti-ZAP70 antibody to control for the loading. C, the expression level of PTEN depends on the concentration of DOX. Two representative clones were cultured at the indicated concentration of DOX for 24 h. D, the expression level of PTEN depends on the time of the induction. Two representative clones were cultured in 1 µg/ml DOX for the time indicated. In C and D, whole cell lysates were subjected to Western blot using an anti-PTEN antibody. All of the blots are representative of three experiments.

 
PTEN Suppresses Cell Growth and Decreases Cell Size in Jurkat.
To evaluate whether the restoration of PTEN had any effect on Jurkat growth, vector control, WT, or G129R stable clones were seeded at the same density and cultured in the absence or presence of DOX for 6 days. The number of live cells daily was determined by trypan blue exclusion. All three clones had indistinguishable morphology and growth rate in the absence of DOX. Upon induction, however, WT PTEN caused potent growth suppression in Jurkat cells, whereas the vector control or phosphatase-inactive mutant had no effect. The growth inhibition was detected as early as 48 h after PTEN induction, increased with time, and reached nearly 90% at day 6 (Fig. 2A)Citation . The expression of PTEN at day 6 was verified by Western blot (Fig. 2B)Citation . The inhibitory effect was dependent on the concentration of DOX and thus the level of PTEN in the cell. At 0.2 µg/ml, DOX addition resulted in 50% growth suppression (data not shown). Because the PI3K pathway has been implicated in T-cell survival, we also tested the effect of a PI3K inhibitor, LY294002, on the growth of Jurkat cells. Similar to PTEN, LY294002 treatment caused a dose-dependent growth-inhibitory effect in Jurkat cells. LY294002, at 3 µM, could block >90% of cell growth at day 6 (Fig. 2A)Citation . We further confirmed the growth suppression induced by PTEN using the CellTiter 96 Aqueous One Solution Cell Proliferation assay that assesses metabolic activity (data not shown). Our results demonstrate that PTEN is important in controlling cell proliferation in Jurkat T cells, and this effect is mediated through its phosphatase activity.


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Fig. 2. PTEN suppresses Jurkat growth. Stable clones were left untreated ({diamond}) or treated with either DOX ({diamondsuit}) or 3 µM LY294002 (bullet) for 6 days. A, the number of live cells was determined by trypan blue exclusion at each time point. Each of the data points represents the mean of values from three separate experiments; SD in all cases was <5% of the mean value. B, the expression level of PTEN at day 6 was determined by Western blot. The blot is representative of three experiments.

 
To verify that the phosphatase activity of WT PTEN was not toxic to cells, we first induced PTEN expression; 6 days later, when cell growth was greatly suppressed by PTEN, we washed out DOX from the medium and reseeded the cells at the original density. Western blot analysis showed that PTEN protein started to decrease at day 7 and was undetectable at day 9. Correlating with the protein level, exponential cell growth resumed after a delay of 2 days (Fig. 3A)Citation . The initial lag was presumably attributable to the presence of PTEN during that period (Fig. 3B)Citation . Therefore, growth inhibition is reversible upon withdrawal of PTEN.


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Fig. 3. PTEN-dependent growth suppression is reversible. Stable clones were left untreated (open symbols) or treated with DOX (filled symbols) for 6 days. Cells were then washed and cultured in the absence of DOX for another 6 days. A, the number of live cells was determined by trypan blue exclusion at each time point. Each of the data points represents the mean of values from three separate experiments; SD in all cases was <5% of the mean value. B, the level of PTEN at the time indicated was determined by Western blot. The blot is representative of three experiments.

 
Genetic studies in Drosophila and more recently in mice suggest that PTEN plays a role in controlling cell size. PTEN mutant flies show an autonomous increase in cell size, which is coordinated with changes in the actin cytoskeleton (29, 30, 31, 32) . Similarly, mutant cells from brain-specific PTEN knockout mice display a cell-autonomous increase in neuronal soma size (33 , 34) . Conditional deletion of the PTEN gene in neural stem/progenitor cells also leads to cell enlargement (35) . Genetic epistasis tests in Drosophila further demonstrate that PI3K, PTEN, and Akt comprise a signaling cassette to regulate cell size (32) . In our study, we compared the forward scatter by flow cytometry and found that PTEN-positive Jurkat cells were smaller than PTEN-negative cells. The difference in cell size was detectable 3 days after PTEN induction. At day 6, along with the block of cell growth, the reduction in cell size became more striking (Fig. 4)Citation . To investigate whether PI3K was involved in determining cell size in our system, we treated PTEN-negative cells with 3 µM LY294002 and observed a similar phenomenon (Fig. 4)Citation . Therefore, our results have defined a novel function of the PI3K/PTEN pathway in Jurkat T cells, i.e., cell size control. The role of PI3K in regulating cell and organ size in mammals has also been reported in transgenic mice expressing constitutively active or dominant-negative mutants of PI3K in the heart (36) .


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Fig. 4. PTEN reduces cell size of Jurkat. Stable clones were left untreated (shaded histogram) or treated with either DOX or 3 µM LY294002 (open histogram) for 6 days. At day 6, cell size was determined by flow cytometry. The histograms are representative of three experiments.

 
PTEN Does Not Significantly Influence the Cell Cycle Profile of Jurkat.
The growth suppression after PTEN restoration in PTEN-null glioma cell lines is associated with cell cycle arrest at the G1 phase (12 , 37 , 38) . To address the mechanism for growth suppression, we first examined the cell cycle progression in Jurkat cells by labeling them with BrdUrd at different time points after PTEN induction. The percentage of cells in each phase of the cell cycle was determined by staining the nuclei with anti-BrdUrd antibody and propidium iodide. As shown in Fig. 5ACitation , except for a slight increase in sub-G1 population in the presence of DOX, the cell cycle distribution was essentially the same regardless of PTEN expression, suggesting that PTEN had no effect on cell cycle progression. Quantitation of the percentage of cells in each phase confirmed that the presence of PTEN for 6 days did not result in a significant change in the cell cycle profile (Fig. 5B)Citation . The same was true for earlier time points (data not shown). Even after cell cycle synchronization with nocodazole, which resulted in a more obvious PTEN-mediated G1 arrest in glioma cells (12) , expression of PTEN in Jurkat cells did not alter the cell cycle profile (data not shown). Finally, we checked the expression level of p27Kip1, a G1 cyclin-dependent kinase inhibitor that regulates G1-S progression. Expression of PTEN in several PTEN-deficient cell lines causes a significant elevation of the level of p27Kip1, which provides a mechanism for the G1 arrest in those cells (37 , 38) . In Jurkat cells, however, the level of p27Kip1 was not influenced by PTEN (Fig. 5C)Citation . This observation further supports the notion that PTEN is not linked to cell cycle control in Jurkat cells.


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Fig. 5. PTEN does not have a significant effect on the cell cycle profile of Jurkat. Stable clones were cultured in the absence and presence of DOX for 6 days. Cells were then labeled with 10 µM BrdUrd for 2 h. The percentage of cells in each phase of the cell cycle was determined by staining the nuclei with anti-BrdUrd antibody and propidium iodide (PI). A, the staining pattern of a WT PTEN clone with or without DOX at day 6. B, the percentage of cells in each phase. Each of the columns represents the mean of values from three separate experiments; bars, SD. C, the level of p27Kip1 at day 6 was determined by Western blot. The blot is representative of three experiments.

 
PTEN Induces Cell Death in Jurkat Cells.
Although we did not see an effect of PTEN on cell cycle, the slight increase in sub-G1 population in cells expressing WT PTEN suggested that PTEN might regulate apoptosis in Jurkat T cells (Fig. 5ACitation , right panel). To explore this possibility, we first examined the rate of cell death using trypan blue staining. Fig. 6ACitation shows that PTEN triggered cell death as early as 48 h after its expression. Dead cells accumulated over time until nearly half of the cells were dead at day 6. In contrast, only 3% of the cells were not viable in the absence of PTEN during the entire 6-day period. LY294002, at 3 µM, induced cell death in a manner similar to WT PTEN. The increase of dead cells correlated well with the decrease of live cells, suggesting that cell death was the major mechanism for the growth suppression mediated by PTEN. We then performed TUNEL assays to address whether apoptosis was the cause of cell death. As shown in Fig. 6BCitation , we detected 5-fold more apoptotic cells 48 h after the induction of PTEN or addition of LY294002, which increased to 12-fold by day 6. The fold increase in apoptosis was comparable with that in cell death from days 1 to 6, suggesting that the mechanism responsible for the observed increase in cell death was apoptosis. The number of apoptotic cells was less than that of dead cells, presumably because the TUNEL assay detects apoptotic cells within a narrower temporal window than trypan blue staining. Finally, we found no difference in apoptosis in cells expressing the G129R mutant (data not shown), indicating that cell death caused by PTEN requires its phosphatase activity.


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Fig. 6. PTEN induces cell death in Jurkat cells. A stable clone of WT PTEN was left untreated ({diamond}) or treated with either DOX ({diamondsuit}) or 3 µM LY294002 ({circ}) for 6 days. At each time point, the number of dead cells was determined by trypan blue exclusion (A), and the percentage of cells undergoing apoptosis was determined by TUNEL assay (B). Each of the columns represents the mean of values from three separate experiments; bars, SD.

 
PTEN Regulates the Sensitivity of Jurkat Cells to Apoptotic Stimuli.
Two findings suggest that PTEN plays a role in apoptosis in T lymphocytes; the impaired Fas mediated apoptosis in PTEN+/– mice and the resistance to apoptosis induced by several stimuli in PTEN–/– T cells (8 , 9 , 20) . Therefore, we investigated whether the induction of PTEN in Jurkat cells would lead to an increased sensitivity to apoptosis. We tested three different conditions known to cause apoptosis: anti-Fas antibody, activation of T cells via the TCR (39) , and serum withdrawal (23) :

(a) We examined the sensitivity to Fas-mediated apoptosis by treating cells with different doses of anti-Fas antibody 24 h after PTEN induction. After another 24 h, the percentage of apoptotic cells was determined by TUNEL assay. Fig. 7ACitation shows that anti-Fas antibody elicited a much stronger apoptotic response in PTEN-expressing cells, which was most apparent at low doses of anti-Fas antibody.


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Fig. 7. PTEN regulates the sensitivity of Jurkat cells to apoptotic stimuli. A stable clone of WT PTEN was left untreated ({square}) or treated with DOX ({blacksquare}) for 24 h and then subjected to the following treatment for another 24 h: A, anti-Fas antibody at the concentration indicated; B, medium or anti-TCR stimulation; and C, medium with reduced serum at the percentage indicated. In each case, the percentage of cells undergoing apoptosis was determined by TUNEL assay. Each of the columns represents the mean of values from three separate experiments; bars, SD.

 
(b) We checked the sensitivity of PTEN-expressing cells to TCR-mediated apoptosis. Activation through the TCR leads to the up-regulation of the Fas-ligand and subsequent apoptosis (39) . After stimulation for 24 h with the anti-TCR antibody, PTEN-positive cells showed a 15-fold increase in the percentage of apoptotic cells, whereas PTEN-negative cells only had a 4.5-fold increase (Fig. 7B)Citation . These results provide further evidence for a role of PTEN in apoptosis in T cells.

(c) We assayed apoptosis in limiting concentrations of serum. Serum contains various mitogenic components that are known to activate PI3K (22 , 23 , 40, 41, 42) . To test whether PTEN enhances the apoptotic effect induced by serum withdrawal in Jurkat cells, PTEN-positive and -negative cells were cultured for 24 h in reduced serum. As shown in Fig. 7CCitation , 2% serum alone induced a 5-fold increase in the number of TUNEL-positive cells. When PTEN was introduced, this effect was further elevated to 10-fold. Similar results were obtained at 0.5 and 0.2% serum (Fig. 7C)Citation .

In all three experiments, the increase in apoptosis correlated well with the decrease in growth (data not shown), supporting our conclusion that apoptosis accounts for the loss of cells in the presence of PTEN. The above effects required phosphatase activity because the phosphatase-inactive mutant did not affect any of these processes.

PTEN Regulates the Downstream Effectors of PI3K in Jurkat Cells.
To elucidate the mechanism for the effect of PTEN, we first measured the levels of 3' phosphorylated inositol phospholipids, the products of PI3K. Fig. 8ACitation shows that the levels of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 were greatly reduced in cells expressing PTEN, indicating that PTEN functions as a lipid phosphatase and regulates the basal level of phospholipids in Jurkat T cells.


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Fig. 8. PTEN regulates the downstream effectors of PI3K in Jurkat cells. A, PTEN reduces the level of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in Jurkat cells. Cells from a WT PTEN clone were cultured in the absence or presence of DOX for 24 h and then labeled with 32P-Pi. The phospholipids were extracted, deacylated, and resolved by anion exchange with an increasing gradient of NaH2PO4, pH 3.8. Fractions were collected and counted for 32P radioactivity. The identity of the peaks was determined by resolving phospholipid standards on the same column. B, PTEN changes the subcellular localization of Akt. Cells from either a WT or a G129R PTEN clone were incubated in the absence or presence of DOX for 24 h and then subjected to fractionation. Immunoprecipitation was performed with the membrane fraction using an antibody against the PH domain of Akt (#1), followed by Western blot using an anti-Akt antibody that recognizes all three Akts (#3; doublets on the blot). The membrane fraction was probed with an anti-CD45 antibody to control for protein level. C and D, PTEN down-regulates the basal activity (C) and phosphorylation (D) of Akt. Cells from a WT PTEN clone were incubated in the absence or presence of DOX for 24 h and then treated with either anti-TCR antibody for the time indicated or 10 µM LY294002 for 15 min. Whole cell lysates were subjected to immunoprecipitation using anti-Akt antibody (#4), and the immune complex was assayed for Akt activity using the peptide substrate Crosstide. The data shown are representative of two separate experiments. Whole cell lysates were also subjected to Western blot analysis using an anti-phosphoAkt (Ser-473) antibody (#2). The same blot was subsequently reprobed with an antibody that recognizes all three Akts (#3; doublets on the blot) to control for protein level. All of the blots are representative of three separate experiments.

 
The activation of Akt, a key regulator of cell survival and proliferation, is dependent on PI3K products. Akt binds phospholipids via its PH domain and translocates to the plasma membrane, where it becomes phosphorylated and activated (21, 22, 23) . We reasoned that the localization of Akt would be altered in PTEN-expressing cells as a result of reduced phospholipid levels. Indeed, the amount of Akt in the plasma membrane fraction of resting Jurkat cells was greatly reduced when WT PTEN, but not the phosphatase-inactive mutant, was induced (Fig. 8B)Citation . To control for protein loading, the membrane fraction was probed with an anti-CD45 antibody (Fig. 8B)Citation . The quality of the cytosolic and membrane fractions was evaluated by immunoblotting for CD45 (membrane) and extracellular signal-regulated kinase (cytosol; data not shown).

Consistent with its influence on the localization of Akt, PTEN down-regulated the basal activity of Akt by 3.6-fold as shown in an in vitro Akt kinase assay using a peptide substrate (Fig. 8C)Citation . We also checked the phosphorylation status of Ser-473 that has been implicated in the activation of the kinase (21 , 23) . Using an antibody specific for the phosphorylated Ser-473 (#2), we detected a significant reduction of the basal level of phospho-Akt after PTEN expression (Fig. 8D)Citation . Pretreatment of the cells with the PI3K inhibitor LY294002 decreased the basal activity and phosphorylation of Akt to a similar extent (Fig. 8, C and D)Citation . We confirmed that the level of total Akt was equivalent in all samples using an anti-Akt antibody (#3; Fig. 8DCitation , lower panel). As predicted, phosphatase-inactive PTEN failed to change either phospholipid levels or Akt activation (data not shown). The effect of PTEN expression on phospholipids and Akt in the basal state provides a potential explanation for the growth suppression and apoptosis seen in PTEN-expressing Jurkat T cells. It is interesting to note that although PTEN expression reduced the basal and the peak phosphorylation and activity of Akt, it did not eliminate the activation of Akt by anti-TCR treatment (Fig. 8, C and D)Citation .

Constitutively Active Akt Efficiently Reverses the Effects Caused by PTEN.
To confirm that PTEN acts via the PI3K/Akt pathway to inhibit cell growth, we attempted to reverse the effects of PTEN by using an activated form of Akt, i.e., myristylated Akt (Myr-Akt), which is constitutively localized to the membrane (21 , 23) . A stable clone of WT PTEN was cotransfected with truncated CD25 and either Myr-Akt or the vector control. Cells were then cultured in the absence or presence of DOX for 4 days. Cell growth and death were determined based on the number of CD25+ live cells and the percentage of trypan blue-positive cells, respectively. As shown in Fig. 9, A and BCitation , cell growth was greatly inhibited, accompanied by a dramatic increase in cell death, when PTEN was induced in vector-transfected cells. In contrast, coexpression of Myr-Akt rescued PTEN-expressing cells from growth suppression and cell death with an efficiency of >90%. We also noticed that PTEN expression reduced cell size in cells transfected with the vector but not in those expressing Myr-Akt (data not shown). Therefore, the expression of a constitutively active Akt could efficiently reverse the effects caused by PTEN, i.e., growth suppression, cell death, and reduced cell size.


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Fig. 9. Constitutively active Akt efficiently rescues the growth suppression caused by PTEN. Cells from a WT PTEN clone were transfected with a control vector ({diamond}, {diamondsuit}) or Myr-Akt ({triangleup}, {blacktriangleup}) and a truncated CD25 construct. Cells were then cultured in the absence (open symbols) or presence (filled symbols) of DOX for 4 days. A and B, Myr-Akt efficiently rescues the growth suppression (A) and cell death (B) caused by PTEN. At each time point, cells were stained with an anti-CD25 antibody and analyzed by flow cytometry. The number of live and dead cells was determined by trypan blue exclusion. A and B show the number of CD25+ cells and the percentage of dead cells, respectively. Each of the data points represents the mean of values from three separate experiments; SD in all cases was <5% of the mean value. C, the expression of Myr-Akt was examined by Western blot using anti-Akt antibody (#3) at the time indicated. The blot is representative of three separate experiments.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
To study the function of PTEN in T cells, we have established Jurkat (PTEN-null) stable clones that inducibly express either WT or a phosphatase-inactive form of PTEN and performed all our studies on PTEN at a level equivalent to the endogenous level in the Hut78 T cell line. Expression of PTEN at a physiological level suppressed cell growth, induced apoptosis, decreased cell size, and rendered the cells more susceptible to apoptotic stimuli. The effects of PTEN were dependent on its phosphatase activity. An explanation for these effects was provided by its regulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 levels and Akt activity in Jurkat cells. Moreover, a constitutively active form of Akt efficiently rescued the growth inhibition, cell death, and reduced cell size mediated by PTEN.

Consistent with its identification as a tumor suppressor, PTEN is not expressed in many tumor-derived cell lines. Transient overexpression of PTEN in PTEN-null cells has been shown to lead to growth inhibition (10, 11, 12, 13, 14, 15 , 37 , 43 , 44) . Despite extensive effort, PTEN has been difficult to express stably in PTEN-deficient cells, presumably because of its growth-suppressive effect. The inducible system we describe here is ideal to study a protein like PTEN because it avoids any potential adaptation or compensation during the generation of stable clones that might influence the behavior of the cells. In addition, the tight control of expression makes it possible to study a protein at a physiological level rather than overexpression. Therefore, our stable clones provide a powerful tool to study this tumor suppressor gene in a null background in a T-cell line.

Using the inducible clones, we addressed two key questions regarding PTEN, the first of which was the mechanism of growth suppression. We have shown that PTEN induced apoptosis but did not significantly change the cell cycle profile of Jurkat T cells. The effect of PTEN on apoptosis in Jurkat T cells was also noted in a transient overexpression study (45) . However, its effects on cell cycle and cell size were not addressed because of the limitation of the transient transfection method. Similar results have been obtained in a variety of cell lines, including glioblastoma, breast cancer, and prostate cancer cells (11 , 13 , 43) . However, in several glioblastoma and renal carcinoma cell lines, G1 cell cycle arrest appears to account for the majority of the observed growth inhibition, which is correlated with an increase in the stability of p27Kip1 (37 , 38) . In another study, PTEN was inducibly overexpressed in a PTEN+ breast cancer line, and the resulting growth suppression was attributed to an initial G1 arrest followed by a combination of cell cycle arrest and apoptosis (46) . In the study reported here, we also followed the time course of apoptosis and cell cycle distribution. In Jurkat cells, apoptosis was evident as early as 48 h after the induction of PTEN, whereas the cell cycle profile remained the same throughout the experiment. The correlation between the increase in cell death and decrease in cell growth suggests that apoptotic cell death could account for all of the growth inhibition mediated by PTEN, at least in Jurkat T cells. The differential effect of PTEN could be attributable to either overexpression versus physiological level of expression or the presence of endogenous PTEN in the breast cancer line. Our data support the hypothesis that the function of PTEN depends on the cell type, the developmental status, and the culture conditions used. Along these lines, overexpression of PTEN in Drosophila prevents cell cycle progression in proliferating cells while promoting apoptosis in differentiating cells during eye development (31) . The effect of PTEN could be dependent on its dosage as well. We have observed that the extent of growth inhibition depends upon the level of PTEN in the cell. It is also possible that the behavior of PTEN could be influenced by other oncogenic pathways that are deregulated during neoplastic transformation in a given cell line. In fact, a link between PTEN and the retinoblastoma pathway has been suggested by the observation that PTEN does not induce G1 arrest in retinoblastoma–/– cells (47) . Moreover, not retinoblastoma all tumor-derived cell lines are PTEN deficient, despite their ability to proliferate autonomously. For example, Hut78 T cells express endogenous PTEN; in contrast, the same level of PTEN causes growth inhibition in Jurkat cells. Consistent with the differential effect of PTEN on cell growth in these two cell lines, a low dose of the PI3K inhibitor LY294002 triggers apoptosis in Jurkat cells but not in Hut78 cells (26) .

The second question concerns the regulation of cell size by PTEN. Although several groups have shown that PTEN controls cell size in Drosophila and in mouse brain (29, 30, 31, 32, 33, 34, 35) , such a phenomenon has not been observed in human tumor cell lines. It is technically difficult to assess cell size in a heterogeneous population of transiently transfected cells. In the case of constitutive stable clones, smaller cells are likely to be considered as clonal variation rather than an effect caused by PTEN. Only the inducible expression of PTEN in a PTEN-null clone could help define its role in size determination. Indeed, we found that cells expressing PTEN appeared smaller 3 days after PTEN induction, and this difference became more dramatic with time. Genetic analysis in flies indicates that PTEN regulates cell size by regulating the PI3K/Akt pathway (29, 30, 31, 32) . Consistent with that, we showed that a PI3K inhibitor, LY294002, had a similar effect on cell size. In addition, Myr-Akt not only rescued the growth suppression but also prevented the decrease in cell size. Therefore, we conclude that Jurkat T cells use a similar signaling machinery containing PI3K, PTEN, and Akt to control cell size. Our conclusion is also supported by recent transgenic mouse models showing that the expression of constitutively active PI3K in cardiac myocytes or Akt1 in pancreatic ß-cells causes cell enlargement (36 , 48) . A likely downstream effector of PI3K and Akt in cell size control is the 40S ribosomal protein S6 kinase. Gene disruption of 40S ribosomal protein S6 kinase in mice and flies is associated with smaller cells and a reduction in body size (49 , 50) . However, the exact mechanism of cell size determination remains unresolved. Interestingly, cell size change is a well-observed phenomenon during T-cell development and activation. It is known that naïve T cells rely on extrinsic signals such as growth factors and TCR signaling to avoid death by neglect. In the absence of these signals, nutrient use is insufficient to maintain either size or viability of primary T cells (51) . This process is thought to play important roles in maintaining T-cell homeostasis and shaping T-cell repertoire. Conversely, disrupting this process would result in tumorigenesis and autoimmunity. This theory is supported by our observations that ectopic expression of PTEN, a tumor suppressor and a repressor of autoimmunity, in the Jurkat leukemia line decreases cell size and promotes apoptosis.

Our study has established a role of PTEN in survival, proliferation, and size control in Jurkat T cells. The findings that the PI3K inhibitor LY294002 could mimic the effects of PTEN on growth, viability, and size and a constitutively active form of Akt efficiently countered the action of PTEN suggest that PTEN exerts its function by inhibiting the PI3K/Akt pathway in Jurkat cells. Nevertheless, the detailed molecular basis of how PTEN might function is largely unknown. To identify downstream effectors of PTEN in Jurkat cells, cDNA microarray analysis is being used with our inducible stable clones before and after PTEN expression. This experiment may also reveal novel genes related to oncogenic transformation of the Jurkat leukemic line and contribute to the understanding of how PTEN might influence intracellular signaling pathways, such as the Fas pathway. In fact, results from mice in which the PTEN gene is inactivated have implicated PTEN in this pathway. T cells from T cell-specific PTEN-deficient mice show increased resistance to apoptosis induced by a variety of stimuli (9) . PTEN+/– mice with impaired autoimmunity also display an impaired Fas response in splenic T and B cells (8) . Consistent with these observations, we found that PTEN increased the susceptibility of Jurkat cells to apoptotic stimuli. Mechanistic studies are needed to elucidate the role of PTEN in Fas-mediated apoptosis in T cells. The lack of an effect in all of the assays using a phosphatase-inactive mutant suggests that PTEN acts as a tumor suppressor via its lipid phosphatase activity. Transiently transfecting the G129E mutant that specifically ablates the lipid phosphatase activity in other cell lines leads to the same conclusion (12 , 14) . Whether PTEN also dephosphorylates protein substrates and regulates other cellular processes in T cells remains unclear. To address this question, mutants that lack only the protein phosphatase activity need to be generated and tested for their biological function.

Interestingly, although PTEN inhibited the basal activity of Akt, it did not prevent the activation of Akt by TCR stimulation in Jurkat cells. Other groups have noticed that the induced activation of Akt by platelet-derived growth factor or insulin is not affected by PTEN, despite the down-regulation of its basal activity in glioblastoma cells (14) . Because Akt activation depends on the intracellular level of phospholipids, the above phenomenon could be attributed to either functional inactivation of PTEN or dominant activity of PI3K over PTEN upon TCR stimulation, or both. This leads to an interesting yet poorly understood question: the regulation of PTEN. Studies from non-T-cell models suggest that PTEN can be regulated at the level of protein expression, stability, posttranslational modification, or access to its substrates (52, 53, 54, 55, 56, 57, 58, 59) . For example, the C2 domain that binds to the membrane phospholipids in vitro has been implicated in stabilizing the protein and productively positioning the catalytic site with respect to the membrane-bound phosphoinositide substrates (53) . Phosphorylation of serine and threonine residues within the 50-amino acid COOH-terminal tail has been linked to protein-protein interaction as well as PTEN stability (54 , 55) . Association of PTEN with PDZ domain proteins via its COOH-terminal PDZ binding site has also been reported to influence its function (56, 57, 58, 59) . Whether these regulatory mechanisms apply to T cells awaits further analysis.

In summary, our study demonstrates a pivotal role of PTEN in controlling the survival and proliferation of T lymphocytes, which implicates PTEN in modulating immune responses as well as in maintaining peripheral T-cell homeostasis. Our stable clones will be a useful model system to investigate the downstream targets and the regulatory mechanism controlling PTEN function in T cells. Further characterization of PTEN will provide valuable insight into the process of tumorigenesis in the immune system and pathogenesis of autoimmune disorders. It may also reveal novel pharmaceutical targets for the control of T-cell tumors or T-cell responses.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Antibodies and Reagents.
The anti-PTEN antibody used in this study was from Cascade Bioscience, except that in Fig. 1ACitation an antibody from Dr. Charles Sawyers (University of California, Los Angeles, CA) was also used. Antibodies against Akt were from the following: #1, anti-PH domain from Upstate Biotechnology, Inc.; #2, anti-phospho-Ser-473 from New England Biolabs; #3, anti-Akt monoclonal antibody from Transduction Lab; and #4, a rabbit polyclonal antibody against Akt, as described previously (60) . Other antibodies were from the following: anti-Jurkat TCRß monoclonal antibody C305 (61) , anti-CD45 antibody 9.4 (American Type Culture Collection; HB10508), anti-ZAP70 antibody (62) , anti-p27Kip1 antibody (Transduction Lab), and anti-tubulin antibody (Sigma Chemical Co.). BrdUrd, nocodazole, and DOX were from Sigma. Anti-Fas and FITC-conjugated anti-BrdUrd antibodies were from Becton-Dickinson. The Tet system-proved FCS was from Clontech. G418 and hygromycin B were from Life Technologies, Inc. EGF-stimulated A431 cell lysate was from Upstate Biotechnology. LY294002 was from Calbiochem. Trypan blue and propidium iodide were from Roche.

Plasmids and Cells.
The plasmid pUHD172-1neo (encoding the reverse tetracycline-controlled transactivator; Ref. 63 ) was a kind gift from Dr. H. Bujard (Zentrum fur Molekulare Biologie, Heidelberg, Germany). pBI-EGFP and pTK-Hyg (encoding the hygromycin-resistant gene) were from Clontech. Myristylated Akt (Myr-Akt) and the truncated CD25 construct were described previously (64 , 65) . Jurkat T cells were maintained in RPMI 1640, supplemented with 5% FCS, 2 mM glutamine, penicillin, and streptomycin.

Transfection of Jurkat Cells.
Jurkat cells in the logarithmic growth phase were transfected by electroporation. Cells (2 x 107) were resuspended in 400 µl of serum-free RPMI 1640. Plasmid DNA was mixed with the cells in a 4-mm gap electroporation cuvette and pulsed at 250 V and 960 µF using the Gene Pulser (Bio-Rad Laboratories). The cells were then transferred to culture flasks and incubated in complete medium.

The Generation of Stable Clones of Jurkat.
A Glu-tag (MEYMPME) was engineered at the NH2 terminus of either WT or phosphatase-inactive (G129R) PTEN cDNA. The tagged cDNA was cloned into the pBI-EGFP expression vector. This vector allows the simultaneous regulation of both a gene of interest and EGFP by one central tetracycline response element. The pBI-EGFP vector or two PTEN constructs were cotransfected with pTK-Hyg into Jrt TA7–6, a Jurkat subline that is stably transfected with pUHD172–1neo. Hygromycin-resistant clones were tested for the induced expression of EGFP in the initial screen and subsequently that of PTEN. Stable clones were maintained in RPMI 1640 supplemented with 10% Tet system-proved FCS and 2 mg/ml G418 and 300 µg/ml hygromycin B.

Cell Stimulation and Lysis.
Cells were harvested by centrifugation, washed once in warm PBS, and resuspended in warm PBS at 108 cells/ml. After incubation at 37°C for 20 min, cells were either left unstimulated or stimulated with C305 (1:500 culture supernatant) for the indicated duration. Cells were lysed immediately in 4°C lysis buffer [10 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 1 mM EDTA, and a mixture of phosphatase and proteinase inhibitors]. After 20 min at 4°C, lysates were prepared by a 15-min centrifugation at 4°C and 32,000 x g. The lysates were either directly analyzed by Western blot or subjected to immunoprecipitation, followed by immunoblotting or a kinase assay.

Immunoprecipitation and Western Blot Analysis.
Whole cell lysates were incubated with an antibody to the PH domain of Akt (#1) and protein G-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4°C. Immunoprecipitates were washed four times with the above lysis buffer. Whole cell lysates and immunoprecipitates to be analyzed by immunoblotting were subjected to electrophoresis on SDS-PAGE and transferred to Immobilon-P membrane (Millipore) according to the manufacturer’s instructions. Concentrations for blotting antibodies varied according to the manufacturer’s recommendations. The blots were developed with the ECL system from Amersham Pharmacia Biotech.

Cell Growth Assay.
The number of viable and dead cells at the indicated time points was determined by trypan blue exclusion. The CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega) was also used to assay cell growth. Cells (100 µl) from the culture were plated on 96-well plates and processed according to the manufacturer’s instructions. Triplicates of each sample were assayed.

Cell Cycle Analysis.
Cells were labeled with 10 µM BrdUrd for 2 h at 37°C and fixed in 70% ethanol overnight at 4°C. Fixed cells (1 x 106) were treated with 3 ml of 0.08% pepsin in 0.1 N HCl at 37°C for 20 min, followed by another 20 min at 37°C in 1.5 ml 2 N HCl. Single nuclei were first stained with a FITC-conjugated anti-BrdUrd antibody for 30 min in the dark on ice. After 30 min treatment at 37°C with RNase A, the nuclei were stained with propidium iodide for 15 min in the dark on ice. Cell cycle distribution was determined by flow cytometry analysis.

TUNEL Assay.
The in situ cell death detection kit TMR Red (Roche) was used according to the manufacturer’s instructions. Samples were analyzed by flow cytometry.

Determination of Inositol Phospholipid Levels.
The phospholipid assay was performed as described previously (66) . Briefly, cells were labeled with 400 µCi of 32P-Pi (Amersham) in phosphate-free RPMI (Life Technologies, Inc.) containing 10% dialyzed FCS for 2 h at 37°C. The cells were lysed in 1 M HCl containing 5 mM tetrabutyl ammonium hydrogen sulfate. The phospholipids were extracted with chloroform:methanol (1:2), deacylated in methylamine for 30 min at 53°C, and then resolved on an anion exchange column (Spherisorb S5SAX, Waters PSS832715) with an increasing gradient of NaH2PO4, pH 3.8 (66) . Fractions were collected and counted for 32P radioactivity. Phospholipid standards were also resolved to confirm the identity of the peaks.

In Vitro Akt Kinase Assay.
The kinase assay was performed as described previously (60) . Briefly, after PTEN induction for 24 h, cells were either stimulated with C305 for the indicated duration or left unstimulated. Lysate from 2 x 107 cells was subjected to immunoprecipitation using a rabbit anti-Akt antibody (#4; Ref. 60 ). The immune complex was assayed for Akt activity using the peptide substrate Crosstide.

Preparation of Cytosolic and Membrane Fractions.
Cells (1 x 108) were washed twice with cold PBS and resuspended in 5 ml of cold hypotonic lysis solution [20 mM HEPES (pH 7.4), 5 mM sodium PPi, 5 mM EGTA, 1 mM MgCl2, and a mixture of phosphatase and proteinase inhibitors] and incubated on ice for 30 min. The cells were disrupted by Dounce homogenization, followed by centrifugation at 100,000 x g for 45 min at 4°C. The supernatant was saved as the cytosolic fraction and the pellet was solubilized in 1 ml of solubilization buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 1% Triton X-100, and a mixture of phosphatase and proteinase inhibitors]. After 30 min on ice, the solubilized pellet was centrifuged at 100,000 x g for 45 min at 4°C. The supernatant was collected as the membrane fraction.


    Acknowledgments
 
We thank Dr. Thea Tlsty for help with cell cycle analysis, Drs. Charles Sawyers, H. Bujard, and Jeffery Leiden for reagents, and members of the Weiss lab for assistance.


    Footnotes
 
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.

1 Supported in part by the Rosalind Russell Medical Research Center for Arthritis and RO1-CA79548 (to D.S.). Back

2 To whom requests for reprints should be addressed, at Departments of Medicine and of Microbiology and Immunology and the Howard Hughes Medical Institute, University of California, San Francisco, 3rd and Parnassus Avenues, San Francisco, CA 94143-0795. Phone: (415) 476-1291; Fax: (415) 502-5081; E-mail: aweiss{at}medicine.ucsf.edu Back

3 The abbreviations used are: PTEN, phosphatase and tensin homologue deleted on chromosome 10; PtdIns(3,4)P2, phosphatidylinositol (3,4)-biphosphate; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate; PI3K, phosphoinositide 3-kinase; PH, pleckstrin homology; EGF, epidermal growth factor; WT, wild type; DOX, doxycycline; BrdUrd, bromodeoxyuridine; TUNEL, TdT-mediated dUTP nick end labeling; TCR, T-cell receptor; EGFP, enhanced green fluorescent protein. Back

Received for publication 2/15/02. Revision received 5/27/02. Accepted for publication 6/ 3/02.


    References
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 Results
 Discussion
 Materials and Methods
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