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Cell Growth & Differentiation Vol. 12, 371-378, July 2001
© 2001 American Association for Cancer Research

PTEN/MMAC1 Overexpression Decreases Insulin-like Growth Factor-I-mediated Protection from Apoptosis in Neuroblastoma Cells1

Cynthia M. van Golen, Tracy S. Schwab, Kathleen M. Woods Ignatoski, Stephen P. Ethier and Eva L. Feldman2

University of Michigan Department of Neurology, Neuroscience Program [C. M. v. G., T. S. S., E. L. F.], and Department of Radiation Oncology [K. M. W. I., S. P. E.], Ann Arbor, Michigan 48109


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Insulin-like growth factor I (IGF-I) protects cells from apoptosis primarily through the action of phosphatidylinositol-3 kinase and the downstream serine/threonine kinase Akt. The PTEN gene product, a protein which dephosphorylates phosphatidylinositol lipids, prevents activation of Akt and regulates several cellular functions, including cell cycle progression, cell migration, and survival from apoptosis. In this study, PTEN overexpression decreases IGF-I-induced Akt activity, enhances serum withdrawal-induced apoptosis, and decreases IGF-I protection and cell growth in SHEP cells. The PTEN lipid phosphatase mutant G129E fails to inhibit IGF-I-stimulated Akt activity and protection from apoptosis. The C124S mutation, which abolishes both lipid and protein phosphatase activity, fails to inhibit Akt activity and IGF-I protection against hyperosmotic-induced apoptosis but still inhibits growth and IGF-I protection against serum withdrawal-induced apoptosis. These data suggest a role for PTEN in modulating the effect of IGF-I on Akt activity, neuroblastoma cell growth, and protection against apoptotic stimuli.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The IGF3 system plays a role in several aspects of tumorigenesis, including growth, transformation, and survival from apoptosis (1) . The biological effects of the IGF ligands during tumor formation are mediated through two major signaling pathways downstream from IGF-IR, the MAPK pathway, and the PI-3K pathway (2) . Although both pathways are critical for IGF-mediated effects, the predominant pathway involved in protection from apoptosis is the PI-3K pathway (3 , 4) . PI-3K is a protein comprised of two subunits, a p85 regulatory subunit and a p110 catalytic subunit (2) . Upon activation of the two subunits through binding to the insulin receptor substrate, PI-3K phosphorylates phosphatidylinositide lipids at the 3 position in the inositol ring (2 , 5) . The multiphosphorylated forms of the phosphatidylinositides then activate downstream targets (5) .

One of the principal downstream effectors for PI-3K is the serine/threonine kinase Akt. Akt is activated either through direct binding to the lipid products of PI-3K (6 , 7) or through the intermediate action of the phosphoinositide-dependent kinases (8) . Akt is important for protection of several cell types against apoptosis (9, 10, 11) . One way in which antiapoptotic factors such as interleukin-3 and IGF-I protect cells from death is through Akt-mediated phosphorylation of the proapoptotic bcl-2 family member BAD (12 , 13) .

Recently, a dual phosphatase, PTEN (phosphatase and tensin homologue deleted on chromosome 10)/mutated in multiple advanced cancers, was found to dephosphorylate PIP3, acting as an antagonist of PI-3K (14) . PTEN is implicated in the regulation of several cellular functions, including cell cycle progression (15 , 16) , survival from apoptosis (17, 18, 19, 20) , and motility (21 , 22) . Whereas PTEN has protein phosphatase activity (23 , 24) , many of the biological effects of PTEN occur through its lipid phosphatase activity (14 , 25 , 26) . Through dephosphorylation of the phosphatidylinositol lipids, PTEN decreases Akt activity resulting in increased levels of apoptosis (26, 27, 28) . In contrast, mutation of PTEN is associated with increased levels of Akt (28 , 29) and increased cell survival (28) . Therefore, PTEN could negatively regulate IGF-mediated protection from apoptosis by decreasing Akt phosphorylation and activation.

Previously, we reported that IGF-I rescues SHEP human neuroblastoma cells from hyperosmotic-induced apoptosis (30) . In the Caenorhabditis elegans system, the PTEN homologue daf-18 can negatively regulate the insulin receptor-like and PI-3K signaling pathways, resulting in developmental abnormalities and a shortened life span (31, 32, 33, 34) . On the basis of the similarities between the downstream signaling of the insulin and IGF-I receptors (2) and the well-characterized role of Akt in IGF-I rescue of cells from apoptosis, we investigated the role of PTEN on IGF-I protection of SHEP neuroblastoma cells from hyperosmotic-induced apoptosis. We report that overexpression of PTEN decreases Akt activity, enhances hyperosmotic-induced apoptosis, decreases the protective effect of IGF-I, and decreases cell growth. Two mutant PTEN constructs, including a mutant with no lipid phosphatase activity, G129E, and a catalytically inactive mutant with neither lipid nor protein phosphatase activity, C124S, were transfected into SHEP cells. These two mutations fail to inhibit Akt activity and the protective effect of IGF-I but also do not restore cell growth. These studies suggest that IGF-I mediates SHEP cell survival through the PI-3K pathway and that PTEN, through its lipid phosphatase domains, can regulate this biological effect.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IGF-I Rescues SHEP Cells from Apoptosis via the PI-3K Pathway and Activates Akt.
The PI-3K pathway is the predominant pathway involved in IGF-I rescue of cells from apoptosis (3 , 4) . There is evidence, however, that the second major signaling pathway downstream from the IGF-IR, the MAPK pathway, may also play a role in IGF-I rescue of some cell types (35) . Therefore, we first investigated the effect of blocking the PI-3K pathway on IGF-I rescue of SHEP neuroblastoma cells from hyperosmotic-induced apoptosis. When SHEP cells are exposed to a hyperosmotic environment created by the addition of 300 mM mannitol to the media (DMEM), ~44% of the cells undergo apoptosis (Fig. 1A)Citation . The addition of WTM, a drug which blocks PI-3K activity (36) , does not affect the level of apoptosis, demonstrating that WTM is not toxic to these cells. When 10 nM IGF-I is added to the media, only 22% of the cells undergo apoptosis; IGF-I therefore rescues 50% of the cells from apoptosis, consistent with previous results (37 , 38) . In the presence of both IGF-I and WTM, however, 56% of cells undergo apoptosis, which is not statistically different from 300 mM mannitol alone (P < 0.01), indicating that WTM blocks the protective effect of IGF-I. When PD98059, an inhibitor of MAPK kinase, is added, IGF-I rescue is not prevented and is actually slightly enhanced, although not to statistically significant levels (Fig. 1B)Citation . Therefore, the PI-3K pathway is important for IGF-I rescue of SHEP cells from hyperosmotic-induced apoptosis.



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Fig. 1. IGF-I rescues SHEP cells from hyperosmotic-induced apoptosis via PI-3K. In A, serum-deprived SHEP cells are incubated with DMEM (D) ± 100 nM WTM or 300 mM mannitol (H) ± 10 nM IGF-I (I) ± 100 nM WTM. Cells exposed to conditions with WTM are pretreated with WTM for 1 h before the addition of experimental conditions. WTM is replenished every 6 h, and IGF-I is replenished every 12 h. After 24 h, cells are collected and prepared for flow cytometry. The percentage of apoptotic cells represents the percentage of DNA in the sub-G0 population as measured by propidium iodide staining for flow cytometry. In B, serum-deprived SHEP cells were exposed to DMEM (D) ± 10 µM PD98059 (PD) or 300 mM mannitol (H) ± 10 nM IGF-I (I) ± 10 µM PD. Cells exposed to PD are pretreated with the inhibitor for 1 h before the addition of experimental conditions. After 24 h, cells are prepared for flow cytometry. For both A and B, * = P < 0.01 compared with DMEM and ** = P < 0.01 compared with 300 mM mannitol.

 
One of the principal downstream effectors of the PI-3K pathway is Akt (39, 40, 41) . Akt is phosphorylated and activated downstream from PI-3K, primarily through phosphorylation of phosphatidylinositol 4,5-bisphosphate to PIP3 (42) . Akt expression and phosphorylation were measured in untransfected SHEP cells treated with DMEM alone or DMEM + 10 nM IGF-I for 0–60 min. Akt protein expression remains constant in both treatments over the 60-min period (Fig. 2, C and D)Citation . Akt phosphorylation on S473 is detected by 15 min in the presence of IGF-I and continues through 60 min, with peak phosphorylation occurring at 30 min (Fig. 2A)Citation . In the absence of IGF-I, little S473 phosphorylation is detected (Fig. 2B)Citation . Because peak Akt phosphorylation on S473 is seen at 30 min with IGF-I present, the remaining Akt studies were performed at this time point.



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Fig. 2. IGF-I induces phosphorylation of Akt on S473. Serum-deprived SHEP cells are exposed to DMEM (B and D) or DMEM + 10 nM IGF-I (A and C). After 0, 5, 15, 30, 45, and 60 min, cell lysates are collected and run on a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and Western immunoblotted for either P-S473-Akt (A and B) or Akt (C and D).

 
PTEN Overexpression and Mutation Affects Akt Phosphorylation and Activation.
PTEN dephosphorylates the lipid PIP3, which in turn decreases S473 phosphorylation and activation of the Akt kinase (14) . We therefore investigated Akt expression, phosphorylation, and kinase activity in several transfected SHEP cell lines: (a) a vector mock transfectant (SHEP/PTP); (b) wtPTEN (SHEP/wtPTEN); (c) a PTEN construct with a mutation in the catalytic domain abolishing all phosphatase activity (SHEP/C124S); and (d) a lipid phosphatase mutant (SHEP/G129E). The four transfected cell lines, SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E, were exposed to IGF-I for 30 min, and whole cell lysates were collected. Western blot analysis using a monoclonal antibody to PTEN confirms both low endogenous expression of PTEN and that transfection with the PTEN constructs increases PTEN protein expression over SHEP/PTP control cells (Fig. 3D)Citation . When serine phosphorylation on S473 of Akt is measured using an antiphospho-S473-Akt antibody, the SHEP/PTP cells exhibit a robust Akt-S473 phosphorylation with IGF-I treatment (Fig. 3A)Citation . Overexpression of wtPTEN completely abolishes the phosphorylation of Akt in SHEP cells. As expected, the catalytically inactive mutant PTEN/C124S and the lipid phosphatase mutant G129E show control levels of S473 Akt phosphorylation (Fig. 3A)Citation . Results of Akt activity studies using GSK-3 as an in vitro substrate parallel the phosphorylation studies, except that a small amount of GSK-3 phosphorylation is seen even in the presence of wtPTEN (Fig. 3B)Citation . Akt expression is equal among all of the cell lines, indicating no change in protein expression (Fig. 3C)Citation . Therefore, the changes in Akt phosphorylation and activation seen are not attributable to changes in Akt protein expression but are the result of post-translational modification. Taken together, these data demonstrate that PTEN overexpression prevents IGF-I-mediated Akt phosphorylation and activation, and mutations of the catalytic or lipid phosphatase domains of PTEN restore both Akt phosphorylation and activation.



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Fig. 3. PTEN overexpression and mutation regulate Akt phosphorylation on S473 and Akt activity. In A, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by Western analysis using a P-S473-Akt antibody. In B, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by an in vitro kinase assay using GSK-3 as a substrate and Western analysis performed using an antibody to P-GSK-3. In C, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by Western analysis for Akt. In D, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cell lysates are collected, followed by Western analysis for PTEN using a PTEN monoclonal antibody.

 
PTEN Overexpression Has No Effect on IGF-I-mediated Erk1/2 Phosphorylation.
IGF-I activates both the PI-3K pathway and the MAPK pathway upon activation of the IGF-IR (2) . Although the inhibitor data shown above indicate the importance of the PI-3K pathway in IGF-I-mediated rescue of neuroblastoma cells from apoptosis, signaling cross-talk between the PI-3K and MAPK pathways has been reported. Therefore, to investigate the possible effect of PTEN expression on MAPK activation, SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells were exposed to either DMEM alone or DMEM supplemented with IGF-I for 30 min, and whole cell lysates were collected. Western blot analysis for the phosphorylated form of Erk1/2, the most downstream kinases in the MAPK cascade, revealed no change in either Erk1/2 phosphorylation (Fig. 4A)Citation or Erk1/2 protein expression (Fig. 4B)Citation with overexpression of wtPTEN or C124S/PTEN. There was a very slight decrease in ERK1/2 phosphorylation in the SHEP/G129E cells, indicating a potential effect of increased PI-3K activation on MAPK activation.



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Fig. 4. PTEN overexpression has no effect on Erk1/2 phosphorylation or expression. In A, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by Western analysis using a P-Erk1/2 antibody. In B, serum-deprived SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to 10 nM IGF-I for 30 min, followed by Western analysis for Erk1/2.

 
PTEN Overexpression and Mutation Affect IGF-I-mediated Survival from Apoptosis.
Survival signaling by several growth factors, including platelet-derived growth factor, brain-derived neurotrophic factor, and IGF-I, is mediated through the PI-3K/Akt pathway (41 , 43 , 44) . Given the effect of PTEN overexpression and mutation on Akt activity levels, we next investigated the effect of PTEN overexpression and mutation on apoptosis and IGF-I rescue. SHEP/PTP, SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells are exposed to DMEM, DMEM + 10 nM IGF-I, 300 mM mannitol, or 300 mM mannitol + 10 nM IGF-I for 24 h and then analyzed by flow cytometry. In serum withdrawal conditions, 37% of SHEP/PTP cells undergo apoptosis (Fig. 5A)Citation . When the cells are exposed to a hyperosmotic environment (300 mM mannitol) in the media, the percentage of apoptotic cells increases to 72%. IGF-I rescues ~50% of the cells in both conditions. In the SHEP/wtPTEN cells, the number of apoptotic cells in serum withdrawal conditions increases to 56% (Fig. 5B)Citation . In hyperosmotic conditions, 79% of SHEP/PTEN cells are apoptotic. Thus, overexpression of wtPTEN significantly increases the amount of apoptosis induced by serum withdrawal but only slightly increases the number of apoptotic cells in hyperosmotic conditions (Fig. 5B)Citation . In PTEN overexpressing cells, IGF-I fails to protect cells from apoptotic death induced by either serum withdrawal or hyperosmotic conditions (Fig. 5B)Citation . In the C124S mutants, in which the catalytic domain of PTEN is mutated, the number of cells undergoing apoptosis induced by either apoptotic stimulus is essentially the same as SHEP/PTP vector controls, with 39% of cells undergoing serum withdrawal apoptosis and 72% of cells undergoing hyperosmotic-induced apoptosis (Fig. 5C)Citation . IGF-I protects a significant number of SHEP/C124S cells from hyperosmotic-induced apoptosis, yet the number of apoptotic SHEP/C124S cells in serum withdrawal conditions remains the same even in the presence of IGF-I. The lipid phosphatase mutant SHEP/G129E (Fig. 5D)Citation cells show a significant reduction in the percentage of apoptotic cells produced by either serum withdrawal or hyperosmotic stress. As shown in control cells, IGF-I rescues the G129E cells from both hyperosmotic-induced and serum withdrawal-induced apoptosis (Fig. 5E)Citation . In summary: (a) in hyperosmotic conditions, IGF-I can rescue SHEP cells containing any of the PTEN mutants, suggesting a requirement for lipid phosphatase activity for PTEN to be effective in these conditions; and (b) in serum withdrawal conditions, lipid phosphatase activity of PTEN negatively affects IGF-I rescue, but the protein phosphatase activity may be necessary for IGF-I-mediated protection (Table 1)Citation .



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Fig. 5. In A–D, PTEN overexpression decreases the protective effect of IGF-I. Serum-deprived SHEP/PTP (A), SHEP/wtPTEN (B), SHEP/C124S (C), and SHEP/G129E (D) cells are exposed to DMEM (D) + 10 nM IGF-I (I) or 300 mM mannitol (H) + 10 nM IGF-I for 24 h, followed by flow cytometry analysis as described in Fig. 1Citation . * = P < 0.01 compared with SHEP/PTP cells. ** = P < 0.01 compared with the corresponding condition without IGF-I.

 

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Table 1 Summary of PTEN mutation effects on apoptosis and growth in SHEP neuroblastoma cells

 
PTEN Inhibits Neuroblastoma Cell Growth.
PTEN overexpression can induce cell cycle arrest in several cell types (15 , 16 , 45) , thus preventing an increase in cell number in otherwise normal growth conditions. We therefore investigated the effect of PTEN overexpression on cell growth using the MTT assay. SHEP/PTP cells show an expected increase in growth in normal serum-containing media over the 3-day time course (Fig. 6)Citation . SHEP/wtPTEN, SHEP/C124S, and SHEP/G129E cells show very little increase in cell number over the 3-day period, resulting in fewer cells than control conditions at days 2 and 3. These data suggest that in the presence of growth media, PTEN does not induce apoptosis but does slow growth in SHEP cells. PTEN has this effect regardless of the mutation status of the cells, suggesting that phosphatase activity is not necessary to inhibit normal growth in SHEP neuroblastoma cells.



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Fig. 6. PTEN overexpression decreases neuroblastoma cell growth. Cells (20,000) were plated onto 96-well plates and grown for 3 days. MTT and lysis buffer were added to each well and measured using a plate reader at 570 nm. Results are represented as the mean absorbance ± SE for each day.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
IGF-IR activation can signal through the PI-3K pathway to regulate survival from apoptosis (3 , 4) . In the current study, the two major signaling pathways, PI-3K and MAPK, were inhibited pharmacologically downstream from the IGF-IR in SHEP cells to confirm that the PI-3K pathway was responsible for IGF-I-mediated protection. Blocking PI-3K with WTM prevents IGF-I protection of neuroblastoma cells from hyperosmotic-induced apoptosis, whereas blocking MAPK kinase using PD98059 does not. In support of our results, rat pheochromocytoma (PC-12) cells are protected from apoptosis by IGF-I through the PI-3K pathway (46) , and PI-3K is required for both nerve growth factor and platelet-derived growth factor-mediated survival of these same cells (47) .

PI-3K activates several downstream effectors, including Akt. The role of Akt in PI-3K-mediated survival signaling is now well known (10) . IGF-I protects cerebellar neurons from potassium withdrawal-induced apoptosis through PI-3K activation of Akt (9) . In parallel, epidermal growth factor survival signaling in epithelial cells requires Akt (48) , and PI-3K and Akt inhibit c-myc-induced apoptosis in fibroblasts (49) .

Our results support a role for Akt in IGF-I-mediated survival of neuroblastoma cells. Akt is phosphorylated within 15 min of IGF-I stimulation and is sustained for 60 min. PTEN decreases Akt kinase activity by dephosphorylating the lipid PIP3, which in turn decreases S473 Akt phosphorylation (14) . Because PTEN activity prevents Akt activation in a variety of cell types (50) , we speculated that overexpression of PTEN would decrease Akt activation in neuroblastoma cells. As predicted, in the current study, PTEN overexpression completely prevented IGF-I-mediated Akt phosphorylation in neuroblastoma cells.

To better understand the role of PTEN in neuroblastoma survival, the effects of PTEN mutations were assessed in SHEP neuroblastoma cells. The C124S mutation, which abolishes both PTEN protein and lipid phosphatase activity, and the G129E mutation, which prevents only PTEN lipid phosphatase activity, fail to prevent Akt activation. Akt protein expression remains unchanged in all of the PTEN cell lines, suggesting that the effect of PTEN on Akt activation over this time course results from a decrease in Akt phosphorylation rather than a decrease in Akt expression.

Given the ability of PTEN to block PI-3K-mediated Akt activation and the role of Akt in IGF-I-mediated survival, we investigated the effects of PTEN on apoptosis and IGF-I rescue of neuroblastoma cells. PTEN overexpression increases serum withdrawal-induced apoptosis by 20% in SHEP cells; however, PTEN overexpression does not enhance hyperosmotic-induced apoptosis. Osmotic shock maintains Akt in a dephosphorylated state (51) . Therefore, PTEN overexpression may not produce an additional effect on hyperosmotic-induced apoptosis because mannitol may already cause the dephosphorylation of Akt. In contrast, serum withdrawal-induced apoptosis may be increased by PTEN overexpression because of a decrease in basal Akt phosphorylation. IGF-I could not significantly prevent apoptosis in SHEP/wtPTEN cells induced by either apoptotic stimulus. Because the PI-3K pathway is necessary for IGF-I-mediated survival in SHEP cells, potentially through the phosphorylation of Akt, it is expected that the continuous presence of PTEN, and therefore continuous dephosphorylation of Akt, impedes the protective effect of IGF-I. Taken together, these data support the hypothesis that PTEN overexpression decreases IGF-I-mediated survival and increases serum withdrawal-induced apoptosis through its effect on Akt activation.

Previously published reports support our finding that PTEN interferes with Akt-mediated survival. Fibroblasts taken from PTEN-deficient mouse embryos show increased survival in the presence of apoptotic stimuli such as UV irradiation, incubation with tumor necrosis factor, or sorbitol exposure (17) . When PTEN is transfected into NIH 3T3 cells, the cells undergo apoptosis, whereas cotransfection with Akt prevents this apoptosis (17) . PTEN also induces apoptosis in breast cancer cell lines, and Akt or bcl-2 transfection rescues these cells (18) . Finally, LNCaP prostate cancer cells, which do not normally express functional PTEN protein, show increased apoptosis and decreased growth when transfected with PTEN (19) .

Akt can affect the survival of cells from apoptosis through several downstream targets. One way in which growth factors such as IGF-I rescue cells in an Akt-dependent manner is through the phosphorylation of the proapoptotic bcl-2 family member BAD (12 , 13) . Upon phosphorylation, BAD is sequestered in the cytoplasm by the protein 14-3-3, and the antiapoptotic bcl proteins can then exert their protective effect (12 , 13) . Therefore, PTEN could affect apoptotic signaling by reversing this process. Recent evidence also suggests that Akt promotes survival by inhibiting several members of the Forkhead family of transcription factors through phosphorylation (52) . PTEN inhibition of Akt could prevent this phosphorylation, allowing Forkhead transcription factors to transcribe apoptosis-related genes, such as Fas ligand.

To understand how the lipid and protein phosphatase activity of PTEN reduces IGF-I protection, we used two PTEN mutant cell lines: the C124S and the G129E mutations. The G129E mutation lowers apoptosis induced by serum withdrawal and hyperosmotic shock to below control levels. This mutant construct has the ability to act as dominant negative construct, preventing the action of endogenous PTEN present in SHEP cells. This likely explains the low amount of apoptosis in the SHEP/G129E cells below that in the control cells. This mutation also demonstrates IGF-I-mediated survival better than control levels. These data suggest that lipid phosphatase activity of PTEN is crucial for producing an antiapoptotic effect in SHEP neuroblastoma cells. The C124S mutation, which prevents all PTEN phosphatase activity yet allows PTEN binding to lipid substrates, reestablishes serum withdrawal- and hyperosmotic-induced apoptosis to control levels and restores IGF-I-mediated survival of neuroblastoma cells from hyperosmotic-induced apoptosis. However, IGF-I rescue of these cells from serum withdrawal-induced apoptosis is still prevented. Also, although IGF-I rescues >50% of the cells from hyperosmotic-induced cell death, the remaining percentage of apoptotic cells equals the percentage of cells that undergoes serum withdrawal-induced death. Therefore, IGF-I cannot rescue SHEP cells from serum withdrawal-induced death when both protein and lipid phosphatase activity of PTEN is inactivated. Because Akt activity is high in these cells, we cannot attribute this effect of the C124S mutation of PTEN to its effect on Akt. This could indicate a need for PTEN protein phosphatase activity to allow IGF-I rescue of SHEP cells from serum withdrawal-induced apoptosis. Another possibility is that in serum-free conditions, multiphosphorylated phosphoinositides are bound by the C124S mutant PTEN and sequestered, preventing these second messengers from having an effect.

In summary, overexpression of PTEN significantly inhibits IGF-I-mediated survival from apoptosis and decreases cell growth. IGF-I protection of SHEP cells requires intact lipid phosphatase activity of PTEN. The protein phosphatase activity of PTEN is required for IGF-I-mediated rescue from serum withdrawal-induced cell death. These data support a PI-3K-mediated pathway for the protection of SHEP cells from apoptosis.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Materials.
Tissue culture plastic was purchased from Corning (Corning, NY). DMEM, HBSS, and trypsin-EDTA were purchased from Life Technologies, Inc. (Gaithersburg, MD). CS was purchased from HyClone (Logan, UT). IGF-I was provided by Cephalon, Inc. (West Chester, PA) and stored in 10 mM acetic acid at -80°C. Polyclonal antibodies to phospho-S473-Akt, Akt, phospho-Erk1/2, and Erk 1/2 were purchased from New England Biolabs (Beverly, MA). The PTEN monoclonal antibody and all secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.

Cell Culture.
Construction of the wtPTEN, C124S, and G129E mutants has been described previously (15 , 25) and were kindly provided by Dr. Jack Dixon and Tomohiko Maehama, Department of Biological Chemistry, University of Michigan. For retroviral PTEN constructs PTP/wtPTEN, PTP/C124S, and PTP/G129E, FLAG-tagged PTEN genes were excised from FLAG/PTEN/CMV5 (14) with EcoRI and BamHI. The ends of each PTEN gene were blunted and ligated into PTP2000. DNA (5 µg) were transfected into {Phi}NX-A cells by calcium phosphate precipitation. Forty-eight h after transfection, conditioned medium was collected and pelleted, and the supernatant was passed through a 0.45-µm syringe filter, before storage at -80°C. Viral supernatants were thawed, and SHEP cells were infected in the presence of Polybrene for 24 h before selection in 500 µg/ml gentamicin (G418) for 2 weeks.

SHEP cells were grown in DMEM + 10% CS at 37°C in a humidified atmosphere with 10% CO2. SHEP/PTP, SHEP/PTEN, SHEP/C124S, and SHEP/G129E cells were grown in DMEM + 10% CS + 250 µg/ml G418 (Life Technologies, Inc.). All cell lines were routinely subcultured by rinsing with HBSS, trypsinizing, and replating.

Flow Cytometry.
Quantitative measurements of apoptosis were performed using analysis of DNA content by flow cytometry (30 , 37) . Cells were plated in six-well tissue culture plates and grown to near confluence. After 4 h of serum deprivation, cells were incubated in experimental conditions and collected using trypsinization at the indicated times. After collection, cells were rinsed in HBSS, fixed in ice-cold 70% ethanol, and stored at 4°C. Cells were stained for 2–12 h with 18 µg/ml PI containing 40 µg/ml RNase A at 4°C, then analyzed using an Epics Elite flow cytometry system (Coulter Cytometry, Hialeah, FL). DNA content of the PI-stained cells was measured and separated into phases of the cell cycle on the basis of PI fluorescence. The percentage of apoptotic cells was taken as %-sub-G0 DNA, and results were expressed as the mean of three separate experiments ± the SE.

Western Immunoblotting.
Western blot analyses were performed as described previously (53) . Briefly, whole cell lysates were collected using radioimmunoprecipitation assay buffer [20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, aprotinin, leupeptin, phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate]. Protein (70 µg) was run on a SDS-PAGE gel and transferred to nitrocellulose membranes, and Western immunoblotting was performed. Blots were developed using ECL (Amersham, Piscataway, NJ) and exposed to film (Hyperfilm-ECL; Amersham). Blots shown are representative of three independent experiments.

Akt Activity Assay.
Akt kinase assays were purchased from New England Biolabs and performed according to kit instructions. Briefly, whole cell lysates were collected using lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na PPi, 1 mM ß-glycerophosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride] and incubated with Akt antibody conjugated to agarose beads. The immunoprecipitates were rinsed with kinase buffer [25 mM Tris (pH 7.5), 5 mM ß-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, and 10 mM MgCl2], incubated with GSK-3 substrate and ATP for 30 min at 30°C, then run on a 12.5% SDS-PAGE gel. After transfer to nitrocellulose, Western immunoblotting was performed using an antiphospho-GSK-3 antibody. Blots were developed using ECL and exposed to film. Blots shown are representative one of three independent experiments.

MTT Assay.
Cells were trypsinized and plated at a cell density of 20,000 cells/well (100 µl final volume) in sterile 96-well plates. Twenty-five µl of 5 mg/ml MTT in sterile 1 x PBS (pH 7.4) was added to each well and incubated for 2 h in a 37°C humidified incubator at days 1–3. One-hundred µl of lysis buffer [0.5 x N,N-dimethyl formamide and 20% SDS (pH 7.4)] was added to each well and incubated at 37°C overnight. Each plate was read the following morning using a Bio-Rad model 2550 EIA plate reader at 570 nm using wells containing media only as the blank.


    Acknowledgments
 
We thank Judy Boldt for expert secretarial assistance; Drs. James Russell, Catherine Delaney, and Kenneth van Golen for helpful discussions; and Dr. Hsin-Lin Cheng for figure preparation. We also thank Dr. Jack Dixon and Tomohiko Maehama for providing the PTEN constructs.


    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 by National Cancer Institute Cancer Biology Training Program Grant 3 T32 CA09676 (to C. M. v. G.), NIH CA70354 (to K. M. W. I. and S. P. E.), and NIH RO1 NS36778 and NIH RO1 NS38849 (to E. L. F.). Back

2 To whom requests for reprints should be addressed, at University of Michigan, Department of Neurology, 4414 Kresge III, 200 Zina Pitcher Place, Ann Arbor, MI 48109. Phone: (734) 763-7274; Fax: (734) 763-7275; E-mail: efeldman{at}umich.edu Back

3 The abbreviations used are: IGF, insulin-like growth factor; IGF-IR, type I IGF receptor; MAPK, mitogen-activated protein kinase; PI-3K, phosphatidylinositol-3 kinase; PIP3, phosphatidylinositol 3,4,5-triphosphate; WTM, Wortmannin; wtPTEN, wild-type PTEN; GSK, glycogen synthase kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CS, calf serum; PI, propidium iodide; ECL, enhanced chemiluminescence. Back

Received for publication 3/ 9/01. Revision received 5/ 7/01. Accepted for publication 5/16/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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