Human Molecular Genetics, 2001, Vol. 10, No. 3 237-242
© 2001 Oxford University Press
PTEN induces apoptosis and cell cycle arrest through phosphoinositol-3-kinase/Akt-dependent and -independent pathways
1Clinical Cancer Genetics and Human Cancer Genetics Programs, Comprehensive Cancer Center, and Division of Human Genetics, Department of Internal Medicine, The Ohio State University, Columbus, OH 43210, USA and 2Cancer Research Campaign Human Cancer Genetics Research Group, University of Cambridge, Cambridge CB2 2QQ, UK
Received 13 October 2000; Revised and Accepted 23 November 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The tumour suppressor PTEN inhibits cell growth through multiple mechanisms. We have previously demonstrated that overexpression of PTEN in MCF-7 breast cancer cells causes G1 arrest followed by cell death, the latter of which is believed to be mediated by the phosphoinositol-3-kinase (PI3K) and Akt/PKB pro-apoptotic pathways. In this present study, we show that culture in the presence of low levels of growth factors increased PTEN-mediated growth suppression through the enhancement of PTEN-induced cell death. The caspase 9-specific inhibitor, ZVAD, blocked PTEN-induced cell death without altering the effect of PTEN on cell cycle distribution. Depending on the level of expression, overexpression of dominant-negative Akt induces more cell death and has less effect on the cell cycle or induces similar or decreased cell death without affecting the cell cycle compared with effects on cell death and the cell cycle when overexpressing PTEN. These observations in sum suggest that, in MCF-7 breast cancer cells, the apoptotic cells induced by the overexpression of PTEN did not derive from the G1-arrested cells. Further, the effect of PTEN on cell death is mediated through the PI3K/Akt pathway whereas PTEN-mediated cell cycle arrests are through PI3K/Akt-dependent and -independent pathways.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The tumour suppressor gene PTEN/MMAC1/TEP1 (1–3) has been implicated in a variety of human cancers and three inherited harmatoma tumour syndromes, Cowden syndrome, which has a high risk of breast, thyroid and other cancers (4–6), Bannayan–Zonana syndrome (7,8) and a Proteus-like syndrome (9). Overexpression of PTEN suppresses tumourigenicity and cell growth (10,11).
Early studies have suggested a tissue-specific effect of PTEN. It was observed that overexpression of PTEN in tumour cell lines results in either cell cycle arrest in glioma cell lines (11–13) or cell death in breast cancer lines (14). This raised the possibility that the tumour-suppressive effect of PTEN is dependent on other signaling pathways that are regulated in a tissue-specific manner. However, recent studies have revealed that PTEN induction of cell cycle arrest and apoptosis are not necessarily mutually exclusive in a single cell type: overexpression of PTEN can lead to both cell death and cell cycle arrest in the same cell line derived from breast cancer (15,16), glioma (17) and prostate cancer (18).
Cell growth requires both proliferation signals and survival signals. Many oncogenes and tumour suppressor genes are implicated in the regulation of both the cell cycle and cell death (19,20). Removal of growth factor leads to cell cycle arrest and subsequent cell death in many cells (21–27). The phosphoinositol-3-kinase (PI3K) pathway, the well known downstream target pathway of PTEN (28), plays an essential role in transmitting signals from growth factors to cell death and cell cycle machineries (29). In MCF-7 breast cancer cells, apoptosis induced by overexpression of PTEN occurs much later than cell cycle arrest (15) or requires serum-free conditions in which cells are already arrested at G1 phase (16). It is also noticed that in prostate cancer, cell death only can be detected 4 days after PTEN infection using a viral delivery system (18). The plausible model for the explanation of those observations would be that PTEN exerts its tumour-suppressive effect initially through the inhibition of cell cycle progression and the induction of apoptosis as a consequence, depending on the level of survival signals. But, so far, no clear evidence exists for a straightforward link between PTEN-mediated cell cycle arrest and apoptosis as a consequence. Although cell death and the cell cycle are tightly linked, they are controlled by distinct cellular machineries. On the one hand, apoptosis can occur independently of cell cycle arrest under certain circumstances. For instance, p53 can induce apoptosis but not G1 arrest in cells from p21CIP1/WAF1-deficient mice (30,31). Further, it has been noted that apoptotic cells can derive from cells in any cell cycle phase (32). On the other hand, G1 arrest is not always a necessary precedent of increased cell death: pRB-deficient SOAS cells transfected with pRB undergo G1 arrest and are protected from apoptosis after irradiation (33). Thus, viewing PTEN-induced apoptosis as a consequence of G1 arrest is probably too simplistic. Besides the well documentated lipid phosphatase activity, PTEN can also dephosphorylate protein substrates (34,35). It is conceivable that PTEN may target several downstream pathways including the PI3K/Akt pathway and others, each of which can regulate the cell cycle or cell survival individually or coordinately. In this report, we provide evidence that PTEN induces apoptosis through blocking the PI3K/Akt pathway and coordinates cell cycle arrest through PI3K/Akt-dependent and -independent pathways
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RESULTS |
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Low growth factor culture enhances PTEN-mediated growth suppression
To determine whether the intensity of available growth factors in culture medium could influence the growth inhibitory function of PTEN, we examined the effect of PTEN on cell growth in both regular culture medium [Dulbecco’s modified Eagle’s medium (DMEM)/10% fetal bovine serum (FBS)] (Fig. 1, Reg.S) and low growth factor medium (Fig. 1, Ch.S). For achieving low growth factor culture conditions, cells were grown for 3 days in the presence of tetracycline in oestrogen-free medium composed of MEBM/phenol red-free (36), a MCDB170-based medium for human breast epithelial cells (37) plus 10% charcoal/dextran-treated FBS (38). Subsequently, cells were grown in the same medium without tetracycline to induce PTEN expression or with tetracycline to serve as a control. As Figure 1 shows, induction of PTEN expression in regular medium (Fig. 1, WT/Reg.S) resulted in <10% growth inhibition at 48 h and 30% growth inhibition at 96 h. Low growth factor culture conditions greatly enhanced the growth-suppressive effect of PTEN: the growth inhibition can be observed as early as 36 h and achieves 20 and 70% growth inhibition at 48 and 96 h, respectively (Fig. 1, WT/Ch.S), levels of inhibition over twice those of regular medium. The C124S mutant promoted cell growth, acting as a dominant-negative form (Fig. 1, CS/Ch.S).
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Low growth factor culture conditions enhance PTEN-induced cell death
We have previously reported that overexpression of PTEN in MCF-7 breast cancer cells suppresses cell growth through the combination of G1 arrest and apoptosis (15). To investigate whether the enhanced growth suppression observed in low growth factor culture was due to the increase in cell death or to cell cycle inhibition, or both, we compared the effect of PTEN on cell survival and the cell cycle under low growth factor culture conditions with that under regular culture conditions (Fig. 2A). Induction of PTEN expression in regular culture medium (Fig. 2A, RC) resulted in 15% more cell death at 96 h but no increase in cell death at 48 h, consistent with our previous report (15). In contrast, under low growth factor culture conditions, overexpression of PTEN led to 10–15% cell death at 48 h and 40–45% cell death at 96 h (Fig. 2A, LGFC/T– versus LGFC/T+). Overexpression of PTEN under low growth factor culture conditions resulted in an overall increase in the G1 population (Fig. 2B, LGFC/T– versus LGFC/T+), but did not alter the effect of PTEN on the G1 population, suggesting that the low growth factor culture enhances the growth-suppressive effect but mainly through the increase in cell death and not G1 arrest.
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Caspase 9 inhibitor ZVAD blocks PTEN-induced cell death without altering the effect of PTEN on the cell cycle
Activation of caspases are the common distal events of apoptosis. To see whether caspase 9 is involved in PTEN-induced cell death in MCF-7 cancer cells, we examined the ability of ZVAD, the caspase 9-specific inhibitor, to block PTEN-induced cell death. Induction of PTEN expression for 48 h in the presence of ZVAD led to a 15% increase in cell number (Fig. 3A, ZVAD versus DMSO), consistent with the number of dead cells detected in the absence of ZVAD (Fig. 2A). No significant increase in dead cells can be detected when inducing PTEN in the presence of ZVAD (data not shown). Having shown that ZVAD can block PTEN-induced cell death, we next examined whether ZVAD had any effect on PTEN-mediated G1 arrest (Fig. 3B). Overexpression of PTEN resulted in an increase in the G1 population (Fig. 3B, T–/DMSO versus T+/DMSO), consistent with previous observations (39). Overexpression of PTEN in the presence of ZVAD revealed an identical cell cycle distribution to that in the absence of ZVAD (Fig. 3B, T–/ZVAD versus T–/DMSO), suggesting that dead cells resulting from PTEN-induced apoptosis did not derive from cells that were arrested in the G1 phase.
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The effect of dominant-negative Akt on the cell cycle and cell death
It is well documented that PTEN can block Akt activation through dephosphorylation of phosphoinositide-3,4,5-triphosphate (PIP3), the product of PI3K, which is required for the translocation of Akt to the cell membrane where Akt is phosphorylated and activated by upstream kinases. To investigate the role of the inactivation of Akt in PTEN-mediated G1 arrest and apoptosis, we evaluated the relative contribution of Akt activity to cell cycle progression and cell survival by reducing the Akt activity in MCF-7 cells and comparing cell cycle distribution with cell survival between the cells overexpressing PTEN and those with low Akt activity. MCF-7 cells with reduced Akt activity were generated by stable expression of kinase-inactive mutant Akt. Three clones (MCF-7/m-Akt1, -3 and -5) that expressed various levels of exogenous Akt, which migrates more slowly than endogenous Akt due to myrisolation (Fig. 4A, top), were chosen for further studies. Overexpression of PTEN under low growth factor culture conditions increased cell death from 4% (Fig. 4B, control) to 18.5% (Fig. 4B, PTEN) and reduced the S/G1 ratio from 0.425 (Fig. 4C, control) to 0.215 (Fig. 4C, PTEN). Under these same culture conditions, depending on the level of dominant- negative Akt expression, overexpression of each of the three kinase-dead myrisolated Akt constructs resulted in one of three outcomes compared with PTEN overexpression alone: an identical percentage of cell death with an increased S:G1 ratio, i.e. no cell cycle inhibition (Fig. 4B and C, M-Akt1 versus PTEN); less cell death without cell cycle inhibition (Fig. 4B and C, M-Akt5 versus PTEN); or more cell death but a lesser effect on the cell cycle (Fig. 4B and C, M-Akt3 versus PTEN). These observations suggest that reduction of Akt activity has profound effects on cell survival, and these effects appear to be tightly titrated. However, reduction of Akt activity seems to have little effect on the cell cycle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Numerous studies have demonstrated that PTEN suppresses cell growth through the negative regulation of the cell cycle and cell survival. It has been postulated that PTEN blocks the PI3K/Akt pathway, which leads to G1 arrest and cell death sequentially, with cell death dependent on cell cycle arrest, based on the following observations (28). First, PTEN can dephosphorylate PIP3, the product of PI3K, which is required for activation of Akt, the well known cell survival factor. Second, overexpression of PTEN leads to the up-regulation of p27, the negative regulator of cyclin-dependent kinases. The effects of PTEN on the level of p27 appear to be dependent on the inhibition of the PI3K/Akt pathway (13,16). Third, overexpression of PTEN in several cancer cell lines results in G1 arrest early and cell death later or in cell death in low growth factor culture conditions (15,16,18). These initial insights into the downstream effectors of PTEN, apoptosis and G1 arrest, seemed to explain the protean manifestations of Cowden syndrome, Bannayan–Riley–Ruvalcaba syndrome and other PTEN hamartoma tumour syndromes (PHTSs) (5,8,9). The hallmark phenotype of PHTSs is hamartomas affecting derivatives of all three germ cell layers: harmatomas and malignancy are the direct results of PTEN dysfunction and, presumably, via these downstream effectors. What cannot be explained to date, whether by genetic analysis or by functional studies, is why malignancies in PHTSs develop predominantly in certain organs, namely, the breast, thyroid and endometrium, and not others.
Cell death and the cell cycle are tightly coupled but distinctive processes (20,40). Apoptosis is often accompanied by growth arrest, and checkpoint proteins have been shown to be involved in the apoptotic response. However, cell cycle arrest does not always necessarily lead to cell death and cell death can be induced without the onset of cell cycle arrest (20). Many tumour suppressor genes and oncogenes are implicated in the regulation of both the cell cycle and cell survival (41,42). In general, apoptosis occurs when the timing and order of cell cycle events are out of control or the damage of DNA is incapable of repair to eliminate the abnormal cells (40). But the circumstances in which an effect on one without affecting the other does exist. It would, therefore, appear that the final outcome depends on the status of other oncogenic factors as well as environmental conditions.
Two lines of evidence from this study suggest that the G1 arrest and cell death induced by the overexpression of PTEN is differentially regulated, at least in the MCF-7 breast cancer model. First, if the induction of cell death by PTEN is solely the consequence of G1 arrest, one would expect that blocking PTEN-induced cell death should result in an increase in the population of cells in the G1 phase. This is not the case, because our data strongly suggest that the apoptotic cells resulting from overexpression of PTEN in the MCF-7 breast cancer cells derive from cells distributed randomly throughout the cell cycle. Second, overexpression of dominant-negative Akt at high levels induces more cell death but had little effect on the cell cycle compared with the effects of PTEN expression alone. Overexpression of dominant-negative Akt at the levels that induce cell death identical to or less than that of PTEN had no effect on the cell cycle, suggesting that inactivation of Akt cannot fully account for the G1 arrest mediated by PTEN. We have found that overexpression of PTEN leads to down-regulation of the cyclin D1 level, which is probably mediated by blocking MAPK phosphorylation by its protein phosphatase activity (L.P. Weng and C. Eng, unpublished data). Therefore, it is likely that PTEN induces cell death by blocking the PI3K/Akt signalling pathway and coordinates G1 arrest by up-regulating p27 and down-regulating cyclin D1 through PI3K/Akt-dependent and -independent pathways, respectively.
Our data strongly suggest that PTEN-mediated G1 arrest can be achieved via both PI3K/Akt-dependent and -independent pathways, at least in the MCF-7 breast cancer model. MCF-7 was chosen as a model because it is a very well characterized breast cancer cell line and breast cancer is a major component cancer of Cowden syndrome. Further, when using mutant clones (e.g. C124S-PTEN mutant), this line mimics the Cowden syndrome and sporadic breast cancer situation more closely with regard to PTEN structure: the great majority of these tumours have at least one intact PTEN allele. Even in Cowden tumours, loss of the remaining allele is not prominent (43). Nonetheless, we suspect that our observations in this paper might be more universal. Another hypothesis would be cell type-specific dependency of PI3K/Akt for mediating PTEN-associated G1 arrest. The challenge, therefore, remains to determine the precise mechanisms involved for this dependency and to determine what other specific pathways are involved in PI3K/Akt-independent PTEN-mediated G1 arrest.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture
The MCF-7 breast cancer cells expressing wild-type and the phosphatase-dead mutant C124S-PTEN were generated as described previously (15). This mutant was chosen because several Cowden syndrome/Bannayan–Riley–Ruvalcaba syndrome patients and primary sporadic tumours harbour missense mutations at C124, a key residue in the phosphatase core motif (8,44). Further, C124S has been shown to be phosphatase dead (34). The stable MCF-7 clones expressing a kinase-inactive mutant Akt, MCF-7/m-Akt cells, was generated by transfecting MCF-7 cells with pLNCX-m-Akt that carries Akt with lysine 179 converted to methionine (45) and GAG sequences that can be myrisolated and confer on the Akt protein the ability to be translocated to the cell membrane. Transfections were done using lipofectamine (Gibco BRL) and selected by hygromycin (Sigma) at 400 µg/ml for 2 weeks. Cells were maintained in DMEM/10% FBS (Gibco BRL Life Technologies) with 100 U/ml penicillin G (Sigma), 100 µg/ml streptomycin sulfate (Sigma similar medium plus 100 mg/ml Geneticin and 1 µg/ml tetracycline). m-Akt clones were maintained in the same medium plus 1 µg/ml insulin.
Induction of PTEN expression
Subconfluent stock cells were washed twice with phosphate-buffered saline (PBS), trypsinized by phenol red-free typsin–EDTA (Gibco BRL Life Technologies) and diluted at a 1:3 ratio into either oestrogen-free culture media, which contain MEBM (phenol red-free; Clonetech), 5% charcoal/dextran-treated FBS (HyClone) or regular DMEM (Gibco BRL) with 5% regular FBS for 3 days. Tetracycline (1 µg/ml) was included. Then equal numbers of cells were plated into the same medium without tetracycline to induce PTEN expression and into medium containing 1 µg/ml tetracycline (Tet+) as a control and cultured for 48 h, unless otherwise noted.
Cell growth assay
Cell growth was measured by methylene blue. Equal numbers of cells were plated into Tet+ and Tet– media in 12-well plates and cultured for 48 h. After incubation, medium was removed and cells were washed with PBS and fixed with 12.5% glutaraldehyde (Fisher) for 20 min at room temperature. Cells were rinsed with distilled water and incubated with 0.05% methylene blue (Sigma) for 30 min, again rinsed with water and then incubated with 800 µl of 0.33 M HCl for 30 min to extract the methylene blue. Absorption was measured at 595 nm. The ratio of the absorption in Tet– cultures to the absorption in Tet+ cultures at each time point was calculated and presented as percentage of cell growth.
FACS analysis
At the end of incubation, cells were trypsinized and washed in ice-cold PBS. Cells were fixed by adding them dropwise into ice-cold 80% ethanol while vortexing, followed by incubation on ice for 60 min. The fixed cells were washed with cold PBS and incubated at 37°C for 30 min in 0.5 ml of PBS containing 10 µg/ml propidium iodine (Sigma) and 5 µg/ml RNase A (New England Biolabs). DNA content was determined by FACS scan analysis (Becton Dickinson).
Cell death assay
Dead cells were determined by TUNEL assay. Both floating cells and trypsinized attached cells were collected. TUNEL analysis of DNA fragmentation was performed using an in situ apoptosis detection kit (ApopTag) following the procedures recommended by the manufacturer (Intergen).Cell death was presented as the percentage of TUNEL-positive cells versus total cells.
Protein extraction and immunoblotting
After PTEN induction, cells were washed twice with ice-cold PBS and lysed in cold lysis buffer (15). For insulin stimulation, cells were grown in serum-free medium for 24 h followed by incubation with 10 µg/ml insulin (Gibco BRL) for 30 min at 37°C. Twenty-five micrograms of protein of cell lysates was used for western blot analysis (15). The anti-PTEN monoclonal antibody 6H2.1 was raised against the C-terminus of PTEN (46). The polyclonal anti-Akt antibody (New England Biolabs) was used at 1:1000 dilution. Monoclonal anti-
-tubulin (Sigma) was used at 1:5000 dilution.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Tom Roberts for providing the Akt constructs. This work is partially funded by the American Cancer Society (RPG98-211-01CCE to C.E.), the Susan G. Komen Breast Cancer Research Foundation (BCTR 2000 462 to C.E.) and the National Cancer Institute (P30CA16058 to the Ohio State University Comprehensive Cancer Center).
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Ohio State University Human Cancer Genetics, 420 West 12th Avenue, Room 690C TMRF, Columbus, OH 43210, USA. Tel: +1 614 292 2347; Fax: +1 614 688 3582; Email: eng-1@medctr.osu.edu
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