The Phosphoinositol Phosphatase Activity of PTEN Mediates a Serum-Sensitive Gl Growth Arrest in Glioma Cells

(CANCER RESEARCH 58. 5002-5008. November 15. I998| Advances in Brief

The Phosphoinositol Phosphatase Activity of PTEN Mediates a Serum-sensitive Gl Growth Arrest in Glioma Cells

Frank B. Furnari,1 H-J. Su Huang, and Webster K. Cavenee

Ludwig Institute for Cancer Research IF. B. F.. H-J. S. H., W. K. C.I, Department nf Medicine Â¡H-J.S. H.. W. K. C.]. Center for Molecular Genetics Â¡W.K. C.I and Cancer Center Â¡W.K. C.I. University of California-San Diego. IM Jolla. California 920Vj-0660

Abstract Three mechanisms of growth control by tumor suppressor genes have been demonstrated and include cell cycle regulation, apoptosis, The PTEN gene (also called MMACl and TEPl) at chromosome 10q23 and angiogenesis. The CDKN2A gene is an example of a tumor is mutated in a variety of predominantly late-stage tumors and has been suppressor whose normal function is to regulate cyclin-dependent shown to suppress glioma cell growth in vitro and in vivo. Here we sought kinase activity and thus entry into the S phase of the cell cycle (17). tu determine the mechanism by which PTEN mediates growth inhibition. Using the mutant PTEN glioma cell line, U87MG, as a transfection recip In contrast, the TP53 gene has been shown to regulate the cell cycle (18-20), as well as apoptosis (21), and angiogenesis (22) under ient for a series of PTEN alÃeles, we provide direct evidence that this capacity requires phosphatase activity. Mutations mapping upstream, certain conditions. Whether the PTEN gene can regulate growth by within, and downstream of the catalytic domain ablated activity toward a any or all of these mechanisms has not been tested to date. Here we 3' phosphorylated phosphoinositide substrate of PTEN, whereas alÃeles show that PTEN mediated growth suppression by a G, cell cycle with mutations flanking the catalytic domain retained activity toward the arrest that is sensitive to growth factor/serum concentration. Further acidic protein polymer substrate, Glu4Tyr,. Thus, catalytic activity to more, although mutant alÃelesof PTEN that lack growth-suppressive ward phosphoinositide substrates was required for growth suppression, activity were similarly defective for 3' phosphoinositide phosphatase whereas activity toward the protein substrate was dispensable for growth activity, some of these mutants retained activity against the acidic suppression. Finally, we used apoptotic and cell proliferation analyses to tyrosine-phosphorylated substrate, polyGlu4Tyr,. These data indicate show that /'// Y mriliatril growth inhibition under reduced serum con that PTEN phosphoinositide phosphatase activity is essential for gli ditions was due to a (., cell cycle block rather than to an induction of oma growth control and may suggest that various substrates of PTEN apoptosis. play roles in differing cellular processes.

Introduction Materials and Methods The PTKN tumor suppressor gene encodes a 403-amino acid cyto- Cell Lines. U87MG and U178 glioma cell lines used in this study were plasmic protein with extensive NH,-terminal homology to tensin and described previously (23-25). Both cell lines were grown in DMEM media auxilin as well as a central domain with perfect homology to protein supplemented with \Q7r cosmic calf serum (Hyclone. Logan. UT) and main tyrosine phosphatases (1-3). PTEN enzymatic activity has been dem tained at 37Â°Cin an 8% CO, environment. onstrated against an acidic tyrosine-phosphorylated polymer substrate Plasmids, Vector Construction, and Cloning of Mutant PTEN AlÃeles. (4), the lipid second messenger, phosphatidylinositol 3,4,5-triphos- The plasmids pCDKN2WT (26). pCMVlFLAG-DR4 (27). and pH2B-GFPNl phate, and inositol 1,3,4,5-tetrakisphosphate (5). This suggests that (28) were described previously; pGreen Lantern-2 was obtained from Life PTEN recogni/.es disparate cellular substrates such as phosphoinositi- Technologies, Inc., Gaithersburg. MD. Construction of wild-type PTEN, des and protein, the latter of which is exemplified by the FAK2 (6). PTEN-\\\. and HA-tagged PTEN mutants d55/70 (lacking amino acids 55- 70), Ãœ237/239(lacking amino acids 237-239), and GI29R in pBP was de PTEN maps to chromosome 10q23, a region of frequent loss in a scribed previously (14). Site-directed mutagenesis by recombinant PCR was number of predominantly late-stage sporadic cancers including pros used to construct the HA-tagged PTEN point mutants D92A, C124G, and tate, breast, thyroid, glioma, endometrial, and melanoma (7). As such. G129E. The RISS. R151, and C105F point mutants were derived from glioma PTEN has been found to be mutated in these cancers, with the highest cell lines LN340. LN319, and LN215. respectively, and constructed as de frequency occurring in high-grade glioma (44%; Ref. 8) and endo- scribed previously (14). metrioid type endometrial cancer (55%; Refs. 9 and 10). In addition, Transfection Assays and Western Blot Analysis. Calcium phosphate germ-line PTEN mutations have been detected at high frequency transfections for growth inhibition assays, puromyocin selection, and anti-HA (~80%) in the autosomal dominant cancer predisposition disorders, Western blot analysis were performed as described previously (29, 30). Cowden disease (11, 12) and Bannayan-Zonana syndrome (13). Its Bacterial Expression of PTEN. Expression vectors for PTEN alÃeleswere constructed by ligating Â£raRI fragments from pBP-PTEN plasmids into the role as a tumor suppressor gene has been shown by both in vitro (14, Â£Â«)RIsite of pGEX-KG (31). Subsequently, COOH-terminal HA tags were 15) and in vivo ( 16) demonstration of growth inhibition of glioma and removed from pGEX-KG vectors by digestion with Nhel/Sall and replaced melanoma cells. with the stop codon-containing Nhe/Sd/I fragment of pBP-PTEN. The vectors were used to transform Escherichia coli strain BL21. Protein expression and Received 8/6/98: accepted 10/1/98. purification was carried out as described (4), with the exception that fusion The costs of publication of this article were defrayed in part by the payment of page proteins were retained on glutathione-Sepharose beads prior to storage in 50 charges. This article must therefore be hereby marked advertisement in accordance with mM HEPES and 30% glycerol (pH 8.0) at -80Â°C. Protein concentrations and 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Ludwig Institute for Cancer the integrity of fusion proteins were determined by SDS-PAGE and by com Research. San Diego Branch 3080 CMM-Easl. 9500 Oilman Drive. La Jolla. CA 92093- parison with known concentrations of BSA. 0660. Phone: (619)534-7808: Fax: (619)534-7816: E-mail: [email protected]. Â¡nVitro Phosphatase Assays. Standard PTEN protein and phosphoinosit : The abbreviations used are: FAK. focal adhesion kinase: HA. hemagglutinin antigen: ide phosphatase assays were performed with polyGlu4Tyr, (Sigma Chemical pBP. pBABE-puro: TÃšNEL, terminal deoxynucleotidyl transferase-medialed nick end Co., St. Louis, MO) and ['H]Ins (1,3,4,5)P4 (New England Nulcear, Boston, labeling: GFP. green fluorescence protein: Ins (1.3.4.5)P4. inositol 1.3.4,5-letrakisphos- phate: BrdUrd. bromodeoxyuridine; GST. glutathione S-transferase: BES. W./V-bis(2- MA) as described previously (4. 5), except that labeling of polyGIUjTyr, was hydroxyethyll-2-aminoethanesulfonic acid. achieved with src kinase (Upstate Biotechnology, Lake Placid, NY) according 5002

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1998 American Association for Cancer Research. PTEN GROWTH SUPPRESSION IN GLIOMA CELLS to the manufacturer's directions, and 5 mM glutathione was included in all cells were subsequently counted. All mutant alÃeleswere defective for phosphatase assay buffers. Assays were performed in triplicate, and results are growth inhibition and yielded cell numbers comparable with vector- presented with standard errors. transfected cells (Fig. 1, upper panel). In contrast, and consistent with TÃšNEL Analyses. U87MG cells were seeded at 8 x 10" per glass cover- previous results (14), transfection with wild-type PTEN-HA caused a slips in triplicate. Twenty-four h after seeding, cells were cotransfected with ~70% decrease in cell number. Protein lysates were prepared from expression constructs and pH2B-GFPNl (which expresses nuclear localized cells 4 days after transfection, immunoblotted, and probed with an- GFP) at a 4:1 ratio, for a total of 5 p.g of plasmid DNA, using the BES ti-HA antibodies; all expression constructs expressed comparable transfection method (32). Transfected cells were indirectly detected by the presence of nuclear GFP. Cells were allowed to recover for 48 h in 2% cosmic levels of exogenous protein (Fig. 1, lower panel). When identical calf serum, followed by fixation in 4% paraformaldehyde/PBS. TÃšNEL anal experiments were performed in the presence of 10% serum, PTEN- ysis was performed with the Apoptosis Detection kit (Upstate Biotechnology) mediated growth suppression was minimal (data not shown). following the manufacturer's protocol with the following alterations. Strepta- Enzymatic Activities of PTEN AlÃeles.To test whether the in vidin-lissamine rhodamine (Molecular Probes, Eugene, OR) at 5 fxg/ml was ability of the mutant PTEN alÃelesto suppress in vitro growth was due substituted for streptavidin-FITC for the detection of terminal deoxynucleoti- to their loss of phosphatase activity, each alÃelewas engineered into dyl transferase-incorporated biotin-dUTP. thus allowing the simultaneous de the pGEXKG plasmid and expressed as a GST fusion protein for tection of TUNEL-positive and nuclear GFP-expressing cells. Nuclei were phosphatase assays. Two substrates have been shown to be preferred stained with Hoechst 33258 at 2.5 /Â¿g/mlin PBS, and the coverslips were by PTEN, the Glu4Tyr, acidic protein polymer (4) and phosphoinosi- placed into antifade medium, mounted, and viewed with a Zeiss fluorescent microscope. Three hundred GFP-positive (transfected) and 300 GFP-negative tols phosphorylated on position three of the inositol ring (5). When (nontransfected) nuclei were scored per coverslip in triplicate for the presence assayed for phosphatase activity against Glu4Tyr,, mutations either of apoptosis, and the results are presented with standard error. upstream (R15S and R151) or downstream (d237/239) had little con BrdUrd Proliferation Assays. U87MG cells were transfected and fixed as sequence for dephosphorylation activity (Fig. 2, upper panel). Each above for TÃšNEL analysis except, pGreen-Lantern-2 (cytoplasmic GFP local mutation within the catalytic domain, with the exception of G129E, ization) was used in place of pH2B-GFPNl. and BrdUrd was added to the caused at least a 10-fold decrease in phosphatase activity (Fig. 2, media for a 16-h pulse prior to coverslip processing. Cells were permeabili/ed upper panel), in agreement with previous data (4). In contrast, all in 0.3% Triton X-100/PBS for 15 min at room temperature, and immunoflu- orescence incubations were performed with a monoclonal rat anti-BrdUrd (Accurate Scientific) diluted 1:500 in blocking buffer (PBS/0.5% NP40/5 mg/ml BSA) with 20 mM MgCI2 and 20 units/ml RNase-free DNase I (Boeh- 140n ringer Mannheim, Indianapolis, IN) for l h at 37Â°C.Coverslips were washed four times with PBS and incubated with a 1:100 dilution of lissamine-rhodamine-conjugated donkey anti-rat (Jackson Immunoresearch, West Grove. PA) 120- and Hoechst 33258 at 2.5 /Â¿g/mlin blocking buffer for l h at 37Â°C.Cells were 0 washed, mounted, viewed, and scored as above. Flow Cytometry Analysis of the Cell Cycle. U87MG and U178 cells were 100- seeded at 7 X IO5 in 10-cm plates and cotransfected 24 h later with expression constructs and pH2B-GFPNl at a 4:1 ratio, for a total of 20 ^g of plasmid DNA, using the BES transfection method. Transfections were terminated after 80 12 h, and cells were allowed to recover for 36 h in 2 or 10% cosmic calf serum, followed by an additional 30-h incubation in the presence of nocoda/ole CD (U87MG, 70 ng/ml; U178, 40 ng/ml). Cells were trypsinized, collected in PBS, Ãœ 60- and fixed for l h in 80% cold ethanol. Propidium iodide DNA staining was CD then used to determine the cell cycle distribution of at least 20,000 GFP- positive and GFP-negative gated cells. 40- Results CD Growth-inhibitory Capabilities of PTEN AlÃeles.We had dem onstrated previously that exogenous wild-type PTEN but not some mutant alÃelescould suppress the growth of cells with endogenous mutant, but not wild-type, PTEN (14). To pursue the structural basis 0 for this, a series of PTEN alÃelesthat have been identified in tumors or cell lines were assessed for enzymatic activities and growth- suppressive abilities. These include: the R15S and R151 mutations within the tensin/auxilin region, which occur in primary gliomas (2) CVJ and the glioma cell lines LN319 and LN340 (14); the C105F mutation within the catalytic domain, which occurs in the LN215 glioma cell 66kDa line (14); and, the G129E mutation, which has been described pri marily in Cowden families with the exception of a single non-Cowden a-HA individual with endometrial cancer (33). In addition, the D92A and C124G mutations were designed based on highly conserved amino Fig. I. Growth-suppressive effect of PTEN alÃelesin U87MG cells. Upper panel, acids in the catalytic domain of all tyrosine phosphatases (34). AlÃeles empty vector (pBP), or vector containing wild-type, PTTi/V-HA, or mutant alÃelestagged d55/70, G129R, and d237/239 were described previously (1, 2, 14). with HA were transfected into the human glioma cell line. U87MG. and selected with puromyocin. Viable cells were counted after 7 days, and results arc presented normalized All alÃeleswere engineered at the COOH terminus with the HA and to vector being 100%. A typical transfection is shown; results were reproduced in three cloned into the retroviral expression plasmid, pBP (35). Twenty-four independent experiments and are presented as the means; bars. SD. Unverpanel, anti-HA Western blot analysis of 30 /Agof protein lysate generated from HA-tagged PTEN alÃeles h after transfecting the U87MG glioma cell line, cells were selected in at 4 days after transfection. Protein lysates were generated from parallel transfection plates media containing 2% serum and puromyocin for 7 days, and surviving used for the above growth suppression assay. 5003

crease in cell adhesion on fibronectin. Whereas mutations of Asp 92 and Cys 124 eliminated this effect on cell adhesion, the GI29E mutant behaved identically to wild type (6). By extrapolation, our results may N indicate that the phosphoinositol phosphatase activity of PTEN may C not be essential for cell adhesion while being required for growth O inhibition. Ability of PTEN AlÃelesto Affect Apoptosis or Cell Prolifera tion. To determine whether the /TEW-mediated growth inhibition of U87MG cells in vitro was due to programmed cell death, cell cycle arrest, or both, TÃšNEL analyses and BrdUrd proliferation assays were O) E performed. To examine the subpopulation of cells that had been "e transfected, nuclear GFP expression was used as an identifier (see "Materials and Methods"). For apoptotic analysis, cells were trans fected on glass coverslips, allowed to recover for 48 h in 2% serum, I and processed for TÃšNEL. The level of apoptosis due to PTEN-HA "o expression was indistinguishable from that in empty vector-trans- E fected cells (Fig. 3). In comparison, in the positive control transfection e of the DR4 receptor, a molecule that has been shown to induce apoptosis when overexpressed (27), the expected marked increase in apoptotic death in the same cells was detected (Fig. 3). For BrdUrd proliferation assays, U87MG transfected cells were similarly prepared except with the addition of a 16-h BrdUrd pulse to measure cells progressing through S phase. /TfiW-HA-expressing cells, as indicated by GFP detection, when compared with vector- transfected and R15S mutant-expressing cells, demonstrated a clear osoN decrease in BrdUrd incorporation (Fig. 4). Because U87MG cells have been shown to be mutant for the cyclin-dependent kinase inhib itor pi6 and its ectopie expression to cause severe growth arrest (26), 40-0)iTlns(1,3,4,5)P4 its transfection was used here as a positive control. All of these data indicate that suppression of cell proliferation was specific to wild-type PTEN (Table 1) and that each mutant alÃeleeliminated this activity consistent with the growth data in Fig. 1. Thus, fTOV-mediated growth inhibition of U87MG cells appears to arise from a decrease or block in cell proliferation and not by apoptotic death. Cell Cycle Analysis of PrKV-mediated Growth Arrest of U87MG and U178 Cells. To define the phase of the cell cycle at which PTEN exerts its growth inhibition, the DNA content of trans fected U87MG cells was determined by fluorescence-activated cell sorter analysis. To gate and thus analyze transfected cells separately from nontransfected cells, expression constructs were cotransfected with pH2B-GFPNl. After transfection, cells were allowed to recover in either 2 or 10% serum-containing media for 48 h and prepared for flow cytometry. A comparison of G,, S, and G,-M cell cycle phases between vector and /TEW-HA-transfected cells under these condi tions did not show an appreciable cell cycle block (Fig. 5A). However, because even control cells grown in 2% serum contained a high G, content (70%), any additional block in this phase of the cell cycle due to PTEN could be difficult to detect (Fig. 5A). To overcome this situation, we increased the sensitivity of the assay by synchronizing Fig. 2. Protein and phosphoinositol phosphala.se activity of PTEN alÃeles.Two fig of GST-PTEN proteins were tested lor phosphalase activity against the protein polymer transfected cells in mitosis with the microtuble-stabilizing agent, polyGlujTyr, (upperpanel] and Â¡nositolphosphate. Ins ( l.3.4.5)P4 (lowerpanel). Activity nocodazole (36). When cells transfected with vector and the GÃŒ29R is expressed as the mean nmol of substrate dephosphorylatcd [Ins ( UASlP.,] (5) or mutant alÃelewere grown in 2% serum and then treated in this fashion, phosphate released (polyGlu^Tyr, ) (4) per min per mg GST protein. Burs. SD. the G, portion of the cell cycle was diminished greatly (Fig. 5/4), and the majority of the cells were synchronized in G2-M. In sharp contrast, mutant alÃelesincluding G129E, had an 8-10-fold or greater decrease cells transfected with wild-type PTEN-HA. or pl6 were also blocked in phosphatase activity toward Ins (1, 3, 4, 5)P4 (Fig. 2, lower panel), in G,. When identical experiments were performed in 10% serum, no a water-soluable phosphoinositide shown previously to be a suitable fTÂ£W-specific G, block could be detected, whereas, predictably, the substrate for PTEN by virtue of a phosphate group on the 3' position pl6 arrest was as effective as in 2% serum. Thus, PTEN inhibits of the inositol ring (5). These results indicate that phosphatase activity proliferation by imposing a block on the G,-S transition of the cell is essential for PTÃ¯W-mediated growth inhibition and that certain cycle. To confirm that these results were not specific to U87MG, mutations may inactivate dephosphorylation for only specific types of U178 cells that contain a PTEN frameshift mutation at codon 248 and substrates and not others. Support for this notion comes from the have been shown previously to be growth inhibited by PTEN (14) recent finding that PTEN can associate with FAK and cause a de were similarly tested. They also arrested in G, upon PTEN transfec- 5004

A DR4 Vector PTEN

TUNEL

nucGFP

â€¢1

Hoechst ..t1u,...1â€¢ I @ S . @.% , â€¢4â€¢!@â€¢1

Cl) C) C.)

>a) C,)a w-J z 1@ I-

nucGFP

Fig. 3. Apoptotic effect of wild-type PTEN in U87MG cells. In A, pBP (Vector), pBP-PTEN-HA (PTEW)or the positive control plasmid pCMV1FLAG-DR4 (DR4) were cotransfected with the nuclear GFP expression construct, pH2B-GFPN1, and TUNEL analysis was performed as described in â€œMaterialsandMethods.â€•Whitearrowheads, nuclear GFP@/TUNEL@cells;yellow arrowheads, GFP11'UNEL@ cells. Small fragmented nuclei, as seen by Hoechst staining, are indicative of cells undergoing apoptosis. A representative fieldis shownforeachtransfectioncondition.B,GFP-positiveand-negativeU87MGcellsfromeachconditioninAwerescoredforTUNELandarepresentedaspercentageofcells undergoing apoptosis. Results are expressed as the mean; bars, SD.

5005

GFP BrdU Hoechst

Vector

PTEN

R15S

p16

Fig. 4. Effect of wild upe PTEN on LI87MG proliferation. pBP (IViwr). pBP-PTEN-HA (PTEN). pBP-RI5S-HA (RISS), or pCDKN2WT (p!6) were cotransfected with pGreen Lantcm-2, and BrdUrd piohÃŽL-nilionassays were performed as deserihed in "Materials and Methods." While nn-nwheiiils. GFP VBrdUrd4 cells; \ellow urniwheiuls, GFP^/BrdUrd cells. A representative field is shown for each Iransfection condition. lion, and this was also dependent on low serum concentrations (Fig. cancers, it is possible that the reduced serum conditions we used in SB}. vitro may reflect cellular stress conditions imposed at later stages of tumorigenesis in vivo. Discussion PTEN appears to possess two phosphatase activities: one against the 3'

The PTEN gene is the first phosphatase that plays a role in certain position of phosphoinositides Ins(l,3,4,5)P4 and PtdIns(3,4,5)P, (5); and sporadic cancers and inheritable-cancer predisposition syndomes (2, another against protein substrates, as reflected by activity toward the 3, 11-13). Here we show that its ability to mediate growth suppression acidic protein polymer Glu4Tyr,. Consistent with the latter activity. in vitro is due to its ability to effect a serum-sensitive G, arrest. PTEN has been demonstrated to cause a decrease in fibronectin-mediated Because the PTEN gene is mutated more frequently in advanced FAK. phosphorylation and also to physically interact with this kinase (6). Thus, PTEN may function to regulate cell migration/adhesion through its protein phosphatase activity and to regulate cellular growth through its Table I PTLN itietlittiea ti decrease in cell prolifÃ©rationin UK7MG celia GFP-positive and -negative U87MG cells from each plasmid transfection condition lipid phosphatase activity, which in turn could regulate Akt function (37). were scored as described in "Materials and Methods." and the results are presented as Our data support this model in that all alÃelestested, including the percentage of cells incorporating BrdUrd. The ratio of transfected to nontransfecled cells incorporating BrdUrd is indicated as GFP+:GFP~. Results are expressed as means Â±SD. prevalent Cowden syndrome GÃŒ29EalÃele,lacked both the ability to suppress growth and to dephosphorylate Ins(l,3,4,5)P4, whereas some 7( BrdUrd incorporation alÃelesretained the ability to dephosphorylate the Glu4Tyr, polymer but (GFP+)79.0 ExpressionplasmidVectorr/fiPTEN-H&PTENKI5SRI51Â¿55/70D92AC)83.886.090.586.188.589.184.686.982.484.589.083.52.22.80.71.42.11.42.30.20.84.91.43.582.0(GFP clearly were not growth suppressive. This finding may be of clinical 0.213.5 importance because genotype-phenotype analysis of Cowden families 2.143.4 indicated that the number of organs showing signs of disease was related 0.634.5 1.283.1 to the type of PTEN mutation (38). Such a severity of disease phenotype 2.377.7 being dictated by the mutation spectrum is well documented for tumor 2.479.7 suppressor genes and is, perhaps, best illustrated by the von Hippel- Â±2.478.5 Â±2.174.7 Lindau gene in which families with pheochromocytomas almost exclu 105FC124CG129RG129EJ237/239TransfectedÂ±0.976.6 sively have missense mutations in von Hippel-Lindau (39) and by ade- Â±0.277.9 Â±0.774.3 nomatous polyposis coli in which mutations in the most proximal region Â±3.370.5 of the adenomatous polyposis coli gene are associated with an attenuated Â±0.7Nontransfected 1.4GFP+:GFP0.940.150.470.400.940.870.940.900.910.910.860.890.86form of the cancer (40, 41). 5006

-noe +noc

Vector PTEN Vector PTEN G129R

2% serum

rPhaseG1SG2+Mpercent:l * | Phase percent Phase percent PhasepercentG1SG2+M5.715.179.1PhasepercentG1oG2+M30.725.943.4PhasepercentG1SG2+M6.512.481.1TF G1 : 70 G1 : 74 58.5: S : 13.7 S : 14.9 16.9: G2+M: 15.8 G2+M: 10.7 24.6

10% serum

jtjzanEEâ€¢¿il11iPhaseG1SG2+M'-J-percent4.28.787.2Phase 1)11141Phase percentG1sG2+M7.512.779.8T"T^^^T"percentG1sG2+M1.510.288.2PhaseG1SG2+Mpercent56.315.128.6 Phase percent Phase percent G1 : 57.8 G1 : 61.5 S : 28.8 S : 27.3 G2+M : 13.4 G2+M: 11.2 B +noc

Vector PTEN G129R

2% serum

Phase percent Phase percent G1 : 7.3 24.2 G1 : 7.5 S : 11.9 10.0 S : 9.9 G2+M : 80.8 65.8 G2+M : 82.6

Fig. 5. Flow cytometry analysis of fTE/V-lransfected U87MG and U178 cells. A, the ability of pBP (Vector). pBP-PTEN-HA (PTEN). pBP-G129R-HA (C129R). and p>CDKN2WT (pl6) to induce cell cycle Gt arrest in transient transfection assays was assessed by flow cytometry. U87MG cells were cotransfected with the indicated expression constructs and pH2B-GFPNl. propagated in 2 or 10% serum and with or without nocodazole. B. U178 cells were transfected as in A and propagated in 2% serum with nocodazole. GFP* cells were analyzed, and their cell cycle profiles and percentages of cells in each phase are shown.

The fact that PTEN-mediated G, arrest was detected in both the Acknowledgments U87MG and U178 cell lines under limited growth conditions could We thank T. Kanda and G. Wahl for pH2B-GFPNl. K. Knudsen and E. imply two possibilities relative to PTEN activity: (a) that PTEN is a Knudsen for helpful advice with BrdUrd proliferation and cell cycle analyses, weak growth inhibitor and therefore requires dilution of positive T. Maehama and N. Tonks for advice on in vitro phosphatase assays, H. Lin growth factors present in high serum to effect growth suppression; or for technical assistance, and members of the Ludwig Institute for helpful (b) that PTEN function is directly modulated by a serum component. discussions. This modulation could be in the form of inhibitory or activating phosphorylation of P TEN itself. Precedent for phosphorylation control References of phosphatase activity has been seen for PTP1B (42) and cdc25 1. Li, J., Yen, C., Liaw, D., Podsypanina. K.. Bose, S.. Wang, S. !.. Puc, J., Miliaresis. activation in response to DNA damage by chkl kinase (43-45). C, Rodgers, L., McCombie, R., Bigner, S. H., Giovanella, B. C., Ittmann, M., Tycko, 5007

B., Hibshoosh, H., Wigler. M. H., and Parsons, R. PTEN. a putative protein tyrosine 24. Van Meir, E.. Ceska. M.. Effenberger. F.. Walz, A., Grouzmann, E., Desbaillets, I., phosphatase gene muiated in human brain, hreasi. and prostate cancer. Science Frei, K., Fontana, A., and de Tribolet, N. lnterleukin-8 is produced in neoplastic and (Washington DC). 275: 1943-1947. 1997. infectious diseases of the human central nervous system. Cancer Res.. 52: 4297- 2. Sleek. P. A.. Pershouse. M. A.. Jasser. S. A.. Yung. W. K.. Lin. H.. LigÃ³n, A. H., 4305, 1992. Langford, L. A.. Baumgard. M. L.. Haitier. T.. Davis, T., Frye. C.. Hu, R.. Swedlund, 25. Carlsson. J.. and Acker, H. Influence of the oxygen pressure in the culture medium on B., Teng, D. H., and Tavtigian. S. V. Identification of a candidate tumour suppressor the oxygÃ©nationof different types of multicellular spheroids. Int. J. RadiÃ¢t.Oncol. gene. MMACI. al chromosome 10q23.3 that is mutated in multiple advanced cancers. Biol. Phys.. //: 535-546. 1985. Nat. Genet., IS: 356-362. 1997. 26. Arap. W.. Nishikawa. R., Furnari. F. B.. Cavenee. W. K.. and Huang, H-J. S. 3. Li. D. M., and Sun. H. TEP1, encoded by a candidate tumor suppressor locus, is a Replacement of the pl6/CDKN2 gene suppresses human glioma growth. Cancer Res.. novel protein tyrosine phosphatase regulated by transforming growth factor ÃŸ.Cancer 55: 1351-1354, 1995. Res., 57: 2124-2129. 1997. 27. Pan. G.. O'Rourke. K.. Chinnaiyan. A. M.. Gentz. R., Ebner. R.. Ni. J.. and Dixit. 4. Myers. M. P.. Stolarov. J. P., Eng, C.. Li. J.. Wang. S. !.. Wigler. M. H.. Parsons. R.. and Tonks. N. K. P-TEN. the tumor suppressor from human chromosome I0q23, is V. M. The receptor for the cytotoxic ligand TRAIL. Science (Washington DC). 276: a dual-specificity phosphatase. Proc. Nati. Acad. Sci. USA, 94: 9052-9057, 1997. 111-113. 1997. 5. Maehama, T.. and Dixon. J. E. The tumor suppressor. PTEN/MMACI, dephospho- 28. Kanda. T.. Sullivan, K. F.. and Wahl. G. M. Histone-GFP fusion protein enables rylates the lipid second messenger, phosphatidylinositol 3,4,5-triphosphate. J. Biol. sensitive analysis of chromosome dynamics in living mammalian cells. Curr. Biol.. X: Chcm., 273: 13375-13378. 1998. 377-385, 1998. 6. Tamura. M., Gu. J.. Matsumoto. K.. Aota. S., Parsons. R.. and Yamada, K. M. 29. Furnari. F. B.. Adams. M. D.. and Pagano. J. S. Regulation of the Epstein-Barr virus Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor DNA polymerase gene. J. Virol.. 66: 2837-2845. 1992. PTEN. Science (Washington DC). 2X0: 1614-1617. 1998. 30. Krieger. M. DNA transfer. In: M. Krieger (ed.). Gene Transfer and Expression: A 7. Mertens. F., Johansson. B.. Hoglund, M., and Mitelman, F. Chromosomal imbalance Laboratory Manual, pp. 96-98. New York: Stockton Press, 1990. maps of malignant solid tumors: a eytogenetie survey of 3185 neoplasms. Cancer 31. Guan. K. L.. and Dixon. J. E. Eukaryotic proteins expressed in Escherichiu coli: an Res.. 57: 2765-2780. 1997. improved thrombin cleavage and purification procedure of fusion proteins with 8. Wang, S. I.. Puc. J.. Li. J.. Bruce. J. N., Caims, P., Sidransky, D.. and Parsons. R. glutathione S-lransferase. Anal. Biochem., 192: 262-267. 1991. Somatic mutations of PTEN in glioblastoma multiforme. Cancer Res.. 57: 4183- 32. Chen, C.. and Okayama. H. High-efficiency transformation of mammalian cells by 4186. 1997. plasmid DNA. Mol. Cell. Biol.. 7: 2745-2752, 1987. 9. Tashiro. H.. Blazes. M. S.. Wu. R.. Cho. K. R., Bose. S.. Wang. S. I.. Li, J.. Parsons. 33. Risinger. J. !.. Hayes. A. K.. Berchuck. A., and Barrett, J. C. PTEN/MMACI R.. and Ellenson. L. H. Mutations in PTEN are frequent in endometrial carcinoma but mutations in endometrial cancers. Cancer Res.. 57: 4736-4738, 1997. rare in other common gynecological malignancies. Cancer Res.. 57: 3935-3940, 34. Barford. D.. Jia. Z.. and Tonks. N. K. Protein tyrosine phosphatases take off. Nat. 1997. Struct. Biol.. 2: 1043-1053. 1995. 10. Kong. D.. Suzuki, A., Zou, T. T.. Sakurada. A., Kemp. L. W., Wakatsuki, S., Yokoyama. T.. Yamakawa, H., Furukawa, T., Sato, M., Ohuchi. N., Sato. S.. Yin, J.. 35. Morgenstern. J. P., and Land. H. Advanced mammalian gene transfer: high litre retroviral vectors with multiple drug selection markers and a complementary helper- Wang. S.. Abraham. J. M.. Souza, R. F.. Smolinski, K. N.. Meltzer, S. J.. and Horii. free packaging cell line. Nucleic Acids Res.. IH: 3587-3596, 1990. A. PTEN I is frequently mutated in primary endometrial carcinomas. Nat. Genet.. 17: 143-144, 1997. 36. Zhu. L.. Enders, G., Lees, J. A.. Beijersbergen. R. L.. Bernards. R.. and Harlow, 11. Nelen, M. R., van Staveren, W. C. G.. Peelers. E. A. J., Ben Massel. M.. Gorlin. R. J.. E. The pRB-related protein pl07 contains two growth suppression domains: Hamm. H.. Lindboe, C. F.. Fryns. J-P.. Sijmons. R. H.. Woods. D. G.. Mariman. independent interactions with E2F and cyclin/cdk complexes. EMBO J., 14: E. C. M., Padberg. G. W.. and Kremer. H. Germline mutations in the PTEN/MMACI 1904-1913. 1995. gene in patients with Cowden disease. Hum. Mol. Genet.. 6: 1383-1387, 1997. 37. Downward. J. Mechanisms and consequences of activation of protein kinase B/Akt. 12. Liaw, D., Marsh. D. J., Li, J., Dahia. P. L.. Wang. S. I., Zheng. Z., BÃ¶se,S.. Call, Curr. Opin. Cell Biol.. 10: 262-267. 1998. K. M., Tsou. H. C.. Peacocke. M., Eng. C., and Parsons, R. Germline mutations of the 38. Marsh, D. J.. Coulon. V.. Lunetta. K. L.. Rocca-Serra. P.. Dahia, P. L.. Zheng. Z.. PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat. Liaw. D.. CarÃ³n, S.. Duboue. B.. Lin. A. Y., Richardson. A. L.. Bonnetblanc, J. M., Genet.. 16: 64-67, 1997. Bressieux, J. M.. Cabarrot-Moreau. A.. Chompret. A.. DÃ©mange,L., Eeles, R. A., 13. Marsh. D. J.. Dahia. P. L. M., Zheng, Z., Liaw, D., Parsons, R., Gorlin, R. J., and Eng, Yahanda, A. M., Fearon, E. R., Flicker, J. P.. Gorlin. R. J., Hodgson, S. V., Huson, C. Germline mutations in PTEN are present in Bannayan-Zonana syndrome. Nat. S., Lacombe, D., LePrat. F.. Toulouse, C.. Olopade. O. I., Sobol, H.. Tishler. S., Genet., 16: 333-334, 1997. Woods. C. G.. Robinson, B. G.. Weber. H. C.. Parsons. R., Peacocke, M., Longy. M., 14. Furnari, F. B., Lin. H.. Huang. H. S.. and Cavenee. W. K. Growth suppression of and Eng. C. Mutation spectrum and genotype-phenotype analyses in Cowden disease glioma cells by PTEN requires a functional phosphatase catalytic domain. Proc. Nati. and Bannayan-Zonana syndrome, (wo hamartoma syndromes with germline PTEN Acad. Sci. USA, 94: 12479-12484, 1997. mutation. Hum. Mol. Genet., 7: 507-515, 1998. 15. Robertson. G. P., Furnari. F. B., Miele. M. E.. Glendening. M. J.. Welch. D. R., Fountain. J. W., Lugo, T. G.. Huang. H-J. S., and Cavenee, W. K. In vitro loss of 39. Crossey. P. A., Richards, F. M., Foster, K., Green, J. S., Prowse. A., Latif, F., Lerman, M. !.. Zbar. B.. Affara, N. A., Ferguson-Smith. M. A., and MÃ¤her,E. R. Identification helerozygosity targets the PTEN/MMACI gene in melanoma. Proc. Nati. Acad. Sci. USA, 95:9418-9423. 1998. of intragenic mutations in the von Hippel-Lindau disease tumour suppressor gene and 16. Cheney. I. W.. Johnson. D. E.. Vaillaneourt. M-T.. Avanzini. J.. Morimoto, A.. correlation with disease phenolype. Hum. Mol. Genet.. 3: 1303-1308. 1994. Demers, G. W.. Wills. K. N.. Shabram. P. W., Bolen. J. B.. Tavtigian. S. V.. and 40. Paul, P.. Letteboer, T., Gelbert, L., Groden. J.. White. R.. and Coppes. M. J. Identical Bookslein. R. Suppression of tumorigenicity of glioblastoma cells by adenovirus- APC exon 15 mutalions result in a variable phenotype in familial adenomatous mediated MMACI/PTEN gene transfer. Cancer Res.. 58: 2331-2334. 1998. polyposis. Hum. Mol. Genel., 2: 925-931. 1993. 17. Serrano. M., Hannon. G. J.. and Beach. D. A new regulatory motif in cell-cycle 41. Spino. L., Olschwang, S.. Groden. J.. Robertson, M., Samowilz. W.. Joslyn. G.. control causing specific inhibition of cyclin D/CDK4. Nature (Lond.), 366: 704-707, Gelbert. L.. Thliveris. A.. Carlson, M.. Ollerud, B., Lynch, H.. Walson, P.. Lynch, P.. 1993. Laurent-Puig, P.. Burt, R., Hughes. J. P.. Thomas. G.. Leppert, M., and White. R. 18. el-Deiry. W. S., Tokino. T., Velculescu. V. E.. Levy, D. B.. Parsons. R.. Trent, J. M.. AlÃelesof the APC gene: an attenuated form of familial polyposis. Cell, 75: 951-957. Lin. D.. Mercer, W. E.. Kinzler, K. W.. and Vogelslein, B. WAF1, a potential 1993. mediator of p53 tumor suppression. Cell. 75: 817-825. 1993. 42. Flint, A. J.. Gebbink, M. F.. Pranza. B. R., Jr., Hill, D. E., and Tonks, N. K. Multi-site 19. Harper. J. W.. Adami. G. R.. Wei. N., Keyomarsi, K., and Elledge. S. J. The p21 phosphorylation of the protein tyrosine phosphatase. PTP1B: identificalion of cell Cdk-interacting protein Cipl is a potent inhibitor of GI cyclin-dependent kinases. Cell, 75: 805-816. 1993. cycle regulated and phorbol ester stimulated sites of phosphorylation. EMBO J., 12: 1937-1946, 1993. 20. Xiong. Y.. Hannon. G. J., Zhang, H.. Casso. D., Kobayashi, R., and Beach. D. p21 is a universal inhibitor of cyclin kinases. Nature (Lond.). 366: 701-704, 1993. 43. Furnari, B., Rhind, N., and Russell. P. Cdc25 mitotic inducer targeted by chkl DNA 21. Yonish-Rouach. E., Resnitzky. D., Lotem, J.. Sachs. L.. Kimchi. A., and Oren, M. damage checkpoint kinase. Science (Washington DC). 277: 1495-1497, 1997. Wild-type p53 induces apoplosis of myeloid leukaemic cells that is inhibited by 44. Sanchez. Y., Wong, C.. Thoma, R. S.. Richman, R.. Wu. Z.. Piwnica-Worms. H.. and interleukin-6. Nature (Lond.). 352: 345-347, 1991. Elledge. S. J. Conservation of the Chkl checkpoint pathway in mammals: linkage of 22. Van Meir, E. G., Polverini, P. J.. Chazin, V. R., Su Huang. H. J., de Tribolet, N.. and DNA damage to Cdk regulation through Cdc25. Science (Washington DC). 277: Cavenee. W. K. Release of an inhibitor of angiogenesis upon induction of wild type 1497-1501. 1997. p53 expression in glioblastoma cells. Nat. Genet.. 8: 171-176. 1994. 45. Peng, C. Y., Graves, P. R., Thoma, R. S.. Wu. Z., Shaw. A. S.. and Piwnica-Worms. 23. Van Meir, E. G.. Kikuchi. T., Tada, M.. Li. H., Diserens, A. C.. Wojcik. B. E.. Huang. H. Mitotic and G2 checkpoint conlrol: regulation of 14-3-3 protein binding by H. J., Friedmann. T.. de Tribolet. N.. and Cavenee. W. K. Analysis of the p53 gene phosphorylation of Cdc25C on serine-216. Science (Washington DC). 277: 1501- and its expression in human gtioblastoma cells. Cancer Res., 54: 649-652. 1994. 1505. 1997.

5008

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1998 American Association for Cancer Research. The Phosphoinositol Phosphatase Activity of PTEN Mediates a Serum-sensitive G 1 Growth Arrest in Glioma Cells

Frank B. Furnari, H-J. Su Huang and Webster K. Cavenee

Cancer Res 1998;58:5002-5008.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/58/22/5002

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/58/22/5002. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.