(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 ¡H-J.S. H.. W. K. C.]. Center for Molecular Genetics ¡W.K. C.I and Cancer Center ¡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 alíeles, 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 alíeles 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 alíelesof 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 37°Cin an 8% CO, environment. onstrated against an acidic tyrosine-phosphorylated polymer substrate Plasmids, Vector Construction, and Cloning of Mutant PTEN Alíeles. (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 alíeleswere constructed by ligating £raRI fragments from pBP-PTEN plasmids into the role as a tumor suppressor gene has been shown by both in vitro (14, £«)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 -80°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]. ¡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: TÚNEL, 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 alíeleswere 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 TÚNEL 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. TÚNEL 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 Alíeles.To test whether the in vidin-lissamine rhodamine (Molecular Probes, Eugene, OR) at 5 fxg/ml was ability of the mutant PTEN alíelesto suppress in vitro growth was due substituted for streptavidin-FITC for the detection of terminal deoxynucleoti- to their loss of phosphatase activity, each alíelewas engineered into dyl transferase-incorporated biotin-dUTP. thus allowing the simultaneous de the pGEXKG plasmid and expressed as a GST