Functional Evaluation of P53 and PTEN Gene Mutations in Gliomas1

Vol. 6, 3937–3943, October 2000 Clinical Cancer Research 3937

Functional Evaluation of p53 and PTEN Gene Mutations in Gliomas1

Hideaki Kato, Shunsuke Kato, INTRODUCTION Toshihiro Kumabe, Yukihiko Sonoda, GBM3 is the most common as well as the most aggressive Takashi Yoshimoto, Satoshi Kato, primary brain tumor and is clinically separable into two sub- Shuang-Yin Han, Takao Suzuki, types. One type is primary or de novo GBM, which is characterized by later onset, rapid tumor growth and a short clinical Hiroyuki Shibata, Ryunosuke Kanamaru, and course. The other type is secondary or “progressive” GBM, 2 Chikashi Ishioka which arises from a less malignant precursor lesion, including Department of Clinical Oncology, Institute of Development, Aging astrocytoma or AA, and is characterized by earlier onset, slow and Cancer, Tohoku University, Sendai 980-8575 [H. K., Sh. K., tumor growth, and less aggressive clinical features (for reviews, Sa. K., S-Y. H., T. S., H. S., R. K., C. I.], and Department of see Refs. 1–3). Although the two types are generally indistin- Neurosurgery, Tohoku University School of Medicine, Sendai 980-8574 [H. K., T. K., Y. S., T. Y.], Japan guishable histologically, recent molecular genetic analyses have provided evidence to support at least two distinct pathways contributing to the tumorigenesis of GBM. Primary GBM is ABSTRACT closely associated with the absence of p53 mutation and the We screened mutations of two major tumor suppressor presence of gene amplification such as that of EGFR, whereas genes, p53 and PTEN, in 66 human brain tumors using a secondary GBM is associated with the presence of p53 mutation yeast-based functional assay and cDNA-based direct se- and the absence of gene amplification (2–4). As well as these quencing, respectively. The frequency of p53 mutations was alterations, loss of chromosome 10q and/or 10p occurs in the 28.8% (19 of 66) and was higher in anaplastic astrocytoma majority of GBMs and AAs and is associated with both de novo (9 of 14, 64.3%,) than in glioblastoma multiforme (GBM; 7 and progressive GBMs (5–8). This suggests that there may be of 27, 25.9%,), supporting previous speculation that there unknown tumor suppressor gene(s) on chromosome 10 that may are at least two genetic pathways leading to GBM, a de novo be involved in the tumorigenesis of either of the two GBM pathway without p53 mutation and a “progressive” pathway subtypes. At chromosome 10q23, the PTEN gene (also called with p53 mutation. PTEN mutation was observed in 8 of 64 MMAC1 and TEP1) was recently identified as a putative tumor tumors (12.5%), mainly GBMs (7 of 26, 26.9%), both with suppressor gene, and mutations of this gene have been reported and without p53 mutation. These results suggest that muta- in human glioma and other tumors (9–18). In addition, germ- tion of the PTEN gene is a later event than that of the p53 line PTEN mutations have been found in the dominant cancer gene in glioma progression and is associated with both the susceptibility syndromes Cowden disease and Bannayan- genetic pathways. All of the detected PTEN missense muta- Zonana syndrome (19, 20). Furthermore, enforced expression of tions and an in-frame small deletion inactivated PTEN phos- PTEN cDNA suppresses tumor cell growth both in vitro and in phoinositide phosphatase activity in vitro. Because the tu- vivo (21–23). These results strongly suggest that PTEN acts as mors containing PTEN mutations also showed loss of a tumor suppressor gene in GBM and other tumors, although the heterozygosity in the chromosome 10q23 region flanking the functional significance of the detected mutations has not been PTEN gene, our data clearly indicate that inactivation of tested. Recent studies have shown that PTEN protein acts as a both PTEN alleles occurs in a subset of high-grade gliomas, phosphoinositol phosphatase and negatively controls the phos- therefore confirming the previous idea that PTEN acts as a phatidylinositol 3Ј-kinase/Akt pathway by dephosphorylating tumor suppressor gene. phosphoinositides at the 3 position (23–27). This biochemical action may contribute to the regulation of cell growth and survival (23, 26, 28). To investigate how PTEN mutations are involved in the tumorigenesis of glioma, we examined glioma samples for both p53 and PTEN mutations and evaluated the Received 5/30/00; revised 7/26/00; accepted 7/26/00. The costs of publication of this article were defrayed in part by the functional significance of PTEN mutations by in vitro phosphoi- payment of page charges. This article must therefore be hereby marked nositide phosphatase assay and examination of LOH at chromo- advertisement in accordance with 18 U.S.C. Section 1734 solely to some 10q23. indicate this fact. 1 Supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare, Japan. 2 To whom requests for reprints should be addressed, at Department of Clinical Oncology, Institute of Development, Aging and Cancer, To- 3 The abbreviations used are: GBM, glioblastoma multiforme; AA, hoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. anaplastic astrocytoma; LOH, loss of heterozygosity; RT-PCR, reverse

Phone: 81-22-717-8547; Fax: 81-22-717-8548; E-mail: chikashi@ transcription-PCR; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; nt, idac.tohoku.ac.jp. nucleotide.

MATERIALS AND METHODS CCCTGTCATCTTCT-3Ј,5Ј-GCCCCTCCTCAGCATCTTAT- Tissue Specimens and Preparation. Sixty-six brain tu- 3Ј, and 5Ј-GGAAGAGAATCTCCGCAAGA-3Ј, were used. For mor samples from Japanese patients with glioma were collected PTEN sequencing, four primers, 5Ј-CACAGCTAGAACTTAT- Ј Ј Ј Ј from frozen surgical materials archived at the Department of CAAACC-3 ,5-TGCACATATCATTACACCAGTT-3 ,5- Ј Ј Neurosurgery, Tohoku University School of Medicine. These GGATTATAGACCAGTGGCAC-3 , and 5 -AGCATTTGCA- Ј were quickly frozen in liquid nitrogen after resection and were GTATAGAGCGT-3 , were used. The reactions were carried kept frozen at Ϫ80°C until nucleic acid extraction. None of the out in an automated DNA analyzer (ABI Prism 310; PE Bio- samples had been examined previously for genetic alterations. systems). The same tissue samples were also examined histopathologi- LOH Analysis. Genomic DNA was extracted from tu- cally and classified according to the WHO classification of mor tissues and the paired peripheral blood lymphocytes of 20 and 21 patients with grade III and IV tumors, respectively, using tumors of the central nervous system (29). Peripheral blood Sepagene (Sanko Junyaku, Tokyo, Japan). Three highly poly- samples for extraction of genomic DNA were available from morphic microsatellite markers flanking the PTEN gene, most of the tumor patients. D10S579, D10S215, and D10S541 (Research Genetics, Hunts- RT-PCR. mRNA was extracted from frozen tumor tissue ville, AL), were used to determine allelic imbalance of the using a Micro-Fast Track mRNA Isolation Kit (Invitrogen, PTEN locus (10q23). PCR was performed in a 10-␮l reaction Carlsbad, CA). Random hexamer-primed cDNA was synthe- mixture containing 2 ␮l of genomic DNA, 1 ␮lof10ϫ Ex Taq sized using a First-Strand cDNA Synthesis Kit (Amersham reaction buffer, 0.1 mM deoxynucleotide triphosphate, 0.5 unit Pharmacia Biotech, Piscataway, NJ). To amplify the p53 and the of Ex Taq polymerase (Takara Shuzo), and 0.5 ␮M forward ␮ PTEN cDNA, PCR was performed in a 20- l of reaction mix- primer with a fluorescent label and reverse primer using a ␮ ␮ ϫ ture containing 2 l of cDNA reaction, 2 lof10 native Pfu PC-800 programmed temperature control system (Astec). The reaction buffer, 1 unit of native Pfu polymerase (Stratagene, La PCR conditions were as follows: an initial 8 min at 94°C; 35 ␮ Jolla, CA), and 0.5 M each primer using a PC-800 programmed cycles of 30 s at 94°C, 30 s at 55°C to 58°C, and 45 s at 72°C; temperature control system (Astec, Fukuoka, Japan) for 4 min at and a 3-min final elongation at 72°C. A suitable amount of PCR 94°C; 35 cycles of 30 s at 94°C, 30 s at 60°C, and 2 min (with product (0.02ϳ4.0 ␮l) was mixed with a Gene Scan-500 4 s/cycle of extension time) at 72°C; followed by a 5-min final TAMRA size standard (PE Applied Biosystems) and deionized elongation at 72°C. The p53-specific primers covering the open formamide and then denatured for 2 min at 95°C. The reactions reading frame were 5Ј-ACGGTGACACGCTTCCCTGGAT- were carried out in an automated DNA analyzer (ABI Prism TGG-3Ј and 5Ј-CTGTCAGTGGGGAACAAGAAGTGGAGA- 310; PE Applied Biosystems). 3Ј. The PTEN-specific primer pairs covering the open reading Bacterial Expression and Purification of PTEN. To frame were 5Ј-TTCTGCCATCTCTCTCCTCC-3Ј and 5Ј-TT- construct a histidine-tagged PTEN [(His)6-PTEN] expression TCATGGTGTTTTATCCCTC-3Ј. vector, the full-length open reading frame of the PTEN cDNA To amplify the p53 cDNA from yeast transformants, PCR was amplified by PCR using Pfu DNA polymerase (Stratagene) was performed in a 50-␮l of reaction mixture containing ϳ103 and primers 5Ј-TACGCGGATCCATGACAGCCATCATCA- yeast cells, 2 ␮lof10ϫ Ex Taq reaction buffer, 3.5 units of Ex AAGAG-3Ј with the BamHI site (shown in italic) and 5Ј- Taq polymerase (Takara Shuzo, Kyoto, Japan), and 0.5 ␮M each AGCCCAAGCTTTCAGACTTTTGTAATTTGTGTATGC-3Ј primer using a PC-800 programmed temperature control system with the HindIII site (shown in italic). Using Escherichia coli (Astec) for 10 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at strain JM109 (Toyobo, Osaka, Japan), the PCR product was 58.5°C, and 2 min at 72°C; followed by an 8-min final elonga- inserted into the BamHI and HindIII sites of the pQE30 vector tion at 72°C. The primers for PCR were 5Ј-CTCGTCATTGT- (Qiagen, Hilden, Germany), generating pHK101. PTEN cDNA TCTCGTTCC-3Ј and 5Ј-CGGGACAAAGCAAATGGAAG-3Ј. with missense mutations (C71Y, R130G, Y155C, and F341V) Yeast-based p53 Functional Assay. To detect function- or an in-frame 3-bp deletion (M199del) listed in Table 1 was ally inactivated p53 mutations, a yeast based-transactivation derived from tumors and introduced into the BamHI/HindIII assay called functional analysis of separated alleles in yeast sites of the pQE30 vector, generating pHK102 for C71Y, (FASAY) was performed as described previously (30). When pHK103 for R130G, pHK104 for Y155C, pHK105 for F341V, more than 20% of the yeast transformants showed a HisϪ and pHK106 for M199del. These vectors are identical to phenotype, we considered them positive for p53 mutation and pHK101, except for the specific mutations. DNA sequencing of carried out further p53 sequencing (see below). the PTEN coding sequences confirmed all of the specific mu-

DNA Sequencing. For sequencing of p53, p53 cDNA tations. To induce expression of (His)6-PTEN, the plasmid was derived from six independent HisϪ yeast transformants was transformed into E. coli strain M15 harboring pREP4 (Qiagen). used as a template (see above), which was considered likely to The resulting transformant was cultured in 50 ml of LB medium ϭ ␤ contain tumor-derived p53 mutations. For sequencing of PTEN, at 37°C by mid-log phase (A600 nm 0.6). Isopropyl -D- the PTEN cDNA derived from tumors was used directly as a thiogalactopyranoside was then added to the culture at a con- template. The PCR products were separated by 1% agarose gel centration of 0.2 mM, and incubation was continued for an electrophoresis, and the excised DNA bands were purified using additional6hat25°C. The bacterial cells were harvested by Suprec-01 (Takara Shuzo). All sequencing reactions were per- centrifugation, the supernatant was removed, and the bacterial formed using a Big Dye Terminator Cycle Sequencing Kit (PE pellet was frozen at Ϫ80°C. The frozen pellet was resuspended

Biosystems, Foster City, CA). For sequencing of p53, four in 1 ml of ice-cold 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, 5 primers, 5Ј-TTGTTGAGGGCAGGGGAGT-3Ј,5Ј-CTGGC- mM imidazole, 5 mM 2-mercaptoethanol, and 1 mM phenylmeth-

Table 1 Mutations in p53 and PTEN genes and LOH at chromosome 10q23 in human gliomas LOH at 10q23f Age FASAY Tumor (yr) Sex Histologya Gradeb %HisϪc p53 mutationd PTEN mutatione D10S579 D10S215 D10S541 74.1 1 F GG I 4 (Ϫ)(Ϫ) nenene 68 14 M GG I 4 (Ϫ)(Ϫ) nenene 69 44 F GG I 0 (Ϫ)(Ϫ) nenene 58.1 14 F PA I 8 (Ϫ)(Ϫ) nenene 95 60 M MG I 4 (Ϫ)(Ϫ) nenene 36 26 M A II 0 (Ϫ)(Ϫ) nenene 46 31 F A II 16 (Ϫ)(Ϫ) nenene 33 55 M A II 12 (Ϫ)(Ϫ) nenene 83 14 M E II 4 (Ϫ)(Ϫ) nenene 22 60 F E II 12 (Ϫ)(Ϫ) nenene 62 59 F CN II 0 (Ϫ)(Ϫ) nenene 84 69 F AMG II 8 (Ϫ)(Ϫ) nenene 5 4 F AA III 76 R249G(AGG3GGG) E7X(GAG3TAG) NI (ϩ)NI 9 29 M AA III 84 M243L(ATG3CTG) (Ϫ) NINI(Ϫ) 44 31 M AA III 92 R273C(CGT3TGT) (Ϫ)(Ϫ)(ϩ)NI 7 36 F AA III 92 H179Y(CAT3TAT) (Ϫ) nenene 89 36 M AA III 76 R273C(CGT3TGT) (Ϫ)(Ϫ)(ϩ)NI 92 37 M AA III 88 S240G(AGT3GGT) (Ϫ)(Ϫ)(Ϫ)NI 85 38 M AA III 96 C135G(TGC3GGC) (Ϫ)(ϩ)(ϩ)* NI 1 39 M AA III 92 P222L(CCG3CTG), (Ϫ) NI* NI* (ϩ) R337C(CGC3TGC) 14 46 F AA III 100 P177R(CCC3CGC), loss of expression (Ϫ) NI* (Ϫ) D186G(GAT3GGT) 66 57 M AA III 96 P152L(CCG3CTG) (Ϫ)(Ϫ)(ϩ)NI 94 26 M AA III 8 (Ϫ)(Ϫ) NI* NI NI 82 37 M AA III 16 (Ϫ)(Ϫ)(ϩ)* NI* (ϩ) 61 41 M AA III 4 (Ϫ)(Ϫ)(Ϫ)(ϩ)NI 64 46 F AA III 12 (Ϫ)(Ϫ)(ϩ)(ϩ)NI 65 68 M AA III 12 (Ϫ)(Ϫ)(ϩ)(Ϫ)NI 98 2 F APA III 4 (Ϫ)(Ϫ) nenene 97.2 70 F APA III 8 (Ϫ)(Ϫ) NI* (Ϫ)NI 26 48 F AO III 16 (Ϫ)(Ϫ)(Ϫ)(ϩ)(ϩ) 34.1 53 M AO III 13 (Ϫ)(Ϫ)(Ϫ)(ϩ)NI 15 57 F AO III 16 (Ϫ)(Ϫ) nenene 80 20 F AE III 8 (Ϫ)(Ϫ)(Ϫ)NI(Ϫ) 77 36 M AE III 4 (Ϫ)(Ϫ)(ϩ)(ϩ)NI 67 18 M AGG III 0 (Ϫ)(Ϫ)(Ϫ)(ϩ)NI 93 23 F GBM IV 100 D259V(GAC3GTC) (Ϫ) NI* NI NI* 38 27 M GBM IV 56 R273C(CGT3TGT) (Ϫ)(Ϫ)(Ϫ)(Ϫ) 76 30 M GBM IV 84 G245D(GGC3GAC) (Ϫ)(Ϫ) NI* (Ϫ)* 42.1 49 F GBM IV 100 Y234C(TAC3TGC) (Ϫ)(Ϫ)(ϩ)NI 70.3 58 F GBM IV 80 R248W(CGG3TGG) C71Y(TGT3TAT) (ϩ) NI* NI 20 66 F GBM IV 84 H179R(CAT3CGT) (Ϫ) nenene 2.1 76 F GBM IV 32 R273H(CGT3CAT) R130G(CGA3GGA) (ϩ)(ϩ)NI 87 24 F GBM IV 12 (Ϫ)(Ϫ)NI(ϩ)(Ϫ) 23.2 28 M GBM IV 0 (Ϫ)(Ϫ) nenene 59 28 M GBM IV 4 (Ϫ)(Ϫ) nenene 37 36 M GBM IV 12 (Ϫ)(Ϫ)(Ϫ)(ϩ)NI 60.2 46 M GBM IV 12 (Ϫ) nt.742insC(CCT3CCCT) ne ne ne 28.1 52 F GBM IV 4 (Ϫ)(Ϫ)(ϩ)(ϩ)(ϩ) 55 53 M GBM IV 4 (Ϫ) F341V(TTT3GTT) (Ϫ)(ϩ)(Ϫ) 4 54 M GBM IV 4 (Ϫ)(Ϫ)(Ϫ)(Ϫ)(Ϫ) 29 54 M GBM IV 0 (Ϫ)(Ϫ) nenene 12.2 57 F GBM IV 12 (Ϫ)(Ϫ) nenene 13 60 F GBM IV 8 (Ϫ) nt.510–514del(GTCAGAG3GG) (ϩ)(ϩ)(ϩ) 6 61 M GBM IV 0 (Ϫ)(Ϫ) nenene 24 61 M GBM IV 0 (Ϫ) loss of expression ne ne ne 81 62 M GBM IV 4 (Ϫ)(Ϫ)(Ϫ)(ϩ)(ϩ) 56 63 M GBM IV 4 (Ϫ)(Ϫ)(ϩ)(ϩ)NI 45 64 F GBM IV 0 (Ϫ)(Ϫ) nenene 3 65 M GBM IV 12 (Ϫ)(Ϫ) NI NI* NI 86 65 F GBM IV 12 (Ϫ) M199del (ϩ)(ϩ)* (Ϫ) 71 67 F GBM IV 16 (Ϫ) Y155C(TAT3TGT) NI (ϩ)NI 63 74 M GBM IV 8 (Ϫ)(Ϫ)(Ϫ)(ϩ)NI 91 13 M MD IV 44 R175H(CGC3CAC) (Ϫ) nenene 96 1 M MD IV 12 (Ϫ)(Ϫ) NININI 88 2 M MD IV 4 (Ϫ)(Ϫ)(ϩ) NI* NI 53.2 3 M PNET IV 88 S241F(TCC3TTC), (Ϫ)(Ϫ)(ϩ)(Ϫ) L348X(TTG3TAG) a Histological type: GG, ganglioglioma; PA, pilocytic astrocytoma; MG, meningioma; A, astrocytoma; E, ependymoma; CN, central neurocytoma; AMG, atypical meningioma; APA, anaplastic pilocytic astrocytoma; AO, anaplastic oligodendroglioma; AE, anaplastic ependymoma; AGG, anaplastic ganglioglioma; MD, medulloblastoma; PNET, primitive neuroectodermal tumor. b WHO histological grade from Kleihues et al. (29). c Result of FASAY. When more than 20% of the yeast transformants showed HisϪ phenotype, p53 sequencing was performed. FASAY, functional analysis of separated alleles in yeast. d,e Predicted amino acid substitution (nucleotide change); Ϫ, not detected; loss of expression, no transcript by RT-PCR. f ϩ, LOH; Ϫ, retention of heterozygosity; NI, not informative; ne, not examined; *, replication error.

ylsulfonyl fluoride, and bacteriolysis was performed by sonica- Table 2 Frequencies of mutations in p53 and PTEN genes and LOH tion until the cell suspension became transparent. After the at 10q23 addition of 10 ␮l of Tween 20, the lysate was incubated on ice Mutation frequency (%) for 30 min and then centrifuged at 15,000 ϫ g for 20 min. The Tumor type p53 PTENa LOH at 10q23b supernatant was mixed with 50 ␮l of Ni-NTA agarose (Qiagen) ␮ Adult AA 9/14 (64.3) 0/13 (0) 9/13 (69.3) for 30 min at 4°C; washed three times with 250 l of washing Adult GBM 7/27 (25.9) 7/26 (26.9) 13/18 (72.2) buffer containing 50 mM NaH2PO4 (pH 8.0), 1 M NaCl, and 50 Others 3/25 (12.0) 1/25 (4.0) 7/10 (70.0) Total 19/66 (28.8) 8/64 (12.5) 29/41 (70.7) mM imidazole; and eluted three times with 50 ␮l of the elution a Two cases with loss of PTEN expression were not included. buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and b WHO grade I and II tumors were not examined. 250 mM imidazole. Recovery of the (His)6-PTEN protein was confirmed by SDS-PAGE and Coomassie Blue staining. The eluate was then diluted with 350 ␮l of TED buffer containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM DTT, 300 mM NaCl, and1mM phenylmethylsulfonyl fluoride and applied to Nano- Detection of p53 Mutations Using a Yeast-based Func- cep (Pall Filtron, Northborough, MA), followed by centrifuga- tional Assay. We have previously described a yeast-based tion in a volume of 50 ␮l. TED buffer (450 ␮l) was then added, transcription assay that efficiently detects both germ-line and followed by further centrifugation to a final volume of 50 ␮l. somatic p53 mutations from patients’ lymphocytes, cell lines, The purified protein was stored in the presence of 2% glycerin and tumor tissues (30–32). We successfully amplified the at Ϫ80°C until use in a phosphatase assay (see below). 1.2-kb p53 cDNA by RT-PCR from all of the samples examined, showing that our materials had been suitably collected and Immunoblotting. To confirm the expression of (His)6- PTEN, the purified protein was fractionated by SDS-PAGE and stored without RNA degradation. Among the 66 tumors, p53 transferred electrophoretically to an Immobilon SQ filter (Mil- mutations that inactivated normal p53 function were found in 19 cases (28.8%; Table 2). Among these, p53 mutations were found lipore, Bedford, MA). (His)6-PTEN was detected using a monoclonal antibody, PTEN(A2B1) (Santa Cruz Biotechnology, most frequently in adult AAs (9 of 14, 64.3%), followed by Santa Cruz, CA), that recognizes amino acids 388–400 of adult GBMs (7 of 27, 25.9%) and other tumor types (3 of 25, human PTEN and then visualized using an enhanced chemilu- 12%) including a childhood AA, a medulloblastoma, and a minescence kit (Amersham Life Science, Buckinghamshire, primitive neuroectodermal tumor. These results support previ- United Kingdom). ous observations in other laboratories indicating that p53 muta- Phosphatase Assay. The phosphoinositide phosphatase tion occurs frequently in high-grade (grade III and IV) astrocytic assay described previously (24) was carried out in a buffer (20 tumors, especially AAs (5, 33, 34). Because the frequency of Ͻ ␮l) consisting of 100 mM Tris-HCl (pH 8.0), 10 mM DTT, 60 ␮M p53 mutation was significantly (P 0.05, Fisher’s exact test) 3 ␮ higher in adult AAs than in adult GBMs, our results also support [ H]Ins(1,3,4,5)P4 (0.01 Ci, New England Nuclear, Boston, ␮ the suggestion that there are at least two genetic pathways MA), and 1 g of the purified (His)6-PTEN protein (see above) at 37°C for 30 min. The reaction was terminated by the addition leading to GBM: (a) a primary or de novo pathway without p53 of 1 ml of stop solution consisting of 0.1 M HCOOH and 0.7 M mutation; and (b) a secondary or progressive pathway with p53 HCOONH . To separate the dephosphorylated product [3H]- mutation (2–4). Among the 19 tumors with p53 mutations, 3 4 Ϫ inositol 1,4,5-triphosphate from the substrate, the reaction mix- showed a 100% yeast His phenotype, 13 showed more than Ϫ Ϫ ture was applied to an AG1-X8 column (0.5 ml; Bio-Rad, 75% His phenotype, and 3 showed 20–75% His phenotype Hercules, CA) equilibrated with the stop solution and eluted (data not shown). Considering the contamination of tumor tis- with 5 ml of the stop solution. Radioactivity in the eluate was sues by normal cells, the tumor cells in most of the tumors (at measured using a liquid scintillation counter. least 16 cases) seemed to express only mutant transcripts. Al- though this could have been confirmed by analyzing LOH at the p53 locus, we speculate that both of the p53 alleles were RESULTS AND DISCUSSION inactivated in these tumors. The 66 tumors analyzed in this study are summarized in Detection of PTEN Mutations by RT-PCR-based Direct Table 1. Among them, WHO grade I tumors included three Sequencing. Recent studies have shown that PTEN mutations gangliogliomas, one pilocytic astrocytoma, and one meningi- are also found in a subset of astrocytic tumors. To examine the oma. WHO grade II tumors included three astrocytomas, two possible involvement of the PTEN mutation in our glioma ependymomas, one central neurocytoma, and one atypical me- samples, we chose a cDNA-based direct sequencing method ningioma. WHO grade III tumors included 15 AAs, 2 anaplastic covering the full-length PTEN coding sequence because the pilocytic astrocytomas, 3 anaplastic oligodendrogliomas, 2 ana- majority of the PTEN mutations reported were point mutations plastic ependymomas, and 1 anaplastic ganglioglioma. WHO and mapped within the open reading frame, and also because grade IV tumors included 27 GBMs, 3 medulloblastomas, and 1 direct sequencing is currently the most reliable method for primitive neuroectodermal tumor. The 15 AAs comprised 14 detecting such small mutations in the PTEN gene. In all but 2 of AAs from 11 male and 3 female adults (mean age at operation, the 66 tumors, we successfully amplified the 1.2-kb PTEN 40.5 years; range, 26–68 years) and 1 AA from a girl (age at cDNA. In the remaining two cases, we confirmed loss of PTEN operation, 4 years). The 27 GBMs were obtained from 15 male mRNA expression by repeated RT-PCR when the p53 tran- and 12 female adults (mean age at operation, 52.0 years; range, scripts were amplified. This suggested that both of the PTEN 23–76 years). alleles were inactivated in these cases by a mechanism such as

homozygous deletion at the PTEN locus, promoter mutation, or methylation of the gene, although this has yet to be confirmed. PTEN mutations were detected in 8 of the remaining 64 cases (12.5%; Tables 1 and 2), including four (50%) missense mutations (C71Y, R130G, Y155C, and F341V), two frameshift mutations (nt 510–514 del, and nt 742 ins C), one nonsense mutation (E7X), and one in-frame 3-bp deletion with a single amino acid elimination (M199del). Six of these eight mutations (with the exception of R130G and nt 742 ins C) were previously unreported mutations. The high frequency (50%) of missense mutations in this gene was consistent with the results of previous studies. Among these, PTEN mutations were found in 7 of 26 (26.9%) adult GBMs and in 1 childhood AA. No PTEN mutations were found in adult AAs and other tumors. These results are consistent with recent reports indicating that PTEN mutations occur in high-grade gliomas but not in low-grade gliomas and that the frequency of such mutations is higher in GBM than in AA (11, 35, 36), suggesting that PTEN mutation occurs at a later stage of glioma progression. Because PTEN mutations were detected in adult GBMs both with (two cases) and without (five cases) p53 mutations (Table 1), PTEN mutations seem to be involved in both the progressive and de novo pathways. Although a previous study suggested that PTEN and p53 mutations are exclusive events (14), our data indicated no Fig. 1 Bacterial expression and purification of PTEN. A, (His)6-PTEN correlation between them, consistent with the results of Zhou et expressed from plasmids pHK101 (WT), pHK102 (C71Y), pHK103 (R130G), pHK105 (F341V), and pHK106 (M199del)inE. coli was al. (35). purified using Ni-NTA agarose as described in “Materials and Meth- LOH at the PTEN Locus. Three microsatellite markers ods.” Approximately 1–2 ␮g of the proteins were separated by SDS- flanking the PTEN gene (chromosome 10q23), D10S579, PAGE and visualized by Coomassie Blue staining. M, molecular mark- D10S215, and D10S541, were used to evaluate allelic loss in 41 er; null, negative control using pQE30 vector; WT, wild-type PTEN. B, an experiment similar to A. Y155C was expressed from pHK104. C, high-grade gliomas. The frequency of LOH in informative cases immunoblotting analysis of (His)6-PTEN (wild-type) protein using a was 36.4% (12 of 33) for D10S579, 66.7% (24 of 36) for monoclonal antihuman PTEN antibody. D10S215, and 35.3% (6 of 17) for D10S541 (Table 1). Overall, LOH at one or more loci was found in 29 cases (70.7%; Table 2). There was no significant difference in the frequencies between adult AAs (9 of 13, 69.2%) and adult GBMs (13 of 18, Y155C, and F341V) and a small in-frame deletion (M199del) is 72.2%), suggesting that LOH at the 10q23 locus was an earlier problematic because the pathogenic effects of such mutations event than PTEN mutation. Seven of the eight tumors with cannot be elucidated until these mutations are tested for PTEN PTEN mutations (Table 1) were subjected to LOH analysis. function. Recently, it has been shown that PTEN appears to Each of these tumors with PTEN mutations also showed LOH at negatively control the phosphatidylinositol 3Ј-kinase/Akt sig- the 10q23 locus, suggesting that both alleles of the PTEN gene naling pathway that regulates cell growth and survival by de- were inactivated by a classical two-hit mechanism (37). Among phosphorylating phosphoinositides at the 3 position (23–27). the 22 cases of LOH in adult AAs and GBMs, PTEN mutations This phosphoinositol phosphatase activity of both wild-type were found in only seven tumors (31.8%). At present, we and mutant PTEN proteins has been analyzed in vitro using speculate that some of these cases may have had homozygous a bacterially expressed glutathione S-transferase-PTEN fu- deletion at the PTEN locus, which may have been underesti- sion protein and phosphatidylinositol 3,4,5-triphosphate or mated, especially in those with larger homozygous deletions Ins(1,3,4,5)P4 as a substrate, and the results have shown that beyond the three microsatellite markers. In addition, mutations tumor-derived missense mutations (including R15S, R15I, in the promoter region or methylation in the gene may also be C105F, C124S, G129R, and R129E) inactivate this phosphatase involved in inactivation of the PTEN gene. In fact, two tumors activity (23, 24, 27). To test whether the four missense muta- (see above) had loss of PTEN expression, possibly through the tions and the in-frame deletion detected in this study also above-mentioned mechanism. Alternatively, another unknown inactivate normal PTEN function, we purified (His)6-PTEN tumor suppressor gene within chromosome 10 might be respon- protein expressed in E. coli. The expression of PTEN was sible for the earlier stage of glioma formation (38). confirmed by SDS-PAGE followed by immunoblotting using a Effect of PTEN Mutations on Normal PTEN Function. PTEN-specific antibody (Fig. 1). The purified wild-type PTEN

Among the eight mutations in the PTEN coding sequence (Table protein dephosphorylated Ins(1,3,4,5)P4, whereas all of the 1), the protein-truncating mutations (E7X, nt 510–514 del, and PTEN mutants failed to do so (Fig. 2). These results indicate nt 742 ins C) are recognized to result in functional loss because that all of the missense mutations and the small in-frame dele- they may remove a potentially functional domain of the PTEN tion found in this study inactivate normal PTEN function when product. Interpretation of missense mutations (C71Y, R130G, they are translated. Combined with the results of RT-PCR,

acts as a tumor suppressor gene, which is inactivated in a manner similar to that of other tumor suppressor genes.

ACKNOWLEDGMENTS We thank Kentaro Nakayama for technical assistance with LOH analysis.

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Hideaki Kato, Shunsuke Kato, Toshihiro Kumabe, et al.

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