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Clinical Cancer Research Vol. 6, 3937-3943, October 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Functional Evaluation of p53 and PTEN Gene Mutations in Gliomas1

Hideaki Kato, Shunsuke Kato, Toshihiro Kumabe, Yukihiko Sonoda, Takashi Yoshimoto, Satoshi Kato, Shuang-Yin Han, Takao Suzuki, Hiroyuki Shibata, Ryunosuke Kanamaru and Chikashi Ishioka2

Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575 [H. K., Sh. K., Sa. K., S-Y. H., T. S., H. S., R. K., C. I.], and Department of Neurosurgery, Tohoku University School of Medicine, Sendai 980-8574 [H. K., T. K., Y. S., T. Y.], Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
We screened mutations of two major tumor suppressor genes, p53 and PTEN, in 66 human brain tumors using a yeast-based functional assay and cDNA-based direct sequencing, respectively. The frequency of p53 mutations was 28.8% (19 of 66) and was higher in anaplastic astrocytoma (9 of 14, 64.3%,) than in glioblastoma multiforme (GBM; 7 of 27, 25.9%,), supporting previous speculation that there are at least two genetic pathways leading to GBM, a de novo pathway without p53 mutation and a "progressive" pathway with p53 mutation. PTEN mutation was observed in 8 of 64 tumors (12.5%), mainly GBMs (7 of 26, 26.9%), both with and without p53 mutation. These results suggest that mutation of the PTEN gene is a later event than that of the p53 gene in glioma progression and is associated with both the genetic pathways. All of the detected PTEN missense mutations and an in-frame small deletion inactivated PTEN phosphoinositide phosphatase activity in vitro. Because the tumors containing PTEN mutations also showed loss of heterozygosity in the chromosome 10q23 region flanking the PTEN gene, our data clearly indicate that inactivation of both PTEN alleles occurs in a subset of high-grade gliomas, therefore confirming the previous idea that PTEN acts as a tumor suppressor gene.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
GBM3 is the most common as well as the most aggressive primary brain tumor and is clinically separable into two subtypes. One type is primary or de novo GBM, which is characterized by later onset, rapid tumor growth and a short clinical course. The other type is secondary or "progressive" GBM, which arises from a less malignant precursor lesion, including astrocytoma or AA, and is characterized by earlier onset, slow tumor growth, and less aggressive clinical features (for reviews, see Refs. 1, 2, 3 ). Although the two types are generally indistinguishable histologically, recent molecular genetic analyses have provided evidence to support at least two distinct pathways contributing to the tumorigenesis of GBM. Primary GBM is closely associated with the absence of p53 mutation and the presence of gene amplification such as that of EGFR, whereas secondary GBM is associated with the presence of p53 mutation and the absence of gene amplification (2, 3, 4) . As well as these alterations, loss of chromosome 10q and/or 10p occurs in the majority of GBMs and AAs and is associated with both de novo and progressive GBMs (5, 6, 7, 8) . This suggests that there may be unknown tumor suppressor gene(s) on chromosome 10 that may be involved in the tumorigenesis of either of the two GBM subtypes. At chromosome 10q23, the PTEN gene (also called MMAC1 and TEP1) was recently identified as a putative tumor suppressor gene, and mutations of this gene have been reported in human glioma and other tumors (9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . In addition, germ-line PTEN mutations have been found in the dominant cancer susceptibility syndromes Cowden disease and BannayanZonana syndrome (19 , 20) . Furthermore, enforced expression of PTEN cDNA suppresses tumor cell growth both in vitro and in vivo (21, 22, 23) . These results strongly suggest that PTEN acts as a tumor suppressor gene in GBM and other tumors, although the functional significance of the detected mutations has not been tested. Recent studies have shown that PTEN protein acts as a phosphoinositol phosphatase and negatively controls the phosphatidylinositol 3'-kinase/Akt pathway by dephosphorylating phosphoinositides at the 3 position (23, 24, 25, 26, 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 functional significance of PTEN mutations by in vitro phosphoinositide phosphatase assay and examination of LOH at chromosome 10q23.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Tissue Specimens and Preparation.
Sixty-six brain tumor samples from Japanese patients with glioma were collected from frozen surgical materials archived at the Department of Neurosurgery, Tohoku University School of Medicine. These were quickly frozen in liquid nitrogen after resection and were kept frozen at -80°C until nucleic acid extraction. None of the samples had been examined previously for genetic alterations. The same tissue samples were also examined histopathologically and classified according to the WHO classification of tumors of the central nervous system (29) . Peripheral blood samples for extraction of genomic DNA were available from most of the tumor patients.

RT-PCR.
mRNA was extracted from frozen tumor tissue using a Micro-Fast Track mRNA Isolation Kit (Invitrogen, Carlsbad, CA). Random hexamer-primed cDNA was synthesized using a First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech, Piscataway, NJ). To amplify the p53 and the PTEN cDNA, PCR was performed in a 20-µl of reaction mixture containing 2 µl of cDNA reaction, 2 µl of 10x native Pfu reaction buffer, 1 unit of native Pfu polymerase (Stratagene, La Jolla, CA), and 0.5 µM each primer using a PC-800 programmed temperature control system (Astec, Fukuoka, Japan) for 4 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 60°C, and 2 min (with 4 s/cycle of extension time) at 72°C; followed by a 5-min final elongation at 72°C. The p53-specific primers covering the open reading frame were 5'-ACGGTGACACGCTTCCCTGGATTGG-3' and 5'-CTGTCAGTGGGGAACAAGAAGTGGAGA-3'. The PTEN-specific primer pairs covering the open reading frame were 5'-TTCTGCCATCTCTCTCCTCC-3' and 5'-TTTCATGGTGTTTTATCCCTC-3'.

To amplify the p53 cDNA from yeast transformants, PCR was performed in a 50-µl of reaction mixture containing ~103 yeast cells, 2 µl of 10x Ex Taq reaction buffer, 3.5 units of Ex Taq polymerase (Takara Shuzo, Kyoto, Japan), and 0.5 µM each primer using a PC-800 programmed temperature control system (Astec) for 10 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 58.5°C, and 2 min at 72°C; followed by an 8-min final elongation at 72°C. The primers for PCR were 5'-CTCGTCATTGTTCTCGTTCC-3' and 5'-CGGGACAAAGCAAATGGAAG-3'.

Yeast-based p53 Functional Assay.
To detect functionally inactivated p53 mutations, a yeast based-transactivation assay called functional analysis of separated alleles in yeast (FASAY) was performed as described previously (30) . When more than 20% of the yeast transformants showed a His- phenotype, we considered them positive for p53 mutation and carried out further p53 sequencing (see below).

DNA Sequencing.
For sequencing of p53, p53 cDNA derived from six independent His- yeast transformants was used as a template (see above), which was considered likely to contain tumor-derived p53 mutations. For sequencing of PTEN, the PTEN cDNA derived from tumors was used directly as a template. The PCR products were separated by 1% agarose gel electrophoresis, and the excised DNA bands were purified using Suprec-01 (Takara Shuzo). All sequencing reactions were performed using a Big Dye Terminator Cycle Sequencing Kit (PE Biosystems, Foster City, CA). For sequencing of p53, four primers, 5'-TTGTTGAGGGCAGGGGAGT-3', 5'-CTGGCCCCTGTCATCTTCT-3', 5'-GCCCCTCCTCAGCATCTTAT-3', and 5'-GGAAGAGAATCTCCGCAAGA-3', were used. For PTEN sequencing, four primers, 5'-CACAGCTAGAACTTATCAAACC-3', 5'-TGCACATATCATTACACCAGTT-3', 5'-GGATTATAGACCAGTGGCAC-3', and 5'-AGCATTTGCAGTATAGAGCGT-3', were used. The reactions were carried out in an automated DNA analyzer (ABI Prism 310; PE Biosystems).

LOH Analysis.
Genomic DNA was extracted from tumor tissues and the paired peripheral blood lymphocytes of 20 and 21 patients with grade III and IV tumors, respectively, using Sepagene (Sanko Junyaku, Tokyo, Japan). Three highly polymorphic microsatellite markers flanking the PTEN gene, D10S579, D10S215, and D10S541 (Research Genetics, Huntsville, AL), were used to determine allelic imbalance of the PTEN locus (10q23). PCR was performed in a 10-µl reaction mixture containing 2 µl of genomic DNA, 1 µl of 10x Ex Taq reaction buffer, 0.1 mM deoxynucleotide triphosphate, 0.5 unit of Ex Taq polymerase (Takara Shuzo), and 0.5 µM forward primer with a fluorescent label and reverse primer using a PC-800 programmed temperature control system (Astec). The PCR conditions were as follows: an initial 8 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 55°C to 58°C, and 45 s at 72°C; and a 3-min final elongation at 72°C. A suitable amount of PCR product (0.02~4.0 µl) was mixed with a Gene Scan-500 TAMRA size standard (PE Applied Biosystems) and deionized formamide and then denatured for 2 min at 95°C. The reactions were carried out in an automated DNA analyzer (ABI Prism 310; PE Applied Biosystems).

Bacterial Expression and Purification of PTEN.
To construct a histidine-tagged PTEN [(His)6-PTEN] expression vector, the full-length open reading frame of the PTEN cDNA was amplified by PCR using Pfu DNA polymerase (Stratagene) and primers 5'-TACGCGGATCCATGACAGCCATCATCAAAGAG-3' with the BamHI site (shown in italic) and 5'-AGCCCAAGCTTTCAGACTTTTGTAATTTGTGTATGC-3' with the HindIII site (shown in italic). Using Escherichia coli strain JM109 (Toyobo, Osaka, Japan), the PCR product was inserted into the BamHI and HindIII sites of the pQE30 vector (Qiagen, Hilden, Germany), generating pHK101. PTEN cDNA with missense mutations (C71Y, R130G, Y155C, and F341V) or an in-frame 3-bp deletion (M199del) listed in Table 1Citation was derived from tumors and introduced into the BamHI/HindIII sites of the pQE30 vector, generating pHK102 for C71Y, pHK103 for R130G, pHK104 for Y155C, pHK105 for F341V, and pHK106 for M199del. These vectors are identical to pHK101, except for the specific mutations. DNA sequencing of the PTEN coding sequences confirmed all of the specific mutations. To induce expression of (His)6-PTEN, the plasmid was transformed into E. coli strain M15 harboring pREP4 (Qiagen). The resulting transformant was cultured in 50 ml of LB medium at 37°C by mid-log phase (A600 nm = 0.6). Isopropyl ß-D-thiogalactopyranoside was then added to the culture at a concentration of 0.2 mM, and incubation was continued for an additional 6 h at 25°C. The bacterial cells were harvested by centrifugation, the supernatant was removed, and the bacterial pellet was frozen at -80°C. The frozen pellet was resuspended in 1 ml of ice-cold 50 mM NaH2PO4 (pH 8.0), 500 mM NaCl, 5 mM imidazole, 5 mM 2-mercaptoethanol, and 1 mM phenylmethylsulfonyl fluoride, and bacteriolysis was performed by sonication until the cell suspension became transparent. After the addition of 10 µl of Tween 20, the lysate was incubated on ice for 30 min and then centrifuged at 15,000 x g for 20 min. The supernatant was mixed with 50 µl of Ni-NTA agarose (Qiagen) for 30 min at 4°C; washed three times with 250 µl of washing buffer containing 50 mM NaH2PO4 (pH 8.0), 1 M NaCl, and 50 mM imidazole; and eluted three times with 50 µl of the elution buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 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 µl of TED buffer containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM DTT, 300 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride and applied to Nanocep (Pall Filtron, Northborough, MA), followed by centrifugation in a volume of 50 µl. TED buffer (450 µl) was then added, followed by further centrifugation to a final volume of 50 µl. The purified protein was stored in the presence of 2% glycerin at -80°C until use in a phosphatase assay (see below).


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  		Table 1 %Mutations in p53 and PTEN genes and LOH at chromosome 10q23 in human gliomas

 
Immunoblotting.
To confirm the expression of (His)6-PTEN, the purified protein was fractionated by SDS-PAGE and transferred electrophoretically to an Immobilon SQ filter (Millipore, Bedford, MA). (His)6-PTEN was detected using a monoclonal antibody, PTEN(A2B1) (Santa Cruz Biotechnology, Santa Cruz, CA), that recognizes amino acids 388–400 of human PTEN and then visualized using an enhanced chemiluminescence kit (Amersham Life Science, Buckinghamshire, United Kingdom).

Phosphatase Assay.
The phosphoinositide phosphatase assay described previously (24) was carried out in a buffer (20 µl) consisting of 100 mM Tris-HCl (pH 8.0), 10 mM DTT, 60 µM [3 H]Ins(1 ,3 ,4 ,5) P4 (0.01 µCi, New England Nuclear, Boston, MA), and 1 µg of the purified (His)6-PTEN protein (see above) at 37°C for 30 min. The reaction was terminated by the addition of 1 ml of stop solution consisting of 0.1 M HCOOH and 0.7 M HCOONH4. To separate the dephosphorylated product [3 H]inositol 1,4,5-triphosphate from the substrate, the reaction mixture was applied to an AG1-X8 column (0.5 ml; Bio-Rad, Hercules, CA) equilibrated with the stop solution and eluted with 5 ml of the stop solution. Radioactivity in the eluate was measured using a liquid scintillation counter.


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
The 66 tumors analyzed in this study are summarized in Table 1Citation . Among them, WHO grade I tumors included three gangliogliomas, one pilocytic astrocytoma, and one meningioma. WHO grade II tumors included three astrocytomas, two ependymomas, one central neurocytoma, and one atypical meningioma. WHO grade III tumors included 15 AAs, 2 anaplastic pilocytic astrocytomas, 3 anaplastic oligodendrogliomas, 2 anaplastic ependymomas, and 1 anaplastic ganglioglioma. WHO grade IV tumors included 27 GBMs, 3 medulloblastomas, and 1 primitive neuroectodermal tumor. The 15 AAs comprised 14 AAs from 11 male and 3 female adults (mean age at operation, 40.5 years; range, 26–68 years) and 1 AA from a girl (age at operation, 4 years). The 27 GBMs were obtained from 15 male and 12 female adults (mean age at operation, 52.0 years; range, 23–76 years).

Detection of p53 Mutations Using a Yeast-based Functional Assay.
We have previously described a yeast-based transcription assay that efficiently detects both germ-line and somatic p53 mutations from patients’ lymphocytes, cell lines, and tumor tissues (30, 31, 32) . We successfully amplified the 1.2-kb p53 cDNA by RT-PCR from all of the samples examined, showing that our materials had been suitably collected and stored without RNA degradation. Among the 66 tumors, p53 mutations that inactivated normal p53 function were found in 19 cases (28.8%; Table 2Citation ). Among these, p53 mutations were found most frequently in adult AAs (9 of 14, 64.3%), followed by adult GBMs (7 of 27, 25.9%) and other tumor types (3 of 25, 12%) including a childhood AA, a medulloblastoma, and a primitive neuroectodermal tumor. These results support previous observations in other laboratories indicating that p53 mutation occurs frequently in high-grade (grade III and IV) astrocytic tumors, especially AAs (5 , 33 , 34) . Because the frequency of p53 mutation was significantly (P < 0.05, Fisher’s exact test) higher in adult AAs than in adult GBMs, our results also support the suggestion that there are at least two genetic pathways leading to GBM: (a) a primary or de novo pathway without p53 mutation; and (b) a secondary or progressive pathway with p53 mutation (2, 3, 4) . Among the 19 tumors with p53 mutations, 3 showed a 100% yeast His- phenotype, 13 showed more than 75% His- phenotype, and 3 showed 20–75% His- phenotype (data not shown). Considering the contamination of tumor tissues by normal cells, the tumor cells in most of the tumors (at least 16 cases) seemed to express only mutant transcripts. Although this could have been confirmed by analyzing LOH at the p53 locus, we speculate that both of the p53 alleles were inactivated in these tumors.


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  		Table 2 %Frequencies of mutations in p53 and PTEN genes and LOH at 10q23

 
Detection of PTEN Mutations by RT-PCR-based Direct Sequencing.
Recent studies have shown that PTEN mutations are also found in a subset of astrocytic tumors. To examine the possible involvement of the PTEN mutation in our glioma samples, we chose a cDNA-based direct sequencing method covering the full-length PTEN coding sequence because the majority of the PTEN mutations reported were point mutations and mapped within the open reading frame, and also because direct sequencing is currently the most reliable method for detecting such small mutations in the PTEN gene. In all but 2 of the 66 tumors, we successfully amplified the 1.2-kb PTEN cDNA. In the remaining two cases, we confirmed loss of PTEN mRNA expression by repeated RT-PCR when the p53 transcripts were amplified. This suggested that both of the PTEN 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 1Citation and 2Citation ), 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)Citation , 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 correlation between them, consistent with the results of Zhou et al. (35) .

LOH at the PTEN Locus.
Three microsatellite markers flanking the PTEN gene (chromosome 10q23), D10S579, D10S215, and D10S541, were used to evaluate allelic loss in 41 high-grade gliomas. The frequency of LOH in informative cases was 36.4% (12 of 33) for D10S579, 66.7% (24 of 36) for D10S215, and 35.3% (6 of 17) for D10S541 (Table 1)Citation . Overall, LOH at one or more loci was found in 29 cases (70.7%; Table 2Citation ). There was no significant difference in the frequencies between adult AAs (9 of 13, 69.2%) and adult GBMs (13 of 18, 72.2%), suggesting that LOH at the 10q23 locus was an earlier event than PTEN mutation. Seven of the eight tumors with PTEN mutations (Table 1)Citation were subjected to LOH analysis. Each of these tumors with PTEN mutations also showed LOH at the 10q23 locus, suggesting that both alleles of the PTEN gene were inactivated by a classical two-hit mechanism (37) . Among the 22 cases of LOH in adult AAs and GBMs, PTEN mutations were found in only seven tumors (31.8%). At present, we speculate that some of these cases may have had homozygous deletion at the PTEN locus, which may have been underestimated, especially in those with larger homozygous deletions beyond the three microsatellite markers. In addition, mutations in the promoter region or methylation in the gene may also be involved in inactivation of the PTEN gene. In fact, two tumors (see above) had loss of PTEN expression, possibly through the above-mentioned mechanism. Alternatively, another unknown tumor suppressor gene within chromosome 10 might be responsible for the earlier stage of glioma formation (38) .

Effect of PTEN Mutations on Normal PTEN Function.
Among the eight mutations in the PTEN coding sequence (Table 1)Citation , the protein-truncating mutations (E7X, nt 510–514 del, and nt 742 ins C) are recognized to result in functional loss because they may remove a potentially functional domain of the PTEN product. Interpretation of missense mutations (C71Y, R130G, Y155C, and F341V) and a small in-frame deletion (M199del) is problematic because the pathogenic effects of such mutations cannot be elucidated until these mutations are tested for PTEN function. Recently, it has been shown that PTEN appears to negatively control the phosphatidylinositol 3'-kinase/Akt signaling pathway that regulates cell growth and survival by dephosphorylating phosphoinositides at the 3 position (23, 24, 25, 26, 27) . This phosphoinositol phosphatase activity of both wild-type and mutant PTEN proteins has been analyzed in vitro using a bacterially expressed glutathione S-transferase-PTEN fusion protein and phosphatidylinositol 3,4,5-triphosphate or Ins(1 ,3 ,4 ,5) P4 as a substrate, and the results have shown that tumor-derived missense mutations (including R15S, R15I, C105F, C124S, G129R, and R129E) inactivate this phosphatase activity (23 , 24 , 27) . To test whether the four missense mutations and the in-frame deletion detected in this study also inactivate normal PTEN function, we purified (His)6-PTEN protein expressed in E. coli. The expression of PTEN was confirmed by SDS-PAGE followed by immunoblotting using a PTEN-specific antibody (Fig. 1)Citation . The purified wild-type PTEN protein dephosphorylated Ins(1 ,3 ,4 ,5) P4, whereas all of the PTEN mutants failed to do so (Fig. 2)Citation . These results indicate that all of the missense mutations and the small in-frame deletion found in this study inactivate normal PTEN function when they are translated. Combined with the results of RT-PCR, sequencing, and LOH studies, these results confirm the fact that PTEN function is frequently inactivated in adult GBMs by a small mutation plus loss of the remaining allele or by loss of expression. One interesting observation is that not only missense mutations in the NH2-terminal phosphatase domain but also missense mutations in the COOH-terminal phosphatase domain (F341V) affect normal PTEN phosphatase activity. Recently, Georgescu et al. (39) analyzed the phosphatase activity and structural stability of two missense mutations (L345Q and T348I) close to F341V, located in one of the two predicted ß-strands of the COOH-terminal PTEN (between amino acids 342 and 349). They showed that these missense mutations probably affected the phosphatase activity as a result of conformational changes in PTEN. Although we did not analyze the effect of F341V on the predicted ß-strand structure, F341V mutation may also inactivate PTEN function through a mechanism similar to L345Q and T348I mutations.



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  		Fig. 1. Bacterial expression and purification of PTEN. A, (His)6-PTEN expressed from plasmids pHK101 (WT), pHK102 (C71Y), pHK103 (R130G), pHK105 (F341V), and pHK106 (M199del) in E. coli was purified using Ni-NTA agarose as described in "Materials and Methods." Approximately 1–2 µg of the proteins were separated by SDS-PAGE and visualized by Coomassie Blue staining. M, molecular marker; null, negative control using pQE30 vector; WT, wild-type PTEN. B, an experiment similar to A. Y155C was expressed from pHK104. C, immunoblotting analysis of (His)6-PTEN (wild-type) protein using a monoclonal antihuman PTEN antibody.

 


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  		Fig. 2. Phosphoinositol phosphatase activity of PTEN. One µg of (His)6-PTEN protein was assayed for phosphoinositol phosphatase activity against [3H]Ins(1,3,4,5)P4 as described in "Materials and Methods." The radioactivity of the dephosphorylated product was counted, and the results from two independent experiments were normalized to wild-type PTEN as 100%. WT, wild-type.

 
In summary, we screened mutations of p53 and PTEN in human brain tumors using a yeast-based functional assay and cDNA-based direct sequencing, respectively. The frequency of p53 mutation was higher in adult AAs than in adult GBMs, supporting the previous suggestion that there are at least two genetic pathways leading to GBM: (a) a primary or de novo pathway without p53 mutation; and (b) a secondary or progressive pathway with p53 mutation. The frequency of PTEN mutation was higher in adult GBMs than in adult AAs, suggesting that in glioma progression, the mutation event in the PTEN gene occurs later than that in the p53 gene. The detected PTEN missense mutations and in-frame small deletion were further evaluated by in vitro phosphatase assay using bacterially expressed PTEN proteins. We observed that all of the examined mutations inactivated normal PTEN phosphatase activity. Because the tumors containing PTEN mutations also presented LOH at the PTEN locus, our data clearly indicate that inactivation of both PTEN alleles occurred in a subset of high-grade gliomas. Our findings confirmed the previous idea that PTEN 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.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture and the Ministry of Health and Welfare, Japan. Back

2 To whom requests for reprints should be addressed, at Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8547; Fax: 81-22-717-8548; E-mail: chikashi{at}idac.tohoku.ac.jp Back

3 The abbreviations used are: GBM, glioblastoma multiforme; AA, anaplastic astrocytoma; LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; nt, nucleotide. Back

Received 5/30/00; revised 7/26/00; accepted 7/26/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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