REDUCED EXPRESSION OF THE Aa SUBUNIT OF PROTEIN PHOSPHATASE2A IN HUMAN GLIOMAS IN THE ABSENCE OF MUTATIONS IN THE Aa ANDAb SUBUNIT GENESStefano COLELLA

1, Hiroko OHGAKI1, Ralf RUEDIGER

2, Fan YANG1, Mitsutoshi NAKAMURA

1, Hironori FUJISAWA1,

Paul KLEIHUES1 and Gernot WALTER

2*1International Agency for Research on Cancer (IARC), Lyon, France2Department of Pathology, University of California at San Diego, La Jolla, CA, USA

Protein phosphatase 2A (PP2A) consists of 3 subunits: thecatalytic subunit, C, and the regulatory subunits, A and B.The A and C subunits both exist as 2 isoforms (a and b) andthe B subunit as multiple forms subdivided into 3 families, B,B* and B(. It has been reported that the genes encoding theAa and Ab subunits are mutated in various human cancers,suggesting that they may function as tumor suppressors. Weinvestigated whether Aa and Ab mutations occur in humangliomas. Using single strand conformational polymorphismanalysis and DNA sequencing, 58 brain tumors were investi-gated, including 23 glioblastomas, 19 oligodendrogliomas and16 anaplastic oligodendrogliomas. Only silent mutationswere detected in the Aa gene and no mutations in the Abgene. However, in 43% of the tumors, the level of Aa wasreduced at least 10-fold. By comparison, the levels of the Baand Ca subunits were mostly normal. Our data indicate thatthese tumors contain very low levels of core and holoenzymeand high amounts of unregulated catalytic C subunit.© 2001 Wiley-Liss, Inc.

Key words: PP2A; subunit expression; oligodendroglioma; glioblas-toma

Protein phosphatase2A (PP2A) isan abundant serine/threonine-specific phosphatase, which is involved in many basic cellularprocesses including cell growth control (for review see Mumbyand Walter1). PP2A exists in cells as 2 abundant forms, coreenzyme made up of a 36-kDa catalytic C subunit and 1 65-kDaregulatory A subunit and holoenzyme composed of core enzymeand 1 of several regulatory B subunits. The A and C subunits bothexist as 2 isoforms (a and b ), whereas the B subunits fall into 3families designated B,2–5, B9 (also called B56)6–10 and B0.11–14Amodel demonstrating how the A, B and C subunits interact in theholoenzyme is shown in Figure 1.15,16 The existence of so manyregulatory subunits suggests that the various forms of PP2A havedistinct functions. In addition, numerous interactionsof PP2A withgrowth regulatory viral17–19 and cellular20 proteins have beenreported, underscoring its regulatory role.

Based on the observation that okadaic acid is both a stronginhibitor of PP2A and a potent tumor promoter, it has beensuggested that PP2A might be atumor suppressor.1,17 Additionalevidence for such a role came from the recent discovery that thegene encoding the Ab subunit of PP2A, located on chromosome11q23 in a region showing a high frequency of loss of heterozy-gosity (LOH), was mutated in 15% of primary lung tumors, 6% oflung tumor-derived cell lines and 15% of primary colon tumors.21

In addition, both the Aa and Ab subunit isoforms of PP2A werefound to be genetically altered in a variety of primary humancancers,22 and mutations were described in the Ab subunit gene in4 colon cancer cases.23 Further support for a roleof PP2A in tumorsuppression comes from the finding that the B9 subunit of PP2Abinds to the tumor suppressor adenomatous polyposis coli protein(APC) and downregulates the abundance of b-catenin in the Wntsignaling pathway, resulting in growth inhibition. Therefore, lossof PP2A may activate the Wnt pathway and stimulate growth.24

Glioblastomas are the most frequent and most malignant neo-plasmsof thehuman nervoussystem. Based on clinical and geneticcriteria, they fall into 2 classes: primary and secondary glioblas-tomas. Primary glioblastomas arise rapidly de novo without evi-

dence of a less malignant precursor, whereas secondary glioblas-tomas develop more slowly and originate from low-grade oranaplastic astrocytomas. Primary and secondary glioblastomas de-velop through different genetic pathways.25 Primary glioblastomasare characterized by epidermal growth factor receptor (EGFR)amplification, phosphatase and tensin homolog deleted on chro-mosome ten (PTEN) mutation and p16 deletion, with MDM2amplifications and p53 mutations being rare. Secondary glioblas-tomas, on the other hand, typically contain p53 and PTEN muta-tions.25 Oligodendrogliomas, which originate from differentiatedoligodendrocytes, appear either as well-differentiated neoplasms[World Health Organization (WHO) grade II ] or as highly mitoticand undifferentiated anaplastic tumors.26 The most frequent ge-netic alterations found in gradeII tumorsareLOH on theshort armof chromosome 1 and on chromosome 19q.26 In addition to thesechanges, anaplastic oligodendrogliomas frequently show loss ofalleles on the short arm of chromosome 9, and less frequently onchromosome10. Overexpression of theEGFR isseen in 50% of alloligodendrogliomas (grade II and III). 26

The reports that the Aa and Ab subunits of PP2A are mutatedin human cancer prompted us to examine whether such mutationsmight also occur in human gliomas, in particular because the geneencoding the Aa subunit maps to chromosome 19q13.1-13.4, aregion where LOH is observed in up to 70% of oligodendroglio-mas and in 25% of glioblastomas.27 By analyzing a large numberof tumors, we found only silent mutations in the Aa gene and nomutations in the Ab gene. However, the level of the Aa subunitwas strongly reduced in approximately 40% of both glioblastomasand oligodendrogliomas. In contrast, the levels of the Ba and Csubunits were rather constant, suggesting a loss of core and ho-loenzyme and ahigh abundance of unregulated catalytic C subunitin these tumors.

MATERIAL AND METHODS

Brain tissueThe surgical specimens of 58 gliomas were obtained from

patients treated at the Department of Neurosurgery at the Univer-sity Hospital Zurich, Switzerland. These tumors were classified asglioblastomas (WHO grade IV, 23 cases), oligodendrogliomas(WHO grade II , 19 cases) and anaplastic oligodendrogliomas(WHO grade III , 16 cases), according to WHO classification.

Grant sponsor: Tobacco-Related DiseaseResearch Program; Grant num-ber: 8RT-0037; Grant sponsor: Public Health Service; Grant number:CA-36111.

*Correspondence to: Department of Pathology 0612, University of Cal-ifornia, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0612, USA.Fax: 858-534-8942. E-mail: gwalter@ucsd.edu

Received 15 February 2001; Revised 26 April 2001; Accepted 8 May2001

Published online 19 July 2001

Int. J. Cancer: 93, 798–804 (2001)© 2001 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

Samples were frozen in liquid nitrogen and kept at280°C. RNA,DNA and protein were simultaneously extracted from about 200mg of the same portions of tumors, using Trizol (Gibco-BRL,Rockville, MD) following the manufacturer’s instruction.

LOH analyses on chromosome 19For 32 tumors (5 oligodendrogliomas, 2 anaplastic oligodendro-

gliomas and 25 glioblastomas) DNA samples from blood wereavailable and microsatellite analyses were carried out to assessLOH on chromosome 19q. All 10 microsatellite markers(D19S425, D19S178, D19S219, D19S412, D19S902, D19S606,D19S596, D19S246, D19S180 and D19S418) were purchasedfrom Research Genetics (Huntsville, AL). PCR was carried outusing a Genius DNA Thermal Cycler (Techne, Cambridge, UK) ina total volume of 10ml, consisting of 10 ng genomic DNA, 2mlof 53 PCR buffer, 6 pmol of each primer, 1.5 mM MgCl2, 1.25mM dNTPs, 0.225 U Taq polymerase (Sigma, St. Louis, MO), and0.5mCi [a-33P]dCTP (ICN Biomedicals, Costa Mesa, CA; specificactivity 3,000 Ci/mmol). Initial denaturation for 2 min at 95°C wasfollowed by 30–40 cycles with denaturation at 94°C for 45 sec,annealing at 57–61°C for 45 sec and extension at 72°C for 1 min.A final extension step for 7 min at 72°C was added. PCR products(10 ml) were mixed with an equal amount of loading dye (95%formamide, 20 mM EDTA, 0.05% xylene cyanol and 0.05%bromophenol blue), denatured at 95°C for 5 min and separated on7% denaturing polyacrylamide gels run at 70 W for 3.5–6 hr. Gelswere dried and exposed to X-ray film (BioMax, Kodak, Rochester,NY). For quantification of allelic loss, signal intensity was ana-lyzed using densitometry (Bio-Rad model GS-670) or a Phospho-rimager (Molecular Dynamics, Sunnyvale, CA). When a signalwas present in a normal DNA sample, a greater than 50% signal

reduction in the corresponding tumor sample was interpreted asLOH.28

Mutations in Aa subunit cDNAFirst-strand cDNA of Aa subunit mRNA was synthesized as

follows: 5 mg of total RNA in a volume of 10ml was heated to90°C for 2 min and then incubated at 37°C for 1 hr followingaddition of 10ml of a mixture containing 13 first-strand cDNAbuffer (Gibco-BRL), 10 mM DTT, 1 mM dNTPs, 25mg oligo dTprimers and 200 U Moloney murine leukemia virus reverse tran-scriptase (M-MuLV RT, Gibco-BRL). cDNA samples were thenheated to 95°C for 5 min, made up to 40ml with water and kept at220°C.

Screening for mutations was carried out by single strand con-formational polymorphism (SSCP) analysis of the entire Aa sub-unit coding region using the primers designed to amplify 9 over-lapping products from cDNA (primer sequences available uponrequest). Briefly, PCR was performed in a volume of 10mlcontaining 1–2ml of cDNA solution, 0.5 U Taq DNA polymerase(Sigma), 0.5mCi [a-33P]dCTP (ICN Biomedicals, specific activity3,000 Ci/mmol), 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.4mMof both sense and antisense primers, 10 mM Tris-HCl (pH 8.3) and50 mM KCl. PCR amplification was performed in a Genius Ther-mal Cycler (Techne) as follows: initial denaturation for 2 min at94°C, 30–40 cycles with denaturation at 94°C for 1 min, anneal-ing at 58-61°C for 1 min and extension at 72°C for 1 min and afinal extension step for 7 min at 72°C. After PCR, 5ml of productswere mixed with 12.5ml loading buffer (95% formamide, 20 mMEDTA, 0.05% xylene cyanol and bromophenol blue), denatured at95°C for 10 min and quenched on ice. Of the above mixture, 4mlwas analyzed on a nondenaturing 6% polyacrylamide gel contain-ing 8% glycerol run at 4 W for 14 hr at room temperature or on anondenaturing 6% polyacrylamide gel containing 6% glycerol runat 40 W for 4–5 hr with cooling by a fan. Gels were dried at 80°Cand autoradiographed for 12–48 hr.

Five samples that showed mobility shifts in the SSCP analysiswere analyzed further by direct DNA sequencing. For this purpose,full-length Aa subunit coding region was amplified in a mixturecontaining 1–2ml of cDNA mix, 13 Buffer II (Sigma), 1.25 mMMgCl2, 0.2 mM dNTPs, 20 mM of each of the required primersand 1 U Taq DNA polymerase (Sigma), in a total volume of 20ml.In a RoboCycler Gradient 96 (Stratagene, La Jolla, CA) the PCRreaction was carried out at 95°C for 5 min, followed by 35 cyclesof denaturation at 95°C for 1 min, annealing at 61°C for 70 sec andpolymerization at 72°C for 90 sec, followed by a final extension at72°C for 5 min. PCR products were purified on Micro Spin S-300HR Columns (Pharmacia Biotech) and sequenced using internalprimers with the DNA sequencing kit dRhodamine Terminator

FIGURE 1 – Model of the PP2A holoenzyme. The A subunit consistsof 15 repeats. Each repeat is composed of 2a helices connected by anintra-repeat loop. Adjacent repeats are connected by inter-repeat loops.The repeats are stacked to form an extended molecule. The B subunitsbind to repeats 1–10 whereas the C subunit binds to repeats 11–15.Polyomavirus small and middle T antigens bind to repeats 2–8,whereas SV40 small T binds to repeats 3–6. Major sites of subunitinteraction are the intra-repeat loops.

FIGURE 2 – Sequence analysis of Aa subunit cDNA of a normal control, case 228 and case 300. The * indicates the third nucleotide of codon574 (Asp), which is GAC in the wild-type control, heterozygous GAT in case 228 and homozygous GAT in case 300.

799Aa SUBUNIT OF PP2A REDUCED IN GLIOMAS

Cycle Sequencing Ready Reaction (Perkin Elmer, Norwalk, CT)and an ABI377 PRISM DNA sequencer.

Mutations in the PP2A Ab subunit gene

The SSCP analysis of the Ab subunit gene was performed byPCR amplification of genomic DNA. The PCR amplification of the15 exons was performed using the set of primers described by

Baysalet al.29 Briefly, the PCR was carried out in a volume of 10ml containing 1–2ml of cDNA solution, 0.5 U Taq DNA poly-merase (Sigma), 0.5mCi [a-33P]dCTP (ICN Biomedicals, specificactivity 3,000 Ci/mmol), 1.5 mM MgCl2, 0.2 mM of each dNTP,0.4mM of both sense and antisense primers, 10 mM Tris-HCl (pH8.3) and 50 mM KCl. Then 35 cycles of denaturation at 94°C for45 sec, annealing at 50-60°C for 1 min and extension at 72°C for1 min were performed in a RoboCycler Gradient 96 (Stratagene).

TABLE I – MUTATIONS IN THE GENE ENCODING THE Aa SUBUNIT OF PP2A AND EXPRESSION OF PP2AIN GLIOBLASTOMAS AND OLIGODENDROGLIOMAS

Sample # Case # Glioma type Base change Codon # Allele status A B C A/C

1 201 GB C1530T Ala 462 Hetero 0.2 40 15 0.013 x2 258 GB — — — 5 20 20 0.254 290 GB — — — 0.2 40 5 0.04 x5 296 GB — — — 0.2 20 5 0.04 x7 233 GB — — — 0.5 20 7 0.07 x8 288 GB C1866T Asp 574 Hetero 0.5 20 7 0.07 x9 292 GB C1866T Asp 574 Hetero 0.5 40 7 0.07 x

10 302 GB — — — 5 40 20 0.2511 314 GB C621T Ser 159 Hetero 5 10 15 0.3312 257 GB — — — 5 20 20 0.2513 344 GB C1866T Asp 574 Hetero 7 20 30 0.2314 287 GB — — — 2 20 15 0.1316 299 GB — — — 5 20 30 0.1717 295 GB C696T Ala 184 Hetero 10 20 30 0.3319 294 GB — — — 5 20 15 0.3320 234 GB — — — 5 40 15 0.3321 298 GB — — — 20 20 20 122 227 OL — — — 5 20 15 0.3323 369 OL — — — 0.2 20 15 0.013 x24 289 GB — — — 1 20 20 0.05 x25 72 GB — — — 7 20 15 0.4626 230 OL C621T Ser159 Hetero 7 20 20 0.3527 232 AOL — — — 0.1 20 5 0.02 x28 231 OL — — — 0.5 40 10 0.05 x29 256 GB — — — 1 20 10 0.1 x30 229 OL — — — 20 20 30 0.6731 228 AOL C621T Ser 159 Hetero 0.2 20 7 0.03 x

C1866T Asp 574 Hetero32 303 GB 7 20 20 0.3533 301 GB C621T Ser 159 Hetero 7 20 20 0.3534 300 GB C1866T Asp 574 Homo 5 20 20 0.2535 345 AOL — — — 0.5 20 20 0.025 x36 346 AOL — — — 1 20 20 0.05 x37 247 AOL — — — 2 20 20 0.1 x38 348 OL — — — 1 20 20 0.05 x39 349 OL — — — 0.2 20 7 0.03 x40 350 AOL — — — 1 5 7 0.1441 351 AOL — — — 2 10 15 0.1342 80 OL — — — 0.2 20 7 0.03 x43 352 AOL — — — 0.1 20 5 0.02 x44 353 OL — — — 0.5 20 10 0.05 x45 354 OL — — — 1 20 10 0.1 x46 355 OL — — — 10 10 10 148 356 OL — — — 15 5 10 1.549 357 OL C1866T Asp 574 Hetero 20 5 15 1.3350 358 OL — — — 1 5 15 0.07 x51 346 AOL — — — 2 5 10 0.252 359 OL — — — 1 10 10 0.1 x53 360 OL — — — 0.1 20 15 0.006 x54 361 OL — — — 2 10 10 0.255 14 AOL — — — 2 20 10 0.256 362 AOL C1866T Asp 574 Hetero 5 10 15 0.3357 363 AOL C1866T Asp574 Hetero 20 10 20 158 364 AOL — — — 2 20 15 0.1359 365 OL — — — 15 10 20 0.7560 366 OL C1866T Asp574 Hetero 10 10 2 561 92 AOL — — — 0.2 20 2 0.1 x62 317 AOL — — — 2 20 10 0.263 368 AOL — — — 2 10 5 0.4

Sample #: Note that the sample numbers go from 1 to 63 while the total number of gliomas analyzed is 58 (samples 3, 6, 15, 18 and 47 werenot available for analysis). The amounts of A, B and C were determined as described in the legend to Fig. 3. Sample 21, containing the sameamounts of A, B and C as normal brain tissue and cultured cells, was used as a standard. A/C ratios# 0.1 are marked with an x. GB,glioblastoma; OL, oligodendroglioma; AOL, anaplastic oligodendroglioma; Hetero, heterozygous; Homo, homozygous.

800 COLELLA ET AL.

After PCR, 5ml of products was mixed with 12.5ml loading buffer(95% formamide, 20 mM EDTA, 0.05% xylene cyanol and bro-mophenol blue), denatured at 95°C for 10 min and quenched onice. Of the above mixture, 4ml were analyzed on a nondenaturing6% polyacrylamide gel containing 8% glycerol run at 4 W for 14hr at room temperature or on a nondenaturing 6% polyacrylamidegel containing 6% glycerol run at 40 W for 4–5 hr with cooling bya fan. Gels were dried at 80°C and autoradiographed for 12–48 hr.

Western blot analysisProtein extracts from glioma tissue and some of the normal

brain samples (hippocampus) were prepared by the Trizol method(see above). The extracts were washed with 95% ethanol, dried,Dounce-homogenized in SDS-PAGE sample buffer (20ml per mgof tissue), incubated at 50°C for 1 hr, boiled, spun and the super-natants were stored at270°C. Frozen cortex and white matterfrom epileptic patients were Dounce-homogenized, incubated at50°C for 1 hr, boiled, spun and the supernatants were stored at270°C. Whole mouse brains were Dounce-homogenized, boiled,spun and the supernatants were stored at270°C. 10T1/2 mousefibroblasts were washed with PBS, lysed with SDS-PAGE samplebuffer, boiled and the supernatants were stored at270°C. Theprotein content was determined by precipitation with 10% trichlo-roacetic acid and use of the Micro BCA protein assay (Pierce).Equal amounts of protein were separated on 10% SDS polyacryl-amide gels and transferred to PVDF membrane (Immobilon-P,Millipore). To detect the A, B and C subunits, the followingprimary antibodies were used: rat monoclonal anti-A subunit(6G3),30 rabbit anti-Ba subunit produced against the peptideKGAVDDDVAEADY, 31 mouse monoclonal anti-C subunit from

Marc Mumby and rabbit anti-Ab subunit produced against thepeptide MAGASELGTGPGK coupled to keyhole limpet hema-cyanin with glutaraldehyde. Appropriate secondary antibodiescoupled to horseradish peroxidase (Jackson ImmunoResearch)were used as well as Renaissance chemiluminescence reagent(NEN Life Science Products). To compare protein patterns invarious samples, duplicate gels were run in parallel to the gels usedfor Western blotting and stained with 0.3 g/L Coomassie.

RESULTS

Mutational analysis of Aa cDNA and Ab genes in glioblastomasand oligodendrogliomas

SSCP analysis of cDNA encoding the Aa subunit revealedmobility shifts in 14 brain tumors. Direct sequencing identified 4different silent mutations in 8 glioblastomas, 3 oligodendroglio-mas and 3 anaplastic oligodendrogliomas (Fig. 2 and Table I). Aheterozygous C621T base change was detected at codon 159 (Ser)in glioblastoma cases 314 and 301, in oligodendroglioma 230, andin anaplastic oligodendroglioma 228. A heterozygous C696Tchange at codon 184 (Ala) was found in glioblastoma 295 and aC1530T change at codon 462 (Ala) in glioblastoma 201. AC1866T change at codon 574 (Asp) was observed in oligodendro-gliomas 357 and 366, anaplastic oligodendrogliomas 228, 362 and363, and glioblastomas 288, 292, 344 and 300. Except for glio-blastoma 300, the normal sequence was detected together with themutated base. Although we were unable to analyze RNAs fromnormal tissues from the patients with these tumors, we assume thatthese alterations are likely to be polymorphisms, as they did notaffect the protein sequence. SSCP analysis of the Ab genes re-

FIGURE 3 – Strong reduction of Aa subunit expression in 43% of brain tumors. Protein extracts from 58 gliomas, labeled 1–63 on the top, wereanalyzed by SDS-PAGE and Western blotting with antibodies specific for the A, Ba, and C subunits as described in Material and Methods. Eachextract contained 20mg of total protein in 20ml. In addition, 1, 2, 5 and 10ml (1, 2, 5 and 10mg) of sample # 21 were loaded to estimate byvisual comparison the amounts of A, B and C subunits in the various samples. The results of this comparison are summarized in Table I. Sample# 21 was chosen as a reference because its ratio of A to B to C is the same as that of normal brain and 10T1/2 cells (Fig. 4). To determine theabsolute amounts of A subunit in the tumor samples, 1, 2, 4, 8, 16 and 32 ng of purified A subunit were also loaded. All 58 glioma samples werequantitated twice by SDS-PAGE and Western blotting with very similar results.

801Aa SUBUNIT OF PP2A REDUCED IN GLIOMAS

vealed no mobility shift in any of the 58 brain tumors investigated,suggesting the absence of mutations in the Ab subunit gene.

Reduced expression of the Aa subunitAlthough mutations altering the sequence of the Aa and Ab

subunits were not found, it seemed possible that their expressionwas changed at the transcriptional or posttranscriptional level. Toinvestigate this possibility, equal amounts of total protein from all58 gliomas were analyzed by Western blotting using antibodiesspecific for the A, Ba and C subunits. As shown in Figure 3, theAa subunit was highly expressed in some tumors (e.g., #10–22)but almost absent in others (e.g., #4–9), whereas the levels of theB and C subunits were remarkably constant with the exception ofsamples #60 and #61. To quantitate the signals of the Aa, Ba andC subunits in the different tumor samples, the signals were com-pared to serial dilutions of tumor sample #21 shown on the left ofFigure 3. This sample was used as a standard because it has similarsubunit amounts and ratios as normal human and mouse brains aswell as 10T1/2 mouse fibroblasts (Fig. 4). We have previouslydetermined that all normal tissues and cell lines tested containsimilar amounts of core and holoenzyme, and an A/C ratio ofapproximately 1. As shown in Table I, Aa levels were stronglyreduced in 8 of 23 (35%) glioblastomas, 10 of 19 (53%) oligo-dendrogliomas and 7 of 16 (44%) anaplastic oligodendrogliomas.In 43% of all tumors (marked by an x), the Aa levels were at least10-fold lower than the corresponding C subunit levels, indicatedby an A/C ratio of 0.1 or less. This finding implies a dramatic lossof core and holoenzyme, and a high level of free catalytic Csubunit in these tumor cells.

To exclude general protein degradation as a cause of reducedAa subunit levels, we analyzed tumor samples by SDS-PAGE andstaining with Coomassie blue (Fig. 5). Importantly, we found thattumors with a strongly reduced Aa subunit level exhibited iden-tical overall protein patterns as tumors with a normal level of Aasubunit. Examples are #7 for low expression and #10 for high(normal) expression, or #9 for low expression and #13 for high(normal) expression. These data suggest that the reduced Aa

subunit expression in 43% of gliomas was not due to overallprotein degradation. To check whether the Aa subunit might besubject to postmortem proteolysis, a normal adult mouse wassacrificed and kept at room temperature for 6 hr before the brainwas harvested in SDS-PAGE sample buffer. Western blotting ofthis sample showed no sign of Aa subunit degradation in compar-ison to a brain sample that was harvested immediately after sac-rifice (Fig. 4). Therefore, the reduced level of the Aa subunit islikely due to reduced expression rather than proteolysis.

Different amounts of purified Aa subunit (1–32 ng) were alsoloaded in Figure 3. From these samples we determined that theabsolute amount of Aa subunit in tumors expressing normalamounts and in normal brain is in the order of 0.1% of total cellprotein in accordance with previous findings. Because Aa is gen-erally expressed at 40 times higher levels than Ab,32 the signals onthe Western blots reflect mainly expression of the Aa subunit.Using an Ab-specific polyclonal antibody, we were unable todetect Ab in the gliomas, although this finding does not exclude avery low level of expression.

LOH on chromosome 19A total of 300 loci were tested and 231 (77%) were found to be

informative. LOH on chromosome 19q was detected in 2 of 5(40%) oligodendrogliomas, 1 of 2 (50%) anaplastic oligodendro-gliomas and 4 of 23 (17%) glioblastomas. There was no correlationbetween reduced Aa subunit expression and LOH on chromosome19q13 (p 5 0.669).

DISCUSSION

Our studies demonstrate that genetic changes altering the aminoacid sequence of the Aa and Ab subunits did not take place in 58gliomas. However, we discovered that the expression of the Aasubunit was reduced 10-fold or more in almost half of all gliomas.The small amount of Aa subunit in these tumors may come fromsome residual normal tissue in the tumor sample. If this were thecase, our data would indicate that expression of the Aa subunit iscompletely turned off in these tumors. Preliminary data fromRT-PCR using a low number of cycles indicate that tumors with

FIGURE 4 – Expression of the Aa subunit in normal human brain.Protein extracts of 13 samples from 7 normal (noncancerous) humanbrains (lanes 2–14) were analyzed by SDS-PAGE and Western blot-ting with antibodies specific for the A, Ba and C subunits. Per lane, 10mg of protein were loaded. For comparison, 10mg of glioma # 21, 10mg of mouse brain harvested either 0 or 6 hr postmortem and 10mg ofa 10T1/2 cell lysate were loaded. Lane 2, case ZH1A hippocampus;lane 3, ZH2A hippocampus; lane 4, 1851B hippocampus; lane 5,3451A white matter; lane 6, 3451B cortex; lane 7, 3500 cortex; lane 8,3501 white matter; lane 9, 3543 cortex; lane 10, 3544 white matter;lane 11, 3549 cortex; lane 12, 3550 white matter; lane 13, 3581 cortex;lane 14, 3582 white matter. The average A/C ratio in the 13 normalhuman brain samples (lanes 2–14) was 16 0.15. Absence of post-mortem Aa subunit degradation is shown in lanes 15 and 16; 10T1/2in lane 17. The samples analyzed in lanes 5–14 were generouslyprovided by Dr. Otmar Wiestler.

FIGURE 5 – Coomassie blue staining patterns of brain tumor sampleswith high (normal) and low expression of Aa subunit. Samples withvirtually identical protein patterns (#7 and #10; #9 and #13) differmarkedly in their Aa subunit levels [#7 and #9 low; #10 and #13 high(normal), see Fig. 3].

802 COLELLA ET AL.

reduced Aa subunit expression contain normal amounts of Aasubunit mRNA. This finding may suggest that the Aa subunitexpression is downregulated in tumors by translational or post-translational mechanisms. The finding that the C and B subunitlevels were relatively constant suggests that the reduction in Aa isnot simply caused by nonspecific degradation. This interpretationis also supported by the observation that tumors with low levels ofAa subunit show no sign of general protein degradation in com-parison to tumors with a high (normal) level of Aa subunit.Furthermore, we found that the Aa subunit was stable in postmor-tem mouse brain for at least 6 hr at room temperature. Therefore,it is not likely that the Aa subunit was degraded postmortem in thehuman brains.

It has been reported recently that the Aa subunit gene is mutatedin lung carcinoma, breast carcinoma and melanoma.22 Importantly,we have shown that in every case the mutation renders the Aasubunit defective in binding B or C subunits, depending onwhether the mutation is located in the N-terminal B subunit bind-ing domain (repeats 1–10) or in the C-terminal C subunit bindingregion (repeats 11–15) (Fig. 1).33 Furthermore, cancer-associatedmutations were found in the gene encoding the Ab subunit.21–23Asin the case of Aa, most of the mutant Ab subunits were defectivein subunit interaction.34 Therefore, it appears that A subunit func-tion is hampered in cancer cells either as a result of gene mutationor changes in gene expression. In each case, the result is anabundance of unregulated, free C subunit. Whether the free Csubunit actually promotes growth is an open question.

What might be the consequences of Aa subunit mutation or lackof Aa subunit expression for PP2A activity or function? The Aasubunit is an extended molecule consisting of 15 repeats. B sub-units bind to repeats 1–10 and the C subunit to repeats 11–15.15,16

B and C subunits physically interact while bound to Aa (Fig. 1).Importantly, B subunits cannot bind C subunits in the absence ofAa. Therefore, a consequence of a drop in Aa subunit levels is anincrease in the concentration of free unbound C and B subunits,which normally do not exist in cells, and a corresponding reductionin the levels of core and holoenzyme. Because the substrate spec-ificity of the monomeric C subunit is markedly different from coreand holoenzyme,35 we anticipate that the phosphorylation state ofmany PP2A substrates will change dramatically as a result of Aasubunit reduction. Another consequence is that the unbound Bsubunits are unable to target the phosphatase to specific subcellularlocations8,10,35or to mediate interactions with other growth-regu-latory proteins (for review see Virshup20). For example, PP2A hasbeen proposed to function as a tumor suppressor in the Wntsignaling pathway by binding to the tumor suppressor APCthrough a regulatory B9 subunit, resulting in downregulation ofb-catenin.24 A decrease in Aa subunit expression would reduceformation of the B9-containing holoenzyme, causing decreasedbinding to APC as well as increasedb-catenin levels and conse-quently enhanced cell proliferation.

ACKNOWLEDGEMENTS

We thank Dr. O. Wiestler for normal brain tissue samples andDr. M. Mumby for anti-C subunit monoclonal antibody.

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