Affiliations of authors: Departments of Neuropathology (CBK, BB, GR) and Dermatology (JR), Heinrich-Heine-University, Düsseldorf, Germany
Correspondence to: Guido Reifenberger, MD, PhD, Department of Neuropathology, Heinrich-Heine-University, Moorenstr. 5, D-40225 Düsseldorf, Germany (e-mail: reifenberger{at}med.uni-duesseldorf.de)
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
We investigated CTMP gene alterations and mRNA expression in 93 primary glioblastomas and nine glioma cell lines (A172, Hs683, U118MG, U178MG, T98G, CCF-STTG1 [all from American Type Culture Collection, Manassas, VA], U138MG [from German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany], U251MG, and U373MG [both from Dr. V. P. Collins, Department of Pathology, University of Cambridge, Cambridge, U.K.]). Human brain and tumor tissue samples were selected from the archives of the Department of Neuropathology, Heinrich-Heine-University, and investigated as approved by the local institutional review board at Heinrich-Heine-University. DNA and RNA were extracted as described (8). All tumors and cell lines were analyzed for CTMP coding region mutations (GenBank accession No. NM_176853) by using single-strand conformation polymorphism analysis and DNA sequencing (8,9), and for homozygous deletion of CTMP by using duplex polymerase chain reaction (PCR) for the simultaneous amplification of fragments from CTMP and the reference gene adenine phosphoribosyltransferase (APRT; GenBank accession No. NM_000485) (8,10). None of the tumors or cell lines had somatic mutations in CTMP or had a homozygous deletion of CTMP. Peripheral blood samples were available from 57 of the 93 patients. These samples were subjected to loss of heterozygosity analysis at the microsatellite loci D1S2344 and D1S305, which map proximal and distal to CTMP at 1q21, respectively. None of the samples showed allelic loss.
We next assessed the CTMP mRNA levels in the 93 glioblastomas by real-time reverse transcriptionPCR using primers binding to sequences shared by transcript variants 1 and 2 (GenBank accession Nos. NM_053055 and NM_176853). CTMP mRNA levels were normalized to the housekeeping gene ADP-ribosylation factor 1 (ARF1; GenBank accession No. NM_001658) and were compared with the normalized CTMP mRNA levels in non-neoplastic brain tissue samples obtained from an individual at autopsy or from patients undergoing surgery for chronic epilepsy or traumatic brain injury. Compared with those in non-neoplastic brain tissue, CTMP mRNA levels were reduced by at least 50% in 37 of 93 (40%) glioblastomas and in six of nine (67%) glioma cell lines (Figs. 1, A, and 2). To determine whether methylation differences in the CTMP promoter were responsible for the reduced mRNA expression, we selected 15 glioblastomas (10 tumors with CTMP mRNA levels reduced by 50% and five tumors with CTMP mRNA levels of
60% relative to non-neoplastic brain tissue), the nine cell lines, and non-neoplastic brain tissue samples from four different individuals for methylation analysis by sequencing the CTMP 5'-CpG island after sodium bisulfite modification of the DNA (10). For each sample, the genomic CTMP sequence from nucleotides -525 to 233 (GenBank accession No. AJ313515) was amplified in three fragments, each of which was cloned into pCR 2.1 vectors (Invitrogen, Carlsbad, CA). At least five clones of each fragment were sequenced to determine the methylation status. Hypermethylation (i.e., methylation of the majority of the 67 investigated CpG sites per sample) was observed in eight of 10 glioblastomas and in all cell lines with reduced CTMP mRNA levels (Figs. 1, B, and 2, A). By contrast, hypermethylation was observed neither in the non-neoplastic brain samples nor in the tumors and cell lines with normal CTMP mRNA levels (Fig. 2, A). To assess whether hypermethylation was responsible for the decreased mRNA expression, we treated the cell lines A172 and T98G, which were positive and negative for CTMP promoter hypermethylation, respectively, with the demethylating agent 5-aza-2'-deoxycytidine, the histone deacetylase inhibitor trichostatin A, or a combination of both. Relative to untreated A172 cells, CTMP mRNA expression increased in A172 cells treated with either drug (data not shown) or the combination (Fig. 1, D). Furthermore, the treatment of A172 cells with the combination of 5-aza-2'-deoxycytidine and trichostatin A resulted in a partial demethylation of the hypermethylated CTMP CpG island (Fig. 2, A). CTMP mRNA expression was unaltered in T98G cells after treatment with either drug alone (data not shown) or the combination (Fig. 1, D).
|
|
We next compared CTMP hypermethylation and/or reduced mRNA expression with previously published data (11) on PTEN and EGFR gene alterations in the 93 glioblastomas (Fig. 2, B). PTEN mutations were present in similar fractions of glioblastomas with and without reduced CTMP mRNA expression (27% versus 30%; P = .599, chi-square test) and with and without CTMP hypermethylation (32% versus 27%; P = .749, chi-square test). Similarly, five of six cell lines with CTMP hypermethylation (A172, U138MG, U178MG, U251MG, and U373MG) and two of three cell lines without CTMP hypermethylation (T98G and U118MG) carry PTEN mutations (12,13). Thus, we conclude that there is no association between PTEN inactivation and CTMP aberrations in glioblastomas. We also detected no relationship between CTMP hypermethylation and EGFR gene amplification (Fig. 2, B; P = .505, chi-square test).
To investigate the Akt activation status in primary glioblastomas, we performed western blot analyses of 15 selected tumors, including samples with CTMP hypermethylation (GB103D), PTEN mutations (GB47D and GB96D), EGFR amplification (GB98D, GB131D, GB139D, and GB140D), and alteration in none (GB60D, GB105D, GB147D, GB181D, and GS11D) or in any two or three of these genes (GB101D, GB191D, and GS3D). Relative to Akt protein levels in non-neoplastic brain tissue, all tumors showed increased levels of serine 473phosphorylated Akt protein (Fig. 1, E). In agreement with previous studies (4,6), these findings indicate that Akt is activated in the majority of glioblastomas, including those tumors with alterations in expression or function of PTEN, CTMP, and/or EGFR.
We found that CTMP mRNA levels were reduced by 50% or more compared with non-neoplastic brain tissue in 40% of primary glioblastomas and in 67% of glioma cell lines. Our findings suggest reduced CTMP expression as a novel molecular mechanism involved in the pathogenesis of glioblastomas. Reduced CTMP expression was closely associated with CTMP 5'-CpG island hypermethylation and could be restored by 5-aza-2'-deoxycytidine and trichostatin A treatment. These findings suggest a role for epigenetic DNA modification in the regulation of CTMP promoter activity. We found that CTMP gene promoter hypermethylation and reduced mRNA expression in glioblastomas are not associated with PTEN mutations and EGFR amplification. Whether our findings regarding CTMP expression and regulation are unique to glioblastomas or are also relevant to other tumors remains to be determined.
![]() |
NOTES |
---|
![]() ![]() ![]() ![]() |
---|
All PCR primer sequences used in this study are available on request from G. Reifenberger.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
1 Holland EC. Gliomagenesis: genetic alterations and mouse models. Nat Rev Genet 2001; 2: 1209.[CrossRef][ISI][Medline]
2 Collins VP. Cellular mechanisms targeted during astrocytoma progression. Cancer Lett 2002; 188: 17.[CrossRef][ISI][Medline]
3 Knobbe CB, Merlo A, Reifenberger G. Pten signaling in gliomas. Neuro-oncol 2002; 4: 196211.[ISI][Medline]
4 Holland EC, Celestino J, Dai C, Schaefer L, Sawaya RE, Fuller GN. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nat Genet 2000; 25: 557.[CrossRef][ISI][Medline]
5 Sonoda Y, Ozawa T, Aldape KD, Deen DF, Berger MS, Pieper RO. Akt pathway activation converts anaplastic astrocytoma to glioblastoma multiforme in a human astrocyte model of glioma. Cancer Res 2001; 61: 66748.
6 Choe G, Horvath S, Cloughesy TF, Crosby K, Seligson D, Palotie A, et al. Analysis of the phosphatidylinositol 3'-kinase signaling pathway in glioblastoma patients in vivo. Cancer Res 2003; 63: 27426.
7 Maira SM, Galetic I, Brazil DP, Kaech S, Ingley E, Thelen M, et al. Carboxyl-terminal modulator protein (CTMP), a negative regulator of PKB/Akt and v-Akt at the plasma membrane. Science 2001; 294: 37480.
8 van den Boom J, Wolter M, Kuick R, Misek DE, Youkilis AS, Wechsler DS, et al. Characterization of gene expression profiles associated with glioma progression using oligonucleotide-based microarray analysis and real-time reverse transcription- polymerase chain reaction. Am J Pathol 2003; 163: 103343.
9 Boström J, Cobbers JM, Wolter M, Tabatabai G, Weber RG, Lichter P, et al. Mutation of the PTEN (MMAC1) tumor suppressor gene in a subset of glioblastomas but not in meningiomas with loss of chromosome arm 10q. Cancer Res 1998; 58: 2933.[Abstract]
10 Wolter M, Reifenberger J, Blaschke B, Ichimura K, Schmidt EE, Collins VP, et al. Oligodendroglial tumors frequently demonstrate hypermethylation of the CDKN2A (MTS1, p16INK4a), p14ARF, and CDKN2B (MTS2, p15INK4b) tumor suppressor genes. J Neuropathol Exp Neurol 2001; 60: 117080.[ISI][Medline]
11 Knobbe CB, Reifenberger G. Genetic alterations and aberrant expression of genes related to the phosphatidylinositol-3'-kinase/protein kinase B (Akt) signal transduction pathway in glioblastomas. Brain Pathol 2003; 13: 50718.[ISI][Medline]
12 Schmidt EE, Ichimura K, Goike HM, Moshref A, Liu L, Collins VP. Mutational profile of the PTEN gene in primary human astrocytic tumors and cultivated xenografts. J Neuropathol Exp Neurol 1999; 58: 117083.[ISI][Medline]
13 Ishii N, Maier D, Merlo A, Tada M, Sawamura Y, Diserens AC, et al. Frequent co-alterations of TP53, p16/CDKN2A, p14ARF, PTEN tumor suppressor genes in human glioma cell lines. Brain Pathol 1999; 9: 46979.[ISI][Medline]
Manuscript received August 14, 2003; revised December 24, 2003; accepted January 12, 2004.
This article has been cited by other articles in HighWire Press-hosted journals:
![]() |
||||
|
Oxford University Press Privacy Policy and Legal Statement |