TP53 promoter methylation in human gliomas
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- Amatya, V.J., Naumann, U., Weller, M. et al. Acta Neuropathol (2005) 110: 178. doi:10.1007/s00401-005-1041-5
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Methylation of the promoter region of tumor suppressor genes may be associated with transcriptional silencing and tumor progression. The 5′ region of the TP53 gene does not contain a CpG island, but a basal promoter region of 85 bp is essential for its full promoter activity. In the present study, we assessed whether TP53 promoter methylation is present in malignant glioma cells and whether this is associated with reduced TP53 expression. Methylation-specific PCR revealed TP53 promoter methylation in three (U87MG, LNT-229, T98G) out of six malignant glioma cell lines studied. Treatment with 5-aza-2’-deoxycytidine (5-aza-dC) led to up-regulated expression of TP53 mRNA and protein in U87MG and T98G cells, suggesting that promoter methylation is associated with reduced expression in some malignant glioma cells. We then assessed TP53 promoter methylation in primary tissue of low-grade gliomas, and observed TP53 promoter methylation in 29/48 (60%) low-grade astrocytomas, 11/18 (61%) oligoastrocytomas, and 31/42 (74%) oligodendrogliomas. Promoter methylation of the p14ARF gene, another gene involved in the TP53 pathway, was detected by methylation-specific PCR in 5/49 (10%) low-grade astrocytomas, 7/18 (39%) oligoastrocytomas, and 15/41 (37%) oligodendrogliomas. Our previous and present data show alterations of at least one of TP53 promoter methylation, p14ARF promoter methylation, and TP53 mutations in 43/49 (88%) of low-grade astrocytomas, 15/18 (83%) of oligoastrocytomas, and 35/42 (83%) oligodendrogliomas, suggesting that disruption of the TP53/p14ARF pathway is frequent in all histological types of low-grade glioma.
KeywordsTP53 mutationTP53 methylationp14ARF methylationLow-grade astrocytomaOligodendrogliomas
Although the 5′ region of the TP53 gene does not contain a CpG island , it has a promoter region with 16 CpG dinucleotides [nucleotides (nt) 631–950, provided the major transcriptional site as no. 843; NCBI Gene Bank database accession no. X54156] , which contains a basal promoter region (85 bp; nt 760–844) that is essential for full promoter activity . Schroeder and Mass  have shown that methylation in the promoter region of the p53 gene reduces reporter gene activity. They found down-regulation of p53 in cultured cells transfected with a plasmid incorporating a p53 promoter containing methylated CpG dinucleotides . Using multiple methylation-sensitive restriction enzymes and a PCR-based assay for methylation status, Pogribny et al.  have also shown that alterations in single-site CpG methylation in the p53 promoter region can lead to initiation of de novo methylation and progressive spreading of methylation associated with transcriptional inactivation of the p53 gene in the rat. This was associated with reduced p53 expression . However, whether TP53 promoter methylation is associated with reduced protein expression in human neoplasms has remained unclear. In the present study, we assessed whether promoter methylation is present in malignant glioma cells and whether this is associated with reduced expression of TP53 mRNA and protein.
Low-grade gliomas (WHO grade II) are well-differentiated and slowly growing gliomas. Low-grade diffuse astrocytoma is genetically characterized by frequent TP53 mutations (50–70%) [23, 36], and consistently shows a tendency to progress into glioblastoma [16, 22], whereas the genetic hallmark of low-grade oligodendroglioma is frequent (70–80%) loss of heterozygosity (LOH) on chromosome 1p and 19q [23, 27, 33]. Oligoastrocytomas are tumors showing a conspicuous mixture of two distinct neoplastic cell types morphologically resembling the tumor cells of oligodendroglioma and diffuse astrocytoma . Oligoastrocytomas with LOH on chromosome 1p (25–50%) [18, 23] and/or 19q (45–70%) [18, 23] are genetically related to oligodendroglioma, and exhibit histologically a dominant oligodendroglioma component, whereas oligoastrocytomas with TP53 mutations (30–45%) are genetically related to astrocytoma and have a dominant astrocytoma component histologically [18, 23].
The TP53 gene is the principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage . Its multiple functions include regulation of gene transcription, induction of G1/S arrest, and promotion of apoptosis . The MDM2 protein binds to mutant and wild-type p53 proteins to inhibit their transcriptional activity, resulting in proteolytic degradation of p53 by the ubiquitin-proteasome pathway . p53 stimulates the expression of MDM2 protein, so that p53 and MDM2 form an autoregulatory feedback loop [24, 39]. p14ARF protein binds to the p53/MDM2 complex and inhibits MDM2-mediated degradation of p53, acting as an upstream positive regulator of p53 targeting MDM2, resulting in activation of p53 function at the G1/S transition. p53 down-regulates p14ARF expression, establishing an autoregulatory feedback loop between p53, MDM2, and p14ARF . Therefore, disruption of the TP53 pathway may occur through alterations of any of the p53, MDM2 or p14ARF genes. Of these, TP53 mutations and p14ARF methylation have been frequently reported [23, 36], but alterations of the MDM2 gene have rarely been seen in low-grade gliomas [11, 36].
In the present study, we assessed the frequencies of disruption of the p53/p14ARF pathway (TP53 mutation, TP53 promoter methylation, and p14ARF promoter methylation) in low-grade astrocytomas, oligoastrocytomas and oligodendrogliomas.
Materials and methods
Patients and tumor samples
Formalin-fixed, paraffin-embedded sections from low-grade gliomas (WHO grade II) were obtained from 109 patients (58 male, 51 female) treated at the University Hospital, Zurich, Switzerland. These tumors were classified according to the WHO classification of tumors of the nervous system : 49 were low-grade astrocytomas (40 fibrillary astrocytomas and 9 gemistocytic astrocytomas), 42 were oligodendrogliomas, and 18 were oligoastrocytomas. DNA was extracted from samples as previously described .
Glioblastoma cell lines and treatment with 5-aza-2′-deoxycytidine (5-aza-dC)
Six human malignant glioma cell lines (U138MG, LN-308, LNT-229, LN-18, U87MG, T98G), kindly provided by Dr. N. de Tribolet (Lausanne, Switzerland) [12, 37] were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 2 mM glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin. U87MG and LNT-229 cells have been reported to have wild-type TP53, while U138MG, LN-18, and T98G contained TP53 missense mutations, and LN-308 showed a heterozygous deletion of the TP53 gene [12, 37].
The cells were seeded at 105 cells in six-well plates, allowed to attach for 24 h and treated with 0, 10 or 30 µM of the demethylating agent, 5-aza-dC (Sigma Chemical Co., St. Louis, MO) in serum-containing medium for 6 days. Medium and 5-aza-dC were changed every 48 h. Genomic DNA was extracted using the Qiagen DNA extraction kit (Qiagen, Hilden, Germany).
Protein lysates were prepared as described elsewhere , and protein levels were assessed by immunoblot using TP53 (Santa Cruz sc-263, Bp53–12) and β-actin (Santa Cruz sc-1616, i19) antibodies.
TP53 promoter methylation
DNA methylation patterns in the CpG islands of the promoter region of the TP53 gene were determined by methylation-specific PCR (MSP) . Sodium bisulfite modification was performed using the CpGenome DNA Modification Kit (Chemicon International, Temecula, CA) according to the manufacturer’s protocol with minor modifications. Briefly, 1 µg DNA was denatured with NaOH (final concentration, 0.2 M) for 15 min at 50°C. Freshly prepared sodium bisulfite solution at pH 5 (550 µl) was added and incubated at 50°C for 16–20 h. The modified DNA was purified, treated with NaOH (final concentration, 0.3 M) for 5 min at room temperature, followed by ethanol precipitation. DNA was resuspended in deionized water, and used immediately or stored at −20°C.
Primer sequences have been reported previously : 5′-TTGGTAGGTGGATTATTTGTTT-3′ (sense) and 5′-CCAATCCAAAAAAACATATCAC-3′ (antisense) for the unmethylated reaction (PCR product 247 bp), and 5′-TTCGGTAGGCGGATTATTTG-3′ (sense) and 5′-AAATATCCCCGAAACCCAAC-3′ (antisense) for the methylated reaction (product 193 bp). The 5′ positions of forward primers for unmethylated and methylated PCRs correspond to nt 703 and 702 of NCBI GenBank accession no. X54156. PCR was carried out in a 10-µl mixture, containing 1× PCR buffer (10 mM TRIS pH 8.3, 50 mM KCl), 1.5–2.0 mM MgCl2, dNTPs (250 µM each), primers (0.5 µM each), 0.5 U Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) and approximately 40 ng bisulfite-modified DNA in a Biometra T3 Thermocycler (Göttingen, Germany). The cycling conditions consisted of an initial denaturation at 95°C for 7 min, 40 cycles of denaturing at 95°C for 45 s, annealing at 58–60°C for 45 s, and extension at 72°C for 1 min, followed by final extension for 5 min at 72°C.
CpGenome universal methylated control DNA (Chemicon) and blood DNA of healthy individuals were treated with bisulfite as mentioned above and used as methylated and unmethylated controls. Amplified products were electrophoresed on 3% agarose gels, and visualized under ultraviolet light after staining with ethidium bromide.
Sequencing of methylation-specific PCR products
After PCR amplification, PCR products from the positive methylated control (universal methylated control DNA), DNA from methylated malignant glioma cells and low-grade gliomas and unmethylated controls (blood DNA from normal healthy subjects) were subjected to direct sequencing. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Courtaboeuf, France) and sequenced with each sense and antisense primer on a Genetic Analyzer (ABI PRISM 3100, Applied Biosystems, CA) using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits (Perkin-Elmer Applied Biosystems).
p14ARF promoter methylation
Primer sequences of p14ARF for the methylated and unmethylated PCR were previously reported . PCR was carried out in a 10-µl mixture containing 1× PCR buffer (10 mM TRIS pH 8.3, 50 mM KCl), 1.5–2.0 mM MgCl2, dNTPs (250 µM each), primers (0.5 µM each), 0.5 U Platinum Taq DNA polymerase (Invitrogen) and approximately 40 ng bisulfite-modified DNA in a Biometra T3 Thermocycler. The cycling conditions consisted of an initial denaturation at 95°C for 7 min, 40 cycles of denaturing at 95°C for 45 s, annealing at 64°C (methylated) or 65°C (unmethylated) for 45 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min.
CpGenome universal methylated control DNA and blood DNA of healthy individuals were treated with bisulfite as mentioned above and were used as methylated and unmethylated controls. Amplified products were electrophoresed on 3% agarose gels, and visualized under ultraviolet light after staining with ethidium bromide.
Methylation of the promoter region and expression of the TP53 gene in malignant glioma cells before and after treatment with 5-aza-dC
TP53 promoter methylation was not detected in U138MG, LN-18 and LN-308 malignant glioma cell lines. There was no significant change in TP53 mRNA expression after treatment with 5-aza-dC in these cells (Fig. 1).
Sequencing of methylation-specific PCR products
TP53 promoter methylation in low-grade gliomas
Profile of low-grade glioma with TP53 and p14ARF promoter methylation
TP53 promoter methylation
p14ARF promoter methylation
p14ARF promoter methylation in low-grade gliomas
Promoter methylation of the p14ARF gene was detected in 15/41 (37%) oligodendrogliomas, 7/18 (39%) oligoastrocytomas, and 5/49 (10%) low-grade astrocytomas. The frequency of p14ARF methylation in low-grade astrocytomas was significantly lower than that in oligodendrogliomas and oligoastrocytomas (P=0.0052) (Table 1, Fig. 3).
Alterations in the p14ARF/p53 pathway in low-grade gliomas
The data on TP53 mutations have been reported previously . At least one of p14ARF methylation, TP53 methylation, or TP53 mutation was found in 43 of 49 (88%) low-grade astrocytomas, in 15 of 18 (83%) oligoastrocytomas, and in 35 of 42 (83%) oligodendrogliomas.
There was an inverse correlation between p14ARF methylation and TP53 mutations: low-grade gliomas with mutant TP53 had a significantly lower frequency of p14ARF methylation (6/41, 15%) than those with wild-type TP53 (21/63, 33%; P=0.04; Table 1).
Methylation of the TP53 promoter region in several human neoplasms has been reported. Using bisulfite sequencing, Kang et al.  showed TP53 promoter methylation in 3 of 19 (16%) breast carcinomas with wild-type TP53. Using methylation-sensitive restriction enzymes, Agirre et al.  observed TP53 promoter methylation in 8 of 25 cases (32%) of acute lymphoblastic leukemia. Pogribny and James  used a methylation-sensitive single nucleotide primer extension assay to show that hepatocellular carcinomas with wild-type TP53 exhibit TP53 promoter methylation and this was associated with reduced levels of mRNA (41% of normal liver tissues) .
In the present study, we found TP53 promoter methylation in three out of six malignant glioma cell lines (U87MG, LNT-229, T98G). Promoter methylation appears to be partial or to be present in only a fraction of the cells, since unmethylated sequences were also detected in these cells. However, treatment with 5-aza-dC reversed the methylation status, and this was associated with increased expression of TP53 mRNA and protein in U87MG (wild-type TP53) and T98G cells (mutated TP53). These results suggest that TP53 promoter methylation may be associated with reduced TP53 expression in tumors both with and without TP53 mutations. However, it remains to be clarified whether TP53 promoter methylation in TP53 mutated cells has biological significance.
In LNT-229 cells, treatment with 5-aza-dC led to increased TP53 protein expression without increasing the TP53 mRNA level. This may be explained by a nonspecific effect of 5-aza-dC, which alters the methylation status also in other genes, indirectly increasing TP53 protein expression. Alternatively, as was shown by Karpf et al. , 5-aza-dC may have induced a p53-dependent DNA damage-response pathway independent from the TP53 promoter methylation status without increasing transcript levels.
The present study further shows that TP53 promoter methylation is frequent in low-grade gliomas. Methylation-specific PCR revealed TP53 promoter methylation in 60% of low-grade astrocytomas, 61% of oligoastrocytomas and 73% of oligodendrogliomas. These results are in contrast to previously reported low frequencies of TP53 methylation in astrocytomas (2/24, 8%), glioblastomas (1/43, 2%) , and oligodendroglial tumors (0/41) . The reason for this discrepancy remains to be clarified, in particular since the primers used for methylation-specific PCR were identical in the present and previous studies [2, 8]. However, it should be noted that we also designed other PCR primers for the methylation-specific reaction (nt 632–894 for methylated PCR and nt 636–915 for unmethylated PCR) and found similar frequencies of TP53 methylation in low-grade gliomas (data not shown).
Loss of p14ARF expression in human neoplasms may be due to homozygous deletion or promoter methylation, while mutational inactivation is rare . Homozygous deletion of p14ARF has been reported in 25–44% of glioblastomas [11, 19, 21] and promoter methylation in 14–31% of cases, the overall frequency of homozygous deletion and/or methylation of the p14ARF gene being 50–55% [19, 21]. Homozygous p14ARF deletion was rare in low-grade gliomas [34, 36], while p14ARF promoter methylation was observed in low-grade astrocytomas (8–33%) [2, 8, 10, 34] and in oligodendrogliomas (21–45%) [2, 19, 38, 40]. In the present study, p14ARF promoter methylation was detected in 15 of 42 (36%) oligodendrogliomas and 5 of 49 (10%) low-grade astrocytomas. These data are consistent with the previously published data [2, 8, 38]. We also found that p14ARF promoter methylation is common in oligoastrocytomas (7/18, 39%).
Alterations of the p14ARF/TP53 pathway have been reported in gliomas [7, 11, 35]. Fulci et al.  found that TP53 mutations and p14ARF deletions were largely mutually exclusive in glioblastomas, and that the TP53 pathway was disrupted in 82% of malignant gliomas, either by TP53 mutations (32%) or by p14ARF deletions (55%) . Ichimura et al.  reported that 76% of glioblastomas, 72% of anaplastic astrocytomas, and 67% of astrocytomas had the TP53 pathway deregulated either by TP53 mutations, MDM2 amplification, or p14ARF homozygous deletion/mutation. In 50% of anaplastic oligodendrogliomas, the TP53 pathway was disrupted through either promoter methylation or homozygous deletion of the p14ARFgene, TP53 mutations or MDM2 amplification . Watanabe et al.  assessed alterations of the TP53/MDM2/p14(ARF) pathway in 46 low-grade astrocytomas. This pathway was altered in 32 of 46 cases (70%) by either TP53 mutation (25 cases) or p14(ARF) methylation (9 cases). Wolter et al.  investigated 7 oligodendrogliomas, 11 anaplastic oligodendrogliomas, 8 oligoastrocytomas, and 8 anaplastic oligoastrocytomas, and found p14ARFpromoter methylation in 41% of the tumors. p14ARFpromoter methylation was predominantly found in tumors without a TP53 mutation. In the present study, with a larger numbers of cases, we observed a significant inverse correlation between p14ARF methylation and TP53 mutations in low-grade gliomas, with frequent alteration of the p14ARF/TP53 pathway through either TP53 mutations, TP53 promoter methylation, or p14ARF promoter methylation in all histological types of astrocytic and oligodendroglial low-grade gliomas.
This study was supported by the Jaqueline Seroussi Memorial Foundation for Cancer Research (MW) and the Foundation for Promotion of Cancer Research, Japan.