TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

Abstract

Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway.

Introduction

Glioblastoma (GB) (World Health Organization [WHO], G IV) is the most common primary tumor of the central nervous system in adults, with the most aggressive clinical course and fatal prognosis. Despite a better understanding of genetic alterations linked to GB, there has been little improvement in patient survival over the past decades. Before the era of first-line radio- and chemotherapy with alkylating agents, especially temozolomide (TMZ), median survival was below 1 year (Burger and Green, 1987). At present, by a combination of radiotherapy (RT) with the alkylating cytostatic drug TMZ median overall survival has been increased to 15 months (Stupp et al., 2009).

There are two types of GBs: primary that developed de novo and secondary that developed by progression from low-grade astrocytomas. In primary GB, the most common molecular alterations are LOH 10q (over 70%), EGFR amplification (about 40%), MDM2 amplification, LOH 10p and 10q, and p16^INK4a and PTEN mutation. In secondary GB, the first common molecular event in multistep carcinogenesis is the mutation of IDH1, TP53, and LOH on 17p, 10q, and 19q (Lang et al., 1994; Ohgaki et al., 2004; Ohgaki and Kleihus, 2009).

The TP53 mutation occurs in about 30% of cases of primary GB. However, this alteration is found in 65%–90% of cases of secondary GB, making it the most important molecular indicator, and occurs early in tumorigenesis (59% in low-grade astrocytoma and 53% in anaplastic astrocytoma) (Ohgaki et al., 2005). The TP53 gene encodes a protein playing key roles in the cell cycle, response to DNA damage, cell differentiation, and cell death, and thus maintains genomic stability (Bögler et al., 1995). Mutations of the TP53 gene often cause changes in the conformation of the protein and lead to its inactivation and accumulation of mutated protein in the nuclei of tumor cells (Hainaut et al., 1995).

In primary GB, TP53 point mutations are equally distributed through the 5–8 exons (hot-spot mutations), whereas in secondary GB most frequent mutation point concerns codon 248 and 273 (exon 7 and 8), and in CpG sites (methylation region) (Ohgaki et al., 2004). Interestingly, a mutation at codon 175 has been related to a shorter survival time of the patient (Ohgaki et al., 2004).The altered function of TP53 may result not only in mutations, but also from alterations in MDM2, MDM4, or CDKN2A/p14^ARF, which binds to MDM2 and inhibits MDM2-mediated TP53 degradation. The Cancer Genome Atlas (TCGA) Research Network project study has reported alterations in the TP53/MDM2/CDKN2A pathway in about 87% of investigated GBs (Cancer Genome Atlas Research Network, 2008). The majority of TP53 mutations and examples of TP53 loss of heterozygosity (54%) are found in the proneural type of GB that shows high expression of oligodendrocytic genes, and they were associated with a better prognosis and younger age (Verhaak et al., 2010).

Primary GB, in contrast with secondary, has been associated with the presence of EGFR amplification and absence of TP53 mutation (is considered mutually exclusive) (Ohgaki et al., 2004). It is not known whether changes in TP53 methylation status and expression is also mutually exclusive in GB.

Molecular markers that are related to better outcome of gliobastoma are MGMT promoter methylation and IDH1 mutations. MGMT promoter methylation is associated with a stronger benefit of radiochemotherapy in GBs and IDH1 mutations are a strong and independent predictor of survival in secondary GB (Stupp et al., 2009; Yan et al., 2009). Promoter methylation of MGMT was linked to the presence of G:C to A:T transition mutations in TP53 in many types of cancer, including GB (Bello et al., 2004). IDH1 mutations mainly occurred in secondary GBs (>70%), associated with young age of patients and with an increase in overall survival, but very rare in primary GBs (<5%) and IDH1 mutations is considered as a molecular marker discriminating secondary GBs from primary GBs (Nobusawa et al., 2009).

Methylation in cancer often occurs in the promoter regions of tumor suppressor genes and may lead to inactivation of gene expression (Esteller and Herman, 2002). Hypermethylation of promoters usually occurs at CpG island regions in about 60% of human genes with specific tumor types (Bird, 1996). The association of TP53 methylation and expression in GB is still not well known. Interestingly, the promoter of TP53 does not contain CpG islands in the 5′ region, and a basal promoter region of 85 bp (nt 760–844) is essential for enabling full promoter activity of TP53, either by directly blocking the binding transcription factor or by directing the binding of repressor proteins (Bienz-Tadmor et al., 1985; Tuck and Crawford, 1989). Further, this region has been shown to be methylated in several cancers (Pogribny and James, 2002; Agirre et al., 2003; Hurt et al., 2006). Experimental evidence suggests that the regulation of TP53 expression is complex and involves both translational (mainly in the case of post chronic genotoxic stress) (Reisman and Rotter, 1993), and post-translational mechanisms to control the intracellular levels of the p53 protein after acute DNA damage (Kastan et al., 1991).

Treatment with 5-aza-2′-deoxycytidine (5-aza-dC) has been experimentally shown to lead to upregulated expression of TP53 mRNA and protein in some, but not all, glioma cell lines, suggesting that promoter methylation is associated with reduced expression in some malignant glioma cell lines (Amatya et al., 2005). In addition, in another study, 5-aza-dC may have induced a p53-dependent DNA damage-response pathway independent of TP53 promoter methylation status without increasing transcript levels of the gene (Karpf et al., 2001). Downregulation of p53 transcriptional level in cultured cells transfected with a plasmid incorporating a p53 promoter containing methylated CpG dinucleotides has been shown (Schroeder and Mass, 1997). Moreover, alterations in single-site CpG methylation in the TP53 promoter region have been found to lead to initiation of de novo methylation and progressive spreading of methylation associated with transcriptional inactivation of the TP53 gene, and reduced p53 expression in rats (Pogribny et al., 2000).

Reduced level of TP53 by methylation of promoter CpG dinucleotides has been reported in some neoplasms (Pogribny et al., 2000; Kang et al., 2001; Agirre et al., 2003). However, whether TP53 promoter methylation is associated with reduced transcriptional and translational expression in GB patients remains unclear.

This present study evaluates the status of the TP53 gene, particularly the impact of methylation and mutation on mRNA and protein expression. The search was focussed on any mutual relationship existing between TP53 alterations (methylation, abnormal mRNA level, mutations, and protein overexpression) and other molecular alterations (MGMT methylation and EGFR amplification). We also evaluate the impact of all molecular alterations on the overall survival.

Materials and Methods

Tumor samples and DNA extractions

Tumor brain tissue samples were obtained from 42 patients with primary GB (21 men and 21women) who were treated in the Departments of Neurosurgery of “Copernicus” Memory Hospital in Lodz, Barlicki Clinical Hospital of Medical University of Lodz, Voivodal Specialistic Hospital in Olsztyn, and Perzyna Memorial Hospital in Kalisz, Poland from 2002 to 2005. Informed consent from patients was obtained in every case (agreement of Bioethical Committee of Medical University of Lodz, Poland RNN/192/03/KE). The age of patients ranged from 23 to 76 years, mean age was 59.1±11.8. All tumors were histopathologically examined and classified according to the WHO classification of tumors of the Central Nervous System (CNS) (Kleihus et al., 2007).

The following criteria to determine primary GB has been applied: a short clinical history of less than 3 months and the presence of histopathological features of GB at the first biopsy without any evidence of precursor low-grade astrocytomas (Watanabe et al., 1997). The molecular criteria confirming the primary GB were frequency of EGFR amplification (37.5%), IDH1 mutation (2.44%), and TP53 mutation (26.2%). The results for EGFR amplification, IDH1, and TP53 were partly published before (Jesionek-Kupnicka et al., 2013).

All patients underwent total or partial surgery and RT and seven underwent chemotherapy (TMZ). TMZ was not used in standard protocol therapy in the years 2002–2005 in Poland.

DNA was isolated by standard proteinase K digestion and phenol/chloroform extraction from frozen tumor tissue samples were taken before radio- and/or chemotherapy. Histologically all specimens consisted of at least 80% tumor cells.

TP53 gene sequencing analysis

Four genomic regions of the TP53 gene (exons 5–8) were amplified by polymerase chain reaction (PCR), as described previously (Wojcik et al., 2005). Sequence analysis was performed by the dideoxy termination method using the SequiTherm Excel DNA Sequencing Kit (Epicentre Technologies, Madison, WI) and fluorescent-labeled primers as described previously (Jesien-Lewandowicz et al., 2009). Products of the sequencing reaction were visualized and analyzed using a LiCor automated laser fluorescence sequencer.

IDH1 gene sequencing analysis

Exon 4, including codon 132 of the IDH1 gene, was amplified by PCR and sequenced using the dideoxy termination method and SequiTherm Excel DNA Sequencing Kit (Epicentre Technologies). The primers used for PCR amplification of the DNA sequences were IDH1–5′-GGCACCCATCTTCTGTGTTT-3′ (sense) and 5′-ATATATGCATTTCTCAATTTCA-3′ (antisense). The sequencing primers used were IDH1 exon 4–5′-CGGTCTTCAGAGAAGCCATT-3′ (sense) and IDH1 exon 4–5′-CACATTATTGCCAACATGAC-3′ (antisense). A Li-Cor automatic sequencer system was used for separation and analysis of PCR-sequencing products.

EGFR amplification analysis

Multiplex PCR was performed for evaluation of EGFR amplification with superoxide dismutase 1 (SOD1) used as a reference gene. PCR conditions and primer sequences were used as previously reported (Rieske et al., 2009).

Methylation-specific PCR for TP53 and MGMT promoter methylation analysis

Sodium bisulfite modification of isolated genomic DNA was performed using the CpGenome DNA kit (Chemicon International, Inc., Temecula, CA) according to the manufacturer's protocol. The bisulfite-treated DNA was stored at −80°C until use. CpGenome Universal Methylated DNA (Chemicon International) was used as a methylation-positive control for the methylated alleles, and DNA from peripheral blood leukocytes was used as the control for unmethylated alleles. The methylation-specific PCR (MSP) for TP53 promoter methylation was performed as previously described (Amatya et al., 2005). MSP for MGMT promoter methylation was performed in a two-step approach as previously reported (Jesien-Lewandowicz et al., 2009). For each PCR, methylated and unmethylated DNA was included as positive and negative controls, and water was used as a control for the PCR reaction. PCR products were separated on 3% agarose gels containing ethidium bromide and documented using the Gel Doc1000 Bio Rad Image System. Repeat testing was performed to confirm results.

RNA isolation

RNA was extracted by means of RNeasy Mini Kit (QIAGEN, Venlo, Netherlands) from tumor tissues and three normal brain tissues used as a reference. RNA samples were treated with DNAase. One hundred nanograms of total RNA was reverse-transcribed into single-stranded cDNA in a final volume of 40 μL containing 50 mM DTT, 1.5 μg oligo(dT), 0.5 mM dNTP, 40 U RNase inhibitor, and 200 U M-MLV reverse transcriptase (Promega Corporation, Madison, WI).

Real-time RT-PCR for TP53 mRNA expression

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on a Rotor Gene 6000 instrument (Corbett, Life Sciences, Australia) for the TP53 gene (TaqMan^® Gene Expression Assays no Hs00153340_m1 and no Hs00153349_m1) and GAPDH (TaqMan Gene Expression Assays no Hs99999905_m1) was used as a reference gene for normalization of the target gene expression levels. All experiments were performed in duplicate and gene expression was analyzed using the 2^−ΔΔCT method (Livak and Schmitteng, 2001)

The expression of the TP53 was determined in 38 samples of primary GB (for four cases TP53 expression was not determined). The threshold cycle (CT) indicates the fractional number at which the amount of amplified target reaches a fixed threshold. CT values for particular analyzed genes were normalized to GAPDH and related to control normal brain tissues. The level of TP53 gene expression was increased in 34.2% (13 cases) of GBs considered as fold change greater than two. Diminished expression was defined as fold change lower than 0.5.

Immunohistochemistry for TP53 protein expression

The standard IHC technique was performed to determine the expression of TP53 in tumor samples using monoclonal antibody anti-P53 (clone DO-7, 1:100 dilution; DAKO, Glostrup, Denmark), and processed with EnVision (DAKO) system. The method was previously described in detail (Szybka et al., 2009). The antibody labels wild-type and mutant-type p53 protein and is a useful tool for the identification of TP53 accumulation in human neoplasias. The normal TP53 protein has a very short half-life and is present in small amounts in normal cells (Fig. 1A). In contrast, mutant TP53 protein produced by malignant cells is usually a product of a point mutation in the TP53 gene that significantly prolongs the half-life of the protein and is detected as positive staining (Fig. 1B, C). Tumor sections were examined for TP53 immunoreactivity under the microscope at 20× and 40× magnifications. The expression of TP53 was considered as positive when the proportion of positive cells was more than 10% (Fig. 1B, C) (Nagpal et al., 2006). The negative TP53 protein staining in tumor section is presented on Figure 1D.

FIG. 1.

(A) The lack of immunoreactivity in normal non-neopastic white matter of brain outside the tumor (hematoksylin counterstaining, anti-TP53, DAKO, ×200) scale bar, 10 μm; (B, C) Strong nuclear immunohistochemical overexpression of TP53 protein in GB (hematoksylin counterstaining, anti-TP53, DAKO, ×400 and 200) scale bars, 50 μm and 10 μm; (D) The negative immunoreaction in tumor GB tissue (hematoksylin counterstaining, anti-TP53, DAKO, ×200) scale bar, 10 μm. GB, glioblastoma.

Statistical analysis

Continuous variables were compared using the Mann–Whitney's U test. Factorial analysis of variance (ANOVA) was harnessed to evaluate influence of more than one nominal variable. Normal distribution fitting was assessed with usage of Shapiro–Wilk test and homoscedasticity was assessed with Levene's test. Fisher's exact test was used to test the associations between molecular alterations. Spearman rank test was used for correlations assessments. All results were considered statistically significant when two-sided p was <0.050. STATISTICA 10.0 (Statsoft, Tulsa, OK) software and the VassarStats online calculator (www.vassarstats.net/) were used for statistical analysis.

Results

Associations between molecular alterations

TP53 alterations

The TP53 mutations were identified in 26.2% (11/42) of cases within exons 5, 6, 7, and 8. All the mutations of TP53 were missense. TP53 promoter methylation was detected in 9/42 cases (21.4%). An increased transcriptional level of TP53 was detected in 13/38 cases (34.2%) and a decreased level in 6/38 (15.8%). The remaining samples were classified as neutral expression (Table 1). Four cases were not analyzed for TP53 expression.

Table 1.

Clinical Data and Results of Molecular Analysis

	Valid N	Median (interquartile range)	Missing data (N)
Age (years)	42	61 (51–68)	0
Overall survival (months)	42	11.5 (8–16)	0
qRT-PCR Fold Change of TP53	38	1.3 (0.78–2.83)	4
Sex	Male (N)	Female (N)	0
	21 (50.00%)	21 (50.00%)
TP53 expression	Down	Up Equal	4
	6 (15.79%)	13 (34.21%) 19 (50.00%)
All types of TP53 mutation	Absent	Present	0
	31(73.81%%)	11 (26.19%)
TP53 transition GC>AT	Absent	Present	0
	34 (80.95%)	8 (19.05%)
TP53 methylation	Absent	Present	0
	33 (78.57%)	9 (21.43%)
TP53 protein presence	Absent	Present	10
	20 (62.50%)	12 (37.50%)
MGMT methylation	Absent	Present	0
	12 (28.57%)	30 (71.43%)
EGFR amplification	Absent	Present	2
	25 (62.50%)	15 (37.50%)

qRT-PCR, quantitative reverse transcription polymerase chain reaction.

There was no relation between TP53 mRNA level and promoter methylation (Median for group with methylation (Me) 1.41 (interquartile range [IQR] 0.64–16.00) vs. Me for group without methylation 1.19 (IQR 0.78–2.55); (Fig. 2; Mann–Whitney's U test p=0.5722).

FIG. 2.

No significant relation between TP53 mRNA level and promoter methylation (p=0.5722). Horizontal lines represent medians with lower and upper quartile boundaries. Each individual datapoint is represented on the figure.

Moreover, there was no significant relation between TP53 mRNA expression and any mutation in TP53 (Me for group with mutation 1.49 [IQR 1.04–2.30] vs. Me 1.09 [IQR 0.75–3.04]; Mann–Whitney's U test p=0.9076) or transition mutation in TP53 (Me for group with transition 1.30 [IQR 0.68–7.65] vs. Me 1.34 [IQR 0.78–2.83]; Mann-Whitney's U test p=0.9857). Relation between TP53 promoter methylation occurs irrespective of the TP53 mutation status (Fisher's exact test p=0.2086). There was no significant interaction between methylation and mutation status and TP53 level (Fig. 3; p ANOVA=0.7867).

FIG. 3.

No relation between TP53 mRNA level depending on TP53 mutation status was found (p=0.7867). Horizontal lines represent medians with lower and upper quartile boundaries. Each individual datapoint is represented on the figure.

Protein of TP53 gene was present in 12 cases (37.5%). No significant association between mRNA TP53 level and protein expression were found (Me for group with present protein 1.41 [IQR 1.04–13.00] vs. Me 1.07 [IQR 0.64–3.25]; Mann–Whitney's U test p=0.5612). Significant association between TP53 mutation and TP53 protein expression was found (21.88% of total had both mutation and protein expression vs. 59.38% had none of them; Fisher's exact test p=0.0016) (Fig. 4). The same pattern was observed for group with TP53 transition (15.63% of total had both transition and protein expression vs. 59.38% had none of them; Fisher's exact test p=0.0185) and for TP53 methylation (15.63% of total had both methylation and protein expression vs. 53.13% had none of them; Fisher's exact test p=0.2045) even though the latter was not significant. The results of all alterations are presented in Table 1.

FIG. 4.

Relation between TP53 mutation status and TP53 protein presence (p=0.0016).

EGFR amplification

There was no relation between EGFR amplification and TP53 expression (Me for group with amplification 1.02 [IQR 0.78–1.93] vs. Me 1.49 [IQR 0.71–2.83]; Mann–Whitney's U test p=0.5812), TP53 mutation (5.0% of total had both abnormalities vs. 40.0% had none of the regarded abnormalities; Fisher's exact test p=0.1582), TP53 transition (5.0% of total had both abnormalities vs. 47.5% had none of the regarded abnormalities; Fisher's exact test p=0.6857) or TP53 methylation (2.5% of total had both abnormalities vs. 42.5% had none of the regarded abnormalities; Fisher's exact test p=0.1166). Relation between EGFR amplification and TP53 expression occurs irrespective of the TP53 mutation status (Fisher's exact test p=1.0).

MGMT methylation

There was no relation between MGMT methylation and TP53 methylation (Fisher's exact test p=0.6987) or TP53 mRNA expression (Me for group with MGMT methylation 1.57 [IQR 0.81–7.21] vs. Me 1.07 [IQR 0.59–2.55]; Mann–Whitney's U test p=0.3424). Also no relation was found significant between MGMT methylation and TP53 mutation (23.81% of total had both abnormalities vs. 26.2% had none of the regarded abnormalities; Fisher's exact test p=0.1333), and between MGMT methylation and transition (16.7% of total had both abnormalities vs. 26.2% had none of the regarded abnormalities; Fisher's exact test p=0.4024) or EGFR amplification (25.0% of total had both abnormalities vs. 15.0% had none of the regarded abnormalities; Fisher's exact test p=0.7162).

The TP53 alterations and influence on survival

The TP53 mutation

There was no difference in survival between patients with TP53 mutation (n=11) (median survival 15.0 months; IQR 7–18 months) and without TP53 mutation (n=31) (median survival 12 months; IQR 7–16 months; Mann–Whitey's U test p=0.8976).

TP53 methylation

Median survival in patients with TP53 methylation (n=9) was 11.0 months (IQR 9–15 months). Median survival in patients without TP53 methylation (n=33) was 12 months (IQR 9–17 months). There was no significant difference in median survival between patients with different TP53 methylation status (Mann–Whitney's U test p=0.4079).

Expression of mRNA TP53

Median survival for patients with decreased TP53 mRNA expression (n=6) was 11 months (IQR 8–20 months), for patients with increased mRNA TP53 expression (n=13) was 12 months (IQR 9–12 months) and with unaltered expression (n=19) was 12 months (IQR 8–18 months). The difference between three groups were not significant (Kruskal–Wallis' test, factorial ANOVA p=0.5943).

P53 protein expression

No difference in overall survival between patient with present (n=12) and absent (n=20) TP53 protein was found (Me for group with protein expression 11 months [IQR 5.5–13.0 months] vs. Me 12 months [IQR 8.5–16.5 months]; Mann–Whitney's U test p=0.3403). Relation between TP53 protein expression and EGFR amplification was not significant (13.33% of total had both EGFR amplification and protein expression vs. 30.00% had none of them; Fisher's exact test p=0.4651).

EGFR amplification

EGFR amplification was identified in 37.5% (15/40) (Table 1). Median survival in patients with EGFR amplification (n=15) was 12.0 months (IQR 5–14 months), while in patients without EGFR amplification (n=25), it was 11.0 months (IQR 8–18 months) The difference in survival time was not significant (Mann–Whitney's U test p=0.5388). No significant relation between MGMT expression and EGFR amplification was found (Me for group with amplification 0.018 [IQR 0.008–0.149] vs. Me 0.026 [IQR 0.004–0.074]; Mann–Whitney's U test p=0.4854).

MGMT methylation

Methylation of MGMT promoter gene was detected in 30/42 (71.43%). Median survival in patients with MGMT promoter methylation (n=30) was 12.0 months (IQR 8–14 months), while median survival without MGMT promoter methylation (n=12) was 10 months (IQR 6.5–17 months). The difference between two groups was not significant (Mann–Whitney's U test p=0.6967).

The IDH1 mutation

IDH1 mutation was found only in one of 41 examined cases (2.44%). One case was not available for IDH1 mutation study.

Discussion

It has been experimentally demonstrated that a reduction in the dosage of wild-type TP53 genes without mutation is sufficient to promote tumorigenesis (Venkatachalam et al., 1998), and this is congruent with the possibility that TP53 promoter region methylation and reduced TP53 gene expression contribute to the selection and expansion of preneoplastic cells. Although, a reduced level of TP53 by methylation of promoter CpG dinucleotides has been reported in acute lymphoblastic lymphoma (Agirre et al., 2003) and in hepatocellular carcinoma (Pogribny and James, 2002) such a relationship is not clear in GB.

In our study, the frequency of TP53 promoter methylation in primary GB was found in 21.4%. This report shows the high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 protein and mRNA level. As far as we are concerned there are no other publications concerning the relationship between TP53 mRNA and protein expression and methylation in primary GB patients. These observations suggest that in primary GB TP53 promoter methylation does not play a role in regulation of TP53 expression. These results indicate that in primary GB the transcriptional and translational TP53 level is probably controlled by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway. Our finding that TP53 promoter methylation occurs irrespective of the TP53 mutation status is consistent with previous observations (Amatya et al., 2005). It can be assumed that the mechanism of TP53 level regulation is more complex and TP53 promoter methylation plays only an adjective epigenetic role in GB. Indeed, in whole-genome integrative analysis in GB, other genes with concordant CpG sites have been found to display an inverse association between both promoter methylation and expression level: B3GNT5, FABP7, ZNF217, BST2, OAS1, SLC13A5, GSTM5, ME1, UBXD3, TSPYL5, FAAH, C7orf13, and C3orf14 (Etcheverry et al., 2010). Silencing by DNA hypermethylation of other genes involved in key cellular functions like cell cycle (p16^INK4a and p15^INK4b ), DNA repair and genome integrity (MGMT and MLH1), and tumor suppression (RB1, VHL, EMP3, RASSF1A, BLU), in addition to tumor invasion and apoptosis (DAPK, TIMP3, CDH1, PCDH-gamma-A11, TMS1/ASC) has been involved in gliomagenesis [rev. in Martinez et al. (2009)]. Interestingly, a large cDNA-array study did not reveal any gene cluster separating groups of gliomas on the basis of their TP53 status (Godard et al., 2003); this may be due to the fact that in >70% of all astrocytic gliomas, the TP53 pathway has been found to be inactivated not only by mutational alteration of TP53, but also by a variety of alternative mechanisms: overexpression of MDM2, (a negative regulator of TP53) or inactivation of p14^ARF (a negative regulator of MDM2) or by deletion or promoter methylation of these genes (Ichimura et al., 2000). p14^ARF was found to induce stabilization of the TP53 transcription factor, leading to the expression of critical TP53 target genes, which can mediate cell cycle arrest or induce apoptosis [rev. in Zerrouqi et al. (2012)]. This confirms a recent study of TCGA Research Network, which revealed a cluster of genes whose drastic over- and underexpression is associated with TP53 mutation status (Cancer Genome Atlas Research Network, 2008). Among other things, MDM2 is overexpressed when TP53 is wild type: that is, MDM2 overexpression is mutually exclusive with TP53 mutation (Masica and Karchin, 2011).

The frequency of TP53 methylation in gliomas depends on the used primers. TP53 promoter methylation is frequent in low-grade gliomas but is not well known in GB. In one study, TP53 presented CpG island hypermethylation constituted only 2% in GB, and 8% in diffuse astrocytomas and anaplastic astrocytomas (Gonzalez-Gomez et al., 2003). Using other MSP primers (nt 632–894 and nt 636–915) in Amatya et al. (2005) study, TP53 promoter methylation frequency reached 60% of low-grade astrocytomas, 61% of oligoastrocytomas, and 73% of oligodendrogliomas, but GBs have not been studied. In the present study, using the same primers for the region as mentioned above 21.4% of TP53 promoter methylation was detected. Such high frequency of TP53 promoter methylation in primary GB was not previously mentioned in literature. Although, there was also no significant relation between TP53 mRNA expression and any mutation in TP53 gene, a significant association between TP53 mutation and TP53 protein expression was found, which is consistent with other studies. A correlation between TP53 mutations and the accumulation of TP53 in the nucleus has been observed in many types of neoplasms, including GB (Newcomb et al., 1993; Soong et al., 1996). Stabilization of TP53 is tightly regulated by ubiquitination. TP53 is mainly ubiquitinated by MDM2 ubiquitin but other ubiquitin ligases such as ARF-BP1 and MdmX are also involved in the regulation leading to TP53 proteasomal degradation (Chen et al., 2005; Wang et al., 2011). MDM2 is a key negative regulator of TP53 function. The TP53-MDM2 autoregulatory feedback loop regulates activity of the TP53 protein and expression of MDM2; wild-type TP53 protein induces transcription of the MDM2 gene and MDM2 protein inhibits the ability of TP53 to activate transcription (Momand et al., 1992). This tight control is lost in tumors harboring mutant TP53, which exhibits a significant TP53 stabilization compared with wild-type TP53 tumors. In human cancer cells mutant TP53, despite its ability to interact with MDM2, suffers from lack of ubiquitination and thus diminished TP53 degradation (Li et al., 2011).

TP53 mutations and EGFR amplification were considered as mutually exclusive (Ohgaki et al., 2004). However, in the present study there were two cases with coincidental EGFR amplification and TP53 mutations; these results has been partly published previously (Jesionek-Kupnicka et al., 2007). Indeed, the copresentation of TP53 mutations and EGFR amplification was sporadically noticed in single cases of GB (Ueki et al., 2002; Okada et al., 2003; Gil-Benso et al., 2007). Simultaneous EGFR/TP53 alterations and the CDK4 amplification had the adverse prognostic value in the overall survival along with age, and RT in patients with primary GB (Ruano et al., 2009). In this study there was no relation between EGFR amplification and TP53 expression and methylation status. In this study the frequency of IDH1 mutation was low, which is typical for primary gioblastoma and correspond to other studies (Ichimura et al., 2009; Yan et al., 2009).

In our study, the most common molecular alteration in primary GB was MGMT methylation (71.4%), which is consistent with our previous study (Jesien-Lewandowicz et al., 2009). There was no relation between MGMT methylation and TP53 mutation (including transitions G:C>A:T) in contrast to other studies (Nakamura et al., 2000; Shamsara et al., 2009).

In terms of clinical outcome, there was no impact of mRNA TP53 levels on survival in patients. Interestingly, Blough et al. demonstrated that TP53 status may influence response to TMZ in differentiated cells in a GB. Glioma cell lines that did not express a functional p53 were significantly more sensitive to TMZ than cell lines with functionally intact wild-type p53 expression, an effect that is independent of MGMT status (Blough et al., 2011).

In conclusion, our data suggest that in GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and translational expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway.

Footnotes

Acknowledgments

The study was supported by funding from the Minister of Science and Higher Education Grant no. 2011/01/B/NZ4/03345, and by the funds of the Medical University of Lodz no. and 503/1-034-03/503-01.

Disclosure Statement

No competing financial interests exist.

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