Elimination of wild-type p53 mrna in glioblastomas showing heterozygous mutations of p53

ABSTRACT We screened 50 glioblastomas for _P53_ mutations. Five glioblastomas showed heterozygous mutations, while three were putatively heterozygous. Six of these eight glioblastomas showed

elimination of wild-type _P53_ mRNA. These results strongly suggest that some sort of mechanism(s) favouring mutated over wild-type _P53_ mRNA exists in glioblastoma cells with heterozygous

mutations of this gene. SIMILAR CONTENT BEING VIEWED BY OTHERS ACQUISITION OF ANEUPLOIDY DRIVES MUTANT P53-ASSOCIATED GAIN-OF-FUNCTION PHENOTYPES Article Open access 31 August 2021 MUTANT

P53: IT’S NOT ALL ONE AND THE SAME Article Open access 31 March 2022 GERMLINE _TP53_ MUTATIONS UNDERGO COPY NUMBER GAIN YEARS PRIOR TO TUMOR DIAGNOSIS Article Open access 05 January 2023

MAIN A majority of tumour suppressor genes present homozygous or hemizygous mutations (Sherr, 2004). Intriguingly, in the _P53_ gene, heterozygous mutations have also been detected. Typical

mutations of this gene are of the missense type, leading to P53 protein gain of function (Nigro et al, 1989; Dittmer et al, 1993). However, the effects of at least some heterozygous

mutations cannot be explained only by the gain of _P53_ function (Park et al, 1994). In case of _P53_ mutations such as R249S or R273H, at least three mutated monomers per tetramer appeared

to be required to inactivate the transactivation of MDM2 and p21 CIP1/WAF1 promoters (Chan et al, 2004). In case of R280T mutations, heterotetramers consisting even of three mutated and one

wild-type P53 monomer showed partially but not completely abolished activity compared to P53 homotetramers consisting of wild-type monomers only (Sun et al, 1993). In this context, the

occurrence of heterozygous mutations of _P53_ remains enigmatic, leading to a question of whether mechanisms other than _P53_ mutations or deletions are involved in the elimination of the

wild-type P53 protein. Several nongenomic mechanisms of protein elimination or aberration have been described, including processes operating at the level of transcription (e.g., methylation)

or translation (e.g., miRNA) (Voorhoeve et al, 2006; Watanabe et al, 2007). We examined whether glioblastoma cells with heterozygous mutations of _P53_ contained a mixture of wild-type and

mutated _P53_ mRNA, or predominantly the mutated _P53_ mRNA. Additionally, we also checked the methylation status of the _P53_ promoter. MATERIALS AND METHODS TUMOUR SAMPLES The study

included 50 cases of glioblastoma, diagnosed at Department of Pathology, Medical University of Lodz, according to the World Health Organization criteria for classification of brain tumours

(Louis et al, 2007). The group consisted of 25 females and 25 males, aged from 15 to 76 years (median 59.5). DNA AND RNA ISOLATION The investigations were performed using snap-frozen tissues

stored at −80°. DNA was isolated from tumour tissues and blood samples from each patient. DNA and RNA were coextracted by means of Macherey-Nagel DNA/RNA purification kit. RNA samples were

treated with DNAase. RNA and DNA concentrations were measured spectrophotometrically. In all 100 ng of total RNA was reverse-transcribed into single-stranded cDNA in a final volume of 40 μl

containing 50 mM DTT, 1.5 _μ_g oligo(dT), 0.5 mM dNTP, 40 units RNase inhibitor and 200 units M-MLV reverse transcriptase (Promega). LOSS OF HETEROZYGOSITY AND MICROSATELLITE INSTABILITY

ANALYSES Loss of heterozygosity (LOH) and microsatellite instability (MSI) analyses were performed using paired tumour specimens and corresponding peripheral blood samples, to recognise

tumour samples with minimal contamination by normal cells. The following LOH and MSI markers were used: D1S2734, D1S197, D1S162, D1S156, D9S319, D9S319, D9S162, D10S587, D10S1267, D17S1828,

AFM119, BAT25, BAT26, BAT40. Forward primers were 5′ end fluorescence-labeled. PCR was performed in thermocycling conditions individually established for each pair of primers. PCR products

were denatured and gel electrophoresis in LiCor automatic sequencer system was applied to the separation and analysis of PCR-generated alleles. _P53_ DNA AND CDNA SEQUENCING Exons 5–8 of the

_P53_ gene were amplified by PCR as described before and sequenced using the dideoxy termination method and SequiTherm Excel DNA Sequencing Kit (Epicentre Technologies) (Zakrzewska et al,

2005). The primers used for the PCR amplification of cDNA sequences were: _p53_: 5′-GTGCAGCTGTGGGTTGATT-3′ (sense) and 5′ GCAGTGCTCGCTTAGTGCTC-3′ (antisense); annealing temperature was 53°C.

The sequencing primers were: _p53_ exon 5–8: 5′-GCCATCTACAAGCAGTCACA-3′ (sense), and _p53_ exon 8–5: 5′-CCCTTTCTTGCGGAGATTCT-3′ (antisense); annealing temperature was 55°C. LiCor automatic

sequencer system was applied to the separation and analysis of PCR-sequencing products. To verify the results, a semi-quantitative densitometric analysis was performed in which wild and

mutated band intensity was estimated, and then compared to a neighbouring band in the same sequencing lane for reference. METHYLATION-SPECIFIC PCR (MSP) Sodium bisulphite modification of

genomic DNA was performed using the CpGenome Universal DNA Modification Kit (Chemicon International, Temecula, CA, USA). CpGenome Universal Methylated DNA (Chemicon International) was used

as a methylation-positive control for the methylated _P53_ promoter, and DNA from peripheral blood leukocytes was used as the control for unmethylated alleles of _P53_. The MSP was performed

as previously described (Amatya et al, 2005). RESULTS Genomic DNA and cDNA obtained from fifty glioblastoma samples were sequenced for _P53_ mutations. Mutations of _P53_ were detected in

16 cases, eight of these being heterozygous (showing a weak mutated band or a mutated band as strong as the wild band; Figure 1B). Five of these eight cases were indeed confirmed as

heterozygous when LOH and MSI analyses showed no or negligible contamination of the tested samples by normal cells (cases 1–5, Table 1; Figure 1A). In three additional cases (6–8, Table 1),

sequencing results suggested heterozygous mutations of _P53_. However, we could not exclude a possible contamination of the tumour specimens with normal cells in this instance because no

LOH/MSI was detected in them. We defined these cases as presenting putative heterozygous mutations of _P53_. In six cases (including heterozygous as well as putative heterozygous mutations),

cDNA sequencing revealed a decreased amount, or lack of, the wild (nonmutated) template when compared to the genomic DNA sequencing (cases 1–4, 6–7, Table 1; Figure 1C). A densitometric

analysis of the wild and mutated bands confirmed the above observations (data not shown). MS-PCR revealed _P53_ promoter methylation (Figure 1D) in only three cases. One had a heterozygous

mutation, while another had a putative heterozygous mutation of the _P53_. However, in both of these cases no wild-type cDNA template was detected (case 4 and 7). The third case also

presented a heterozygous mutation of _P53 –_ but without any decrease in the amount of wild-type cDNA template – as shown by cDNA _vs_ genomic DNA sequencing (case 5, Table 1). DISCUSSION

Heterozygous mutations of _P53_ have been widely described (Dittmer et al, 1993). In this study we show that a majority of glioblastomas presenting heterozygous mutations of _P53_ gene

presented no wild-type _P53_ mRNA. These results strongly suggest that glioblastoma cells may have the ability to develop a mechanism(s) which would allow for either (1) the silencing of

wild-type _P53_ transcription, (2) the degradation of wild-type _P53_ mRNA, or (3) the selective overproduction of mutated _P53_ mRNA. Moreover, our results show that heterozygous mutations

of _P53_ gene, elimination of wild-type _P53_ mRNA, or selective production of mutated mRNA can occur during glioblastoma tumorigenesis. An extremely important question thus arises – that

is, ‘what mechanism(s) is (are) responsible for favouring the mutated _P53_ mRNA over the wild-type ones?’ A methylation of DNA regulatory elements – mainly promoters, is one of the

mechanisms of tumour suppressor genes silencing (Watanabe et al, 2007). It was shown that a similar proportion of gliomas with and without _P53_ mutation present _P53_ promoter methylation

(Amatya et al, 2005). Indeed, the lack of wild-type mRNA – despite the presence of wild-type DNA observed by us, could be explained by a selective methylation of DNA regulatory element of

nonmutated _P53_ allele. The primers we used in methylation-specific PCR have already been successfully used by the Ohgaki group in analysing _P53_ promoter methylation in gliomas (Amatya et

al, 2005). Nevertheless, we observed that no _P53_ promoter methylation was detectable with this set of primers in the majority of cases analysed in this study. Collectively, these results

suggest that identification of mechanism(s) responsible for the elimination of wild-type _P53_ mRNA requires more research. Obviously, the elimination of wild-type P53 may have resulted from

a mechanism other than epigenetic changes of _P53_ DNA regulatory elements. Nonetheless, uncovering of this mechanism can be very important for the development of new anti-tumour

therapeutics. In conclusion, we show in this article that glioblastomas presenting heterozygous mutations of _P53_ employ some sort of mechanism(s) to positively select mutated _P53_ mRNA.

We offer a relatively easy procedure for determining whether similar situations also occur in other cancers. The precise mechanism(s) for favouring the mutated type of _P53_ mRNA – however,

still remains to be discovered. CHANGE HISTORY * _ 16 NOVEMBER 2011 This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at

publication _ REFERENCES * Amatya VJ, Naumann U, Weller M, Ohgaki H (2005) TP53 promoter methylation in human gliomas. _Acta Neuropathol (Berl)_ 110: 178–184, doi: 10.1007/s00401-005-1041-5

Article  CAS  Google Scholar  * Chan WM, Siu WY, Lau A, Poon RY (2004) How many mutant p53 molecules are needed to inactivate a tetramer? _Mol Cell Biol_ 24: 3536–3551 Article  CAS  PubMed 

PubMed Central  Google Scholar  * Dittmer D, Pati S, Zambetti G, Chu S, Teresky AK, Moore M, Finlay C, Levine AJ (1993) Gain of function mutations in p53. _Nat Genet_ 4: 42–46, doi:

10.1038/ng0593-42 Article  CAS  PubMed  Google Scholar  * Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO Classification of

tumors of the central nervous system. _Acta Neuropathol (Berl)_ 114: 97–109 Article  Google Scholar  * Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hosteller R, Cleary K, Signer SH,

Davidson N, Baylin S, Devilee P, Glover T, Collins FS, Weslon A, Modali R, Harris CC, Vogelstein B (1989) Mutations in the p53 gene occur in diverse human tumour types. _Nature_ 342:

705–707, doi: 10.1038/342705a0 Article  CAS  PubMed  Google Scholar  * Park DJ, Nakamura H, Chumakov AM, Said JW, Miller CW, Chen DL, Koeffler HP (1994) Transactivational and DNA-binding

abilities of endogenous p53 in p53 mutant cell lines. _Oncogene_ 9: 1899–1906 CAS  PubMed  Google Scholar  * Sherr C (2004) Principles of tumor suppression. _Cell_ 116: 235–246, doi:

10.1016/S0092-8674(03)01075-4 Article  CAS  PubMed  Google Scholar  * Sun Y, Dong Z, Nakamura K, Colburn NH (1993) Dosage-dependent dominance over wild-type p53 of a mutant p53 isolated from

nasopharyngeal carcinoma. _FASEB J_ 7: 944–950 Article  CAS  PubMed  Google Scholar  * Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R, Liu YP, van Duijse J, Drost J,

Griekspoor A, Zlotorynski E, Yabuta N, De Vita G, Nojima H, Looijenga LH, Agami R (2006) A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumours.

_Cell_ 124: 1169–1181, doi: 10.1016/j.cell.2006.02.037 Article  CAS  PubMed  Google Scholar  * Watanabe T, Katayama Y, Yoshino A, Yachi K, Ohta T, Ogino A, Komine C, Fukushima T (2007)

Aberrant hypermethylation of p14ARF and O6-methylguanine-DNA methyltransferase genes in astrocytoma progression. _Brain Pathol_ 17: 5–10, doi: 10.1111/j.1750-3639.2006.00030.x Article  CAS 

PubMed  PubMed Central  Google Scholar  * Zakrzewska M, Wojcik I, Zakrzewski K, Polis L, Grajkowska W, Roszkowski M, Augelli BJ, Liberski PP, Rieske P (2005) Mutational analysis of

hSNF5/INI1 and TP53 genes in choroid plexus carcinomas. _Cancer Genet Cytogenet_ 156: 179–182, doi:10.1016/j.cancergencyto.2004.05.002 Article  CAS  PubMed  Google Scholar  Download

references ACKNOWLEDGEMENTS This work was supported by Medical University of Lodz Grant no 502-11-442 and Ministry of Scientific Research and Information Technology Grant no 2P05A 7929. Mr

Giac Nguyen from the Medical University of Lodz is kindly acknowledged for language assistance. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Pathology, Medical University of

Lodz, Paderewskiego 4, Lodz, 93-509, Poland M Szybka, D Kulczycka, E Jesien, D Kupnicka & R Kordek * Department of Molecular Pathology and Neuropathology, Medical University of Lodz,

Czechoslowacka 8/10, Lodz, 92-216, Poland I Zawlik, E Golanska, R Stawski, S Piaskowski, E Bieniek, M Zakrzewska, P P Liberski & P Rieske * Department of Radiology, Medical University of

Lodz, Czechoslowacka 8/10, Lodz, 92-216, Poland E Jesien Authors * M Szybka View author publications You can also search for this author inPubMed Google Scholar * I Zawlik View author

publications You can also search for this author inPubMed Google Scholar * D Kulczycka View author publications You can also search for this author inPubMed Google Scholar * E Golanska View

author publications You can also search for this author inPubMed Google Scholar * E Jesien View author publications You can also search for this author inPubMed Google Scholar * D Kupnicka

View author publications You can also search for this author inPubMed Google Scholar * R Stawski View author publications You can also search for this author inPubMed Google Scholar * S

Piaskowski View author publications You can also search for this author inPubMed Google Scholar * E Bieniek View author publications You can also search for this author inPubMed Google

Scholar * M Zakrzewska View author publications You can also search for this author inPubMed Google Scholar * R Kordek View author publications You can also search for this author inPubMed 

Google Scholar * P P Liberski View author publications You can also search for this author inPubMed Google Scholar * P Rieske View author publications You can also search for this author

inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to P Rieske. RIGHTS AND PERMISSIONS From twelve months after its original publication, this work is licensed under the Creative

Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Szybka, M., Zawlik, I., Kulczycka, D. _et al._ Elimination of wild-type _P53_ mRNA in glioblastomas showing heterozygous mutations of _P53_. _Br J Cancer_ 98,

1431–1433 (2008). https://doi.org/10.1038/sj.bjc.6604258 Download citation * Received: 13 August 2007 * Revised: 18 December 2007 * Accepted: 18 January 2008 * Published: 18 March 2008 *

Issue Date: 22 April 2008 * DOI: https://doi.org/10.1038/sj.bjc.6604258 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link

Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * P53 * glioblastoma *

methylation * mRNA