eISSN: 1509-572x
ISSN: 1641-4640
Folia Neuropathologica
Current issue Archive Manuscripts accepted About the journal Editorial board Journal's reviewers Abstracting and indexing Subscription Contact Instructions for authors Ethical standards and procedures
SCImago Journal & Country Rank
vol. 43

Materials from XIII Conference of Polish Association of Neuropathologists Warszawa, May 12-14, 2005
Original article
The diagnosis and therapy of brain tumours

Stanisław Nowak
Ryszard Zukiel
Anna-Maria Barciszewska
Jan Barciszewski

Folia Neuropathol 2005; 43 (3): 193-196
Online publish date: 2005/09/30
Article file
- The diagnosis.pdf  [0.08 MB]
Get citation
JabRef, Mendeley
Papers, Reference Manager, RefWorks, Zotero
Communicating author:
Jan Barciszewski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznan, Poland,
phone: +48 61 8528 503, fax: +48 61 8520 532, e-mail: jbarcisz@ibch.poznan.pl

Understanding molecular mechanisms that occur in a normal cell and their possible ways of disregulation that lead to cancer is the prerequisite step in developing an anti-cancer therapy. After human genome sequence had been solved, it became obvious that not only alterations in the gene sequence can be deleterious, but also a damage of the epigenetic control of cell processes. There are several mechanisms of epigenetics: DNA methylation; histone methylation, acetylation and phosphorylation; RNA interference; chromosomal silencing via binding protein complexes or small RNAs; transposition of mobile elements. In this article we will discuss briefly the diagnostic potential and therapeutic application of DNA methylation and RNA interference in brain tumours.

DNA methylation
DNA methylation is a regulatory mechanism of gene activity. This process involves formation of
5-methylcytosine (m5C), which function is a silencing or activation of a gene expression. It is an epigenetic marker and is inherited independently of the nucleotide sequence of DNA. Only 5% of all DNA cytosines are methylated, most of them (70-80%) in CpG islands. CpG dinucleotides usually occur at the 5'-ends of many human genes, most common in promoters and first exons, as well as at the 3'-ends. There are ca. 29 000 CpG islands in the human genome. They are combined with ca. 60% of human genes [1,2,5,14,18,25]. During cancerogenesis both global hypomethylation and local hypermethylation of CpG islands can occur, and that is the basis for the neoplastic process. These changes can be silencing of suppressor genes, a loss of gene imprinting, oncogenes activation, a higher number of point mutations in CpG islands and microsatellite DNA instability [15,22]. Partially a disturbance in the methylation pattern can be due to overexpression of DNA methyltransferases [22].

Molecular markers
The genetic code is the first level of transmission of the hereditary information encoded in the nucleotide sequence. However, there are genetic variations that occur without corresponding changes in DNA sequence. These are called epigenetic and involve informational abilities of nucleic acids, proteins and chemical groups modifying them [5]. Genetic changes occur when nucleic bases are modified e.g. in the reaction with genotoxic chemicals and reactive oxygen species (ROS). However, a damage of m5C with hydroxyl radical (•OH) beyond being a source of various modified nucleotides can also lead to thymine or cytosine, that are normal DNA bases. The final effect of this modification is global hypomethylation, which we analysed using chromatographic separation of [32P]postlabelled components after enzymatic hydrolysis of tumour DNA [29]. We have found that there are significant differences in the content of m5C in DNA of various tumour types: the lowest for WHO grade IV linearly rising while lowering the grade (Fig. 1). That finding makes the m5C an epigenetic marker of DNA damage, which can be used for diagnosis and prognosis in brain tumours.

Silencing with RNA interference
RNA interference (RNAi) is an epigenetic regulatory pathway that serves as a sequence-specific gene-silencer. Besides the role of the defence mechanism of eukaryotic cells against viruses and transposons, RNAi regulates the expression of homologous target-gene transcripts [12]. But sometimes the application of that technology in vertebrates, including mammals, is difficult because of additional dsRNA-triggered pathways that mediate a non-specific suppression of gene expression [7,19,21,27]. However, these non-specific responses are observed in the case of long dsRNAs but not
short-interfering RNAs (siRNAs) [6,8,10,28]. siRNAs therefore seem to be promising reagents for developing gene-specific therapeutics [24]. siRNAs are 19-28nt dsRNA duplexes sometimes with symmetric
2-3nt 3’ overhangs, and 5’-phosphate and 3’-hydroxyl groups [11]. This structure is characteristic of an RNase III-like enzymatic cleavage pattern, which led to the identification of the highly conserved Dicer family of RNase III enzymes as the mediators of dsRNA cleavage [3,4,17]. The process of degradation of the target messenger RNAs is restricted to cytoplasm [13,16,26]. In the first step, Dicer cleaves long dsRNAs to produce siRNAs, which are incorporated into a multiprotein RNA-inducing silencing complex (RISC). There is a strict requirement for the siRNAs to be 5’ phosphorylated in order to enter the RISC [20,23]. siRNAs that lack a
5’ phosphate are rapidly phosphorylated by an endogenous kinase [23]. The duplex siRNA is unwound, leaving the antisense strand to guide the RISC to its homologous target mRNA for endonucleolytic cleavage. The target mRNA is cleaved at a single site in the centre (10 nt from the 5’ end of the siRNA) of the duplex region between the guide siRNA and the target mRNA [10,11]. siRNAs for gene-targeting experiments can only be introduced into cells via classic gene-transfer methods, such as liposome-mediated transfection, electroporation and microinjection, all of which require the chemical or enzymatic synthesis of siRNAs [9]. The efficiency of siRNA uptake is dependent upon the cell type. siRNAs can be synthesized in large quantities and, thus, can be used to analyse large numbers of sequences emerging from genome projects. siRNA-mediated RNAi is a powerful tool for regulating gene function.

We decided to use dsRNA for suppression of human glioblastoma multiforme by inhibition of the expression of tenascin C (TN-C)21. The protein is coded by a single gene and its expression is regulated by a single promoter. TN-C mRNA consists of 7560 nucleotides. The sequence length of 164 nucleotides was selected by computer calculations and match TN-C mRNA close to its 5’ end. ATN-RNA was administered directly. In all cases postoperative wounds healing was per primam intentionem.
1. Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci USA 1993; 90: 11995-11999.
2. Baylin SB, Herman JG. DNA hypermethylation in tumorogenesis: Epigenetics joins genetics. Trends Genet 2000; 16: 168-174.
3. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409: 363-366.
4. Billy E, Brondani V, Zhang H, Muller U, Filipowicz W. Specific interference with gene expression induced by long, double-stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc Natl Acad Sci USA 2001; 98: 14428-14433.
5. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16: 6-21.
6. Bitko V, Barik S. Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses. BMC Microbiol 2001; 1: 34.
7. Caplen NJ, Fleenor J, Fire A, Morgan RA. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene 2000; 252: 95-105.
8. Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001; 98: 9742-9747.
9. Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res 2002; 30: e46.
10. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494-498.
11. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 2001; 15: 188-200.
12. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806-811.
13. Hutvagner G, Zamore PD. A microRNA in a multiple-turnover RNAi enzyme complex. Science 2002; 297: 2056-2060.
14. Issa JP. CpG-island methylation in aging and cancer. Curr Top Microbiol Immunol 2000; 249: 101-118.
15. Jaenisch R, Bird A. Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet Suppl 2003; 33: 245-254.
16. Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA (Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 2003; 31: 700-707.
17. Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev 2001; 15: 2654-2659.
18. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 2551-2569.
19. Nakano H, Amemiya S, Shiokawa K, Taira M. RNA interference for the organizer-specific gene Xlim-1 in Xenopus embryos. Biochem Biophys Res Comm 2000; 274: 434-439.
20. Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 2001; 107: 309-321.
21. Oates AC, Bruce AE, Ho RK. Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo. Dev Biol 2000; 224: 20-28.
22. Robertson KD, Jones PA. DNA methylation: past, present and future directions. Carcinogenesis 2000; 21: 461-467.
23. Schwarz DS, Hutvagner G, Haley B, Zamore PD. Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways. Mol Cell 2002; 10: 537-548.
24. Tuschl T, Borkhardt A. Small interfering RNAs: a revolutionary tool for analysis of gene function and gene therapy. Mol Interv 2002; 2: 158-167.
25. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, Smith HO, Yandell M, Evans CA, Holt RA, et al. The sequence of the human genome. Science 2001; 291: 1304-1351.
26. Zeng Y, Cullen BR. RNA interference in human cells is restricted to the cytoplasm. RNA 2002; 8: 855-60.
27. Zhao Z, Cao Y, Li M, Meng A. Double-stranded RNA injection produces nonspecific defects in zebrafish. Dev Biol 2001; 229: 215-223.
28. Zhou Y, Ching YP, Kok KH, Kung HF, Jin DY. Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA. Nucleic Acids Res 2002; 30: 1664-1669.
29. Zukiel R, Nowak S, Barciszewska AM, Gawronska I, Keith G, Barciszewska MZ. A simple epigenetic method for the diagnosis and classification of brain tumors. Mol Cancer Res 2004; 2: 196-202.
Copyright: © 2005 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
Quick links
© 2021 Termedia Sp. z o.o. All rights reserved.
Developed by Bentus.