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

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



Introduction
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.
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