Epigenetic and Immunomodulatory Changes in Malignant Head and Neck Tumours
A degree of methylation of a gene promoter is an important epigenetic regulator of the gene activity. Although the DNA is globally hypomethylated in malignant tumors, the promoters of tumor suppressor genes are usually hypermethylated and thus, silenced. The best model for studying methylation as the regulatory mechanism of gene transcription are imprinted genes that are always expressed from one parental allele. The key enzyme in establishing and maintaining the DNA methylation is DNA methyltransferase type 1 (DNMT1). Noncovalent binding of the ADP-ribose polymers to DNMT1 is obtained by automodification of poly (ADP-ribose) polymerase (PARP-1), which diminishes its catalytic activity. The substrate for the PARP-1 enzyme is nicotinamide adenin dinucleotide (NAD) which is de novo synthesized in kynurenine pathway of triptophan degradation. The key enzyme in this process is indolamine 2,3, dioxygenase (IDO), whose activity is increased in tumors by action of IFN-gamma; what causes the local anergy of T lymphocytes. Automodificiation of PARP-1 also increases expression of inducible NO synthase (iNOS) by decreasing a degree of methylation of its promotor. Both the aim and importance of this project are understanding the association between locally modified immune response and the imprinting status of selected imprinted genes on the chromosome 11: IGF-2, H19, CDKN1, LIT1, KCNQ1 in human malignant head and neck tumors. The final results should establish a functional connection between the genes/proteins activated in locally modified immunologic response and loss of imprinting (LOI), i.e. discovery of epigenetic changes which affects the gene activity, as well as mechanisms that could cause these changes. The final conclusions will be drawn on the basis of experiments performed at the level of mRNAs and proteins for: IDO, iNOS, PARP-1, IGF2, H19, CDKN1, LIT1, KCNQ1. The loss of heterozygosity will be determined for H19, CDKN1, CTCF and PARP-1, and LOI for all mentioned imprinted genes. A level of tryptophan and NO will be determined in the serum of patients. The role of tryptophan in regulation of iNOS will be investigated in cell cultures. The results will be checked complementary: DNA-RNA-protein, and by use of several methods, in logical order (at the level of mRNA: RT-PCR, quantitative PCR, microarray). The understanding of association between LOI and local immune response opens a new approach in molecular oncology research.