IDO gene expression induction by DNA methylation inhibitors

Sammanfattning: The aim was to study the effect of DNA methylation of CpG motifs in cancer cells and immune cells by use of the DNA methyltransferas (DNMT) inhibitor zebularine. In cancer cells it is known that genes can be silenced by chromosomal deletion(s). It is also known that genes can be silenced by DNA methylation or expression of micro-RNA. In cancer cells genes can also be overexpressed by chromosomal amplification. In creased gene expression in tumor cells can also be caused by DNA demethylation. In other words, gene expression in cancer cells can be caused both by hyper- and hypo-methylation. The immune system has been proven to be important in the regulation of tumor growth and many tumors produce factors that inhibit the immune reactivity against tumors and tumor associated antigens (TAAs). We advocated, that genes that were silenced by DNA methylation might cause immune suppression from tumor cells. We therefore exposed rat colon cancer cells to the DNMT inhibitor Zebularine to investigate if we could generate tumor cells that were less immune suppressive. Surprisingly, the tumor cells became less immune stimulatory. We identified a strong increase in the expression of the immune suppressive indoleamine 2,3-dioxygenase (IDO1) gene. By blocking this gene we could revert the increased immune suppression of the zebularine treated cancer cells. Next, we studied the effect on stimulated immune cells from both rat and human origin. Zebularine not only induced IDO1 in rat cancer cells but also in normal rat and human lymphoid cells. IDO1 is a very important enzyme to control and suppress immune response in pregnancy and in tolerance against auto antigens, i.e., to avoid immune response against the fetus and to avoid autoimmune diseases. Hence, we found molecules to control the immune reactivity that could be used to suppress immune reactions in autoimmune disorders. In cancer we could block the immune supprssion mediated by IDO1 by using IDO1 inhibitors. When studying the DNA methylation we also found that the DNA methylation from Escherichia coli was recognized and maintained in human cells. DNA was isolated from E.coli and transferred to human cells in such a way that it became stably integrated in the human chromosomes. This DNA was investigated for DNA methylation and we demonstrated that the bacterial DNA methylation pattern on cytidine residues were maintained. The find is of importance when planning future gene therapy studies.