engleski

DNA Demethylating agent 5-azacytidine reduces overall tumour growth of experimental teratocarcinoma and teratoma

Experimental rat teratoma contains differentiated tissue derivatives of all three germ-layers while mouse teratocarcinoma at the same time harbours undifferentiated embryonal carcinoma (EC) cells responsible for the malignancy of the tumor. Experimental teratocarcinoma can be induced by transplantation of the gastrulating mouse embryo to extrauterine sites as has been discovered at the Department of Biology, School of Medicine, Zagreb already in the 1960s. These important results were the basis for further extensive reseach worldwide but still is not known what really causes an embryo to become malignant. The mechanism of development of experimental teratocarcinoma seems not to be dependent upon mutations but rather on changes of the epigenetic regulation of gene expression at the ectopic microenvironment. Based on the gene expression profiling it has been recently proposed that cell cycle pathways might be important for malignant transformation of embryonic stem cells (ES) to teratocarcinoma. Epigenetic mechanism that could cause these changes is DNA methylation, responsible for inactivation of gene expression. The DNA demethylating agent 5-azacytidine (5azaC) has been known to change development through alterations in gene expression. It was shown that it is able to induce differentiation of EC cells in vitro. Therefore we started to investigate the unknown effect of 5-azacytidine treatment on development (growth and differentiation) of experimental mouse teratocarcinoma in vivo. Additionally, we started to investigate 5azaC influence in experimental rat teratoma because a number of our previous experiments demonstrated that 5-azaC changes differentiation in culture in vitro of the gastrulating embryo and impairs growth of the rat embryo and placenta during pregnancy. 9, 5-days-old Fisher rat embryos and 7, 5-days-old C3H mouse embryos were microsurgically isolated under the dissecting microscope. Adult males of respective strains were anesthesized and embryos were grafted under the kidney capsule or in testis. 5 mg/kg 5-azacytidine was administered intraperitoneally once or twice a week to experimental animals. Controls were treated with PBS in the same way. Tumors and male gonads were weighted, fixed in St. Marie solution and processed for routine histology or immunohistochemistry. In the rat as well as in the mouse 5azaC significantly reduced tumor growth under the kidney capsule when applied twice a week. Moreover, in the mouse, when 5azaC was applied only once a week, significantly smaller tumors were found too. It seems that in the mouse some grafted embryos did not develop tumors at all, while in controls almost all grafts developed tumors. When embryos were grafted in the testis and treated after a delay of 6 months, in controls a small number of teratocarcinoma was obtained whereas in the experimental group no teratocarcinomas were obtained. In both rat and mouse after 5azaC treatment reduced weight of male gonads was detected. It can be concluded that the DNA demethylating agent 5azaC reduces growth of teratoma and teratocarcinoma in the rat and in the mouse, respectively, but also has a deleterious effect upon male gonads. This investigation points out that the regulation of gene expression through DNA methylation is crucial for development of mammalian teratoma and teratocarcinoma and therefore could also be of importance in human medicine because DNA demethylating agents have alraedy been used in treatment of other malignancies.

5-azacytidine; teratoma; teratocarcinoma; growth

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