Utjecaj 5-azacitidina na razvoj eksperimentalnog teratokarcinoma u miša

Sinčić, Nino (2012) Utjecaj 5-azacitidina na razvoj eksperimentalnog teratokarcinoma u miša. PhD thesis, Sveučilište u Zagrebu.

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Abstract

The aim of this PhD thesis was to investigate the DNA methylation pattern and influence of DNA demethylating agent 5-azacitydine (5azaC) on the development of experimental mouse teratocarcinoma (TCa) obtained by the transplantation of gastrulating mouse embryo (E 7,5) under the kidney capsule of an adult animal. It was discovered that TCa develops from male embryos only. Investigated 8- week long period of TCa development started with subtle growth followed by resting phase. TCa development ended with explosive growth paralleled with concomitant increase in expression of Pcna gene (proliferation marker). In vitro experiments have pinpointed the key role of stemness genes Oct3/4 and Nanog expression since their esiRNA induced inhibition caused the suppression of tumour development from the embryo. Through analysis of DNA methylation pattern we discovered continuing hypomethylation of Nanog gene during TCa development compared with normal testicular tissue. Contrary, compared with embryo development, Oct3/4 gene showed hypermethylation reprogramming overlapping with the period of tumour subtle growth and resting phase period. Achieving the maximum hypermethylation phase, TCa initiated the intensive growth correlating with proportion of Oct3/4 demethylated cells. Sox2 gene, although with low percentage of DNA methylation, showed the promoter methylation reprogramming during TCa development similar to the one established for Oct3/4 gene promoter. Based on the promoter DNA methylation pattern analysis of Scgb3a1, Prss21, Stat3, Brca1, Mgmt, c-Myc, Trrap and Rassf1 genes and established global methylation pattern based on the analysis of Mml, Mmetnltr and B1 repetitive DNA sequences it may be concluded that these genes and repetitive sequences are not involved in teratocarcinoma development. 5azaC applied immediately after the embryo transplantation completely inhibited teratocarcinoma development whereas applied on already developed teratocarcinoma, decreased its growth with statistical significance. Despite the fact that 5azaC causes global hypomethylation, its application did not influence the methylation status and reprogramming of stemness genes Oct3/4, Nanog and Sox2. Quite contrary, treatment with 5azaC inhibited the expression of Oct3/4 and Nanog genes in developed teratocarcinoma by some other mechanisms that did not include DNA methylation change. Our results showed that 5azaC treatment does not cause changes of oxidative stress markers nor the markers associated with the inflammation process. According to our obtained results it seems that 5azaC influence the establishment of methyloma which encourages apoptosis and decreases proliferation of TCa stem cells. Our research reveals the epigenetic platform of TCa development and potentials of 5azaC in the treatment of testicular germ cell tumours.

Abstract in Croatian

Cilj disertacije bio je istražiti epigenetičku platformu i utjecaj DNA demetilacijskog agensa 5- azacitidina (5azaC) na razvoj eksperimentalnog teratokarcinoma miša (TCa) dobivenog transplantacijom gastrulirajućeg zametka (E 7,5) pod čahuru bubrega odrasle životinje. Otkriveno je da se TCa razvija samo iz zametaka muškog spola. Istraživani osmotjedni razvoj TCa započeo je blagim rastom nakon kojeg je uslijedio period mirovanja. TCa je završio razvoj eksplozivnim rastom koji se dogodio istovremeno s povećanjem ekspresije proliferacijskog gena Pcna. Eksperiment in vitro ukazao je na ključnu ulogu ekspresije gena matičnosti Oct3/4 i Nanog u razvoju TCa jer je inhibicijom njihove ekspresije pomoću esiRNA došlo do supresije rasta zametka. Analizom metilacije DNA otkrili smo kontinuiranu hipometilaciju gena matičnosti Nanog tijekom razvoja TCa u usporedbi s testisom. Gen Oct3/4 pokazao je pak hipermetilacijski reprogram u usporedbi s zametkom koji se preklapao s periodom blagog rasta i mirovanja tumora. Postizanjem maksimuma hipermetilacije, TCa je započeo intenzivan rast u korelaciji s udjelom Oct3/4 demetiliranih stanica. Gen Sox2, iako s niskim stupnjem metilacije DNA, pokazao je reprogram metilacije promotora u usporedbi s zametkom tijekom razvoja teratokarcinoma sličan promotoru gena Oct3/4. Analizom metilacijskih obrazaca i njihove promjene na promotorima gena Scgb3a1, Prss21, Stat3, Brca1, Mgmt, c-Myc, Trrap i Rassf1 te globalne metilacije DNA temeljene na analizi repetitivnih sekvenci Mml, Mmetnltr i B1 moglo se zaključiti da navedeni geni i sekvence nisu bili uključeni u razvoj teratokarcinoma. 5azaC primijenjen neposredno nakon transplantacije zametka potpuno je spriječio razvoj TCa, dočim primijenjen na razvijeni TCa, statistički značajno smanjuje rast tumora. Iako je 5azaC uzrokovao globalnu hipometilaciju, nije utjecao na metilacijski status i metilacijske reprograme gena matičnosti Oct3/4, Nanog i Sox2. Dapače, 5azaC inhibirao je ekspresiju gena matičnosti Oct3/4 i Nanog u tretiranih razvijenih TCa mehanizmom koji ne uključuje promjenu metilacije DNA. Naši rezultati pokazali su da 5azaC ne izaziva promjene biljega oksidacijskog stresa, niti onih povezanih s upalnim procesima. Dakle, čini se da 5azaC izaziva uspostavu metiloma koji vjerojatno potiče apoptozu i smanjuje proliferaciju tumorskih matičnih stanica TCa. Naše istraživanje otkriva epigenetičku platformu razvoja tumora i potencijal upotrebe 5azaC u liječenju tumora zametnih stanica testisa.

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