An in vivo rat model to study epigenetic control of cell invasion.

Due to it ability to invade deep into endometrium, as well as into other tissues at ectopic sites (testis, kidney capsule), throphoblast plays an important role in shaping the future placenta. To accomplish this task, it is necessary for throphoblast cells to differentiate into highly invasive throphoblast giant cells (TGC). The behaviour of TGC during implantation resembles that of cancer cells during metastasis. In both cases, the invasive phenotype is to a large degree controlled epigenetically, by DNA methylation, with resulting gene expression silencing. DNA demethylating agents, such as 5-azacitidine (5azaC), reverse the gene expression and change cell behaviour; already used in cancer therapy, 5azaC is also useful experimentally to elucidate epigenetic pathways in normal and malignant cells. In this paper we describe an in vivo rat model of throphoblast cell invasion, in which cells are exposed to 5azaC and transplanted ectopically under kidney capsule. We conclude that temporal variation in exposure to 5azaC, such as the gestation day, affects the throphoblast cells differentiation, and thus changes their invasive properties. We suggest that this in vivo model could be useful to study steps in epigenetic control of both the placental development and cancer cell spread.

GW body disassembly triggered by siRNAs independently of their silencing activity.

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.

The impact of 5-azacytidine on placental weight, glycoprotein pattern and proliferating cell nuclear antigen expression in rat placenta.

During the placentation process, the expression of various glycoproteins plays an important role in embryonal development. Alterations in DNA methylation caused by 5-azacytidine (5azaC) can disturb normal glycoprotein expression as well as the proliferative ability of trophoblast cells. In order to assess this, a single dose of 5azaC was injected intraperitoneally into pregnant rats during days 1-19 of gestation. Animals were euthanised on day 20 and placental weight, as well as glycoprotein composition, was analysed together with immunohistological assessment of the degree of proliferation of the trophoblast cells. The placental weight was found to be significantly smaller in animals treated by 5azaC during days 4 to 14 of gestation (p<0.01, Student's t-test). The treatment on days 4, 5, and 6 resulted in a lack of labyrinth with the strong proliferative activity of the cells in the basal layer. Expression of glycoproteins with molecular mass smaller than 60 kDa was reduced with treatment on day 6. The 5azaC administered from days 7 to 10 completely disturbed the placental structure and the proliferation of trophoblast cells was poor. During these days GP70 exhibited stronger expression in treated animals, contrary to GP40, which was stronger in controls. A natural border between the labyrinth and the basal layer was established on days 11 and 12. The basal layer was dominant with a lower proliferation of trophoblast cells compared with the controls. With the establishment of the labyrinth on day 13, the expression of GP40 was restored. Proliferation of the trophoblast cells from days 13 to 15 was higher compared with the controls. The changes in placental mass and the proliferative ability of trophoblast cells in rat placenta exposed to 5azaC represent more proof of the importance of epigenetics in the regulation of placental development.