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Acta Physiologica 2012; Volume 206, Supplement 693
Joint FEPS and Spanish Physiological Society Scientific Congress 2012
9/8/2012-9/11/2012
Santiago de Compostela, Spain
IMMORTALIZATION OF MOUSE EMBRYO ASTROCYTES (MEAS) IS ASSOCIATED WITH A DECREASE IN P19 AND P21
Abstract number: P248
gonzalez-mosquera1 t, seoane1 m, arce1 vm
1physiology, university of santiago de compostela
Objectives:
Our main aim was to develop an immortalized line of mouse embryo astrocytes as a suitable cellular model for the study of replicative senescence; as well as to investigate the role of p19 and p21 in the immortalization process. Mouse embryo fibroblasts (MEFs) have been largely considered as the gold-standard cells to investigate a wealth of cellular processes included those related with the regulation of cell proliferation, differentiation and senescence. However, these cells present different drawbacks that make some findings difficult to extrapolate to other cellular systems. In order to overcome this problem, we have developed a novel cell line derived from immortalized mouse embryo astrocytes (MEAs)in order to investigate the process of replicative senescence.
Materials:
Primary astrocyte cultures were established from 13.5 pcd C57/BL6 mouse embryos. Cells were seeded in DMEM supplemented with 10% FBS, and immortalized according with the 3T3 protocol. GFAP expression was evaluated by immunocytochemistry and RT-PCR. Senescence was evaluated with a commercial kit. p19 and p21 protein levels were evaluated by western blot. Statistical analysis was performed with the Mann-Whitney test.
Results:
An immortalized population of primary astrocytes was established by using the 3T3 protocol in primary astrocytes obtained from mouse embryos. Analysis of cellular senescence revealed a progressive increase in the percent of senescent cells, starting at passage 4 and reaching the higher level at passage 5. After this, senescence levels progressively decreased, and no senescence was found in cells after passage 18. When we investigated the changes in p19 and p21 expression, we found that the highest levels of both proteins were present in passage 5 and passage 6 cells, decreasing thereafter to levels similar to those observed in pre-senescent cells.
Conclusions:
Altogether, our findings suggest that a significant population of primary embryo astrocytes is able to overcome the senescence program induced by replicative stress, leading to the establishment of an immortalized cell line. This effect is likely secondary to the capacity of these cells to avoid p19 and p21 upregulation.
To cite this abstract, please use the following information:
Acta Physiologica 2012; Volume 206, Supplement 693 :P248