Estudio sobre el papel de las acuaporinas en proceso de proliferación celular

• Autores: Ana Galan Cobo
• Directores de la Tesis: Miriam Echevarría (dir. tes.)
• Lectura: En la Universidad de Sevilla ( España ) en 2014
• Idioma: español
• Tribunal Calificador de la Tesis: José López Barneo (presid.), Ricardo Pardal Redondo (secret.), Maria da Graça Soveral Rodrigues (voc.), Carmen Sáez Torres (voc.), Gema Frühbeck Martínez (voc.)
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• Resumen

  • ROLE OF AQUAPORINS IN THE CELL PROLIFERATION PROCESS Aquaporin-1 and 3 (AQP1 and AQP3) expression has been associated with tumor formation, particularly with angiogenesis, cell migration and proliferation, but the specific role of these proteins in tumorigenesis and cell proliferation is far unclear. Thus, to better understand the role AQP1 and AQP3 may play in these processes we have explored the effect that stable overexpression of these proteins produce over the proliferation process and cell cycle of PC12 cells as cellular models. Additionally, using the gold (III) complex Auphen, recently described as a very selective and potent inhibitor of AQP3¿s glycerol permeability, we have particularly explored the role of AQP3 in the cell proliferation process. Moreover, in the adult carotid body (CB), an oxygen-sensing organ that grows under chronic hypoxia and in which expression of AQP1 was detected in type I chemoreceptor cells and other CB tissues, we explore the role of AQP1 in the ¿in vivo¿ cell proliferation process that occurs upon exposure to chronic hypoxia, comparing wild type animals (AQP1 +/+) for AQP1 expression to knock out ones (AQP1 -/-).

    Methods: Proliferation measurements and cell cycle analysis were performed over wild type PC12 cells (PC12-wt) and in PC12 with overexpression of either AQP1 (PC12-AQP1) or AQP3 (PC12-AQP3). Cell proliferation was also evaluated in cell lines that either express AQPs in a natural way (A431-epidermoid cells) or cells which were transiently transfected for expression of AQPs (HEK293T), and compare it with cells with low/or none AQP expression (NIH-3T3 and PC12-wt). Cell proliferation rate was evaluated through cell counting and BrdU staining and cell cycle by flow cytometry with propidium iodide (PI) staining. We studied the response to arrest cell cycle drugs as butyrate and nocodazole and explored the resistance to apoptosis by Annexin V staining using nocodazole. In addition, proteomic (western blot) and transcriptomic (Affymetrix) techniques were performed to reveal key molecules implicated in cell proliferation process which expression may be altered by overexpression of AQP1. Finally, mice were either let in normoxia (21% of O2), or maintained for 12 and 21 days in a hypoxic atmosphere (10% of O2), and received BrdU treatment as a proliferation cell marker.

    Immunohistological analysis of the CB was used to quantify the number of TH+, BrdU+ and TH plus BrdU double-positive cells in normoxia and hypoxia conditions. Parenchyma CB total volume and TH+ cell volume were also measured.

    Results: Cells with overexpression of AQP1 and AQP3 showed higher cell proliferation rate and larger percentage of cells in phases S and G2/M of the cell cycle. In the presence of butyrate they showed lower synchronization in G1 and when treated with nocodazole they exhibited less modification of the cell cycle pattern and null apoptotic induction was observed after 24h in the presence of this compound. Western blot analysis performed in PC12-AQP1, showed higher expression of cyclin D1, E1 and ß-catenin, while E-cadherin levels were lower when compared to PC12-wt. Also Affymetrix analysis revealed consistent changes in molecules related with cell proliferation, tumor and cell cycle progression, such as, Zeb2, Jun, NF-kß, Cxcl9, Cxcl10, TNF, and TNF receptors. Treatment with Auphen strongly reduced the proliferation process of cells that express AQP3. Conversely, Auphen did not reduce the proliferation of cells with low or none expression of AQP3. Flow cytometric analysis of the cell cycle phases performed in PC12 overexpressing AQP3 in presence of Auphen showed a strong arrest of the cells in S-G2/M phases compare with PC12-wt. Finally, lower number of total BrdU+ and TH-BrdU+ cells were counted in AQP1 -/- mice after the two times of hypoxia indicated.

    Conclusions: A significant role of AQP1 and AQP3 in the cell proliferation process is demonstrated here. It seems to be connected to increments in the cell cycle turn over, that in turn maybe associated with changes in different tumor canonical pathways such as NF-kß, Jun or EMT pathways. Our results support the view that larger expression of AQP1 and AQP3 confer to the cell a more tumoral like phenotype and contribute to explain the presence of this protein in many different tumors. Overall our results demonstrate a significant role of AQP3 in the cell proliferation process and may indicate a potential therapeutic effect of Auphen over tumorigenesis due to its capacity of interfere in the cell proliferation associated with AQP3, but more studies are necessary to dig inside the molecular basis that underline to this mechanism(s). By other hand, ¿in vivo¿ experiments performed in the KO mice for AQP1 evidence that the lack of this protein impair the cell proliferation process in CB, so AQP1 could has a significant role in non-pathologic proliferation process as well.