GL-3 accumulation occurs primarily in endothelial cells with impairment of vessel reactivity

ype individuals. However, when the remaining functional copy of SdhD was deleted, a significant induction of Vegf was observed in the SDHD-ESR kidney only three weeks after the start of injections, whereas for Glut1 and Phd3 their mRNA levels did not significantly increase further. Moreover, no signs of Hif1a accumulation were observed, with this protein remaining undetected in mutant tissues as assessed by western blot at both time points. As a control, protein extracts from a tissue-specific knock-out mouse for pVHL showed induced expression of Hif1a. In addition, no increase was observed for any of the same tested genes in liver and brain. Western blot of the same tissue extracts against the Hif2a antibody also produced no signal. Together, these data suggest that activation of “pseudo-hypoxic drive”as a consequence of MCII depletion does not take place in a general and obvious manner in the analyzed mouse tissues. The “pseudo-hypoxic drive”in SDHD-ESR-derived cell lines differs from that in tissues. As the diffusion kinetics of the drug throughout the mouse tissues could expand the SdhD deletion on time, thus hampering the detection of transitory Hif1a stabilization and the transcriptional activation of its target genes, we decided to establish cell lines from the SDHD-ESR mouse in which the accessibility of the 22351639 cells to the drug is better controlled. We newly derived and immortalized the commonly used mouse embryonic fibroblasts cell type, as well as the epitheliumderived baby mouse kidney cells, which are more suitable for the study of the biology of epithelial-derived tumors. The BMK cells have recently proved to be useful for identifying synthetic lethal genes with the Krebs cycle enzyme fumarate hydratase, which is closely related to Sdh. Loss of SdhD was confirmed following addition of tamoxifen to the culture medium in two independent immortalized clones per cell type. We tested the pseudo-hypoxia pathway in our immortalized MEF and BMK cell lines after tamoxifen exposure. Whereas in heterozygous lines no differences were found in either Hif1a or Glut1 protein levels with respect to the wild type, the SDHD-ESR mutant line underwent accumulation of both proteins, although with different kinetics of induction. When mRNA levels were determined for some Hif1a-target genes, Phd3 and Glut1 expression were found to be induced in the SDHD-ESR cell line, whereas Vegf expression was not, or was even significantly identifying ONX-0914 site alternative molecular pathways altered upon SdhD deletion, and to determine if some tissue-specific features could condition the transcriptional response to MCII depletion, we performed high-throughput gene expression analysis of kidney and adrenal medulla tissue. The adrenal 22564524 medulla was chosen because it is one of the main pheochromocytoma/paraganglioma target tissues. The kidney was chosen not only because of the more intense effect on SdhD deletion exerted by tamoxifen, but also due to the finding that renal cell carcinoma, although much less frequently, is also associated with Sdh-mutations. To minimize secondary effects on gene expression, we analyzed samples obtained one week after the first tamoxifen injection. At this time point, SdhD mRNA levels in both tissues had already decreased considerably . It is worth to mention that at this time-point no major histological abnormalities were detected for any of the tissues analyzed. For large-scale gene expression analysis we performed two-color microarray hybridiype individuals. However, when the remaining functional copy of SdhD was deleted, a significant induction of Vegf was observed in the SDHD-ESR kidney only three weeks after the start of injections, whereas for Glut1 and Phd3 their mRNA levels did not significantly increase further. Moreover, no signs of Hif1a accumulation were observed, with this protein remaining undetected in mutant tissues as assessed by western blot at both time points. As a control, protein extracts from a tissue-specific knock-out mouse for pVHL showed induced expression of Hif1a. In addition, no increase was observed for any of the same tested genes in liver and brain. Western blot of the same tissue extracts against the Hif2a antibody also produced no signal. Together, these data suggest that activation of “pseudo-hypoxic drive”as a consequence of MCII depletion does not take place in a general and obvious manner in the analyzed mouse tissues. The “pseudo-hypoxic drive”in SDHD-ESR-derived cell lines differs from that in tissues. As the diffusion kinetics of the drug throughout the mouse tissues could expand the SdhD deletion on time, thus hampering the detection of transitory Hif1a stabilization and the transcriptional activation of its target genes, we decided to establish cell lines from the SDHD-ESR mouse in which the accessibility of the cells to the drug is better controlled. We newly derived and immortalized the commonly used mouse embryonic fibroblasts cell type, as well as the epitheliumderived baby mouse kidney cells, which are more suitable for the study of the biology of epithelial-derived tumors. The BMK cells have recently proved to be useful for identifying synthetic lethal genes with the Krebs cycle enzyme fumarate hydratase, which is closely related to Sdh. Loss of SdhD was confirmed following addition of tamoxifen to the culture medium in two independent immortalized clones per cell type. We tested the pseudo-hypoxia pathway in our immortalized MEF and BMK cell lines after tamoxifen exposure. Whereas in heterozygous lines no differences were found in either Hif1a or Glut1 protein levels with respect to the wild type, the SDHD-ESR mutant line underwent accumulation of both proteins, although with different kinetics of induction. When mRNA levels were determined for some Hif1a-target genes, Phd3 and Glut1 expression were found to be induced in the SDHD-ESR cell line, whereas Vegf expression was not, or was even significantly identifying alternative molecular pathways altered upon SdhD deletion, and to determine if some tissue-specific features could condition the transcriptional response to MCII depletion, we performed high-throughput gene expression analysis of kidney and adrenal medulla tissue. The adrenal medulla was chosen because it is one 15130089 of the main pheochromocytoma/paraganglioma target tissues. The kidney was chosen not only because of the more intense effect on SdhD deletion exerted by tamoxifen, but also due to the finding that renal cell carcinoma, although much less frequently, is also associated with Sdh-mutations. To minimize secondary effects on gene expression, we analyzed samples obtained one week after the first tamoxifen injection. At this time point, SdhD 1417961 mRNA levels in both tissues had already decreased considerably . It is worth to mention that at this time-point no major histological abnormalities were detected for any of the tissues analyzed. For large-scale gene expression analysis we performed two-color microarray hybridi