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Madrid, Spain

Luque-Garcia J.L.,Protein Technology Unit | Martinez-Torrecuadrada J.L.,Protein Technology Unit | Epifano C.,Protein Technology Unit | Canamero M.,Comparative Pathology Unit | And 4 more authors.
Proteomics | Year: 2010

Progression to metastasis is the critical point in colorectal cancer (CRC) survival. However, the proteome associated to CRC metastasis is very poorly understood at the moment. In this study, we used stable isotope labeling by amino acids in cell culture to compare two CRC cell lines: KM12C and KM12SM, representing poorly versus highly metastatic potential, to find and quantify the differences in protein expression, mostly at the cell surface level. After biotinylation followed by affinity purification, membrane proteins were separated by SDSPAGE and analyzed using nanoflow LC-ESI-LTQ. A total of 291 membrane and membraneassociated proteins were identified with a p value<0.01, from which 60 proteins were found to be differentially expressed by more than 1.5-fold. We identified a number of cell signaling, CDs, integrins and other cell adhesion molecules (cadherin 17, junction plakoglobin (JUP)) among the most deregulated proteins. They were validated by Western blot, confocal microscopy and flow cytometry analysis. Immunohistochemical analysis of paired tumoral samples confirmed that these differentially expressed proteins were also altered in human tumoral tissues. A good correlation with a major abundance in late tumor stages was observed for JUP and 17-β-hydroxysteroid dehydrogenase type 8 (HSD17B8). Moreover, the combined increase in JUP, occludin and F11 receptor expression together with cadherin 17 expression could suggest a reversion to a more epithelial phenotype in highly metastatic cells. Relevant changes were observed also at the metabolic level in the pentose phosphate pathway and several amino acid transporters. In summary, the identified proteins provide us with a better understanding of the events involved in liver colonization and CRC metastasis. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA. Source


Guillou E.,DNA Replication Group | Guillou E.,Laboratoire Of Biologie Moleculaire Eucaryote | Ibarra A.,DNA Replication Group | Coulon V.,Montpellier University | And 8 more authors.
Genes and Development | Year: 2010

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. © 2010 by Cold Spring Harbor Laboratory Press. Source

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