Dose-dependent effects of Allopurinol on human foreskin fibroblast cell and human umbilical vein endothelial cell under hypoxia

Allopurinol, an inhibitor of xanthine oxidase, has been used in clinical trials of patients with cardiovascular and chronic kidney disease. These are two pathologies with extensive links to hypoxia and activation of the transcription factor hypoxia inducible factor (HIF) family. Here we analysed the effects of allopurinol treatment in two different cellular models, and their response to hypoxia. We explored the dose-dependent effect of allopurinol on Human Foreskin Fibroblasts (HFF) and Human Umbilical Vein Endothelial Cells (HUVEC) under hypoxia and normoxia. Under normoxia and hypoxia, high dose allopurinol reduced the accumulation of HIF-1α protein in HFF and HUVEC cells. Allopurinol had only marginal effects on HIF-1α mRNA level in both cellular systems. Interestingly, allopurinol effects over the HIF system were independent of prolyl-hydroxylase activity. Finally, allopurinol treatment reduced angiogenesis traits in HUVEC cells in an in vitro model. Taken together these results indicate that high doses of allopurinol inhibits the HIF system and pro-angiogenic traits in cells

Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK

Down-Regulation of HtrA1 Activates the Epithelial-Mesenchymal Transition and ATM DNA Damage Response Pathways

Expression of the serine protease HtrA1 is decreased or abrogated in a variety of human primary cancers, and higher levels of HtrA1 expression are directly related to better response to chemotherapeutics. However, the precise mechanisms leading to HtrA1 down regulation during malignant transformation are unclear. To investigate HtrA1 gene regulation in breast cancer, we characterized expression in primary breast tissues and seven human breast epithelial cell lines, including two non-tumorigenic cell lines. In human breast tissues, HtrA1 expression was prominent in normal ductal glands. In DCIS and in invasive cancers, HtrA1 expression was greatly reduced or lost entirely. HtrA1 staining was also reduced in all of the human breast cancer cell lines, compared with the normal tissue and non-tumorigenic cell line controls. Loss of HtrA1 gene expression was attributable primarily to epigenetic silencing mechanisms, with different mechanisms operative in the various cell lines. To mechanistically examine the functional consequences of HtrA1 loss, we stably reduced and/or overexpressed HtrA1 in the non-tumorigenic MCF10A cell line. Reduction of HtrA1 levels resulted in the epithelial-to-mesenchymal transition with acquisition of mesenchymal phenotypic characteristics, including increased growth rate, migration, and invasion, as well as expression of mesenchymal biomarkers. A concomitant decrease in expression of epithelial biomarkers and all microRNA 200 family members was also observed. Moreover, reduction of HtrA1 expression resulted in activation of the ATM and DNA damage response, whereas overexpression of HtrA1 prevented this activation. Collectively, these results suggest that HtrA1 may function as a tumor suppressor by controlling the epithelial-to-mesenchymal transition, and may function in chemotherapeutic responsiveness by mediating DNA damage response pathways

Procollagen Lysyl Hydroxylase 2 Is Essential For Hypoxia-induced Breast Cancer Metastasis

Metastasis is the leading cause of death among patients who have breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies to treat cancer patients. Intratumoral hypoxia, found in the majority of breast cancers, is associated with an increased risk of metastasis and mortality. We found that in hypoxic breast cancer cells, HIF-1 activates transcription of the PLOD1 and PLOD2 genes encoding procollagen lysyl hydroxylases that are required for the biogenesis of collagen, which is a major constituent of the extracellular matrix. High PLOD2 expression in breast cancer biopsies is associated with increased risk of mortality. We demonstrate that PLOD2 is critical for fibrillar collagen formation by breast cancer cells, increases tumor stiffness, and is required for metastasis to lymph nodes and lungs

Procollagen Lysyl Hydroxylase 2 Is Essential For Hypoxia-induced Breast Cancer Metastasis

El presente estudio analiza el valor que la sentencia del Tribunal Supremo de 16 de abril de 2014 ha dado al preaviso que, para los supuestos de distribución irregular, prevé el art. 34.2 del TRLET tras su reforma. Asimismo se analiza el impacto que para la negociación colectiva ha tenido la admisión de la facultad empresarial de distribuir irregularmente hasta un 10% de la jornada ordinaria en los supuestos de falta de acuerdo en convenio o acuerdo.This paper analyzes the value that the decision of the Spanish Supreme Court of April 16th, 2014 has given to the "advance notice" for the irregular distribution of working time after the reform of section 34.2 of the Worker's Statute. It also studies the impact of such reform on collective bargaining since it authorizes the employer to distribute irregularly and unilaterally up to 10% of normal working hours

Inhibitors Of Hypoxia-inducible Factor 1 Block Breast Cancer Metastatic Niche Formation And Lung Metastasis

Intratumoral hypoxia, a frequent finding in metastatic cancer, results in the activation of hypoxia-inducible factors (HIFs). HIFs are implicated in many steps of breast cancer metastasis, including metastatic niche formation through increased expression of lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) proteins, enzymes that remodel collagen at the metastatic site and recruit bone marrow-derived cells (BMDCs) to the metastatic niche. We investigated the effect of two chemically and mechanistically distinct HIF inhibitors, digoxin and acriflavine, on breast cancer metastatic niche formation. Both drugs blocked the hypoxia-induced expression of LOX and LOXL proteins, collagen cross-linking, CD11b(+) BMDC recruitment, and lung metastasis in an orthotopic breast cancer model. Patients with HIF-1 alpha-overexpressing breast cancers are at increased risk of metastasis and mortality and our results suggest that such patients may benefit from aggressive therapy that includes a HIF inhibitor.link_to_OA_fulltex

Collagen Prolyl Hydroxylases Are Essential For Breast Cancer Metastasis

Metastasis is the leading cause of death among patients with breast cancer. Understanding the role of the extracellular matrix in the metastatic process may lead to the development of improved therapies for cancer patients. Intratumoral hypoxia is found in the majority of breast cancers and is associated with an increased risk of metastasis and patient mortality. Here we demonstrate that hypoxia-inducible factor 1 activates the transcription of genes encoding collagen prolyl hydroxylases that are critical for collagen deposition by breast cancer cells. We show that expression of collagen prolyl hydroxylases promotes cancer cell alignment along collagen fibers, resulting in enhanced invasion and metastasis to lymph nodes and lungs. Lastly, we establish the prognostic significance of collagen prolyl hydroxylase mRNA expression in human breast cancer biopsies, and demonstrate that ethyl 3,4-dihydroxybenzoate, a prolyl hydroxylase inhibitor, decreases tumor fibrosis and metastasis in a mouse model of breast cancer