Some proteases possess a membrane receptor that focalizes their proteolytic activity on the cell surface and may mediate a proliferative effect, such as urokinase on glomerular epithelial cells. Since some hypertensive states are associated with high concentrations of renin and proliferation of arteriolar smooth muscle cells, we asked whether renin, an aspartyl-protease, would bind to mesangial cells that are smooth-muscle derived cells, which would induce their proliferation. The binding of 125I labeled recombinant human renin (125I-R) was studied on human primary mesangial cells and mesangial cells immortalized by transfection with SV40-T antigen. At 37 degrees C, the binding of 125I-R was time dependent and reached a plateau after two hours. 125I-R was found to bind in a saturable and specific manner with a Kd = 0.4 nM and 1 nM and 8,000 and 2,000 binding sites/cell, for primary and immortalized cells, respectively. When binding experiments were performed in the presence RO 42-5892, a synthetic inhibitor of renin, RO 42-5892 could inhibit the specific binding of labeled renin only at concentrations 1,000 times superior to the IC 50, indicating that the renin-mesangial receptor interaction did not depend on the active site of renin. Analysis by SDS-PAGE and autoradiography of cross-linking experiments of 125I-R bound to a membrane preparation showed a band of approximately 110 to 120 kDa, suggesting a Mr of 70 to 80 kDa for the renin receptor. Incubation of mesangial cells with 100 nM renin for 24 hours provoked a 100% increase of 3H thymidine incorporation that was not accompanied by an increase of the cell number, even after a seven day period of incubation. However, the incubation of mesangial cells with renin for 24 hours induced a significant increase (170% of control, P = 0.04) of plasminogen activator inhibitor-1 (PAI1) antigen in the conditioned medium. In conclusion, we have shown that human mesangial cells in culture express a specific receptor for renin, and that the binding of renin increases 3H thymidine incorporation independently of renin enzymatic activity. The absence of cell proliferation, the increase of 3H thymidine incorporation and the increase of PAI1 antigen suggest that the binding of renin can induce mesangial cell activation, which is reflected by a change in the fibrinolytic capacity of the cells. The role of this receptor remains to be determined in nephropathies and hypertensive states associated with high plasma/tissue renin concentrations, hypertrophy of mesangial or smooth muscle cells and extracellular matrix remodeling.
The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profiles in sensitive cell lines, including a c-MYC decrease and an HEXIM1 increase. OTX015 exposure also induced a strong decrease of BRD2, BRD4 and c-MYC and increase of HEXIM1 proteins, while BRD3 expression was unchanged. c-MYC, BRD2, BRD3, BRD4 and HEXIM1 mRNA levels did not correlate however with viability following exposure to OTX015. Sequential combinations of OTX015 with other epigenetic modifying drugs, panobinostat and azacitidine have a synergic effect on growth of the KASUMI cell line. Our results indicate that OTX015 and JQ1 have similar biological effects in leukemic cells, supporting OTX015 evaluation in a Phase Ib trial in relapsed/refractory leukemia patients.
Long-term T-cell reconstitution after hematopoietic stem cell transplantation (HSCT) is dependent on patient thymic function and affected by graft-versushost disease (GVHD). To assess the impact of acute GVHD (aGVHD) on thymic function, we followed a cohort of 93 patients who received HSCT from a human histocompatibility leukocyte antigenidentical sibling, mainly for hematologic malignancies. Thymic output was measured by signal-joint T-cell receptor excision circles (sjTREC) real-time polymerase chain reaction. Absolute sjTREC number was lower at 6 months in patients with aGVHD (P ؍ .014), associated with lower absolute counts of naive CD4 T cells at 6 and 12 months (P ؍ .04 and .02), and persistent abnormalities in T-cell repertoire diversity. Age and aGVHD affected thymic function independently in multivariate analysis. In patients less than 25 years of age, thymic function recovered almost totally at 1 year. As a marker of thymocyte proliferation, we quantified the TREC generated during the T-cell receptor -chain recombination, in a group of 20 age-matched patients. Mean TREC level was reduced at 6 months in patients with aGVHD, indicating an impact on early thymic differentiation rather than on intrathymic proliferation. These data show that aGVHD or its treatment has a transient impact on thymic function in younger patients in the first months after HSCT. (Blood. 2009;113:6477-6484)
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