Previously, we discovered that deletion of c-Rel in the Eµ-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Eµ-Myc/cRel−/− cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Eµ-Myc/cRel−/− lymphomas highly resemble wild-type (WT) Eµ-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Eµ-Myc/cRel−/− lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Eµ-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours.
Hepatic iron is known to regulate insulin signaling pathways and to influence insulin sensitivity in insulin resistance (IR) patients. However, the role of insulin on hepatic iron homeostasis remains unexplored. Here, we report that insulin promotes transferrin-bound iron uptake but shows no influence on non transferrin-bound iron uptake in human hepatic HepG2 cells. As a mechanism we detected increased transferrin receptor-1 (TfR1) expression both at protein and mRNA levels. Unaltered stability of protein and transcript of TfR1 suggested the regulation at transcriptional level that was confirmed by promoter activity. Involvement of transcription factor hypoxia inducible factor-1 (HIF-1) was shown by mutational analyses of the TfR1 promoter region and by electrophoretic mobility shift assay. When HepG2 cells were transfected with specific siRNA targeted to 3'UTR of HIF-1α, the regulatory subunit of HIF-1; insulin-induced TfR1 expression and iron uptake were inhibited. Transfection of cDNA expressing stable form of HIF-1α reversed the increased TfR1 expression and iron uptake. These results suggest a novel role of insulin in hepatic iron uptake by a HIF-1 dependent transcriptional regulation of TfR1.
[structure: see text] An artificial macrocyclic fluorescent receptor 1 has been designed and synthesized for the recognition of urea. 1 shows significant fluorescence quenching on complexation with urea and thiourea in chloroform and thus may be used as a synthetic fluorescent molecular sensor for their determination in a nondegradative way.
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