p53 monitors genomic integrity at the G1 and G2/M cell cycle checkpoints. Cells lacking p53 may show gene ampli®cation as well as the polyploidy or aneuploidy typical of many tumors. The pathways through which this develops, however, are not well de®ned. We demonstrate here that the combination of p53 inactivation and c-myc overexpression in diploid cells markedly accelerates the spontaneous development of tetraploidy. This is not seen with either N-myc or L-myc. Tetraploidy is accompanied by signi®cantly higher levels of cyclin B and its associated cdc2 kinase activity. Mitotic spindle poisons accelerate the appearance of tetraploidy in cells either lacking functional p53 or overexpressing c-myc whereas the combination is additive. Restoration of p53 function in cells overexpressing c-myc causing rapid apoptosis, indicating that cells yet to become tetraploid have nonetheless suered irreversible genomic and/or mitotic spindle damage. In the face of normal p53 function, such damage would either be repaired or trigger apoptotis. We propose that loss of p53 and overexpression of c-myc permits the emergence and survival of cells with increasingly severe damage and the eventual development of tetraploidy.
BackgroundRoxarsone (3-nitro-4-hydroxy benzene arsonic acid) is an arsenic compound widely used in the poultry industry as a feed additive to prevent coccidiosis, stimulate growth, and to improve tissue pigmentation. Little is known about the potential human health effects from roxarsone released into the environment from chicken waste or from residual compound in chicken products.ObjectiveThe growth potentiation and enhanced tissue pigmentation suggest that low levels of roxarsone exposure may have an angiogenic potential similar to that of inorganic arsenite (AsIII). The goal of this investigation was to test the hypothesis described above using cultured human aortic and lung microvascular endothelial cells in high-content imaging tube-forming assays and begin developing a molecular level understanding of the process.MethodsWe used a three-dimensional Matrigel assay for probing angiogenesis in cultured human endothelial cells, and a polymerase chain reaction (PCR) array to probe the gene changes as a function of roxarsone or AsIII treatment. In addition, we used Western blot analysis for changes in protein concentration and activation.ResultsRoxarsone was found to exhibit a higher angiogenic index than AsIII at lower concentrations. Increased endothelial nitric oxide synthase (eNOS) activity was observed for roxarsone but not for AsIII-induced angiogenesis. However, AsIII caused more rapid and pronounced phosphorylation of eNOS. Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression.ConclusionsThe results demonstrate that roxarsone and AsIII promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.
The complexity of mitogen-activated protein kinase (MAPK) signaling pathways and their activation by different stimuli makes assaying the activation of particular MAPKs by specific receptors a challenging problem. The multiplexing capability of quantitative high-content screening (HCS) assays enables the simultaneous monitoring and correlation, in the same cell, of an MAPK's specific activation with a particular receptor's post-signaling behavior, such as its internalization. We demonstrate a cell-based HCS assay to quantify the epidermal growth factor (EGF) receptor-specific activation of the MAPK ERK. Activation was quantified by measuring immunofluorescently labeled phosphorylated extracellular signal-regulated protein kinases (ERK) in the nucleus. Specificity of ERK activation by the EGF receptor was simultaneously confirmed in the same cell by quantitatively monitoring fluorescent EGF's internalization and subsequent intracellular degradation. Quantitative analysis of the temporal behavior of these two activities showed that phosphorylated ERK's accumulation in the nucleus peaked at 5 min before falling to basal levels by 30 min. Cellular accumulation of fluorescent EGF was slower, peaking around 30 min, before being degraded. This assay strategy can serve as a paradigm to study other signaling pathways and their activation by specific receptors. The flexibility and multiplexing capability of HCS assays allow the use of additional targets to further qualify the specificity of response by including other MAPKs or receptors, to rule out cross-talk from competing signaling pathways, or to simultaneously monitor toxicity effects of compounds. This automated, non-subjective, easy-to-use assay procedure provides information rich, quantitative results, and demonstrates the potential of the HCS assay approach in deconvolving intracellular signaling pathways.
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