A Novel p53 Mutant Found in Iatrogenic Urothelial Cancers Is Dysfunctional and Can Be Rescued by a Second-site Global Suppressor Mutation

Exposure to herbal remedies containing the carcinogen aristolochic acid (AA) has been widespread in some regions of the world. Rare A→T TP53 mutations were recently discovered in AA-associated urothelial cancers. The near absence of these mutations among all other sequenced human tumors suggests that they could be biologically silent. There are no cell banks with established lines derived from human tumors with which to explore the influence of the novel mutants on p53 function and cellular behavior. To investigate their impact, we generated isogenic mutant clones by integrase-mediated cassette exchange at the p53 locus of platform (null) murine embryonic fibroblasts and kidney epithelial cells. Common tumor mutants (R248W, R273C) were compared with the AA-associated mutants N131Y, R249W, and Q104L. Assays of cell proliferation, migration, growth in soft agar, apoptosis, senescence, and gene expression revealed contrasting outcomes on cellular behavior following introduction of N131Y or Q104L. The N131Y mutant demonstrated a phenotype akin to common tumor mutants, whereas Q104L clone behavior resembled that of cells with wild-type p53. Wild-type p53 responses were restored in double-mutant cells harboring N131Y and N239Y, a second-site rescue mutation, suggesting that pharmaceutical reactivation of p53 function in tumors expressing N131Y could have therapeutic benefit. N131Y is likely to contribute directly to tumor phenotype and is a promising candidate biomarker of AA exposure and disease. Rare mutations thus do not necessarily point to sites where amino acid exchanges are phenotypically neutral. Encounter with mutagenic insults targeting cryptic sites can reveal specific signature hotspots.     

Endothelial cell confluence regulates Weibel-Palade body formation

Secretory granules called Weibel-Palade bodies (WPBs) containing Von Willebrand factor (VWF) are characteristic of the mammalian endothelium. We hypothesized that vascular-specific antigens such as VWF are linked to endothelial identity and proliferation in vitro. To test this idea, the cellular accumulation of VWF in WPBs was monitored as a function of cell proliferation, confluence and passage number in human umbilical vein endothelial cells (HUVECs). We found that as passage number increased the percentage of cells containing VWF in WPBs was reduced significantly, whilst the protein was still detected within the secretory pathway at all times. However, the endothelial-specific marker protein, PECAM-1, is present on all cells even when WPBs are absent, indicating partial maintenance of endothelial identity. Biochemical studies show that a significant pool of immature pro-VWF can be detected in sub-confluent HUVECs; however, a larger pool of mature, processed VWF is detected in confluent cells. Newly synthesized VWF must thus be differentially sorted and packaged along the secretory pathway in semi-confluent versus confluent endothelial cells. Our studies thus show that WPB formation is linked to the formation of a confluent endothelial monolayer.     

Evolution of the VEGF-Regulated Vascular Network from a Neural Guidance System

The vascular network is closely linked to the neural system, and an interdependence is displayed in healthy and in pathophysiological responses. How has close apposition of two such functionally different systems occurred? Here, we present a hypothesis for the evolution of the vascular network from an ancestral neural guidance system. Biological cornerstones of this hypothesis are the vascular endothelial growth factor (VEGF) protein family and cognate receptors. The primary sequences of such proteins are conserved from invertebrates, such as worms and flies that lack discernible vascular systems compared to mammals, but all these systems have sophisticated neuronal wiring involving such molecules. Ancestral VEGFs and receptors (VEGFRs) could have been used to develop and maintain the nervous system in primitive eukaryotes. During evolution, the demands of increased morphological complexity required systems for transporting molecules and cells, i.e., biological conductive tubes. We propose that the VEGF–VEGFR axis was subverted by evolution to mediate the formation of biological tubes necessary for transport of fluids, e.g., blood. Increasingly, there is evidence that aberrant VEGF-mediated responses are also linked to neuronal dysfunctions ranging from motor neuron disease, stroke, Parkinson’s disease, Alzheimer’s disease, ischemic brain disease, epilepsy, multiple sclerosis, and neuronal repair after injury, as well as common vascular diseases (e.g., retinal disease). Manipulation and correction of the VEGF response in different neural tissues could be an effective strategy to treat different neurological diseases.     

A biphasic endothelial stress-survival mechanism regulates the cellular response to vascular endothelial growth factor A

Vascular endothelial growth factor A (VEGF-A) is an essential cytokine that regulates endothelial function and angiogenesis. VEGF-A binding to endothelial receptor tyrosine kinases such as VEGFR1 and VEGFR2 triggers cellular responses including survival, proliferation and new blood vessel sprouting. Increased levels of a soluble VEGFR1 splice variant (sFlt-1) correlate with endothelial dysfunction in pathologies such as pre-eclampsia; however the cellular mechanism(s) underlying the regulation and function of sFlt-1 are unclear. Here, we demonstrate the existence of a biphasic stress response in endothelial cells, using serum deprivation as a model of endothelial dysfunction. The early phase is characterized by a high VEGFR2:sFlt-1 ratio, which is reversed in the late phase. A functional consequence is a short-term increase in VEGF-A-stimulated intracellular signaling. In the late phase, sFlt-1 is secreted and deposited at the extracellular matrix. We hypothesized that under stress, increased endothelial sFlt-1 levels reduce VEGF-A bioavailability: VEGF-A treatment induces sFlt-1 expression at the cell surface and VEGF-A silencing inhibits sFlt-1 anchorage to the extracellular matrix. Treatment with recombinant sFlt-1 inhibits VEGF-A-stimulated in vitro angiogenesis and sFlt-1 silencing enhances this process. In this response, increased VEGFR2 levels are regulated by the phosphatidylinositol-3-kinase and PKB/Akt signaling pathways and increased sFlt-1 levels by the ERK1/2 signaling pathway. We conclude that during serum withdrawal, cellular sensing of environmental stress modulates sFlt-1 and VEGFR2 levels, regulating VEGF-A bioavailability and ensuring cell survival takes precedence over cell proliferation and migration. These findings may underpin an important mechanism contributing to endothelial dysfunction in pathological states.     

Conservation and diversity in families of coated vesicle adaptins

The complete sequence of the beta adaptin subunit of the plasma membrane adaptor complex from coated vesicles has been elucidated. Complementary cDNA clones from human fibroblasts, rat lymphocytes, and bovine lymphocytes have been isolated, sequenced, and compared with each other and with beta adaptin sequences from rat brain (Kirchhausen, T., Nathanson, K.L., Matsui, W., Vaisberg, A., Chow, E.P., Burne, C., Keen, J.H., and Davis, A.E. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2612-2616). Surprisingly, the 937-amino acid beta adaptin polypeptide is totally conserved between species. This remarkable homology contrasts with the absence of significant sequence similarity between the alpha (Robinson, M.S. (1989) J. Cell Biol. 108, 833-842) and beta adaptins of the plasma membrane adaptor complex. Diversity within each adaptin family is created by the expression of different genes and by tissue-specific differential splicing. The structures of the beta and alpha adaptins can both be divided into two globular domains interconnected by a variable and potentially flexible stalk domain.     

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.