Pro- and anti-tumour activities of CD146/MCAM in breast cancer result from its heterogeneous expression and association with epithelial to mesenchymal transition.

CD146, also known as melanoma cell adhesion molecule (MCAM), is expressed in numerous cancers and has been implicated in the regulation of metastasis. We show that CD146 negatively regulates transendothelial migration (TEM) in breast cancer. This inhibitory activity is reflected by a reduction in MCAM gene expression and increased promoter methylation in tumour tissue compared to normal breast tissue.

A research-led flexible cell biology practical for biological sciences undergraduate and postgraduate degree courses.

A challenge in the pandemic era is to implement effective but flexible practical teaching for biological sciences courses. Such teaching needs to deliver conceptual, analytical and practical skills training while having the option to rapidly respond to health and safety issues, local regulations, staff and student concerns. In this paper, we describe a set of cell biology practicals (mini-project) that meets many of these requirements and provides flexibility in providing skills training both through online and in practical laboratory environments.

Acute aerobic exercise-conditioned serum reduces colon cancer cell proliferation in vitro through interleukin-6-induced regulation of DNA damage.

Epidemiological evidence shows that regular physical activity is associated with reduced risk of primary and recurrent colon cancer. However, the underlying mechanisms of action are poorly understood. We evaluated the effects of stimulating a human colon cancer cell line (LoVo) with human serum collected before and after an acute exercise bout vs nonexercise control serum on cancer cell proliferation.

In Vitro Co-culture of Fibroblast and Endothelial Cells to Assess Angiogenesis.

Angiogenesis relies on the spatial and temporal coordination of endothelial migration and proliferation to form new blood vessels. This occurs through synchronous activation of multiple downstream pathways which facilitate vascular development. Proangiogenic growth factors and supporting extracellular matrix allow the formation of capillary-like tubules, reminiscent of microvascular beds, in vitro.

Tumour cell CD99 regulates transendothelial migration via CDC42 and actin remodelling.

Metastasis requires tumour cells to cross endothelial cell (EC) barriers using pathways similar to those used by leucocytes during inflammation. Cell surface CD99 is expressed by healthy leucocytes and ECs, and participates in inflammatory transendothelial migration (TEM). Tumour cells also express CD99, and we have analysed its role in tumour progression and cancer cell TEM.

ATF-2 and Tpl2 regulation of endothelial cell cycle progression and apoptosis.

Cells respond to soluble and membrane-bound factors to activate signalling cascades that control cell proliferation and cell death. Vascular endothelial growth factor A (VEGF-A) is a soluble ligand that modulates a variety of cellular responses including cell proliferation and apoptosis. It is not well understood how VEGF-A signalling pathways regulate cell proliferation and cell death.

Tpl2 is required for VEGF-A-stimulated signal transduction and endothelial cell function.

New blood vessel sprouting (angiogenesis) and vascular physiology are fundamental features of metazoan species but we do not fully understand how signal transduction pathways regulate diverse vascular responses. The vascular endothelial growth factor (VEGF) family bind membrane-bound receptor tyrosine kinases (VEGFRs), which trigger multiple signal transduction pathways and diverse cellular responses. We evaluated whether the MAP3K family member and proto-oncoprotein Tpl2 (MAP3K8) regulates basal and VEGF-A-stimulated signal transduction in endothelial cells.

Genome-wide functional analysis reveals central signaling regulators of lymphatic endothelial cell migration and remodeling.

Lymphatic vessels constitute a specialized vasculature that is involved in development, cancer, obesity, and immune regulation. The migration of lymphatic endothelial cells (LECs) is critical for vessel growth (lymphangiogenesis) and vessel remodeling, processes that modify the lymphatic network in response to developmental or pathological demands. Using the publicly accessible results of our genome-wide siRNA screen, we characterized the migratome of primary human LECs and identified individual genes and signaling pathways that regulate LEC migration.

Mechanical properties of normal versus cancerous breast cells.

A cell's mechanical properties are important in determining its adhesion, migration, and response to the mechanical properties of its microenvironment and may help explain behavioral differences between normal and cancerous cells. Using fluorescently labeled peroxisomes as microrheological probes, the interior mechanical properties of normal breast cells were compared to a metastatic breast cell line, MDA-MB-231. To estimate the mechanical properties of cell cytoplasms from the motions of their peroxisomes, it was necessary to reduce the contribution of active cytoskeletal motions to peroxisome motion.

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear.

Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation.

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes.

Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells.

The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs.

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.

Expression of biologically active human colony stimulating factor-1 in Pichia pastoris.

Colony Stimulating Factor-1 (CSF-1) is involved in proliferation, differentiation, and survival of the mononuclear lineage, in development of the female reproductive system and mammary glands during pregnancy and lactation. It is also implicated in the biology of breast cancer and promotion of its metastasis to bones. Therefore, CSF-1 is required for many applications in cellular and molecular biology studies.

The S100A6 calcium-binding protein regulates endothelial cell-cycle progression and senescence.

Endothelial cells regulate many aspects of vascular physiology, including vasculogenesis and angiogenesis. The S100 family of calcium-binding proteins regulates many aspects of cell function but their roles in vascular physiology are less well understood. Herein, we investigated the expression and function of S100-related family members in endothelial cells.

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.

Efficient introduction of specific TP53 mutations into mouse embryonic fibroblasts and embryonic stem cells.

This protocol describes a rapid, precise method for generating sets of embryonic stem (ES) cells or mouse embryonic fibroblasts (MEFs) harboring point mutations in the p53 tumor suppressor gene (officially known as Trp53). The strategy uses cells from the Trp53 (p53-null) 'platform' mouse, which allows site-specific integration of plasmid DNA into the Trp53 locus. Simple PCR protocols identify correctly targeted clones and immunoblots verify re-expression of the protein.

A VE-cadherin-PAR3-α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A(2)α in endothelial cells.

Phospholipase A(2) enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A(2)α (cPLA(2)α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA(2)α localization and enzymatic activity.

Rapid derivation of genetically related mutants from embryonic cells harboring a recombinase-specific Trp53 platform.

Recombinase-mediated cassette exchange (RMCE) is a powerful method for achieving gene targeting repeatedly at a single mammalian locus. This approach could be applied to the efficient establishment of genetically related cell lines harboring different p53 mutations found in human tumors. To this end we generated a mouse strain called p53 Platform mice (PLF mice), containing PhiC31 integrase-specific attP sequences at the Trp53 locus.

How to become immortal: let MEFs count the ways.

Understanding the molecular mechanisms and biological consequences of genetic changes occurring during bypass of cellular senescence spans a broad area of medical research from the cancer field to regenerative medicine. Senescence escape and immortalisation have been intensively studied in murine embryonic fibroblasts as a model system, and are known to occur when the p53/ARF tumour suppressor pathway is disrupted. We showed recently that murine fibroblasts with a humanised p53 gene (Hupki cells, from a human p53 knock-in mouse model) first senesce, and then become immortalised in the same way as their homologues with normal murine p53.

Wild-type and Hupki (human p53 knock-in) murine embryonic fibroblasts: p53/ARF pathway disruption in spontaneous escape from senescence.

Research on cell senescence and immortalization of murine embryonic fibroblasts (MEFs) has revealed important clues about genetic control of senescence in humans. To investigate senescence and genetic alterations in the p53 pathway that lead to senescence bypass in culture, we compared the behavior of MEFs from wild-type mice with MEFs from Hupki mice, which harbor a humanized p53 gene. We found that humanizing the p53 gene in mice preserved major features of the MEF senescence/immortalization process.

Ligand-stimulated VEGFR2 signaling is regulated by co-ordinated trafficking and proteolysis.

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration.

Rab GTPase regulation of VEGFR2 trafficking and signaling in endothelial cells.

Objective: Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration.