Mary E. Vail

Over-expression of insulin-like growth factor binding protein-related protein-1(IGFBP-rP1/mac25) in the M12 prostate cancer cell line alters tumor growth by a delay in G1 and cyclin A associated apoptosis

In the present study, we examined the effects of over-expression of the potential tumor suppressor gene IGFBP-rP1/mac25 on cell-cycle kinetics in prostate cancer cells. The majority of the high expressing IGFBP-rP1/mac25 cell population was located in the G1 and sub-G0/G1 peaks; synchronizing cells in G2/M with nocodazole demonstrated the high expressing IGFBP-rP1/mac25 clones were delayed in the G1 phase of the cell cycle. Unscheduled expression of cyclin A in the sub-G0/G1 peak occurred in the IGFBP-rP1/mac25 clones. Immunoblots showed decreased cyclin D1 and p21 and increased cyclin E, p16, and p27 in the high expressing IGFBP-rP1/mac25 clones compared to the control cells. Cyclin D1/cdk-4,6 and cyclin E/cdk-2 kinase activities decreased but cyclin A/cdk-2 kinase activity increased for the high expressing IGFBP-rP1/mac25 clones compared to control cells. A pRb immunoprecipitation demonstrated more binding of E2F-1 to pRb in the high expressing IGFBP-rP1/mac25 clones than in control cells. Finally, cell senescence, as assessed by senescence-associated β–galactosidase, demonstrated significantly more staining in the IGFBP-rP1/mac25 cells than control cells. These results suggest that IGFBP-rP1/mac25 alters the cell cycle kinetics of the M12 prostate cell line by delaying the cells in the G1 phase of the cell cycle. In addition, the appearance of cyclin A in the sub-G0/G1 phase of the cell cycle and the increased kinase activity of cyclin A/cdk-2 in the IGFBP-rP1/mac25 clones suggests that cyclin A is associated with the apoptotic cells.

PTP1B regulates Eph receptor function and trafficking

Changes in protein tyrosine phosphatase 1B expression affect duration and amplitude of EphA3 phosphorylation and cell surface concentration.

Bcl-2 expression delays hepatocyte cell cycle progression during liver regeneration

Bcl-2 is the prototype of a family of genes that prevent apoptosis. However, several reports indicate that Bcl-2 may also act as a cell cycle modulator. In several human tumors, Bcl-2 expression correlates with a more favorable prognosis and lower tumor proliferative activity. We have shown that Bcl-2 expression delays liver tumor development in transgenic mice even when the gene is turned on shortly before the time of tumor development. We hypothesized that Bcl-2 may delay liver tumorigenesis by interfering with hepatocyte proliferation. To test whether Bcl-2 expression may act on hepatocyte replication we studied liver regeneration in Bcl-2 transgenic mice and wild-type littermates. DNA replication was delayed by approximately 8 h in Bcl-2 transgenic mice compared to the timing of the response in wild-type littermates. Cyclin D expression showed no alterations in the regenerating liver of Bcl-2 transgenic mice. In contrast, there was a delay in the expression of p107, cyclin E and in the activity of cyclin E/cdk 2 activity. These results show that Bcl-2 expression delays cell cycle progression in hepatocytes and suggests that it acts at a step involving cyclin E and p107.

Targeting EphA3 Inhibits Cancer Growth by Disrupting the Tumor Stromal Microenvironment

Eph receptor tyrosine kinases are critical for cell-cell communication during normal and oncogenic tissue patterning and tumor growth. Somatic mutation profiles of several cancer genomes suggest EphA3 as a tumor suppressor, but its oncogenic expression pattern and role in tumorigenesis remain largely undefined. Here, we report unexpected EphA3 overexpression within the microenvironment of a range of human cancers and mouse tumor xenografts where its activation inhibits tumor growth. EphA3 is found on mouse bone marrow-derived cells with mesenchymal and myeloid phenotypes, and activation of EphA3(+)/CD90(+)/Sca1(+) mesenchymal/stromal cells with an EphA3 agonist leads to cell contraction, cell-cell segregation, and apoptosis. Treatment of mice with an agonistic α-EphA3 antibody inhibits tumor growth by severely disrupting the integrity and function of newly formed tumor stroma and microvasculature. Our data define EphA3 as a novel target for selective ablation of the tumor microenvironment and demonstrate the potential of EphA3 agonists for anticancer therapy.

Antibodies binding the ADAM10 substrate recognition domain inhibit Eph function

Summary The ADAM10 transmembrane metalloprotease cleaves a variety of cell surface proteins that are important in disease, including ligands for receptor tyrosine kinases of the erbB and Eph families. ADAM10-mediated cleavage of ephrins, the ligands for Eph receptors, is suggested to control Eph/ephrin-mediated cell-cell adhesion and segregation, important during normal developmental processes, and implicated in tumour neo-angiogenesis and metastasis. We previously identified a substrate-binding pocket in the ADAM10 C domain that binds the EphA/ephrin-A complex thereby regulating ephrin cleavage. We have now generated monoclonal antibodies specifically recognising this region of ADAM10, which inhibit ephrin cleavage and Eph/ephrin-mediated cell function, including ephrin-induced Eph receptor internalisation, phosphorylation and Eph-mediated cell segregation. Our studies confirm the important role of ADAM10 in cell-cell interactions mediated by both A- and B-type Eph receptors, and suggest antibodies against the ADAM10 substrate-recognition pocket as promising therapeutic agents, acting by inhibiting cleavage of ephrins and potentially other ADAM10 substrates.

EphA3 biology and cancer

Abstract Eph receptor tyrosine kinases control cell-cell interactions during normal and oncogenic development, and are implicated in a range of processes including angiogenesis, stem cell maintenance and metastasis. They are thus of great interest as targets for cancer therapy. EphA3, originally isolated from leukemic and melanoma cells, is presently one of the most promising therapeutic targets, with multiple tumor-promoting roles in a variety of cancer types. This review focuses on EphA3, its functions in controlling cellular behavior, both in normal and pathological development, and most particularly in cancer.

Hypoxia-controlled EphA3 marks a human endometrium-derived multipotent mesenchymal stromal cell that supports vascular growth

Eph and ephrin proteins are essential cell guidance cues that orchestrate cell navigation and control cell-cell interactions during developmental tissue patterning, organogenesis and vasculogenesis. They have been extensively studied in animal models of embryogenesis and adult tissue regeneration, but less is known about their expression and function during human tissue and organ regeneration. We discovered the hypoxia inducible factor (HIF)-1α-controlled expression of EphA3, an Eph family member with critical functions during human tumour progression, in the vascularised tissue of regenerating human endometrium and on isolated human endometrial multipotent mesenchymal stromal cells (eMSCs), but not in other highly vascularised human organs. EphA3 affinity-isolation from human biopsy tissue yielded multipotent CD29+/CD73+/CD90+/CD146+ eMSCs that can be clonally propagated and respond to EphA3 agonists with EphA3 phosphorylation, cell contraction, cell-cell segregation and directed cell migration. EphA3 silencing significantly inhibited the ability of transplanted eMSCs to support neovascularisation in immunocompromised mice. In accord with established roles of Eph receptors in mediating interactions between endothelial and perivascular stromal cells during mouse development, our findings suggest that HIF-1α-controlled expression of EphA3 on human MSCs functions during the hypoxia-initiated early stages of adult blood vessel formation.

An activated form of ADAM10 is tumor selective and regulates cancer stem-like cells and tumor growth

Janes et al. developed an anti-ADAM10 mAb (8C7) that binds to an active form of ADAM10 present in tumors, particularly in stem-like cells. Administration of 8C7 inhibits Notch activity and tumor growth in mouse models, including regrowth after chemotherapy.

Virtual Microscopy and Analysis Using Scientific Workflows

Most commercial microscopes are stand-alone instruments, controlled by dedicated computer systems. These provide limited storage and processing capabilities. Virtual microscopes, on the other hand, link the image capturing hardware and data analysis software into a wide area network of high performance computers, large storage devices and software systems. In this paper we discuss extensions to Grid workflow engines that allow them to execute scientific experiments on virtual microscopes. We demonstrate the utility of such a system in a biomedical case study concerning the imaging of cancer and antibody based therapeutics.

An abstract virtual instrument system for high throughput automatic microscopy

Modern biomedical therapies often involve disease specific drug development and may require observing cells at a very high resolution. Existing commercial microscopes behave very much like their traditional counterparts, where a user controls the microscope and chooses the areas of interest manually on a given specimen scan. This mode of discovery is suited to problems where it is easy for a user to draw a conclusion from observations by finding a small number of areas that might require further investigation. However, observations by an expert can be very time consuming and error prone when there are a large number of potential areas of interest (such as cells or vessels in a tumour), and compute intensive image processing is required to analyse them. In this paper, we propose an Abstract Virtual Instrument (AVI) system for accelerating scientific discovery. An AVI system is a novel software architecture for building an hierarchical scientific instrument – one in which a virtual instrument could be defined in terms of other physical instruments, and in which significant processing is required in producing the illusion of a single virtual scientific discovery instrument. We show that an AVI can be implemented using existing scientific workflow tools that both control the microscope and perform image analysis operations. The resulting solution is a flexible and powerful system for performing dynamic high throughput automatic microscopy. We illustrate the system using a case study that involves searching for blood vessels in an optical tissue scan, and automatically instructing the microscope to rescan these vessels at higher resolution.