Modeling Tumor Development: the Role of L1

L1 is a membrane-bound neural cell adhesion protein involved in the development of the nervous system.  However, L1 has been implicated in mediating some of the invasive properties of several cancers including gliomas and breast cancers when proteolyzed to create a soluble ectodomain, which then binds to cell surface integrin receptors in an autocrine/paracrine manner.  L1 has also been shown to rescue cancerous cells from apoptosis.  It was hypothesized that polyclonal antibodies raised against a 15 amino acid peptide corresponding to a critical integrin binding region of human L1 would slow the migration of rat 9L/LacZ and human T98 G glioma cells in vitro, whereas the peptide alone would stimulate migration.  Incubation of cells in the peptide decreased cellular migration, possibly due to competitive inhibition.  The effects of the antibody on cell migration were inconclusive.  Quail QT6 cells have been stably transfected with an L1 ectodomain-Fc fusion protein (L1-Fc) designed to be secreted into the media.  Once purified, it will be applied to 9L/LacZ and T98 G cells and analyzed using automated time-lapse microscopy.  Additionally, glioma cell migration will be analyzed while on top of QT6 cells secreting L1-Fc.  Both conditions are hypothesized to increase the migration rates of these cell lines by mimicking the activity of endogenous L1 ectodomains.  Supported by HHMI and INBRE.

Stephanie Dreher1, Daphne Salick2, Lisa Haines-Butterick2, and
 Joel P. Schneider2

Honorable Mention in the ASBMB Undergraduate Poster Competition

Hydrogels are porous, physically rigid, three dimensional matrices primarily composed of water which show promise as scaffolds to aid in tissue regeneration. The Schneider laboratory has developed a class of β-hairpin peptides that self-assemble into a hydrogel in response to specific stimuli, such as light, pH, temperature, ionic strength, and cell culture media. The peptide MAX8 consists of alternating valine and lysine residues on each β-strand flanking a type II´ β-turn (-V^DPPT-) with a glutamic acid at position 15. The kinetics of self-assembly for MAX8 under cellular encapsulation conditions are fast enough to result in homogeneous gel-cell constructs. These constructs can be prepared in a syringe and subsequently shear thin delivered to target sites while maintaining cellular homogeneity. Although it has been demonstrated that MAX8 is a suitable scaffold for tissue engineering applications, this sequence must be prepared by chemical synthesis due to the unnatural amino acid ^DProline in the turn region. In an attempt to make this material more cost efficient for large-scale application, a new sequence, BBH8, was designed to contain all natural amino acids which can possibly be overexpressed. However, before overexpression, BBH8 was first chemically synthesized and its bulk material properties characterized under physiological and cell culture conditions. Data show that BBH8 behaves analogously to MAX8. Preliminary data also demonstrate that chondrocytes can be encapsulated with homogeneous cellular distribution, while maintaining viability in the network. This suggests that BBH8 is an excellent candidate for tissue engineering. Funding provided by NSF CHEM0348323, NIH R01 DE016386-01, and the Howard Hughes Medical Institute Undergraduate Science Education Program.

Nicole Gentile, Julio Fuentes, 
and Erica Selva

Department of Biological Sciences

Phenotypic Characterization of the 7H24 Mutant in O-xylosyltransferase

Formation of morphogenetic gradients is a fundamental mechanism of developmental signaling. Establishment of the gradient dictates how tissues respond to extracellular signals to elicit appropriate cellular responses to a secreted ligand. Recently, we have identified a novel mutation in the Drosophila O-xylosyltransferase (oxt) that encodes a key enzyme required for heparan and chondroitin sulfate (HS and CS) biosynthesis, named 7H24.  In Drosophila, HS has been shown to be essential for Wingless (Wg)/Wnt, Hedgehog (Hh) and Decapentaplegic (Dpp)/Bone Morphogenic Protein (BMP) extracellular ligand movement and gradient formation. When HS is absent downstream signaling of these elemental pathways that drive the Drosophila and human developmental program is disrupted. The 7H24 mutation in oxt is a functional null and removal of maternal and zygotic oxt is embryonic lethal with terminal segment polarity cuticle phenotypes suggesting disruption of the Wg and/or Hh signaling pathway. Removal of zygotic oxt causes homozygous mutants to die as pharate adults with small rough eyes and thorax defects that cannot close from the pupal case. We find deficiencies in Wg, Hh and Dpp signaling in the mutant larval wing imaginal discs and evaluation of larval eye phenotypes suggests they do not arise because of defects in cell type specification. 

Brian Grindel1, M. C. Farach-Carson1,2, Joseph Bennett2, Erik Snapp3

1Department of Biological  Sciences
2Center for Translational Cancer Research at the Helen F. Graham Cancer Center
3Albert Einstein College of Medicine of Yeshiva University

Regulation and expression of ERp57 in hepatocellular carcinoma

ERp57/PDIA3, a 1,25D₃ binding protein with thiol-oxidoreductase activity, resides in the endoplasmic reticulum (ER) owing to a C-terminal QEDL.   ERp57 partitions among cell surface, cytoplasm and nucleus with functions including 1,25D₃ binding, ion transport, and redox modulation of transcription. A tissue array immunoscreen of normal and diseased liver sections showed dynamic changes in expression in both cytoplasm and nucleus.  To understand expression of ERp57 in hepatocellular carcinoma (HCC), its steady state distribution was assessed in HepG2 cells using: 1) GFP (green fluorescent protein) tagged ERp57 and, 2) subcellular fractionation.  We also measured the response to various treatments including PMA, 1,25D₃, or TNF-α.  High resolution imaging demonstrates localization of ERp57-GFP in the ER along with additional peri-nuclear staining.  A small (<5%) fraction of ERp57 also exists in the cytosol. Co-treatment with PMA, alone and in combination with 1,25D₃ for times from 15´ to 1 h did not reorganize EPp57-GFP. In contrast, TNF-α treatment of HepG2 cells moved cytoplasmic ERp57-GFP to the nucleus in 15 min.  Ongoing studies will determine if ERp57 binds to NFkB in HepG2 cells or in normal hepatocytes after TNF-a treatment.  These studies will shed light on redox regulation of transcription by nuclear ERp57 during TNF-a signaling in normal and transformed liver cells.  Supported by HHMI and the CTCR-UD.

Investigating Human and Drosophila Sprinter Function

Within developing tissues, the Wingless (Wg)/Wnt signaling pathway acts as a morphogen, where the extracellular concentration of the Wg ligand governs the level of pathway activation in receiving cells.  Regulated downstream target activation within these receiving cells is essential for proper tissue growth and organization during organismal development.  Our laboratory has identified a novel highly conserved transmembrane protein in Drosophila named Sprinter (Srt), which is required by secretory cells for the release of active Wg ligand (1).  The specific mechanism of Srt function is unknown, however without it, Wg is retained and accumulates in the cells that produce it.  Functional Wg cannot be effectively disseminated to target cells without complex posttranslational processing and packaging (2).  Hence, we hypothesize that Srt functions in the secretion of active Wg ligand by supplying a critical activity required for maturation of Wg protein in its route through the secretory pathway.  The focus of this research is to begin to characterize Srt function and determine if the mechanism of function is conserved between humans and Drosophila. Compartment localization studies in Srt-Wg co-transfected cells show that the majority of Drosophila and human Srt reside in an undefined secretory compartment and high level Srt expression causes cells to produce processes that appear to contain vesicles of Wg protein (Figure 1).  Furthermore, through co-immunoprecipitation we have found that Srt exerts its function by direct physical interaction with the Wg protein and this interaction is conserved across species.  These results suggest that Srt is a multifunctional protein that acts through physical interaction with Wg and perhaps promotes cellular morphological changes that support the dissemination of functional Wg from secretory cells. This data also indicates that these functions are conserved across species. Furthermore, these experiments provide the foundation for evaluating Srt function and conservation in developing Drosophila.

 Recipient of an ASBMB Undergraduate Travel Award
First Place Winner (Proteins) in ASBMB Undergraduate Poster Competition

Selected for a symposium presentation