Adult Human Keratinocytes Migrating over Nonviable Dermal Collagen Produce Collagenolytic Enzymes That Degrade Type I and Type IV Collagen

Human adult keratinocytes migrating on a nonviable dermal substrate in cultures without fibroblasts induce thinning and degradation of the collagen substrate beneath the migrating epithelium. Further, unconcentrated conditioned medium from the cultures exhibit collagenolytic activity against both type I and type IV collagen which is inhibited by EDTA but not by phenylmethylsulfonyl fluoride or N-ethylmaleimide. Since the migrating epithelium and dermal substrate do not contain fibroblasts, this study shows that migratory keratinocytes in contact with interstitial collagen are capable of producing collagenases against type I and type IV collagen. Moreover, migratory keratinocytes appear to be similar to highly metastatic cells in their ability to degrade basement membrane collagen

Centrosome defects can account for cellular and genetic changes that characterize prostate cancer progression

Factors that determine the biological and clinical behavior of prostate cancer are largely unknown. Prostate tumor progression is characterized by changes in cellular architecture, glandular organization, and genomic composition. These features are reflected in the Gleason grade of the tumor and in the development of aneuploidy. Cellular architecture and genomic stability are controlled in part by centrosomes, organelles that organize microtubule arrays including mitotic spindles. Here we demonstrate that centrosomes are structurally and numerically abnormal in the majority of prostate carcinomas. Centrosome abnormalities increase with increasing Gleason grade and with increasing levels of genomic instability. Selective induction of centrosome abnormalities by elevating levels of the centrosome protein pericentrin in prostate epithelial cell lines reproduces many of the phenotypic characteristics of high-grade prostate carcinoma. Cells that transiently or permanently express pericentrin exhibit severe centrosome and spindle defects, cellular disorganization, genomic instability, and enhanced growth in soft agar. On the basis of these observations, we propose a model in which centrosome dysfunction contributes to the progressive loss of cellular and glandular architecture and increasing genomic instability that accompany prostate cancer progression, dissemination, and lethality

Lectin-binding domains on laminin

The nature and location of carbohydrate moieties on the laminin molecule were identified by studying the binding affinity of a series of lectins for purified, proteasederived fragments of laminin. Laminin is a cross-shaped molecule containing three short arms (36 nm) and one long arm (76 nm). All arms contain globular end regions by electron microscopy. Purified fragments of laminin were obtained which (a) lacked the long arm of the molecule but retained the intact short arms, or (b) lacked both the long arm and the globular end regions of the short arms. These two types of fragments differed markedly in lectin-binding capacity. Using the known sugar specificities of the lectins and hapten sugar competition for lectin-binding to laminin fragments, the following conclusions were reached: (a) [alpha]--Galactopyranosyl end groups are markedly enriched in the globular end regions of the short arms compared to the rod-shaped portions of the molecule. (b) [alpha]--Mannopyranosyl residues are present on both the globular end regions and the rod-shaped portions of the molecule. (c) Exposed N-acetyl--galactosaminyl end groups are absent or present in low amounts on laminin. (d) (NANA)-(2 --> 6)-[beta]--Gal-(1 --> 4)-[beta]--GlcNAc-(1 --> 2)--Man-terminated oligosaccharide units are enriched on the rod-shaped regions of the short arms compared to the globular end regions.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25066/1/0000497.pd

L-histidine inhibits production of lysophosphatidic acid by the tumor-associated cytokine, autotaxin

BACKGROUND: Autotaxin (ATX, NPP-2), originally purified as a potent tumor cell motility factor, is now known to be the long-sought plasma lysophospholipase D (LPLD). The integrity of the enzymatic active site, including three crucial histidine moieties, is required for motility stimulation, as well as LPLD and 5'nucleotide phosphodiesterase (PDE) activities. Except for relatively non-specific chelation agents, there are no known inhibitors of the ATX LPLD activity. RESULTS: We show that millimolar concentrations of L-histidine inhibit ATX-stimulated but not LPA-stimulated motility in two tumor cell lines, as well as inhibiting enzymatic activities. Inhibition is reversed by 20-fold lower concentrations of zinc salt. L-histidine has no significant effect on the Km of LPLD, but reduces the Vmax by greater than 50%, acting as a non-competitive inhibitor. Several histidine analogs also inhibit the LPLD activity of ATX; however, none has greater potency than L-histidine and all decrease cell viability or adhesion. CONCLUSION: L-histidine inhibition of LPLD is not a simple stoichiometric chelation of metal ions but is more likely a complex interaction with a variety of moieties, including the metal cation, at or near the active site. The inhibitory effect of L-histidine requires all three major functional groups of histidine: the alpha amino group, the alpha carboxyl group, and the metal-binding imidazole side chain. Because of LPA's involvement in pathological processes, regulation of its formation by ATX may give insight into possible novel therapeutic approaches

PO-485 Low abundance circulating proteins in giant cell tumours of bone

Introduction Circulating low-abundance proteins/fragments generating from tumour cells and tissues, represent the most important source of cancer biomarkers useful for early diagnosis and prognosis. Giant cell tumour of bone (GCT) is a benign neoplasm occurring in the long bone and in the axial skeleton of young adults. Approximately 5% of GCT develop pulmonary metastases. Although many biomarkers have been proposed, identification of circulating low abundance molecules may be useful to predict metastasis with a non invasive method. Material and methods The hydrogel nanoparticles technique followed by mass spectrometry was used to detect low molecular weight serum proteins or protein fragments in serum of 20 GCT patients with different clinical course and in 10 healthy sera used as control. The most representative low-abundant de novo or differentially abundant proteins were submitted to String database in order to define protein-protein interaction network. Cluster analysis was performed to identify prognostic groups of patients with similar abundance of proteins that significantly discriminate between the groups. Results and discussions For the 25 low-abundant de novo or differentially abundant proteins identified, we recognised that the top interconnected pathways included protein activation cascade, wound healing, blood coagulation, cell-substrate adhesion. Proteoma cluster analysis separated metastasis-free from metastatic GCT patients in two well-defined groups where serum levels of signalling transduction mediators and regulators of kinase activity presented a high discriminatory power. Increased expression of proteins STAT5B, GRB2 and OXSR1 was related to a higher probability of metastasis. Conclusion In conclusion, using a no invasive technique, we identified differentially abundant serum biomarkers, also providing prognostic information in patients with GCT of bone. Future studies are ongoing to establish the interplay between these biomarkers in order to fully understand the mechanism involved in tumour development and to focus on the planning of tailored therapies that should be more effective and less toxic

The use of nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells

HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients\u27 serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1 detection by concentrating viral proteins and infectious virions from infected samples

Systems analysis of the NCI-60 cancer cell lines by alignment of protein pathway activation modules with "-OMIC" data fields and therapeutic response signatures

The NCI-60 cell line set is likely the most molecularly profiled set of human tumor cell lines in the world. However, a critical missing component of previous analyses has been the inability to place the massive amounts of "-omic" data in the context of functional protein signaling networks, which often contain many of the drug targets for new targeted therapeutics. We used reverse-phase protein array (RPPA) analysis to measure the activation/phosphorylation state of 135 proteins, with a total analysis of nearly 200 key protein isoforms involved in cell proliferation, survival, migration, adhesion, etc., in all 60 cell lines. We aggregated the signaling data into biochemical modules of interconnected kinase substrates for 6 key cancer signaling pathways: AKT, mTOR, EGF receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), integrin, and apoptosis signaling. The net activation state of these protein network modules was correlated to available individual protein, phosphoprotein, mutational, metabolomic, miRNA, transcriptional, and drug sensitivity data. Pathway activation mapping identified reproducible and distinct signaling cohorts that transcended organ-type distinctions. Direct correlations with the protein network modules involved largely protein phosphorylation data but we also identified direct correlations of signaling networks with metabolites, miRNA, and DNA data. The integration of protein activation measurements into biochemically interconnected modules provided a novel means to align the functional protein architecture with multiple "-omic" data sets and therapeutic response correlations. This approach may provide a deeper understanding of how cellular biochemistry defines therapeutic response. Such "-omic" portraits could inform rational anticancer agent screenings and drive personalized therapeutic approaches. © 2013 American Association for Cancer Research