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1.
Clin Proteomics ; 17: 1, 2020.
Article in English | MEDLINE | ID: mdl-31997976

ABSTRACT

The "omics revolution: beyond genomics" satellite meeting, run under the auspices of the Australian Peptide Association, The Human Proteome Organisation (HUPO) and the HUPO Australia/New Zealand Chromosome 7 initiative, was held at the Oaks Resort, Port Douglas, Queensland, Australia, on 8th September 2019, immediately prior to the 13th Australian Peptide Conference. The meeting, which had over 100 participants representing Australasia, Europe and America, focused on recent advances in omics-related technologies, including mass spectrometry, biosensors and CryoEM, which will assist in the clinical translation of proteomics towards precision/personalized medicine. An overview of the conference and a summary of the oral presentations are presented.

2.
Methods Enzymol ; 586: 247-274, 2017.
Article in English | MEDLINE | ID: mdl-28137566

ABSTRACT

Colorectal cancer (CRC) is the third leading cause of cancer mortality for both men and women, and the second leading cause of cancer death for men and women combined. If detected early, before metastasis has occurred, survival following surgical resection of the tumor is >90%. Early detection is therefore critical for effective disease surveillance. Unfortunately, current biomarker assays lack the necessary sensitivity and specificity for reliable early disease detection. Development of new robust, non- or minimally invasive specific and sensitive biomarkers or panels with improved compliance and performance is therefore urgently required. The use of fecal samples offers several advantages over other clinical biospecimens (e.g., plasma or serum) as a source of CRC biomarkers, including: collection is noninvasive, the test can be performed at home, one is not sample limited, and the stool effectively samples the entire length of the inner bowel wall contents (including tumor) as it passes down the gastrointestinal tract. Recent advances in mass spectrometry now facilitate both the targeted discovery and validation of potential CRC biomarkers. We describe, herein, detailed protocols that can be used to mine deeply into the fecal proteome to reveal candidate proteins, identify proteotypic/unitypic peptides (i.e., peptides found in only a single known human protein that serve to identify that protein) suitable for sensitive and specific quantitative multiplexed analysis, and undertake high-throughput analysis of clinical samples. Finally, we discuss future directions that may further position this technology to support the current switch in translation research toward personalized medicine.


Subject(s)
Biomarkers, Tumor/isolation & purification , Colorectal Neoplasms/diagnosis , Proteome/isolation & purification , Amino Acid Sequence , Animals , Biomarkers, Tumor/chemistry , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Chromatography, Reverse-Phase , Early Detection of Cancer , Feces , Humans , Neoplasm Proteins/chemistry , Neoplasm Proteins/isolation & purification , Proteome/chemistry , Proteomics/methods , Sensitivity and Specificity , Sequence Analysis, Protein , Tandem Mass Spectrometry
4.
Cell Death Differ ; 23(4): 616-27, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26794444

ABSTRACT

Interleukin-6 (IL-6) has been demonstrated to be involved in Hepatitis B virus (HBV)-associated hepatocarcinogenesis through activation of the STAT3 pathway. The sustained activation of the IL-6/STAT3 pathway is frequently associated with repression of SOCS3, which is both a target gene and a negative regulator of STAT3. However, the silencing mechanism of SOCS3 in hepatocellular carcinoma (HCC) remains to be elucidated. Here, we showed that the repression of SOCS3 and sustained activation of IL-6/STAT3 pathway in HBV-producing HCC cells were caused by HBV-induced mitochondrial ROS accumulation. Mechanistic studies revealed that ROS-mediated DNA methylation resulted in the silencing of SOCS3. Decreased SOCS3 expression significantly promoted the proliferation of HCC cells and growth of tumor xenografts in mice. Further studies revealed that HBV-induced ROS accumulation upregulated the expression of the transcription factor, Snail, which bound to the E-boxes of SOCS3 promoter and mediated the epigenetic silencing of SOCS3 in association with DNMT1 and HDAC1. In addition, we found that the expression of Snail and SOCS3 were inversely correlated in HBV-associated HCC patients, suggesting that SOCS3 and/or Snail could be used as prognostic markers in HCC pathogenesis. Taken together, our data show that HBV-induced mitochondrial ROS production represses SOCS3 expression through Snail-mediated epigenetic silencing, leading to the sustained activation of IL-6/STAT3 pathway and ultimately contributing to hepatocarcinogenesis.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Transformation, Viral , Gene Expression Regulation, Neoplastic , Gene Silencing , Hepatitis B virus/metabolism , Liver Neoplasms/metabolism , Neoplasm Proteins/metabolism , Reactive Oxygen Species/metabolism , Snail Family Transcription Factors/metabolism , Suppressor of Cytokine Signaling 3 Protein/metabolism , Animals , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Hep G2 Cells , Hepatitis B virus/genetics , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/virology , Male , Mice , Mice, Inbred BALB C , Neoplasm Proteins/genetics , Snail Family Transcription Factors/genetics , Suppressor of Cytokine Signaling 3 Protein/genetics
5.
Cancer Metastasis Rev ; 34(4): 715-34, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26337740

ABSTRACT

The ß6 subunit of the αvß6 integrin heterodimer has long been an enigma in cancer biology though recent research has provided many new insights into its biology. Collectively, these findings include discovery of the transcriptional, translational and cell biological mechanisms by which ß6 acts, the identification of the cellular influences ß6 exerts upon the cell proteome, the characterisation of multiple ß6-centric pro-metastatic signalling systems and the search for pharmacological therapies (industry and academia) targeted against ß6. Once expressional restriction is overcome in early colorectal cancer (CRC), epithelial cell surface restricted αvß6 can physically interact with, and activate, known oncoproteins, and has the potential to enable the cross-talk through non-canonical signal transduction pathways, resulting in the adoption of an invasive/metastatic phenotype. This recent research has identified numerous interconnections and potential feedback loops, highlighting the fact that the expression of the ß6 subunit may initiate a cascade of downstream effects on the CRC cell rather than acting through a single mechanism. We here review these recent studies and postulate that the existence of a cell surface uPAR/αvß6/TGFß "metastasome" interactome in/on a proportion of colorectal cancer cells, where ß6 expression sequesters and activates multiple systems at the invasive front of tumour lesions, promoting cancer metastasis and hence explaining why ß6 has been correlated with reduced patient survival in CRC.


Subject(s)
Antigens, Neoplasm/metabolism , Colorectal Neoplasms/pathology , Integrins/metabolism , Neoplasm Metastasis/pathology , Cell Movement , Chemokine CXCL12/metabolism , Eukaryotic Initiation Factor-4E/metabolism , Humans , Promoter Regions, Genetic/genetics , Transforming Growth Factor beta/metabolism , Urokinase-Type Plasminogen Activator/metabolism
6.
J Proteomics ; 127(Pt B): 300-9, 2015 Sep 08.
Article in English | MEDLINE | ID: mdl-25979773

ABSTRACT

The low molecular weight (LMW; <10kDa)* plasma peptidome has been considered a source of useful diagnostic biomarkers and potentially therapeutic molecules, as it contains many cytokines, peptide hormones, endogenous peptide products and potentially bioactive fragments derived from the parent proteome. The small size of the peptides allows them almost unrestricted vascular and interstitial access, and hence distribution across blood-brain barriers, tumour and other vascular permeability barriers. Therefore, the peptidome may carry specific signatures or fingerprints of an individual's health, wellbeing or disease status. This occurs primarily because of the advantage the peptidome has in being readily accessible in human blood and/or other biofluids. However, the co-expression of highly abundant proteins (>10kDa) and other factors present inherently in human plasma make direct analysis of the blood peptidome one of the most challenging tasks faced in contemporary analytical biochemistry. A comprehensive compendium of extraction and fractionation tools has been collected concerning the isolation and micromanipulation of peptides. However, the search for a reliable, accurate and reproducible single or combinatorial separation process for capturing and analysing the plasma peptidome remains a challenge. This review outlines current techniques used for the separation and detection of plasma peptides and suggests potential avenues for future investigation. This article is part of a Special Issue entitled: HUPO 2014.


Subject(s)
Blood Proteins/metabolism , Peptides/blood , Proteome/metabolism , Proteomics/methods , Humans
7.
Cancer Biomark ; 13(2): 67-73, 2013.
Article in English | MEDLINE | ID: mdl-23838134

ABSTRACT

OBJECTIVE: To determine the usefulness of brain-derived neurotrophic factor (BDNF) as a diagnostic biomarker for colorectal cancer (CRC). MATERIALS AND METHODS: ELISA immunoassay was used to examine BDNF concentrations in the sera of two different retrospective cohorts consisting of CRC patients and age/gender matched controls. Cohort 1 consisted of 99 controls and 97 CRC patients, whereas cohort 2 consisted of 47 controls and 91 CRC patients. RESULTS: In cohort 1, the median concentration of BDNF was significantly (p< 0.0001) lower in CRC patient samples (18.8 ng/mL, range 4.0-56.5 ng/mL) than control samples (23.4 ng/mL, range 3.0-43.1 ng/mL). This finding was validated in an independent patient cohort (CRC patients: 23.0 ng/mL, range 6.0-45.9 ng/mL; control patients: 32.3 ng/mL, range 14.2-62.4 ng/mL). BDNF concentrations did not differ significantly between Dukes' staging in the patient cohort, however patients with Stages A, B, C and D (p< 0.01 for each stage) tumours had significantly reduced BDNF levels compared to healthy controls. Receiver operating characteristic analysis was performed to determine the ability of BDNF to discriminate between healthy controls and those with CRC. At 95% specificity, BDNF concentrations distinguished CRC patients with 25% and 18% sensitivity, respectively, in cohorts 1 and 2 (cohort 1: AUC=0.79, 95% CI 0.70-0.87; cohort 2: AUC =0.69, 95% CI 0.61-0.76). CONCLUSION: The serum levels of BDNF were significantly lower in colorectal cancer patients when compared to a control population, and this did not differ between different Dukes' stages.


Subject(s)
Biomarkers, Tumor/blood , Brain-Derived Neurotrophic Factor/blood , Colorectal Neoplasms/blood , Adult , Aged , Aged, 80 and over , Carcinoembryonic Antigen/blood , Case-Control Studies , Colorectal Neoplasms/diagnosis , Female , Humans , Male , Middle Aged , Neoplasm Staging , ROC Curve , Retrospective Studies , Sensitivity and Specificity
8.
Colorectal Dis ; 15(7): e373-81, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23489866

ABSTRACT

AIM: Annexin A2 (ANXA2) is known to be a tumourigenic molecule and is highly expressed in colorectal cancer (CRC). Its diagnostic and prognostic value is not fully understood. This study was designed to investigate the relationship between ANXA2 expression, clinicopathological characteristics, tumour recurrence and survival. METHOD: Immunohistochemical staining was used to evaluate ANXA2 expression in 150 matched samples from patients with CRC. Overall survival and recurrence were determined by Kaplan-Meier analysis. The Cox proportional hazards model was used to determine independent factors contributing to survival and recurrence. Receiver operating characteristic (ROC) curve and liner correlation analysis were used to estimate the sensitivity and specificity of ANXA2 expression for clinical diagnosis. RESULTS: ANXA2 was found to be strongly expressed in poorly differentiated tumours (P < 0.001), late stage (P = 0.020) and lymph node positivity (P = 0.002). ANXA2 expression was significantly related to recurrence (P < 0.001) and survival (P = 0.002). The Cox proportional hazards model indicated that ANXA2 expression [P < 0.001, hazard ratio (HR) = 1.366, 95% CI 1.232-1.515] and tumour location (P = 0.039, HR = 1.891, 95% CI 1.034-3.456) were independent factors in predicting overall survival while ANXA2 expression (P < 0.001, HR = 1.445, 95% CI 1.222-1.709) were independent factors predicting recurrence. Receiver operating characteristic (ROC) (AUC = 0.768, 95% CI = 0.642-0.894) and liner correlation analysis suggested that ANXA2 was suitable for the clinical diagnosis of CRC. CONCLUSION: These results indicate that ANXA2 is a biomarker with diagnostic and prognostic potential for patients with CRC.


Subject(s)
Annexin A2/metabolism , Biomarkers, Tumor/metabolism , Colorectal Neoplasms/metabolism , Neoplasm Recurrence, Local/metabolism , Adult , Aged , Aged, 80 and over , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/mortality , Disease-Free Survival , Female , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Linear Models , Male , Middle Aged , Prognosis , Proportional Hazards Models , ROC Curve , Sensitivity and Specificity
9.
J Chromatogr A ; 1168(1-2): 190-210; discussion 189, 2007 Oct 19.
Article in English | MEDLINE | ID: mdl-17597136

ABSTRACT

The routine detection of low abundance components in complex samples for detailed proteomics analysis continues to be a challenge. Whilst the potential of multidimensional chromatographic fractionation for this purpose has been proposed for some years, and was used effectively for the purification to homogeneity of trace components in bulk biological samples for N-terminal sequence analysis, its practical application in the proteomics arena is still limited. This article reviews some of the recent data using these approaches, including the use of microaffinity purification as part of multidimensional protocols for downstream proteomics analysis.


Subject(s)
Proteomics , Chromatography, Affinity/methods , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Chromatography, Liquid/methods , Electrophoresis, Gel, Two-Dimensional/methods , Isoelectric Focusing/methods , Proteomics/instrumentation , Proteomics/methods
10.
Biochem Pharmacol ; 71(10): 1422-34, 2006 May 14.
Article in English | MEDLINE | ID: mdl-16522318

ABSTRACT

The tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) is a potent and specific inhibitor of EGFR tyrosine kinase whose favourable preclinical profile supports progression towards clinical trials. Microphysiometric evaluation revealed a short (<24 min) effective inhibition of cellular receptor response to EGF challenge in BaF/ERX cells indicating a need to maintain sustained levels of inhibitor. Initial pharmacokinetic evaluation in mice of novel AG1478 formulations in a beta-cyclodextrin (Captisol) showed monoexponential elimination from plasma (half-life 30 min) following subcutaneous administration. A two-fold dose escalation gave a 2.4-fold increase in the total AUC. Bolus i.v. and 6 h continuous infusion were investigated in rats to mimic a more clinically relevant administration regimen. Drug elimination following bolus i.v. administration was biphasic (terminal elimination half-life 30-48 min). The linear relationship between dose and AUC(0-->infinity) (r2=0.979) enabled the prediction of infusion rates and doses for sustained delivery using continuous 6 h infusions, where steady state was reached in 120 min. Plasma levels of AG1478>10 microM were achieved over the duration of the infusion. At the lowest dose, plasma drug levels after the cessation of infusion declined with a half-life of approximately 43 min. EGFR activity, measured both by autophosphorylation and downstream signalling, was inhibited in a dose-dependent manner by injection of AG1478 in mice bearing xenografts of the human glioblastoma cell line U87MG.delta2-7, which expresses a constitutively active variant of the EGF receptor. Taken together, these experiments provide essential data to assess the anti-tumour efficacy of AG1478 and will assist in the rational design of dose regimens for clinical studies.


Subject(s)
Enzyme Inhibitors/pharmacokinetics , ErbB Receptors/antagonists & inhibitors , Protein Tyrosine Phosphatases/antagonists & inhibitors , Tyrphostins/pharmacokinetics , Animals , Area Under Curve , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Glioblastoma/drug therapy , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Injections, Intravenous , Injections, Subcutaneous , Mice , Molecular Structure , Quinazolines , Rats , Thymidine/metabolism , Tyrphostins/chemistry , Tyrphostins/pharmacology , Xenograft Model Antitumor Assays
11.
J Proteome Res ; 4(5): 1646-56, 2005.
Article in English | MEDLINE | ID: mdl-16212417

ABSTRACT

A biosensor-based micro-affinity purification method to recover protein binding partners and their complexes for down stream proteomics analysis has been developed using the BIAcore 3000 fitted with a prototype Surface Prep Unit (SPU). The recombinant GST-intracellular domain of E-cadherin or the recombinant GST-beta-catenin binding domain of Adenomatous Polyposis Coli (APC) were immobilized onto the SPU and used to affinity purify binding partners from chromatographically enriched SW480 colon cancer cell lysates. A GST- immobilized surface was used as a control. Samples recovered from the SPU were subjected to SDS-PAGE with sensitive Coomassie staining followed by automated in-gel digestion and LC-MS/MS. The results obtained using the SPU were compared with similar experiments performed using Sepharose beads.


Subject(s)
Biosensing Techniques , Proteomics/methods , Blotting, Western , Cadherins/chemistry , Cell Line, Tumor , Chromatography , Chromatography, Ion Exchange , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Humans , Mass Spectrometry , Peptides/chemistry , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Proteins/chemistry , Recombinant Proteins/chemistry , Sepharose/pharmacology , Time Factors , beta Catenin/chemistry
12.
J Microsc ; 217(Pt 3): 265-74, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15725130

ABSTRACT

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.


Subject(s)
Microscopy, Confocal/methods , Microscopy, Interference/methods , Animals , Cell Line, Tumor , Embryo, Nonmammalian/anatomy & histology , Fibroblasts , Humans , Leishmania mexicana , Mast Cells , Mice , NIH 3T3 Cells , Rats , Zebrafish/embryology
13.
Growth Factors ; 20(2): 71-80, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12148565

ABSTRACT

Epidermal Growth Factor (EGF) is a small growth factor containing 53 amino acid residues capable of stimulating the proliferation of both mesenchymal and epithelial cells. Comparison of the amino acid sequences of EGF from several species, and related proteins that can bind to the EGF receptor (e.g. TGFalpha, VGF, heparin-binding EGF, and betacellulin), suggests that Leu47, which is highly conserved, is important for biological function. Additionally, we have shown previously, using a combination of trypsin and carboxypeptidase Y digestion of native murine EGF, that removal of Leu47 results in more than 100-fold decrease in both receptor binding and mitogenic activity. We now describe a micromethod for the rapid generation of C-terminally modified EGFs to investigate further the role of C-terminal residues in determining functional activity. These analogues have been generated by digesting native murine EGF with trypsin, purifying the biologically inactive, but structurally intact, EGF1-45 core by micropreparative RP-HPLC, and then reversing the action of trypsin to couple synthetic peptides (e.g. DL, DI, DF, EL, DLLW) onto the C-terminus of the EGF1-45 core. This enzymic semisynthesis method allows multiple derivatives to be generated rapidly from microgram quantities of EGF1-45 in sufficient quantities for sensitive biological and physicochemical analysis. We have validated the method by regenerating EGF1-47 from EGF1-45 with equivalent mitogenic and receptor binding activity to EGF1-47 generated from wild type EGF by digestion with trypsin and carboxypeptidase Y. We have also investigated the effect of substituting alternative normal or nonphysiological amino acids (e.g. allo-Ile) for Asp46, Leu47 or Arg48. Even small changes in these C-terminal residues reduce the mitogenic potency of the analogue.


Subject(s)
Epidermal Growth Factor/analogs & derivatives , Epidermal Growth Factor/chemical synthesis , 3T3 Cells , Amino Acid Sequence , Amino Acids/chemistry , Animals , Carboxypeptidases/pharmacology , Cathepsin A , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Humans , Kinetics , Leucine/chemistry , Ligands , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Structure-Activity Relationship , Time Factors , Trypsin/pharmacology
15.
J Pept Res ; 58(3): 204-12, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11576326

ABSTRACT

Large numbers of colon tumors stem from mutations in the gene coding for the production of the adenomatous polyposis coli (APC) tumor suppressor protein. This protein contains a coiled-coil N-terminal domain that is known to be responsible for homodimerization. Previous work by others has led to the design of a specific 54-residue anti-APC peptide (anti-APCp1) that dimerizes preferentially with this domain. We have undertaken the chemical synthesis of a modified form of this peptide (anti-APCp2) that bears a biotin moiety at its N-terminus for use in subsequent ligand-binding analysis studies. The peptide was subjected to comprehensive chemical characterization to confirm its purity. Secondary structural analysis by circular dichroism spectroscopy and Fourier transform infrared spectroscopy indicated that the peptide could assume a wide range of potential conformations, depending upon the precise microenvironment. Significantly, a stable alpha-helical structure was generated when the solvent conditions supported intramolecular salt-bridge formation along the helix barrel. The biotinylated anti-APCp2 was immobilized onto a streptavidin sensor surface, in a specific orientation leaving all amino acids available to form a coiled structure. In one experiment, injection of colonic cell lysate extracts (LIM1215) onto a size-exclusion column resulted in the isolation of a high molecular mass protein peak (> 600 kDa) that reacted specifically with the immobilized anti-APCp2 on the biosensor surface. In another experiment, a high molecular mass protein (M(r) > 250 kDa on SDS-PAGE) could be specifically immunoprecipitated from this peak using either the anti-APCp2 peptide or an anti-APC polyclonal antibody. This demonstrates the specific interaction between the anti-APCp2 peptide and native APC and highlights the potential use of the former peptide in a multidimensional micropreparative chromatographic/biosensor/proteomic protocol for the purification of APC alone and APC complexed with different biopolymers in various cell lines, and stages of tumor development.


Subject(s)
Adenomatous Polyposis Coli Protein/isolation & purification , Adenomatous Polyposis Coli/metabolism , Biosensing Techniques/methods , Colonic Neoplasms/chemistry , Molecular Probes/chemistry , Peptides/chemistry , Adenomatous Polyposis Coli Protein/chemistry , Amino Acid Sequence , Chromatography, High Pressure Liquid , Circular Dichroism , Colonic Neoplasms/therapy , Humans , Peptides/analysis , Precipitin Tests/methods , Protein Structure, Secondary , Solutions/chemistry , Spectroscopy, Fourier Transform Infrared , Tumor Cells, Cultured , Water/chemistry
16.
EMBO J ; 20(17): 4762-73, 2001 Sep 03.
Article in English | MEDLINE | ID: mdl-11532940

ABSTRACT

Vascular endothelial growth factor receptor-3 (VEGFR-3/Flt4) binds two known members of the VEGF ligand family, VEGF-C and VEGF-D, and has a critical function in the remodelling of the primary capillary vasculature of midgestation embryos. Later during development, VEGFR-3 regulates the growth and maintenance of the lymphatic vessels. In the present study, we have isolated and cultured stable lineages of blood vascular and lymphatic endothelial cells from human primary microvascular endothelium by using antibodies against the extracellular domain of VEGFR-3. We show that VEGFR-3 stimulation alone protects the lymphatic endothelial cells from serum deprivation-induced apoptosis and induces their growth and migration. At least some of these signals are transduced via a protein kinase C-dependent activation of the p42/p44 MAPK signalling cascade and via a wortmannin-sensitive induction of Akt phosphorylation. These results define the critical role of VEGF-C/VEGFR-3 signalling in the growth and survival of lymphatic endothelial cells. The culture of isolated lymphatic endothelial cells should now allow further studies of the molecular properties of these cells.


Subject(s)
Apoptosis/physiology , Endothelial Growth Factors/pharmacology , Endothelial Growth Factors/physiology , Endothelium, Vascular/physiology , Endothelium/physiology , Lymphatic System/physiology , MAP Kinase Signaling System/physiology , Receptor Protein-Tyrosine Kinases/physiology , Receptors, Growth Factor/physiology , Apoptosis/drug effects , Biosensing Techniques , Capillaries/embryology , Capillaries/physiology , Cell Division , Cell Movement , Cell Survival , Cells, Cultured , Endothelium/cytology , Endothelium, Vascular/cytology , Enzyme Activation , Humans , Kinetics , Lymphatic System/cytology , Microcirculation/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Protein Kinase C/metabolism , RNA, Messenger/genetics , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor , Skin/blood supply , Umbilical Veins , Vascular Endothelial Growth Factor C , Vascular Endothelial Growth Factor D , Vascular Endothelial Growth Factor Receptor-3
17.
J Biol Chem ; 276(36): 34189-98, 2001 Sep 07.
Article in English | MEDLINE | ID: mdl-11448961

ABSTRACT

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-alpha/beta heterodimer. Importin-alpha contains the NLS binding site, whereas importin-beta mediates the translocation through the nuclear pore. We characterized the interactions involving importin-alpha during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-alpha is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-beta (stoichiometry, 1:1; K(D) = 1.1 x 10(-8) m) increases the affinity for NLSs; the importin-alpha/beta complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K(D) = 3.5 x 10(-8) m and 4.8 x 10(-8) m, respectively) comparable with those of a truncated importin-alpha lacking the autoinhibitory domain (T-antigen NLS, K(D) = 1.7 x 10(-8) m; nucleoplasmin NLS, K(D) = 1.4 x 10(-8) m). The autoinhibitory domain (as a separate peptide) binds the truncated importin-alpha, and the crystal structure of the complex resembles the structure of full-length importin-alpha. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-alpha and provide a quantitative description of the binding and regulatory steps during nuclear import.


Subject(s)
Active Transport, Cell Nucleus , Nuclear Proteins/chemistry , Nuclear Proteins/physiology , Animals , Biosensing Techniques , Cell Nucleus/metabolism , Circular Dichroism , Crystallography, X-Ray , Dimerization , Escherichia coli/metabolism , Karyopherins , Kinetics , Ligands , Mice , Models, Biological , Models, Molecular , Nucleoplasmins , Peptide Biosynthesis , Phosphoproteins/chemistry , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Time Factors , Ultracentrifugation
18.
Biochemistry ; 40(30): 8930-9, 2001 Jul 31.
Article in English | MEDLINE | ID: mdl-11467954

ABSTRACT

Murine and human epidermal growth factor receptors (EGFRs) bind human EGF (hEGF), mouse EGF (mEGF), and human transforming growth factor alpha (hTGF-alpha) with high affinity despite the significant differences in the amino acid sequences of the ligands and the receptors. In contrast, the chicken EGFR can discriminate between mEGF (and hEGF) and hTGF-alpha and binds the EGFs with approximately 100-fold lower affinity. The regions responsible for this poor binding are known to be Arg(45) in hEGF and the L2 domain in the chicken EGFR. In this study we have produced a truncated form of the hEGFR ectodomain comprising residues 1-501 (sEGFR501), which, unlike the full-length hEGFR ectodomain (residues 1-621, sEGFR621), binds hEGF and hTGF-alpha with high affinity (K(D) = 13-21 and 35-40 nM, respectively). sEGFR501 was a competitive inhibitor of EGF-stimulated mitogenesis, being almost 10-fold more effective than the full-length EGFR ectodomain and three times more potent than the neutralizing anti-EGFR monoclonal antibody Mab528. Analytical ultracentrifugation showed that the primary EGF binding sites on sEGFR501 were saturated at an equimolar ratio of ligand and receptor, leading to the formation of a 2:2 EGF:sEGFR501 dimer complex. We have used sEGFR501 to generate three mutants with single position substitutions at Glu(367), Gly(441), or Glu(472) to Lys, the residue found in the corresponding positions in the chicken EGFR. All three mutants bound hTGF-alpha and were recognized by Mab528. However, mutant Gly(441)Lys showed markedly reduced binding to hEGF, implicating Gly(441), in the L2 domain, as part of the binding site that recognizes Arg(45) of hEGF.


Subject(s)
Epidermal Growth Factor/metabolism , ErbB Receptors/genetics , ErbB Receptors/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Sequence Deletion , Animals , Binding, Competitive/genetics , Biosensing Techniques , CHO Cells , Cell Line , Chickens , Cricetinae , Dimerization , Epidermal Growth Factor/antagonists & inhibitors , ErbB Receptors/biosynthesis , ErbB Receptors/isolation & purification , Growth Inhibitors/genetics , Growth Inhibitors/metabolism , Humans , Ligands , Mice , Mutagenesis, Site-Directed , Peptide Fragments/biosynthesis , Peptide Fragments/isolation & purification , Plasmids/biosynthesis , Plasmids/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Transfection , Transforming Growth Factor alpha/metabolism
19.
Cancer Res ; 61(11): 4474-82, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11389078

ABSTRACT

The chimeric monoclonal antibody KM871, directed against the G(D3) antigen, is under evaluation for its potential to target melanoma. To facilitate the in vivo evaluation of biodistribution properties and measurement of pharmacokinetics, KM871 was radiolabeled with (125)I via tyrosine residues and with (111)In via the bifunctional metal ion chelator C-functionalized trans-cyclohexyl diethylenetriaminepentaacetic acid (CHX-A"-DTPA) to lysine residues. Using antigen-positive SK-MEL-28 melanoma cells, immunoreactivities of 42 and 40% cell binding were obtained, respectively, for the two radioconjugates. Binding was enhanced in the presence of added unlabeled antibody. A humanized A33 antibody was similarly labeled with the two isotopes and used as a control. To determine and compare in vivo biodistribution characteristics of KM871 radiolabeled with (111)In or (125)I, mixtures of the radioconjugates were injected i.v. into BALB/c nude mice bearing G(D3)-positive-SK-MEL-28 melanoma xenografts. Gamma camera images were acquired; groups of five mice were sacrificed at various time intervals, and tumors, blood, and tissues were analyzed. (111)In-labeled CHX-A"-DTPA-KM871 showed a maximum tumor uptake of 41.9 +/- 7.0% injected dose/g at 72 h with prolonged retention over a 15-day period. The tumor:blood ratio was 3:1 by 72 h, and higher ratios were observed at later time points. No abnormal accumulation of (111)In-labeled conjugate was found in normal tissues. In contrast, there was little accumulation of (125)I-labeled KM871 in the same tumors. The specificity of antibody localization was confirmed by the low tumor uptake values for radiolabeled control antibody. Gamma camera imaging demonstrated excellent uptake of (111)In-labeled CHX-A"-DTPA-KM871 in the xenografts. Chromatographic analyses of xenograft cytosolic extracts demonstrated tumor internalization and catabolism of radiolabeled KM871 with the formation of small molecular weight metabolites. Laser scanning confocal microscopy demonstrated that the majority of intracellular KM871 is localized to lysosomes. Despite the catabolism of the radioconjugate, a dose-dependent increase in KM871 tumor localization was shown through immunohistochemical examination of xenograft biopsies. This study demonstrates for the first time the in vivo localization of a radiolabeled anti-G(D3) monoclonal antibody to G(D3)-expressing xenografts using gamma camera scanning techniques and tumor cell internalization of KM871 tagged with a green fluorescent dye, Alexa Fluor 488, through confocal microscopy. KM871 has potential for targeting tumors in patients with melanoma.


Subject(s)
Antibodies, Monoclonal/pharmacokinetics , Gangliosides/immunology , Immunoconjugates/pharmacokinetics , Melanoma/diagnostic imaging , Melanoma/metabolism , Pentetic Acid/analogs & derivatives , Radiopharmaceuticals/pharmacokinetics , Animals , Antibodies, Monoclonal/immunology , Chelating Agents/chemistry , Chelating Agents/pharmacokinetics , Female , Gamma Cameras , Gangliosides/biosynthesis , Humans , Immunohistochemistry , Indium Radioisotopes/chemistry , Iodine Radioisotopes/chemistry , Isothiocyanates/chemistry , Isothiocyanates/pharmacokinetics , Isotope Labeling , Melanoma/immunology , Mice , Mice, Inbred BALB C , Mice, Nude , Pentetic Acid/chemistry , Pentetic Acid/pharmacokinetics , Radionuclide Imaging , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacokinetics , Tissue Distribution , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
20.
Cancer Immunol Immunother ; 50(2): 102-8, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11401024

ABSTRACT

Bispecific antibodies are currently being used in clinical trials in increasing numbers in the areas of breast cancer, prostate cancer, non-Hodgkin's lymphoma and Hodgkin's lymphoma. We have previously performed two clinical trials in patients with Hodgkin's disease with an anti-CD30/anti-CD16 bispecific antibody and demonstrated a 30% response rate in a cohort of patients otherwise resistant to standard therapeutic modalities. However, no surrogate marker could be defined in these trials indicative of optimal antibody dosing/scheduling or predictive for favorable response. In order to evaluate accurately the potential biodistribution properties of bispecific antibody in patients, we have performed a detailed analysis of the binding properties and animal model in vivo characteristics of these constructs. For this purpose, the parental antibodies (anti-CD30 and anti-CD16) and the bispecific antibody (anti-CD30/anti-CD16) were radiolabeled with either 125I or 111In. Antibody integrity and binding properties after labeling were confirmed by Scatchard plot and Lindmo analysis. 111In-labeled antibodies revealed superior targeting properties in a standard SCID mouse tumor model. Both the bivalent parental anti-CD30 monoclonal antibody and the monovalent anti-CD30/anti-CD16 bispecific antibody showed excellent uptake in CD30+ tumors which did not differ significantly between the two (maximum uptake 16.5%+/-4.2% vs. 18.4%+/-3.8% injected dose/gram tissue). The equivalent targeting properties of the bispecific antibody compared with the parental anti-CD30 antibody encourages the further clinical development of this bispecific antibody, and might help to explain the clinical responses seen with this antibody so far in patients suffering from Hodgkin's disease.


Subject(s)
Antibodies, Bispecific/pharmacokinetics , Antibodies, Monoclonal/pharmacokinetics , Ki-1 Antigen/immunology , Neoplasms, Experimental/therapy , Receptors, IgG/immunology , Animals , Antibodies, Bispecific/therapeutic use , Antibodies, Monoclonal/therapeutic use , Female , Humans , Indium Radioisotopes , Iodine Radioisotopes , Mice , Mice, SCID , Neoplasms, Experimental/metabolism , Tissue Distribution
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