ABSTRACT
Cell-surface receptors can be difficult to express and purify for structural and biochemical studies due to low expression levels, misfolding, aggregation, and instability. Cell-surface receptor ectodomains are more amenable to large-scale production, but this requires designing and testing various truncation constructs. However, since each protein is unique, testing these constructs individually for many targets is a time-consuming process. In this context, we present a high-throughput ELISA fluorescence approach that allows the rapid assessment of numerous recombinant constructs simultaneously. Cell-surface ectodomains are expressed in small scale, enzymatically biotinylated, and detected using a C-terminal His-tag. As an example, we tested the expression of truncation constructs for the neurexin, neuroligin, and latrophilin families and show that the small-scale ELISA allowed us to prioritize well-expressing construct for large-scale production. By employing this method, one can efficiently detect clones with low expression levels, streamlining the process and saving valuable time in identifying optimal candidates for further study.
Subject(s)
Enzyme-Linked Immunosorbent Assay , High-Throughput Screening Assays , Humans , Enzyme-Linked Immunosorbent Assay/methods , High-Throughput Screening Assays/methods , Membrane Proteins/genetics , Membrane Proteins/metabolism , Protein Domains , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/isolation & purification , HEK293 Cells , Gene ExpressionABSTRACT
Integrins are cell surface receptors that mediate the interactions of cells with their surroundings and play essential roles in cell adhesion, migration, and homeostasis. Eight of the 24 integrins bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, comprising the RGD-binding integrin subfamily. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands to fulfill specific functions in cellular processes. Antibodies against individual RGD-binding integrins are desirable for investigating their specific functions, and were selected here from a synthetic yeast-displayed Fab library. We discovered 11 antibodies that exhibit high specificity and affinity toward their target integrins, i.e. αVß3, αVß5, αVß6, αVß8, and α5ß1. Of these, six are function-blocking antibodies and contain a ligand-mimetic R(G/L/T)D motif in their CDR3 sequences. We report antibody-binding specificity, kinetics, and binding affinity for purified integrin ectodomains, as well as intact integrins on the cell surface. We further used these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6 = ß8 > ß3 > ß1 = ß5.
Subject(s)
Saccharomyces cerevisiae , Humans , Saccharomyces cerevisiae/genetics , Integrin beta Chains/immunology , Integrin beta Chains/chemistry , Integrin beta Chains/metabolism , Integrin beta Chains/genetics , Integrin alphaV/immunology , Integrin alphaV/metabolism , Integrins/immunology , Integrins/metabolism , Peptide Library , Cell Surface Display Techniques , Protein Binding , Antibody SpecificityABSTRACT
Solid tumors, especially those with aberrant MYCN activation, often harbor an immunosuppressive microenvironment to fuel malignant growth and trigger treatment resistance. Despite this knowledge, there are no effective strategies to tackle this problem. We found that chemokine-like factor (CKLF) is highly expressed by various solid tumor cells and transcriptionally up-regulated by MYCN. Using the MYCN-driven high-risk neuroblastoma as a model system, we demonstrated that as early as the premalignant stage, tumor cells secrete CKLF to attract CCR4-expressing CD4+ cells, inducing immunosuppression and tumor aggression. Genetic depletion of CD4+ T regulatory cells abolishes the immunorestrictive and protumorigenic effects of CKLF. Our work supports that disrupting CKLF-mediated cross-talk between tumor and CD4+ suppressor cells represents a promising immunotherapeutic approach to battling MYCN-driven tumors.
Subject(s)
Chemokines , MARVEL Domain-Containing Proteins , N-Myc Proto-Oncogene Protein , Neuroblastoma , Humans , Cell Line, Tumor , Chemokines/metabolism , Gene Expression Regulation, Neoplastic , MARVEL Domain-Containing Proteins/metabolism , N-Myc Proto-Oncogene Protein/metabolism , Neuroblastoma/metabolism , Neuroblastoma/pathology , Neuroblastoma/therapy , Tumor MicroenvironmentABSTRACT
Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions. In this study, we discovered 11 antibodies that exhibit high specificity and affinity towards integrins αVß3, αVß5, αVß6, αVß8, and α5ß1 from a synthetic yeast-displayed Fab library. Of these, 6 are function-blocking antibodies containing an R(G/L/T) D motif in their CDR3 sequences. We report antibody binding specificity, kinetics, and binding affinity for purified integrin ectodomains as well as intact integrins on the cell surface. We further employed these antibodies to reveal binding preferences of the αV subunit for its 5 ß-subunit partners: ß6=ß8>ß3>ß1=ß5.
ABSTRACT
The antioxidant- and flavonoid-rich contents of red wine and green tea are reported to offer protection against cancer, cardiovascular disease, and diabetes. Some studies, however, show that flavonoids inhibit GLUT1-mediated, facilitative glucose transport, raising the possibility that their interaction with GLUT1 and subsequent downstream effects on carbohydrate metabolism may also impact health. The present study explores the structure-function relationships of flavonoid-GLUT1 interactions. We find that low concentrations of flavonoids act as cis-allosteric activators of sugar uptake, whereas higher concentrations competitively inhibit sugar uptake and noncompetitively inhibit sugar exit. Studies with heterologously expressed human GLUT1, -3, or -4 reveal that quercetin-GLUT1 and -GLUT4 interactions are stronger than quercetin-GLUT3 interactions, that epicatechin gallate (ECG) is more selective for GLUT1, and that epigallocatechin gallate (EGCG) is less GLUT isoform-selective. Docking studies suggest that only one flavonoid can bind to GLUT1 at any instant, but sugar transport and ligand-binding studies indicate that human erythrocyte GLUT1 can bind at least two flavonoid molecules simultaneously. Quercetin and EGCG are each characterized by positive, cooperative binding, whereas ECG shows negative cooperative binding. These findings support recent studies suggesting that GLUT1 forms an oligomeric complex of interacting, allosteric, alternating access transporters. We discuss how modulation of facilitative glucose transporters could contribute to the protective actions of the flavonoids against diabetes and Alzheimer's disease.
Subject(s)
Flavonoids/pharmacology , Glucose Transporter Type 1/antagonists & inhibitors , Glucose Transporter Type 1/metabolism , Sugars/metabolism , Tea/chemistry , Wine/analysis , Allosteric Regulation/drug effects , Binding, Competitive , Biological Transport/drug effects , Flavonoids/metabolism , Glucose Transporter Type 1/chemistry , HEK293 Cells , Humans , Insulin/metabolism , Molecular Docking Simulation , Protein ConformationABSTRACT
A growing body of evidence demonstrates that GLUT1-mediated erythrocyte sugar transport is more complex than widely assumed and that contemporary interpretations of emergent GLUT1 structural data are incompatible with the available transport and biochemical data. This study examines the kinetic basis of one such incompatibility-transport allostery-and in doing so suggests how the results of studies examining GLUT1 structure and function may be reconciled. Three types of allostery are observed in GLUT1-mediated, human erythrocyte sugar transport: (1) exofacial cis-allostery in which low concentrations of extracellular inhibitors stimulate sugar uptake while high concentrations inhibit transport; (2) endofacial cis-allostery in which low concentrations of intracellular inhibitors enhance cytochalasin B binding to GLUT1 while high concentrations inhibit binding, and (3) trans-allostery in which low concentrations of ligands acting at one cell surface stimulate ligand binding at or sugar transport from the other surface while high concentrations inhibit these processes. We consider several kinetic models to account for these phenomena. Our results show that an inhibitor can only stimulate then inhibit sugar uptake if (1) the transporter binds two or more molecules of inhibitor; (2) high-affinity binding to the first site stimulates transport, and (3) low-affinity binding to the second site inhibits transport. Reviewing the available structural, transport, and ligand binding data, we propose that exofacial cis-allostery results from cross-talk between multiple, co-existent ligand interaction sites present in the exofacial cavity of each GLUT1 protein, whereas trans-allostery and endofacial cis-allostery require ligand-induced subunit-subunit interactions.
Subject(s)
Glucose Transporter Type 1/metabolism , Biological Transport/physiology , Cytochalasin B/metabolism , Erythrocytes/metabolism , Glucose/metabolism , Humans , Kinetics , Protein BindingABSTRACT
Recent structural studies suggest that GLUT1 (glucose transporter 1)-mediated sugar transport is mediated by an alternating access transporter that successively presents exofacial (e2) and endofacial (e1) substrate-binding sites. Transport studies, however, indicate multiple, interacting (allosteric), and co-existent, exo- and endofacial GLUT1 ligand-binding sites. The present study asks whether these contradictory conclusions result from systematic analytical error or reveal a more fundamental relationship between transporter structure and function. Here, homology modeling supported the alternating access transporter model for sugar transport by confirming at least four GLUT1 conformations, the so-called outward, outward-occluded, inward-occluded, and inward GLUT1 conformations. Results from docking analysis suggested that outward and outward-occluded conformations present multiple ß-d-glucose and maltose interaction sites, whereas inward-occluded and inward conformations present only a single ß-d-glucose interaction site. Gln-282 contributed to sugar binding in all GLUT1 conformations via hydrogen bonding. Mutating Gln-282 to alanine (Q282A) doubled the Km(app) for 2-deoxy-d-glucose uptake and eliminated cis-allostery (stimulation of sugar uptake by subsaturating extracellular maltose) but not trans-allostery (uptake stimulation by subsaturating cytochalasin B). cis-Allostery persisted, but trans-allostery was lost in an oligomerization-deficient GLUT1 variant in which we substituted membrane helix 9 with the equivalent GLUT3 sequence. Moreover, Q282A eliminated cis-allostery in the oligomerization variant. These findings reconcile contradictory conclusions from structural and transport studies by suggesting that GLUT1 is an oligomer of allosteric, alternating access transporters in which 1) cis-allostery is mediated by intrasubunit interactions and 2) trans-allostery requires intersubunit interactions.
Subject(s)
Glucose Transporter Type 1/metabolism , Glucose/metabolism , Models, Molecular , Algorithms , Allosteric Regulation , Amino Acid Substitution , Binding Sites , Biological Transport , Dimerization , Glucose Transporter Type 1/chemistry , Glucose Transporter Type 1/genetics , HEK293 Cells , Humans , Hydrogen Bonding , Ligands , Maltose/metabolism , Molecular Docking Simulation , Mutation , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Stochastic Processes , Structural Homology, ProteinABSTRACT
WZB117 (2-fluoro-6-(m-hydroxybenzoyloxy) phenyl m-hydroxybenzoate) inhibits passive sugar transport in human erythrocytes and cancer cell lines and, by limiting glycolysis, inhibits tumor growth in mice. This study explores how WZB117 inhibits the erythrocyte sugar transporter glucose transport protein 1 (GLUT1) and examines the transporter isoform specificity of inhibition. WZB117 reversibly and competitively inhibits erythrocyte 3-O-methylglucose (3MG) uptake with Ki(app) = 6 µm but is a noncompetitive inhibitor of sugar exit. Cytochalasin B (CB) is a reversible, noncompetitive inhibitor of 3MG uptake with Ki(app) = 0.3 µm but is a competitive inhibitor of sugar exit indicating that WZB117 and CB bind at exofacial and endofacial sugar binding sites, respectively. WZB117 inhibition of GLUTs expressed in HEK293 cells follows the order of potency: insulin-regulated GLUT4 â« GLUT1 ≈ neuronal GLUT3. This may explain WZB117-induced murine lipodystrophy. Molecular docking suggests the following. 1) The WZB117 binding envelopes of exofacial GLUT1 and GLUT4 conformers differ significantly. 2) GLUT1 and GLUT4 exofacial conformers present multiple, adjacent glucose binding sites that overlap with WZB117 binding envelopes. 3) The GLUT1 exofacial conformer lacks a CB binding site. 4) The inward GLUT1 conformer presents overlapping endofacial WZB117, d-glucose, and CB binding envelopes. Interrogating the GLUT1 mechanism using WZB117 reveals that subsaturating WZB117 and CB stimulate erythrocyte 3MG uptake. Extracellular WZB117 does not affect CB binding to GLUT1, but intracellular WZB117 inhibits CB binding. These findings are incompatible with the alternating conformer carrier for glucose transport but are consistent with either a multisubunit, allosteric transporter, or a transporter in which each subunit presents multiple, interacting ligand binding sites.
Subject(s)
3-O-Methylglucose/metabolism , Erythrocytes/metabolism , Glucose Transporter Type 1/metabolism , Glucose/metabolism , Hydroxybenzoates/pharmacology , Animals , Binding Sites , Biological Transport , Crystallography, X-Ray , Cytochalasin B/metabolism , Erythrocytes/drug effects , Glucose Transporter Type 1/chemistry , Glucose Transporter Type 3/chemistry , Glucose Transporter Type 3/metabolism , Glucose Transporter Type 4/chemistry , Glucose Transporter Type 4/metabolism , HEK293 Cells , Humans , Kinetics , Mice , Molecular Docking Simulation , Protein ConformationABSTRACT
Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.
Subject(s)
Caffeine/pharmacology , Glucose Transporter Type 1/metabolism , Glucose/metabolism , 3-O-Methylglucose/metabolism , Adenosine Triphosphate/metabolism , Binding Sites , Biological Transport/drug effects , Biological Transport/physiology , Cytochalasin B/metabolism , Erythrocyte Membrane/metabolism , Erythrocytes/drug effects , Erythrocytes/metabolism , Humans , Signal Transduction/drug effectsABSTRACT
Hairpins are common nucleic acid secondary structures that serve many structural and functional roles. Recently, we reported that r(UNCG) and r(GNRA) hairpin families use molecular mimicry and electrostatic factors to attain exceptional thermodynamic stability with a CG closing base pair (cbp). Despite having very different overall folds, these tetraloops present the same functionalities and partial charges to the major groove edge of the CG cbp to achieve stability. Herein, we compare the r(GNRA) tetraloop family to the DNA triloop family d(GNA), which is also exceptionally stable with a CG cbp and possesses the same base pairing between the first and last positions of the loop. Nucleobase and functional group modifications were used to investigate interactions of d(GNA) loops with the cbp, which provided for comparison with similar substitutions in r(GNRA) hairpins. Interruption or deletion of loop-cbp interactions in d(GNA) was consistent with electrostatic interactions identified through nonlinear Poisson-Boltzmann (NLPB) calculations, and loop stability changed in a manner consistent with similar loop-cbp interactions for d(GNA) and r(GNRA) loops. We also compared the relationship of DeltaG degrees (37) and log[Na+] for d(GNA) and r(GNRA) loops and found a decreased dependence of stability on salt for both loop families when a CG cbp was present. The similarity of the loop-cbp interactions shows portability of this loop-cbp motif across polymer type and loop size and indicates convergence on similar molecular solutions for stability in RNA and DNA.
Subject(s)
DNA/chemistry , Models, Molecular , Nucleic Acid Conformation , RNA/chemistry , Base Pairing/genetics , Crystallography, X-Ray , Cytosine/chemistry , DNA/genetics , Guanine/analogs & derivatives , Guanine/chemical synthesis , Guanine/chemistry , Nonlinear Dynamics , Osmolar Concentration , Polymers/chemistry , RNA/genetics , RNA Stability/genetics , Salts/chemistry , Static Electricity , ThermodynamicsABSTRACT
Pulmonary vascular hypertension in general is a progressive, nearly always fatal condition that until recently has had very few treatment options. Our understanding of the pulmonary vascular disease process has opened the window to earlier screening techniques, diagnosis, and treatment options. However, all current treatment options are complex and expensive and therefore require clinical support strategies often necessitating specialized pulmonary hypertension treatment centers. Whether idiopathic or secondary, pulmonary arterial hypertension is characterized by the deregulated proliferation of pulmonary artery endothelial cells and intimal smooth muscle cells, both resistant to cellular apoptosis. Early recognition of such disregulation may lead to earlier diagnosis and treatment and thus alteration in the disease process. Screening of high-risk populations such as those with connective tissue disorders, HIV disease, congenital heart disease, portal hypertension, and those exposed to certain drugs and toxins such as methamphetamines and the diet drugs Dexfenfluramine and Fenfluramine is of utmost importance. Similarly, early symptom recognition in these high-risk groups is essential to earlier diagnosis and treatment.
Subject(s)
Hypertension, Pulmonary/diagnosis , Hypertension, Pulmonary/therapy , Algorithms , Antihypertensive Agents/therapeutic use , Cardiac Surgical Procedures , Clinical Trials as Topic , Diagnostic Imaging , Disease Progression , Drug Therapy, Combination , Electrocardiography , Humans , Phosphodiesterase Inhibitors/therapeutic use , Risk Factors , Vasodilator Agents/therapeutic useSubject(s)
Diabetes Mellitus , Methicillin Resistance , Staphylococcal Skin Infections/diagnosis , Staphylococcus aureus , Aged , Community-Acquired Infections/diagnosis , Community-Acquired Infections/microbiology , Community-Acquired Infections/pathology , Diagnosis, Differential , Humans , Male , Staphylococcal Skin Infections/microbiology , Staphylococcal Skin Infections/pathology , Staphylococcus aureus/isolation & purification , Thigh/pathologyABSTRACT
Mouse monoclonal antibody MX35 was developed against ovarian cancer. The antibody showed homogeneous reactivity with approximately 90% of human ovarian epithelial cancers and with a limited number of normal tissues by immunohistochemistry. Although mAb MX35 has been used in a number of clinical trials in ovarian cancer, it has been difficult to define the molecular identity of MX35. We report here that mAb MX35 recognizes the sodium-dependent phosphate transport protein 2b (NaPi2b) in human cancer cells. This conclusion is based on several lines of experimental evidence, including 1) the identification of SLC34A2, the gene coding for NaPi2b, by immunoscreening an ovarian cancer cell line cDNA expression library with mAb MX35; 2) mass spectrometry sequencing of peptides obtained by fragmentation from mAb MX35 affinity-purified antigen, which show complete sequence homology to amino acid sequences in NaPi2b; 3) selective down-regulation of SLC34A2 gene expression by RNA interference and the resulting loss of mAb MX35 binding to MX35-expressing human cancer cells; and 4) the demonstration of specific mAb MX35 reactivity with recombinant fusion proteins and with synthetic peptides of the putative largest extracellular loop of NaPi2b. We further show that NaPi2b in cancer cells is expressed on the cell surface as a heavily N-glycosylated protein, with evidence of additional post-translational modifications such as palmitoylation and the formation of disulfide bridges in the major extracellular loop. Membrane transporter molecules, such as NaPi2b, represent a new family of potential cell surface targets for the immunotherapy of cancer with monoclonal antibodies.
Subject(s)
Antibodies, Monoclonal , Antibody Specificity , Binding Sites, Antibody , Ovarian Neoplasms/immunology , Sodium-Phosphate Cotransporter Proteins, Type IIb , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibody Specificity/genetics , Antibody Specificity/immunology , Antigen-Antibody Reactions , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Binding Sites, Antibody/genetics , Binding Sites, Antibody/immunology , Female , Humans , Immunohistochemistry , Immunotherapy/trends , Mass Spectrometry , Mice , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIb/antagonists & inhibitors , Sodium-Phosphate Cotransporter Proteins, Type IIb/biosynthesis , Sodium-Phosphate Cotransporter Proteins, Type IIb/genetics , Sodium-Phosphate Cotransporter Proteins, Type IIb/immunologyABSTRACT
Acquired periungual fibrokeratoma (APF) is an uncommon lesion located around the nail and related to acquired digital fibrokeratoma. Histologic features of APF include a core of thick collagen bundles oriented along the longitudinal axis of the tumor and a very characteristic pseudo-nail plate formation. Different terms have been used to describe these lesions, and there has been no consensus on the terminology for some years. It is now believed that trauma may play a role in the pathogenesis of these periungual lesions.
Subject(s)
Keratosis/pathology , Nail Diseases/pathology , Aged , Finger Injuries/complications , Humans , Keratosis/etiology , Keratosis/surgery , Male , Nail Diseases/etiology , Nail Diseases/surgerySubject(s)
Melanoma/diagnosis , Skin Neoplasms/diagnosis , Arm/pathology , Diagnosis, Differential , Female , Forearm/pathology , Genetic Predisposition to Disease , Humans , Knee/pathology , Leg/pathology , Lentigo/diagnosis , Lentigo/genetics , Lentigo/pathology , Melanoma/genetics , Melanoma/pathology , Middle Aged , Neck/pathology , Pedigree , Shoulder/pathology , Skin Neoplasms/genetics , Skin Neoplasms/pathologyABSTRACT
Although ganglioside GD3 levels are highly elevated in malignant melanomas, the role of GD3 in melanomas' malignant properties has not been clearly shown. To investigate this problem, we genetically generated GD3-positive (GD3+) transfectant cells from a GD3-negative (GD3-) mutant line SK-MEL-28-N1 and analyzed the phenotypic changes in the transfected cells. GD3+ cells showed markedly increased cell growth and invasive characteristics. Two bands that underwent stronger tyrosine phosphorylation in GD3+ cell lines than in controls after treatment with FCS were found with molecular masses of 130 and 68 kDa. They were identified as p130Cas and paxillin by sequential immunoprecipitation. Their roles in cell growth and invasion were analyzed with a small interfering RNA (siRNA) approach. Cell growth, as analyzed by BrdUrd uptake, was strongly suppressed in GD3+ cells to near the levels of GD3- cells when treated with siRNA for p130Cas but not when treated with siRNA for paxillin. However, treatment with siRNAs of either p130Cas or paxillin resulted in the marked suppression of the invasive activity of GD3+ cells almost to the levels of control cells. These results suggested that these two molecules function as effectors of GD3-mediated signaling, leading to such malignant properties as rapid cell growth and invasion.
Subject(s)
Cytoskeletal Proteins/metabolism , Gangliosides/metabolism , Melanoma/etiology , Phosphoproteins/metabolism , Proteins/metabolism , Cell Division , Cell Line, Tumor , Crk-Associated Substrate Protein , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Humans , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Neoplasm Invasiveness , Paxillin , Phenotype , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphorylation , Proteins/antagonists & inhibitors , Proteins/chemistry , Proteins/genetics , RNA, Small Interfering/genetics , Retinoblastoma-Like Protein p130 , Sialyltransferases/genetics , Transfection , Tyrosine/chemistryABSTRACT
One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.