Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa.     

Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor

Zinc is an essential trace element for human nutrition and is critical to the structure, stability, and function of many proteins. Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation but down-regulate the extrinsic pathway of coagulation. The protein C pathway plays a key role in blood coagulation and inflammation. At present there is no information on whether zinc modulates the protein C pathway. In the present study we found that Zn²⁺ enhanced the binding of protein C/activated protein C (APC) to endothelial cell protein C receptor (EPCR) on endothelial cells. Binding kinetics revealed that Zn²⁺ increased the binding affinities of protein C/APC to EPCR. Equilibrium dialysis with ⁶⁵Zn²⁺ revealed that Zn²⁺ bound to the Gla domain as well as sites outside of the Gla domain of protein C/APC. Intrinsic fluorescence measurements suggested that Zn²⁺ binding induces conformational changes in protein C/APC. Zn²⁺ binding to APC inhibited the amidolytic activity of APC, but the inhibition was reversed by Ca²⁺. Zn²⁺ increased the rate of APC generation on endothelial cells in the presence of physiological concentrations of Ca²⁺ but did not further enhance increased APC generation obtained in the presence of physiological concentrations of Mg²⁺ with Ca²⁺. Zn²⁺ had no effect on the anticoagulant activity of APC. Zn²⁺ enhanced APC-mediated activation of protease activated receptor 1 and p44/42 MAPK. Overall, our data show that Zn²⁺ binds to protein C/APC, which results in conformational changes in protein C/APC that favor their binding to EPCR.     

Mechanical Stress Is a Pro-Inflammatory Stimulus in the Gut: In Vitro,In Vivo and Ex Vivo Evidence

Aims

Inflammatory infiltrates and pro-inflammatory mediators are found increased in obstructive and functional bowel disorders, in which lumen distention is present. However, what caused the low level inflammation is not well known. We tested the hypothesis that lumen distention- associated mechanical stress may induce expression of specific inflammatory mediators in gut smooth muscle.

Methods

Static mechanical stretch (18% elongation) was applied in vitro in primary culture of rat colonic circular smooth muscle cells (RCCSMCs) with a Flexercell FX-4000 Tension Plus System. Mechanical distention in vivo was induced in rats with an obstruction band placed in the distal colon.

Results

In the primary culture of RCCSMCs, we found that static stretch significantly induced mRNA expression of iNOS, IL-6, and MCP-1 in 3 hours by 6.0(±1.4), 2.5(±0.5), and 2.2(±0.5) fold (n = 6∼8, p<0.05), respectively. However, gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In the in vivo model of colon obstruction, we found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, but not in the sham controls or distal segments. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of NF-κB p65. This treatment further increased mRNA expression of inflammatory mediators in the naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against rat IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators.

Conclusions

Our studies demonstrate that mechanical stress induces gene expression of inflammatory mediators i.e. iNOS, IL-6, and MCP-1 in colonic SMC. Further ex vivo study showed that mechanical stress functions as a pro-inflammatory stimulus in the gut.     

Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation

In rats rendered hyperthyroid by chronic treatment with L-triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]beta-CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 microM, whereas in hyperthyroid rats, it was 83 microM, a 92-fold difference. In control rats, the Ki was 11 microM. These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.     

Neuroprotection by the N-Methyl-d-Aspartate Receptor Antagonist CGP 40116: In Vivo and In Vitro Studies

Abstract: The goal of this study was to evaluate the effects of a novel competitive N -methyl- d -aspartate (NMDA) receptor antagonist, d -( E )-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5-min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8%± 13.1%; CGP 20 mg/kg, 9.9 ± 3.6%; CGP 40 mg/kg, 8.7 ± 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 ± 10.4%; CGP 20 mg/kg, 20.1 ± 6.7%; CGP 40 mg/kg, 27.5 ± 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10-min exposure to 500 µ M NMDA or by 45 min of oxygen-glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration-dependent manner (ED₅₀ of 3.2 µ M against NMDA toxicity and 23.1 µ M against OGD) as measured by lactate dehydrogenase levels 24 h after the insult. The neuroprotective effects of CGP 40116 in vivo and in vitro suggest it may be of potential clinical therapeutic value.     

Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.     

Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H₂O₂-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.     

In Vitro Reactivation of Rat Cortical Tryptophan Hydroxylase Following In Vivo Inactivation by Methylenedioxymethamphetamine

The activity of tryptophan hydroxylase (EC 1.14.16.4) from rat brain was significantly decreased 1 h following a single systemic injection of 3,4-methylenedioxymethamphetamine (MDMA) when assessed ex vivo by radioenzymatic assay or in vivo by the quantitation of 5-hydroxytryptophan accumulation following central L-aromatic amino acid decarboxylase inhibition. Recovery of enzymatic activity in vivo, which occurred within 24 h of low-dose MDMA treatment, appeared not to involve synthesis of new enzyme protein, because the return of enzymatic activity was not prevented by prior cycloheximide. Acutely MDMA-depressed cortical tryptophan hydroxylase activity could be completely restored in vitro by a prolonged (20-24 h) anaerobic incubation in the presence of dithiothreitol and Fe2+ at 25 degrees C; partial reconstitution occurred when 2-mercapto-ethanol was substituted for dithiothreitol. Cortical tryptophan hydroxylase acutely inactivated by methamphetamine or p-chloroamphetamine could be similarly reactivated. MDMA-inactivated cortical tryptophan hydroxylase derived from rats killed later than 3 days after drug treatment could not be significantly reactivated under the conditions described above, indicating the development of irreversible enzymatic damage. Kinetic analysis of enzyme reactivation revealed an approximate doubling of enzyme Vmax with no change in enzyme affinity for either substrate, tryptophan, or pterin cofactor. These studies suggest that MDMA and its congeners inactivate central tryptophan hydroxylase by inducing oxidation of key enzyme sulfhydryl groups. The reactivation capacity of drug-inactivated enzyme at various times after MDMA treatment may provide a means of assessing the development of MDMA-induced neurotoxicity.     

Celiac Disease–Specific TG2-Targeted Autoantibodies Inhibit Angiogenesis Ex Vivo and In Vivo in Mice by Interfering with Endothelial Cell Dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility.     

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca²⁺ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.     

Outer Membrane Vesicles Derived from Escherichia coli Up-Regulate Expression of Endothelial Cell Adhesion Molecules In Vitro and In Vivo

Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1^-/- and TLR4^-/- mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide.     

Blockade of the Glucocorticoid Receptor with RU 486: Effects In Vitro and In Vivo on Human Adipose Tissue Lipoprotein Lipase Activity

Cortisol is known to induce lipoprotein lipase (LPL) activity in human adipose tissue in vitro and in vivo such as in Cushing's syndrome. The significance of the glucocorticoid receptor (GR) for this induction was evaluated in the present study. The synthetic steroid molecule RU 486, a potent glucocorticoid antagonist, was used as a tool to block the GR, in vitro and in vivo. In addition to LPL activity, glucose tolerance, blood pressure and plasma lipids, all variables influenced by Cortisol, were studied in order to evaluate the peripheral antiglucocorticoid activity of RU 486 in vivo, in man. Addition of both Cortisol and RU 486 to incubations of human adipose tissue pieces significantly inhibited the increase in LPL activity that could be induced by Cortisol alone (p<0.01). In a ten-fold molarity excess RU 486 totally abolished Cortisol action (p<0.01). With Cortisol and RU 486 in equimolar concentrations the RU 486 blockade was probably incomplete and LPL activity induced (p<0.05). The results imply that the stimulating effect of Cortisol on LPL activity in human adipose tissue is mediated via the GR. Administration of 400 mg RU 486 at 2200 hours on two consecutive days to healthy men caused a significant rise in serum Cortisol levels measured at 0800 hours (p<0.05). The mean LPL activity in the subcutaneous abdominal adipose tissue remained unchanged. The mean level of serum triglycerides decreased significantly (p<0.01) and there was a negative correlation between change in LPL activity and change in triglyceride levels (r=-0.73, p<0.05). Glucose tolerance and blood pressure were not affected. In conclusion, a total blockade of the GR with RU 486 can be achieved in human adipose tissue in vitro. The blockade abolishes the stimulating effect of Cortisol on LPL activity suggesting that the stimulation is GR dependent. In vivo, with the dose used, the negative pituitary feedback regulation probably compensates for the blockade, at least during the morning hours, thus obviating any peripheral effect of blockade of the GR.     

In Vivo, Ex Vivo, and In Vitro One- and Two-Dimensional Nuclear Magnetic Resonance Spectroscopy of an Intracerebral Glioma in Rat Brain: Assignment of Resonances

Abstract: An in vivo study of intracerebral rat glioma using proton-localized NMR spectroscopy showed important modifications of the spectra in the tumor as compared with the contralateral brain. To carry out the assignment of the resonances of the glioma spectra, tumoral and normal rat brain tissues were studied in vivo, ex vivo, and in vitro by one-dimensional and two-dimensional proton spectroscopy. N -Acetylaspartate was found at an extremely low level in the glioma. The change of peak ratio total creatine/3.2 ppm peak was found to be due to a simultaneous decrease of the total creatine content and an increase of the 3.2 ppm peak. The 3.2 ppm resonance in the glioma spectra has been shown to originate from choline, phosphocholine, glycerophosphocholine, taurine, inositol, and phosphoethanolamine. The increase of the 3.2 ppm peak in the glioma was found to result from the increase of taurine and phosphoethanolamine contents. The peak in the 1.3 ppm region of the glioma spectra was due to both lactate and mobile fatty acids. Moreover, two-dimensional spectroscopy of excised tissues and extracts showed the presence of hypotaurine only in the tumor.     

Regulation of T Cell Motility In Vitro and In Vivo by LPA and LPA2

Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1–6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2−/− CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.     

Effects of Propyzamide on Tobacco Cell Microtubules In Vivo and In Vitro

Treatment with propyzamide at 2 ? 10^-6 M or at higher concentrationsarrested the cell cycleat metaphase in tobacco BY-2 cells. Metaphasecells having disorganized spindle microtubulesand scatteredchromosomes began to appear within several minutes of the additionof propyzamide. Within 30 min, disrupted spindle microtubulesand dispersed chromosomes were seenin all metaphase cells. Propyzamideat 2 ? 10^-6 M or at higher concentrations also disrupted corticalmicrotubules, but disruption of cortical microtubules requiredmore time than disruption of spindle microtubules. The effectof propyzamide on microtubules was found to be readily reversible.The cells arrested at metaphase by 2 ? 10^-6 M propyzamide resumedmitosis within 2 h from the termination of treatment with propyzamide.Spindle microtubules reappeared within 15 min from the terminationof treatment with propyzamide, and the cortical microtubuleswithin 1 h. Tubulin was isolated from tobacco BY-2 cells bycolumn chromatography on ethyl Nphenylcarbamate-Sepharose 4B.On incubation with EGTA, Mg²⁺ and DMSO, the purified tobaccotubulin polymerized into microtubules. Propyzamide at 1 ? 10^-4M completely inhibitedthe polymerization of tobacco tubulin,but did not inhibit polymerization of bovine braintubulin. Tobaccotubulin was adsorbed onto a column of propyzamide-analogue-linkedSepharose 4B and then purified by chromatography on this column. (Received February 15, 1988; Accepted June 29, 1988)     

Ex Vivo Measurement of Brain Tissue Nitrite and Nitrate Accurately Reflects Nitric Oxide Synthase Activity In Vivo

Abstract: The ex vivo tissue concentration of nitrite and nitrate (NO_x) was found to correlate closely with the activity of nitric oxide synthase (NOS; EC 1.14.13.39) in various brain regions. Systemic administration of the nonselective NOS inhibitor N ^ω-nitro- l -arginine ( l -NA) at doses that completely inhibited both central and peripheral NOS, depleted whole-brain and CSF NO_x by up to 75% but had no effect on plasma NO_x. Selective inhibition of central NOS by intracerebroventricular administration of l -NA methyl ester produced similar decreases in levels of whole-brain NO_x. A residual concentration of NO_x of 10–15 µ M remained in all brain regions even after complete inhibition of brain NOS. Brain NO_x content decreased rapidly and in parallel with the inhibition of brain NOS. The ex vivo measurement of levels of brain NO_x was found to reflect the in vivo efficacy of several different types of NOS inhibitor: l -NA, N ^ω-monomethyl- l -arginine, and 7-nitroindazole. Intraperitoneal administration of the NOS substrate l -arginine increased brain NO_x concentrations by up to 150% of control values. These results demonstrate that the ex vivo measurement of levels of brain tissue NO_x is a rapid, reliable, and straightforward technique to determine NOS activity in vivo. This method can be used to assess both the regional distribution and the degree of inhibition of NOS activity in vivo.     

Expression of Epidermal Growth Factor Receptor Detected by Cetuximab Indicates Its Efficacy to Inhibit In Vitro and In Vivo Proliferation of Colorectal Cancer Cells

Cetuximab is a chimeric mouse–human monoclonal antibody that targets the human epidermal growth factor receptor (EGFR). However, EGFR expression determined by immunohistochemistry does not predict clinical outcomes of colorectal cancer (CRC) patients treated with cetuximab. Therefore, we evaluated the correlation between EGFR levels detected by cetuximab and drug sensitivities of CRC cell lines (Caco-2, WiDR, SW480, and HCT116) and the A431 epidermoid carcinoma cell line. We used flow cytometry (FCM) to detect EGFR-binding of biotinylated cetuximab on the cell surface. Subcloned cell lines showing the highest and lowest EGFR expression levels were chosen for further study. Cytotoxic assays were used to determine differential responses to cetuximab. Xenograft models treated with cetuximab intraperitoneally to assess sensitivity to cetuximab. Strong responses to cetuximab were specifically exhibited by subcloned cells with high EGFR expression levels. Furthermore, cetuximab inhibited the growth of tumors in xenograft models with high or low EGFR expression levels by 35% and 10%–20%, respectively. We conclude that detection of EGFR expression by cetuximab promises to provide a novel, sensitive, and specific method for predicting the sensitivity of CRC to cetuximab.