Carcinoembryonic Antigen Molecules Research Articles

Optical fiber sensing probe for detecting a carcinoembryonic antigen using a composite sensitive film of PAN nanofiber membrane and gold nanomembrane

An optical fiber sensing probe using a composite sensitive film of polyacrylonitrile (PAN) nanofiber membrane and gold nanomembrane is presented for the detection of a carcinoembryonic antigen (CEA), a biomarker associated with colorectal cancer and other diseases. The probe is based on a tilted fiber Bragg grating (TFBG) with a surface plasmon resonance (SPR) gold nanomembrane and a functionalized polyacrylonitrile (PAN) PAN nanofiber coating that selectively binds to CEA molecules. The performance of the probe is evaluated by measuring the spectral shift of the TFBG resonances as a function of CEA concentration in buffer. The probe exhibits a sensitivity of 0.46 dB/(µg/ml), a low limit of detection of 505.4 ng/mL in buffer, and a good selectivity and reproducibility. The proposed probe offers a simple, cost-effective, and a novel method for CEA detection that can be potentially applied for clinical diagnosis and monitoring of CEA-related diseases.

Single-Molecule Investigation of the Protein-Aptamer Interactions and Sensing Application Inside the Single Glass Nanopore.

Solid-state nanopores offer a nanoconfined space for a single-molecule sensing strategy. Evaluating the behavior of proteins and protein-related interactions at the single-molecule level is becoming more and more important for a better understanding of biological processes and diseases. In this work, the aptamer-functionalized nanopore was prepared as the sensing platform for kinetic analysis of the carcinoembryonic antigen (CEA) with its aptamers, which is an important cancer biomarker. CEA molecules were captured by the aptamers immobilized on the inner surface of the nanopore, and there was a complicated interaction between the CEA molecules and the aptamer, which is the process of association and dissociation. This could be used to measure the dynamics of aptamer-protein interactions without labeling. The kinetic analysis could be evaluated at the single-molecule level to interpret the dissociation constants of the binding and dissociation processes. Results showed that the translocation of CEA molecules in a functionalized nanopore had a deep blockades degree and long duration compared with nanopore modified with bare gold, which could be used for CEA sensing. This protein and protein-related interaction we designed provides new insights for evaluating the binding affinity, which will be beneficial for protein sensing and immunoassays.

Trimetallic nanoparticle-decorated MXene nanosheets for catalytic electrochemical detection of carcinoembryonic antigen via Exo III-aided dual recycling amplifications

Changes in trace levels of tumor markers are important indicators for diagnosing and treating different diseases, including cancers. On account of a recycled dual amplification approach aided by exonuclease III (Exo III) and the trimetallic nanoparticle-decorated MXene nanosheet (Au-Pd-Pt/Ti3C2Tx)-modified electrode as the catalytic sensing interface, we describe the establishment of a label-free and aptamer-based sensitive carcinoembryonic antigen (CEA) detection assay platform. The target CEA molecules bind the aptamer containing hairpin probes to trigger cyclic cleavage of the secondary hairpins with the assistance of Exo III to release a large amount of ssDNAs, which further hybridize with the G-quadruplex-integrated triple-helix complex (THC) signal probes on the sensor electrode. Exo III then cyclically cleaves the resulting duplexes to liberate many G-quadruplex sequences that can confine hemin on the electrode surface. Subsequent catalytic reduction of H2O2 mediated by hemin on Au-Pd-Pt/Ti3C2Tx-modified sensing interface thus yields drastically enhanced current signals for detecting CEA with high sensitivity between 1 fg mL−1 and 1 ng mL−1 with the detection limit of 0.32 fg mL−1. Such a demonstration of our assay method for CEA indicates that the developed sensing platform can be extended as a promising means for sensitive monitoring of different molecular biomarkers.

Enhanced two-electrode photoelectrochemical biosensing platform amplified by bilirubin oxidase labelling

Herein, a novel and robust two-electrode photoelectrochemical (PEC) biosensing platform amplified by bilirubin oxidase (BOD) labelling was reported. This two-electrode operating system was composed of photoanode and sensing biocathode. Carcinoembryonic antigen (CEA) was utilized as a model for probing. Specifically, Fe3+-doped TiO2 (Fe:TiO2) nanotubes were first prepared and then deposited with ZnIn2S4 (ZIS) nanocrystals to develop the ZIS/Fe:TiO2 photoanode. Toward sensing biocathode, after carbon nanotubes (CNTs) and Au nanoparticles (NPs) were modified on conductive glass, an Au/CNT cathodic substrate was fabricated to anchor the CEA aptamer. Complementary DNA (cDNA) of the CEA aptamer was labelled with BOD to form the cDNA-BOD conjugate that amplified the signal through DNA hybridization. Target detection hinged on an evident decline in the current signal generated by terminated amplification of the BOD labels and evident steric hindrance of the captured CEA molecules. Profiting from excellent PEC property of the ZIS/Fe:TiO2 photoanode and good oxygen-reduction capability of the BOD labels, the two-electrode PEC biosensing platform exhibited high sensitivity. Because of the division of biorecognition with photoanode, the platform also showed satisfactory selectivity in biological matrix. This elegant two-electrode strategy offers a promising path for seeking other robust PEC biosensors applying in complex biological samples.

Integrated MOEMS based cantilever sensor for early detection of cancer

In this paper, we present a novel microcantilever sensor for early detection of cancer in human body using an integrated optical micro ring resonator. Effect of geometrical modification of microcantilever on sensitivity and quality factor of proposed sensor is investigated. Regular rectangular and triangular profile are the two types of microcantilever considered during the analysis. The concept of integration of triangular and regular rectangular microcantilever profile with six hexagonal ring with hexagonal lattice configuration is novel work considered here. Finite element method simulation were carried out to obtain the surface stress of microcantilever and finite difference time domain method are used to obtain the transmission characteristics of photonic resonator structure. Range of pressure exerted on microcantilever due to binding of carcino embryonic antigen molecules on surface microcantilever is calculated as 0–2 MPa for specific number of molecules. High resolution with high quality factor 11,458 and pressure sensitivity of 60 nm/MPa is obtained for triangular microcantilever profile. Regular rectangular microcantilever has shown sensitivity of 0.01 nm/MPa and maximum quality factor of 3805. Both the sensor configuration has shown transmission efficiency above 90%. Thus a novel photonic resonator based microcantilever sensor configurations shows a promising linear characteristic as MOEMS sensor.

A Simple Liquid Crystal-based Aptasensor Using a Hairpin-shaped Aptamer for the Bare-Eye Detection of Carcinoembryonic Antigen

Sensitive detection of carcinoembryonic antigen (CEA) is highly important in clinical diagnosis because CEA is an effective tumor marker associated with gastric, lung, rectal, and breast cancers. Thus, the development of an accurate, simple, and efficient method for the detection of CEA remains a long-term clinical goal. In this study, a liquid crystal-based aptasensor was constructed for the labelfree and sensitive detection of CEA. The aptamer, as a molecule identification element, was immobilized onto (3-aminopropyl)triethoxysilane (APTES)- and dimethyloctadecyl[3-(trimethoxysilyl) propyl]ammonium chloride (DMOAP)-modified surface slides through glutaraldehyde (GA); this aptamer can specifically bind to CEA. In the presence of CEA molecules, the loop-stem structure of the aptamers coated onto the surface of the slides opens, and the aptamer then combines with CEA, leading to the formation of aptamer-CEA complexes, which causes the orientation of the liquid crystal molecules (LCs) to transition from a homeotropic to a random alignment. This transition can easily be observed using polarized light microscopy, based on the change of dark optical images into bright ones. The aptasensor system is quite sensitive, as the detection limit is as low as 0.12 pg/ml, with a linear equation of Grayscale=20.318logCCEA+26.329 (R2=0.9925), for a CEA concentration range of 0.05 pg/ml to 50 ng/ml. Bovine serum albumin (BSA) and human serum albumin (HSA) were also tested in this experiment, indicating that this sensor can selectively distinguish between CEA and other interfering compounds with similar protein structures. Moreover, these results were further confirmed by atomic force microscopy (AFM) and ellipsometry.

Measurement of Carcinoembryonic Antigen in Clinical Serum Samples Using a Centrifugal Microfluidic Device.

Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker used in clinical applications. The primarily clinical method for measuring CEA is based on chemiluminescence in serum during enzyme-linked immunosorbent assays (ELISA) in 96-well plates. However, this multi-step process requires large and expensive instruments, and takes a long time. In this study, a high-throughput centrifugal microfluidic device was developed for detecting CEA in serum without the need for cumbersome washing steps normally used in immunoreactions. This centrifugal microdevice contains 14 identical pencil-like units, and the CEA molecules are separated from the bulk serum for subsequent immunofluorescence detection using density gradient centrifugation in each unit simultaneously. To determine the optimal conditions for CEA detection in serum, the effects of the density of the medium, rotation speed, and spin duration were investigated. The measured values from 34 clinical serum samples using this high-throughput centrifugal microfluidic device showed good agreement with the known values (average relative error = 9.22%). These results indicate that the high-throughput centrifugal microfluidic device could provide an alternative approach for replacing the classical method for CEA detection in clinical serum samples.

Improving biosensing activity to carcinoembryonic antigen with orientated single domain antibodies

Carcinoembryonic antigen (CEA), also referred as CEACAM5, is integral to the adhesion process during cancer invasion and metastasis and is one of the most widely used tumor markers for assisting the diagnosis of cancer recurrence and cancer metastasis. Antibodies against CEA molecules have been developed for detection and diagnostic applications following tumor removal. Single domain antibodies (sdAbs) against CEA isolated from dromedary and llama exhibited high specificity in binding to tumor cells. However, because these CEA sdAbs were not designed to be orientated when conjugated to surface sensors, there is potential for significant improvements in their activity and limit of detection. Herein we modified the CEA sdAbs with two different C-terminal fusions designed to aid with orientation by way of the tail’s charge and biotin binding. A fusion which incorporated the C-terminus addition of a positively charged tail (B5-GS3K) improved biosensor sensitivity to CEA while also retaining the sub-nanomolar binding affinity and thermal stability of the unmodified sdAb. Using our fabricated surfaces on bare gold chips and a multiplexed surface plasmon resonance imager (SPRi), we quantified the specific binding activities, defined as the percentage of bound epitopes to the total immobilized, of the sdAb fusions and anti-CEA mAb. Our results demonstrate that monovalent B5-GS3K exhibited significantly improved binding activity, approximately 3-fold higher than bivalent mAb.

Enhanced detection sensitivity of carcinoembryonic antigen on a plasmonic nanoimmunosensor by transmission grating-based total internal reflection scattering microscopy

Carcinoembryonic antigen (CEA) is a glycoprotein associated with colorectal carcinomas and is commonly used as a clinical tumor marker. Enhanced detection sensitivity for the assay of CEA molecules was achieved on a plasmonic nanoimmunosensor by wavelength-dependent transmission grating (TG)-based total internal reflection scattering microscopy (TIRSM). The plasmonic nanoparticles were placed in an evanescent field layer on a glass nanoimmunosensor that produced evanescent wave scattering by the total internal reflection of light from two lasers. The light scattered by target protein (CEA)-bound 20-nm silver nanoparticles (plasmonic nanoprobes) was collected and spectrally isolated in first-order spectral images (n=+1) by a TG (70 grooves/mm). The combination of evanescent wave scattering and TG ​significantly enhanced the detection sensitivity and selectivity due to the minimized spectroscopic interference and background noise. The TG-TIRSM method detected the CEA molecules at concentrations down to 19.75zM with a wide linear dynamic range of 19.75zM-39.50nM (correlation coefficient, R=0.9903), which was 45 to 1.25×109 times lower than the detection limits and 2×105 to 2×1011 times wider than the dynamic ranges of previous assay methods. In particular, by simply changing the antibody of the target molecule, this technique can be used to detect various disease-related protein biomarkers directly in human biological samples at the single-molecule level.

Molecular-Counting-Free and Electrochemiluminescent Single-Molecule Immunoassay with Dual-Stabilizers-Capped CdSe Nanocrystals as Labels.

Biorelated single-molecule detection (SMD) has been achieved typically by imaging the redox fluorescent labels and then determining each label one by one. Herein, we demonstrated that the capping agents (i.e., mercaptopropionic acid and sodium hexametaphosphate) can facilitate the electrochemical involved hole (or electron) injecting process and improve the stability of the dual-stabilizers-capped CdSe nanocrystals (NCs), so that the CdSe NCs could be electrochemically and repeatedly inspired to excited states by giving off electrochemiluminescence (ECL) in a cyclic pattern. With the CdSe NCs as ECL label and carcinoembryonic antigen (CEA) as target molecule, a convenient single-molecule immunoassay was proposed by simply detecting the ECL intensity of the dual-stabilizers-capped CdSe NCs in a sandwich-typed immune complex. The limit of detection is 0.10 fg/mL at S/N = 3, which corresponds to about 6-8 CEA molecules in 20 μL of serum sample. Importantly, the ECL spectra of both CdSe NCs and its conjugate with probe antigen in the immune complex were almost identical to the photoluminescence spectrum of bare CdSe NCs, indicating that all emissions were originated from the same excited species. The molecular-counting-free and ECL-based SMD might be a promising alternative to the fluorescent SMD.

Development of antibody functionalized magnetic nanoparticles for the immunoassay of carcinoembryonic antigen: a feasibility study for clinical use.

BackgroundMagnetic nanoparticles functionalized antibodies are used for in-vitro assays on bio-markers. This work demonstrates the synthesis of high-quality magnetic nanoparticles coated with antibodies against carcinoembryonic antigen (CEA). Various characterizations, such as particle size, particle suspension, bio-activity and the stability of bio-magnetic nanoparticles suspended in liquid, are studied. The properties for the assay of CEA molecules in serum are also studied. The assay method used is so-called immunomagnetic reduction.ResultsThe results show that the effects of common materials in serum that interfere with detected signals are not significant. The low-detection limit is 0.21 ng/ml, which is well below the clinical threshold of 2.5 ng/ml.ConclusionsThe dynamic range for the assay of CEA molecules in serum is 500 ng/ml. By assaying serum CEA molecules from 24 normal controls and 30 colorectal-cancer patients, the threshold for the serum-CEA concentration to diagnose colorectal cancer is 4.05 ng/ml, which results in a clinical sensitivity of 0.90 and specificity of 0.87.

Detection of a carcinoembryonic antigen using aptamer-modified film bulk acoustic resonators

An AlN-based based film bulk acoustic resonator has been fabricated using a magnetron sputtering method, which was employed as a biosensor for detection of a carcinoembryonic antigen. The resonator consisted of an AlN piezoelectric layer and a Mo/Ti Bragg reflector which had a working resonance near 2.0GHz. The carcinoembryonic antigen binding aptamers were self-assembled on the top gold electrode as the sensitive layer to capture the carcinoembryonic antigen molecules. When the sensitive layer absorbs the target molecules, the resonance frequency of the sensor decreases proportionally in response to the increase of the mass loading. The resonant frequency shifts of various carcinoembryonic antigen concentrations in the range of 0.2–1.0mg/mL were measured. The sensor modified with the carcinoembryonic antigen binding aptamer exhibits a high sensitivity of 2284Hzcm2/ng and Q value around 520, exhibiting a high performance. These positive results suggest that the aptamer-immobilized film bulk acoustic resonator is a promising candidate for detection of the carcinoembryonic antigen.

Carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4) is specifically expressed in medullary thyroid carcinoma cells.

Carcinoembryonic antigen (CEA), an oncofetal cell surface glycoprotein, has been widely used as a human tumor marker due to its high expression in tumors and secretion to serum. It belongs to the immunoglobulin superfamily named CEA-related cell adhesion molecule (CEACAM) family. Members of this family are detected in various cancers and have been shown to be involved in cancer growth and invasion. In this study, we examined the mRNA expression profiles of CEACAM family members including CEACAM1, CEACAM3, CEACAM4, CEACAM5 (CEA), CEACAM6, CEACAM7, and CEACAM8 in various tumor cell lines. Our screening data indicated that the mRNA expression patterns of CEACAMs in TT cells, which are derived from medullary thyroid carcinoma (MTC), were distinct from other tumor cell lines. Additionally, CEACAM4 was only expressed in TT cells, in which two novel splice variants of CEACAM4 were expressed. These findings suggested that production of CEA and CEA-related molecules in MTC may be distinct from other tumor-based production of those molecules and that the specific expression of CEACAM4 would make possible to differentiate between MTC and other CEA-producing tumors.

Isolation of carcinoembryonic antigen N-terminal domains (N-A1) from soluble aggregates

Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellman’s assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.

Epitope mapping of the carcinoembryonic antigen by monoclonal antibodies and establishment of a new improved radioimmunoassay system.

A comprehensive mapping of epitopes on the carcinoembryonic antigen (CEA) molecule has been achieved by analyses of the specificities of 146 monoclonal antibodies (MAbs) from more than 300 hybridomas established recently. The reactivities of MAbs were analyzed by radioimmunoassays (RIA) with highly purified preparations of CEA and related antigens including normal fecal antigen-1 (NFA-1), NFA-2 in normal adult feces, nonspecific cross-reacting antigen (NCA) in lung and NCA-2 in meconium. The MAbs could be divided into five groups: group I, 23 clones directed to the NCA-common part of the CEA molecule; group II, 31 clones directed to the normal fecal cross-reacting antigen (NFCA)-common part; group III, 46 clones directed to the NFA-1-common part; group IV, 33 clones reactive with the heterogeneous carbohydrate part; and group V, 13 clones directed to the CEA-distinctive part which seemed to be highly specific for CEA. Mutual inhibitions of CEA binding between MAbs of the individual groups revealed that at least 25 different subgroups can be defined i.e., 4, 7, 8, 4, and 2 subgroups in groups I to V, respectively. The epitopes recognized by the group IV MAbs were found to be sensitive to oxidation with periodate, while the epitopes defined by MAbs of the other groups were resistant to this treatment. A solid-phase sandwich-type RIA system for CEA was established by using 2 MAbs from groups II and III as the CEA catcher and an MAb of group V as the tracer. This assay was shown to exhibit improved cancer-specificity and accuracy in the estimation of serum CEA levels.

The human carcinoembryonic antigen (CEA) GPI anchor mediates anoikis inhibition by inactivation of the intrinsic death pathway

Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation of CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can have tumorigenic effects including the inhibition of cell differentiation and anoikis, a specific type of apoptosis triggered by the absence of extracellular matrix-cell contacts. In the present work, we investigate the involvement of the caspase cascade in CEA-mediated inhibition of anoikis and the structural requirements for this signal. Expression of CEA and/or a chimeric protein consisting of the NCAM extracellular domain attached to the CEA-GPI anchor correlates with an early inactivation of caspase-9 and activation of the PI3-K/Akt survival pathway, and at later times, inactivation of caspase-8. The CEA-mediated caspase inactivation as well as activation of Akt was not observed by expression of a CEA molecule incapable of self-binding (DeltaNCEA). These results suggest that the intrinsic caspase pathway is involved in the inhibitory effects of anoikis by CEA and this signal is dependent on the presence of self-adhesive extracellular domains and a CEA-GPI anchor.

Antibody response against neem leaf preparation recognizes carcinoembryonic antigen

An immune serum generated in Swiss mice against an aqueous preparation from neem leaf was reactive with carcinoembryonic antigen (CEA) and a peptide sequence derived from it. Using ELISA, we have demonstrated that CEA reactive antibody titer (chiefly IgG2a) was significantly decreased after absorption of the immune sera with CEA. Neem leaf preparation (NLP) generated immune sera was also reactive with CEA in immunoblotting and CEA reactive component in the NLP sera can be immunoprecipitated. Identical recognition of CEA expressed on human colorectal cancer specimens, by anti-CEA monoclonal antibody and NLP sera was documented by immunohistochemistry. NLP generated sera could also react with NLP in ELISA and this reactivity was decreased after absorption of the NLP with anti-CEA antibody. Estimation of protein in NLP revealed the presence of it, at least 10% of the total dry weight. In addition, existence of flavone and quercetin was also evidenced from LC-MS analysis. Further studies are needed to identify the antigenic component may have some homology with CEA molecule. This unique property of neem may be utilized for the immunotherapy of CEA positive tumors.

276. Enhancement of Cytotoxic T-Lymphocyte Responses Against Carcinoembryonic antigen (CEA) Using Genetically Modified Dendritic Cells Expressing CEA and CD40 Ligand

Carcinoembryonic antigen (CEA), one of the well-characterized tumor-associated antigens is commonly expressed by a wide variety of adenocarcinomas. However, as a self-antigen it is poorly immunogenic due to immune tolerance. Although several human-leukocyte-antigen (HLA)-restricted epitopes within the CEA protein that can be recognized by human T lymphocytes have been identified, CEA expressing tumor cells are generally weakly recognized by the immune system. Therefore, development of immunotherapeutic strategies to break its tolerance is necessary to generate effective anti-tumor immunity against CEA expressing malignancies. In this study we evaluated the efficiency of genetically modified human peripheral blood monocyte-derived dendritic cell (MoDC) expressing CEA and immunostimulatory molecule CD40 ligand (CD40L) to generate effective immune response against autologous as well as tumor cell lines expressing CEA. The recombinant human immunodeficiency virus (HIV)-1-based self-inactivating lentiviral vectors expressing the full length CEA (Lenti-CEA) or human CD40L (Lenti-CD40L) under the transcriptional control of human PGK promoter provided a highly efficient reproducible gene transfer into immature MoDC. After single exposure to Lenti-CEA and/or Lenti-CD40L vector, more than 80% of DCs showed cell surface expression of CEA alone and/or CD40L without any toxicity. Notably, the Lenti-CD40L transduced DCs showed increased cell surface expression of maturation marker CD83 and molecules involved in antigen presentation (CD80, CD86, CD54, HLA-ABC and HLA-DR), secreted high amounts of T-cell immunostimulatory cytokine interleukin (IL)-12, and showed enhanced T-cell stimulatory function. Importantly, the CEA/CD40L transduced DCs were able to generate CEA-specific T-cell responses ex vivo, which was higher than that of DC transduced with CEA alone and matured with recombinant soluble CD40L or prostaglandin E-2 either alone or in combination with tumor necrosis factor-a. The T-cell lines generated ex vivo with CEA/CD40L expressing DCs comprised both CD8+ and CD4+ T-cells possessing polyclonal T-cell receptor repertoire, showed increased antigen-specific proliferative response, and secreted high levels of interferon-|[gamma]| and IL-2 upon antigen recall. Importantly, the CEA-specific T-cell lines were able to specifically recognize CEA expressing autologous target cells as well as CEA positive tumor cell lines in MHC restricted manner. Although cytotoxicity toward target cells was mediated primarily by CD8+ T-cells, interestingly both CD8+ and CD4+ T-cells were able to lyse CEA expressing target cells. Taken together, these results suggest that lentiviral vector-mediated expression of CEA and CD40L in DC may be a promising strategy for the rational development of DC vaccines or antigen-specific T-cells for adoptive immunotherapy of CEA expressing malignancies.

The Adhesion and Differentiation-inhibitory Activities of the Immunoglobulin Superfamily Member, Carcinoembryonic Antigen, Can Be Independently Blocked

The external domains of Ig superfamily members are involved in multiple binding interactions, both homophilic and heterophilic, that initiate molecular events leading to the execution of diverse cell functions. Human carcinoembryonic antigen (CEA), an Ig superfamily cell surface glycoprotein used widely as a clinical tumor marker, undergoes homophilic interactions that mediate intercellular adhesion. Recent evidence supports the view that deregulated overexpression of CEA has an instrumental role in tumorigenesis through the inhibition of cell differentiation and the disruption of tissue architecture. The CEA-mediated block of the myogenic differentiation of rat L6 myoblasts depends on homophilic binding of its external domains. We show here that L6 transfectant cells expressing CEA can "trans-block" the myogenesis of juxtaposed differentiation-competent L6 transfectant cells expressing a deletion mutant of CEA (DeltaNCEA). This result implies the efficacy of antiparallel CEA-CEA interactions between cells in the differentiation block. In addition, DeltaNCEA can acquire differentiation blocking activity by cross-linking with specific anti-CEA antibodies, thus implying the efficacy of parallel CEA-CEA interactions on the same cell surface. The myogenic differentiation blocking activity of CEA was demonstrated by site-directed mutations to involve three subdomains of the amino-terminal domain, shown previously to be critical for its intercellular adhesion function. Monovalent Fab fragments of monoclonal antibodies binding to the region bridging subdomains 1 and 2 could both inhibit intercellular adhesion and release the myogenic differentiation block. Amino acid substitutions Q80A, Q80R, and D82N in subdomain 3, QNDTG, however, were found to completely ablate the differentiation blocking activity of CEA but had no effect on intercellular adhesion activity. A cyclized peptide representing this subdomain was the most effective at releasing the differentiation block.

In this issue

Suppression of the immune system is a problem faced by many cancer patients, especially those whose diseases are in an advanced stage. Elucidating the mechanism(s) behind this immunosuppression would be beneficial in trying to counter it and bolster immune response to tumors. To this end, Takahashi et al. are studying the effects of macrophages in tumor-draining lymph nodes (MTDL) on T-cell apoptosis and comparing these effects among patients with early and advanced stage gastric cancer. In their paper on pages 393–399 they report two interesting findings. Firstly, they note that, in patients with advanced stage gastric cancer, MTDL induce T-cell apoptosis, elevate caspase-3 activity in peripheral T-cells, reduce expression of signal transducing TCR ζ molecules and impair production of IFN ζ. Secondly, they note that MTDL from patients with advanced stage gastric cancer are qualitatively different from those with an early stage of the disease: they exhibit an increased production of hydrogen peroxide and elevated production of intracellular IL-10 and IL-12. Hydrogen peroxide had been known to induce T-cell apoptosis; however, the presence of catalase, a selective scavenger of hydrogen peroxide, did not prevent MTDL downregulation of TCR ζ. This then must occur by another mechanism. Even so, this paper supports the notion that normalization of T-cell function by anti-oxidant therapy is an advantageous course of study. Carcinoembryonic antigen (CEA) is a 180- to 200-kDa glycoprotein, expressed by various types of neoplasms, that is widely used as a human tumor marker. Clinical trials have investigated the possibility that CEA may be used as a target antigen for active specific immunotherapy of patients with colorectal carcinoma. Unfortunately, the use of CEA-directed vaccines has thus far been unsuccessful in controlling cancer progression in humans. One hypothesis for this failure is that CEA is expressed heterogeneously in the tumor. Thus, pharmacological or biological agents that may increase the expression of CEA and MHC molecules have been sought. The anti-metabolite 5-fluorouracil (5-FU) has been shown to upregulate CEA and MHC expression in various colon and breast carcinoma cell lines in vitro, although the mechanism(s) responsible for this upregulation remain unknown. In their report on pages 437–445 of this issue, Correale et al. extend the ramifications of these findings by demonstrating that 5-FU treatment enhances cancer cell susceptibility to the lytic effects of CEA peptide-specific cytotoxic T-cells. Kaplan-Meier plotting of overall (p = 0.0012) and disease-free (p = 0.0033) survival of SMAD7 deletion. Boulay et al. studied the influence of each of these genes on the clinical outcome of patients suffering from colorectal cancer, and their results are published on pages 446–449. They had previously defined SMAD4 deletion as a negative predictive marker for a benefit of chemotherapy. They observed no clinical relevance for SMAD2 deletion, but SMAD7 deletion had a low hazard ratio for both relapse and death, while its amplification had a hazardous effect on survival. Given that SMAD7 is the only SMAD specifically inhibitory for TGFβ, these data suggest that SMAD7 deletion may enhance sensitivity to the tumor suppressor activity of TGFβ and confirm the hypothesis that resistance to TGFβ-mediated apoptosis is an important factor in the expansion of colorectal carcinoma. Effect of ATN-161, with or without 5-FU, on vessel counts, tumor cell proliferation and tumor cell apoptosis. On pages 496–503 of this issue, Stoeltzing et al. hypothesize that the blocking of α5β1 binding to fibronectin by a different peptide antagonist, ATN-161, which does not affect cell adhesion, may show the same results. Furthermore, they suggest that α5β1 need not be expressed on the cancer cells to achieve these results, as the more sensitive target is the α5β1 on the activated endothelial cells. They also thought that chemotherapy with the anti-metabolite 5-fluorouracil (5-FU) might synergize with the anti-angiogenic effects of the peptide to inhibit angiogenesis and the growth of liver metastases in a murine model. As their report demonstrates, they were correct in all of their hypotheses; not only did the combination therapy increase tumor cell apoptosis, decrease tumor cell survival and reduce the formation of liver metastases, it improved survival in a murine colon cancer model.