Characterization of lectin resistant cell populations derived from human colon carcinoma: correlation of K-Ras with beta1-6 branching of N-linked carbohydrate and CEA production.

Previous studies of cell lines derived from human colon carcinoma showed that the extent of beta1-6 branching on N-linked carbohydrate was associated with the presence of K-ras mutation and Ras-activation. We observed that the extent of Ras-activation in these cell lines depends not only upon the presence of an activating mutation in K-ras, but also on the amount of total K-Ras protein produced. Here we examined whether negative selective pressure by PHA-L against beta1-6 branching could select for cells having a lower level of K-Ras protein and Ras-activation. PHA-L binds specifically to the beta1-6 branch in N-linked carbohydrate. We utilized a K-ras mutant colon carcinoma cell line, HTB39, which had abundant beta1-6 branching and high levels of K-Ras mutant protein. Lectin resistant cell populations of HTB39 were generated and found to have less beta1-6 branching and less K-Ras protein than their parental counterpart. The lectin resistant cell populations produced lower levels of highly glycosylated CEA, which contributed to the lower level of beta1-6 branching in these cells. PHA-L resistant cell populations were two-fold less sensitive than the parental line to an inhibitor of farnesyl transferase (an enzyme essential for Ras processing and function). This suggested a decrease in dependence on K-ras mediated signaling. Collectively, the data indicated that beta1-6 branching of N-linked carbohydrate and CEA production were linked to K-Ras protein synthesis and activation of the Ras-signaling pathway.     

Carcinoembryonic antigen and CD44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin in shear flow

Selectin-mediated adhesion of tumor cells to platelets, leukocytes, and endothelial cells may regulate their hematogenous dissemination in the microvasculature. We recently identified CD44 variant isoforms (CD44v) as functional P-, but not E- or L-, selectin ligands on colon carcinoma cells. Moreover, an approximately 180-kDa sialofucosylated glycoprotein(s) mediated selectin binding in CD44-knockdown cells. Using immunoaffinity chromatography and tandem mass spectrometry, we identify this glycoprotein as the carcinoembryonic antigen (CEA). Blot rolling assays and flow-based adhesion assays using microbeads coated with CEA immunopurified from LS174T colon carcinoma cells and selectins as substrate reveal that CEA possesses E- and L-, but not P-, selectin ligand activity. CEA on CD44-knockdown LS174T cells exhibits higher HECA-452 immunoreactivity than CEA on wild-type cells, suggesting that CEA functions as an alternative acceptor for selectin-binding glycans. The enhanced expression of HECA-452 reactive epitopes on CEA from CD44-knockdown cells correlates with the increased CEA avidity for E- but not L-selectin. Through the generation of stable knockdown cell lines, we demonstrate that CEA serves as an auxiliary L-selectin ligand, which stabilizes L-selectin-dependent cell rolling against fluid shear. Moreover, CEA and CD44v cooperate to mediate colon carcinoma cell adhesion to E- and L-selectin at elevated shear stresses. The novel finding that CEA is an E- and L-selectin ligand may explain the enhanced metastatic potential associated with tumor cell CEA overexpression and the supportive role of selectins in metastasis.     

Use of Phaseolus vulgaris leukoagglutinating lectin in histochemical and blotting techniques: a comparison of digoxigenin- and biotin-labelled lectins

An increase in the number of 1,6 branches of the trimannosyl core of asparagine-linked oligosaccharides has been shown to be directly correlated with the metastatic potential of cultured tumour cells. The Phaseolus vulgaris leukoagglutinating lectin (PHA-L) binds to 1,6 branches of tri- and tetra-antennary oligosaccharides. We have applied digoxigenin- and biotin-conjugated PHA-L to establish a non-radioactive detection system for 1,6 branches, which can be used in lectin blotting as well as light and electron microscopic cytochemistry. For this purpose the HCT116 human colon carcinoma cell line and colon carcinoma tissue were investigated. Digoxigenin-conjugated PHA-L in conjunction with alkaline phosphatase-conjugated anti-digoxigenin antibodies was superior to biotin-conjugated PHA-L in lectin blotting with respect to sensitivity and specificity. Similarly, the digoxigenin conjugated PHA-L in conjunction with gold-labelled anti-digoxigenin antibodies resulted in more intense specific staining and lower background compared to biotin-conjugated PHA-L visualized with a streptavidin immunogold complex. The specificity of lectin binding in blotting and cytochemical studies was demonstrated by the absence of staining when the lectin was omitted or preabsorbed with glycoprotein, and following pretreatment of the cellular homogenates or tissue sections by N-glycosidase F. Our results demonstrate that digoxigenin-conjugated PHA-L provides high sensitivity and specificity for histochemical and blotting techniques and is amenable for quantification. The technique should have applications in tumour research.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

Purification of two glycoproteins expressing beta 1-6 branched Asn-linked oligosaccharides from metastatic tumour cells.

Increased branching at the trimannosyl core of 'complex-type' Asn-linked oligosaccharides has been observed in both human and murine tumour cells, and appears to be associated with enhanced metastatic potential in several murine tumour models [Dennis, Laferte, Waghorne, Breitman & Kerbel (1987), Science 236, 582-585]. The lectin leucoagglutinin (L-PHA) requires the-GlcNAc beta 1-6Man alpha 1-6Man-linked lactosamine antenna in complex-type oligosaccharides for high-affinity binding and can be used to detect these structures in glycoproteins separated on SDS/polyacrylamide-gel electrophoresis. The major L-PHA-binding glycoproteins in the highly metastatic lymphoid tumour cell line called MDAY-D2 were purified and resolved into two major species, termed P2A (110 kDa) and P2B (130 kDa). P2A had L-PHA-reactive Asn-linked oligosaccharides with polylactosamine sequences as well as a large component of sialylated O-linked carbohydrates. The glycoprotein showed structural characteristics similar to those of leukosialin (i.e. CD43), a glycoprotein previously identified on the surface of leukocytes. Based on monosaccharide compositional analysis and glycosidase digestions, P2B was found to be 50-60% Asn-linked oligosaccharide containing polylactosamine sequences and sialic acid. The N-terminal peptide sequence of P2B was determined to be very similar to that of murine lysosomal membrane glycoprotein (LAMP-1), a ubiquitous glycoprotein found largely in the lysosomal membranes but also in the plasma membrane of several murine and human tumour cell lines.     

GPI-anchored CEA family glycoproteins CEA and CEACAM6 mediate their biological effects through enhanced integrin alpha5beta1-fibronectin interaction

Carcinoembryonic antigen (CEA) and CEA family member CEACAM6 are glycophosphatidyl inositol (GPI)-anchored, intercellular adhesion molecules that are up-regulated in a wide variety of human cancers, including colon, breast, and lung. When over-expressed in a number of cellular systems, these molecules are capable of inhibiting cellular differentiation and anoikis, as well as disrupting cell polarization and tissue architecture, thus increasing tumorigenicity. The present study shows that perturbation of the major fibronectin receptor, integrin alpha5beta1, underlies some of these biological effects. Using confocal microscopy and specific antibodies, CEA and CEACAM6 were demonstrated to co-cluster with integrin alpha5beta1 on the cell surface. The presence of CEA and CEACAM6 was shown to lead to an increase in the binding of the integrin alpha5beta1 receptor to its ligand fibronectin, without changing its cell surface levels, resulting in increased adhesion of CEA/CEACAM6-expressing cells to fibronectin. More tenacious binding of free fibronectin to cells led to enhanced fibronectin matrix assembly and the formation of a polymerized fibronectin "cocoon" around the cells. Disruption of this process with specific monoclonal antibodies against either fibronectin or integrin alpha5beta1 led to the restoration of cellular differentiation and anoikis in CEA/CEACAM6 producing cells.     

Induction of polarized apical expression and vectorial release of carcinoembryonic antigen (CEA) during the process of differentiation of HT29-D4 cells

HT29-D4 clonal cells can be induced to differentiate by a simple alteration of the culture medium, that is, by the replacement of glucose by galactose [Fantini, J., et al. (1986) J. Cell Sci., 83:235-249] as reported for the nonclonal HT29 cells [Pinto, M., (1982) Biol. Cell, 44:193-196]. An essential property of the HT29-D4 cell line is the fact that no cell loss occurs after the medium change, so that the differentiated cells can be considered as the true counterpart of the undifferentiated one. This model is particularly suitable to study morphological and biochemical events associated with the progressive establishment of the differentiation state. We report here that carcinoembryonic antigen (CEA), a 180 kDa glycoprotein originally described as a colon tumor associated antigen, is faintly expressed at the surface of undifferentiated HT29-D4 cells. These cells release a small amount of CEA (2.5 ng/10(6) cells/24 hr) in the culture medium. Fourty-eight hours after glucose substitution by galactose, both CEA cell surface expression and release are strongly enhanced as demonstrated by immunofluorescence and immunoprecipitation studies. Ten days after the medium change, the amount of CEA released reaches a maximum value of 130 ng/10(6) cells/24 hr, which remains stable for differentiated HT29-D4 cells cultured in glucose-free, galactose-containing medium (Gal-medium) for several months. HT29-D4 cells grown in Gal-medium in porous-bottom culture dishes generate leakproof epithelial monolayers. We have successfully performed an independent radioiodination of the apical and basolateral domains of these cells, followed by immunoprecipitation. We demonstrate that CEA is expressed exclusively at the apical surface of differentiated HT29-D4 cells, since the 180 kDa polypeptide was immunoprecipitated only when the radioiodination was performed at the apical side of the monolayer. Leakproof HT29-D4 monolayers cultured in permeable chambers were also used to demonstrate that CEA was exclusively released in the medium bathing the apical side of the cells. In conclusion, this study of cell surface CEA expression and CEA release during the process of differentiation of HT29-D4 cells demonstrated that 1) CEA cell surface expression and CEA release are correlated with cell differentiation; 2) CEA is expressed in the apical brush border membrane of differentiated HT29-D4 cells; and 3) CEA release is exclusively oriented toward the apical side of the polarized monolayer.     

Promotion of differentiation in human colon carcinoma cells by vasoactive intestinal polypeptide

The effects of vasoactive intestinal polypeptide (VIP) and dibutyryl cyclic adenosine 3':5'monophosphate (dbcAMP) on two human colon carcinoma cell lines, HCT 116 and GEO, were investigated. VIP and dbcAMP inhibited the growth of both cell lines in monolayer culture in a dose-dependent manner. Within 6 h of treatment with 1 mM dbcAMP or 0.3 microM VIP, numerous mucin-like droplets were secreted by GEO cells. VIP and dbcAMP also increased carcinoembryonic antigen (CEA) secretion. In both cell lines, a 9-fold increase in conditioned medium CEA levels was observed at 1 mM dbcAMP and a 2.6-fold increase at 1.5 microM VIP. Time- and concentration-dependent evaluation in cAMP levels were elicited by VIP in the two cell lines. Immunocytochemical studies for cell-surface glycoprotein detection in GEO cells showed that VIP induced a morphological and functional organization of mucin-secreting cells. These results indicate that VIP and dbcAMP have antiproliferative and strong differentiation-promoting effects in colon cancer cells. This is the first report of VIP-induced mucin secretion in colon tumor cells.     

Induction of carcinoembryonic antigen expression in a three-dimensional culture system

Summary MIP-101 is a poorly differentiated human colon carcinoma cell line established from ascites that produces minimal amounts of carcinoembryonic antigen (CEA), a 180 kDa glycoprotein tumor marker, and nonspecific cross-reacting antigen (NCA), a related protein that has 50 and 90 kDa isoforms, in monolayer culture. However, MIP-101 produces CEA when implanted into the peritoneum of nude mice but not when implanted into subcutaneous tissue. We tested whether three-dimensional (3D) growth was a sufficient stimulus to produce CEA and NCA 50/90 in MIP-101 cells, because cells grow in 3D in vivo rather than in two-dimensions (2D) as occurs in monolayer cultures. To do this, MIP-101 cells were cultured on microcarrier beads in 3D cultures, either in static cultures as nonadherent aggregates or under dynamic conditions in a NASA-designed low shear stress bioreactor. MIP-101 cells proliferated well under all three conditions and increased CEA and NCA production three- to four-fold when grown in 3D cultures compared to MIP-101 cells growing logarithmically in monolayers. These results suggest that 3D growth in vitro simulates tumor function in vivo and that 3D growth by itself may enhance production of molecules that are associated with the metastatic process.     

Identification of a carcinoembryonic antigen binding protein on monocytes

Tumor-associated monocyte/macrophage cells are important stromal components involved in tumor development. A protein on human monocyte is identified that binds to carcinoembryonic antigen (CEA), a glycoprotein overexpressed in colon tumors. This implicates a role for this protein in CEA processing and establishes a link between monocytes and colon tumor cells. In vitro uptake of 125I-labeled CEA with isolated monocytes showed time and temperature dependence. The binding of 125I-CEA was specific and saturable as it could be inhibited by an excess of unlabeled CEA. To identify the binding protein on monocyte, we used a radiolabeled photoactivable heterobifunctional crosslinking agent and demonstrated that CEA reacts with a 115kDa protein as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. Treatment of human monocytes in vitro with CEA resulted in a several fold increase in the production of proinflammatory cytokines TNF-alpha, IL-1 beta, and IL-6 compared to untreated controls. Binding of CEA to the monocyte protein may have implications in colon tumorigenesis.     

Cell surface-expressed Thomsen-Friedenreich antigen in colon cancer is predominantly carried on high molecular weight splice variants of CD44.

Increased mucosal expression of TF, the Thomsen-Friedenreich oncofetal blood group antigen (galactose beta1-3 N-acetylgalactosamine alpha-) occurs in colon cancer and colitis. This allows binding of TF-specific lectins, such as peanut agglutinin (PNA), which is mitogenic to the colorectal epithelium. To identify the cell surface TF-expressing glycoprotein(s), HT29 and Caco2 colon cancer cells were surface-labeled with Na[(125)I] and subjected to PNA-agarose affinity purification and electrophoresis. Proteins, approximately 110-180 kDa, present in HT29 but not Caco2 were identified by Western blotting as high molecular weight splice variants of CD44 (CD44v). Selective removal of TF antigen by Streptococcus pneumoniae endo-alpha-N-acetylgalactosaminidase substantially reduced PNA binding to CD44v. Immunoprecipitated CD44v from HT29 cell extracts also expressed sialyl-Tn (sialyl 2-6 N-acetylgalactosaminealpha-). Incubation of PNA 15 microg/ml with HT29 cells caused no additional proliferative effect in the presence of anti-CD44v6 mAb. In colon cancer tissue extracts (N = 3) PNA bound to CD44v but not to standard CD44. These data show that CD44v is a major PNA-binding glycoprotein in colon cancer cells. Because CD44 high molecular weight splice variants are present in colon cancer and inflammatory bowel disease tissue but are absent from normal mucosa, these results may also explain the increased PNA reactivity in colon cancer and inflammatory bowel disease. The coexpression of oncofetal carbohydrate antigens TF and sialyl-Tn on CD44 splice variants provides a link between cancer-associated changes in glycosylation and CD44 splicing, both of which correlate with increased metastatic potential.     

Overexpression and activation of the RON receptor tyrosine kinase in a panel of human colorectal carcinoma cell lines

RON is a receptor tyrosine kinase belonging to the MET proto-oncogene family. The purposes of this study are to determine the expression and activation of RON in a panel of human colon carcinoma cell lines. Western blotting showed that RON is barely detectable in normal and SV-40-transformed colon epithelial cells, but highly expressed and constitutively activated in several colon carcinoma cell lines including Colo201, HT-29, HCT116, and SW837. Moreover, a novel RON variant with a molecular mass of 160 kDa (RONDelta160) was identified from HT-29 cells. The cDNA encoding RONDelta160 has an in-frame deletion of 109 amino acids in the extracellular domain of the RON beta chain, which is caused by splicing out of two exons in the RON mRNA. No mutations were found in the kinase domain of the RON gene in five carcinoma cell lines screened. By expressing RON in colon epithelial cells, we found that RON activation increases cell motile-invasive activities and protects cells against apoptotic death. These data suggest that RON expression and activation are deregulated in colon carcinoma cell lines. By abnormal activation of RON, this receptor and its variant may regulate motile-invasive phenotypes of certain colon carcinoma cells in vivo.     

Effects of alpha, beta and gamma recombinant interferons on CEA production from HT-29 human colon adenocarcinoma cell line

The human colon adenocarcinoma derived cell line HT-29 is a good in vitro model for the study of CEA production and release under various experimental conditions. Many studies indicate that CEA secretion is correlated with cell proliferation and seems to depend on the growth conditions and differentiation characteristics induced by the culture medium. The present study demonstrates that recombinant interferons alpha, beta and gamma (rIFN alpha, rIFN beta, rIFN gamma) can modify CEA production and release by HT-29 cell-line. rIFN gamma in particular causes an enhancement of CEA production and release in the culture medium. This dose-depending effect is in some way correlated to cell growth inhibition since the enhancement of CEA expression in the interferon treated cells is evident in the presence of a reduction in cell proliferation. The activity of rIFN alpha and rIFN beta on CEA release is much less remarkable than that demonstrated by rIFN gamma, and is probably only due to the fact that HT-29 colon adenocarcinoma cells respond poorly to the effects of rIFN alpha and rIFN beta at the doses we used. These findings suggest that CEA production, expression and release can be modulated in a variety of ways under the influence of different rIFN treatment and this situation must be taken into account in immunodiagnostic and immunotherapeutic applications of anti-CEA monoclonal antibodies in the cancer patient.     

Immunochemical characterization of two antigens recognized by new monoclonal antibodies against human colon carcinoma

Two different monoclonal antibodies (MAb), called L-D1 and L-C5, were produced after immunization with either intact cells or the methanol phase of glycolipid extracts, respectively, from the same human colon carcinoma line, LoVo. As determined by an antibody-binding radioimmunoassay (RIA) on intact cells, MAb L-D1 and MAb L-C5 were highly reactive with all five colon carcinoma lines tested and with only one out of the 21 cell lines of various tissue origin tested. No reactivity of either MAb was observed with peripheral blood lymphocytes, granulocytes, or erythrocytes from healthy donors of various blood groups. Both MAb were tested in competitive binding experiments with an anti-CEA MAb from our laboratory (CEA 35) and with two previously described anti-colon carcinoma MAb from the Wistar Institute called 1083-17-1A (17-1A) and NS-19.9. In competitive binding experiments, MAb L-D1 was inhibited by MAb 17-1A and reciprocally, whereas MAb L-C5 was not inhibited by any of the other MAb tested. MAb L-D1 precipitated a major protein band with an apparent molecular weight (MW) of 41 kilodaltons (kD); interestingly, MAb 17-1A, which was reported to react with an uncharacterized antigen, precipitated the same protein band of 41 kD. This was confirmed with immunodepletion experiments. Furthermore, after treatment of the colon carcinoma cell line with tunicamycin, both MAb L-D1 and 17-1A precipitated a protein band of 35 kD. This shift of 6 kD suggests that the glycoprotein recognized by these 2 MAb contains two to three N-linked carbohydrate side chains. MAb L-C5 precipitated a group of three to four protein bands ranging from 43 to 53 kD that were not modified by tunicamycin treatment. A preliminary study conducted by using immunoperoxidase labeling on frozen sections of primary colon carcinoma showed that the two new MAb react strongly with these tumors, but also weakly with the normal adjacent mucosa, as did the other anti-colon carcinoma MAb tested.     

Induction of carcinoembryonic antigen and its crossreactive gene products in human colonic adenomas and carcinomas by transforming growth factor-beta

The inducing effect of transforming growth factor-beta (TGF-B) on carcinoembryonic antigen (CEA) secretion and the cellular expression of CEA and CEA crossreactive glycoproteins (CEA-GLY) was examined from a panel of human colonic cell lines with different phenotypic classification. This panel included carcinomas with dissimilar differentiation characteristics and metastatic behavior, and premalignant adenomas derived from colonic polyps. A great degree of heterogeneity was observed in the endogenous levels of CEA secretion and the cellular expression of CEA and CEA-GLY species. The response profiles of the different cell lines to TGF-B treatment were also found to be heterogenous. However, TGF-B was able to induce CEA secretion and up-modulated the cellular expression of CEA and CEA-GLY from a majority of the cells tested. More importantly, TGF-B was able to exert these effects on carcinoma cells that secrete or express minimal or nondetectable amounts of these glycoproteins. These biologic modifying effects of TGF-B may have potential in augmenting the efficacy of CEA as a colon cancer marker, and in antibody-directed radioimaging and therapeutics. Further investigation in vivo in an experimental animal model system is warranted.     

Colonic epithelium reactive monoclonal antibodies

Summary We have produced a small library of colonic mucosa and colorectal carcinoma reactive monoclonal antibodies (MoAbs) by immunizations with extracts of human colon cancer tissue and a human colon cancer cell line. Hybridoma supernatants were tested on (normal and neoplastic) human tissues by immunoperoxidase methods to evaluate organ or tissue specificity. Initial biochemical characterization of the target antigens was performed by gelpermeation chromatography, Western blotting and competition assays.Based upon the immunoreactivity patterns and the characteristics of the antigen four groups of MoAbs could be distinguished. The first group concerns the antibodies PAR-LAM 3, 9 and 10. These antibodies react with an 87 kDa protein moiety in high molecular weight (2–5×10⁶ Da) glycoproteins. In intestinal and colon mucosa these antibodies showed diffuse binding with goblet cells. In colon carcinoma decreased reactivity with these MoAbs was found.The second group consists of antibodies PARLAM 8, 12 and 13. These antibodies react with large (>5×10⁶ Da) glycoproteins, most likely with carbohydrate epitopes. By immunohistochemistry in normal colon mucosa the antibodies all show granular supranuclear reactivity with goblet cells. These antibodies show increased reactivity with colon adenomas and adenocarcinomas.A third group is formed by PARLAM 2, which also reacts with a large (>5×10⁶ Da) glycoprotein, showing a granular distribution in goblet cells. In colon carcinomas more extensive expression is found than in normal colonic mucosa. Finally, the fourth group consists of PARLAM 11, which also reacts with a large (>5×10⁶ Da) glycoprotein, located in the brush border of colonic columnar cells.These antibodies might be useful tools for the analysis of the expression of mucin related glycoproteins in normal, preneoplastic and neoplastic colon mucosa.Supported by grant RL 82-1 of the Netherlands Cancer Foundation, K.W.F.     

Reactivities of an anti-CEA peptide monoclonal antibody.

Synthetic peptides representing different areas of the CEA molecule were used as immunogens for the development of anti-CEA antibodies. Both polyclonal and monoclonal antibodies were generated using peptides composed of CEA amino acid positions 99-128 and 585-613, respectively. One MAb, designated CP4, generated using the CEA peptide 99-128, was chosen for a more detailed analysis of reactivity. MAb CP4 reacts in solid phase RIAs with CEA peptide 99-128 immunogen and purified native CEA. CP4 did not react with purified non-specific cross reacting antigen (NCA), even though there were two single amino acid differences between NCA and CEA in the 29 amino acid peptide. The affinity constants of CP4 for the CEA peptide 99-128 and native CEA are 4.07 x 10(9) M-1 and 5.75 x 10(8) M-1, respectively. When CP4 was reacted with purified CEA in Western blotting experiments, the Mr 180,000 glycoprotein characteristic of CEA was detected, but CP4 reacted to various size entities in tumor cell extracts. The results of liquid competition RIAs showed that the epitope that MAb CP4 recognized on native CEA is not available for binding when CEA is in solution. Physical (adsorption to a solid matrix) or chemical (deglycosylation or formalin-fixation) alteration of CEA is required for binding of CP4 to CEA. MAb CP4 reacted approximately 1,000-fold greater to deglycosylated CEA than native CEA. Immunohistochemical studies using formalin-fixed paraffin-embedded tissue sections demonstrated that, among carcinomas, CP4 reacts selectively with colorectal carcinomas, while normal colon is negative. Although stomach carcinoma is negative, dysplastic lesions and areas of intestinal metaplasia are reactive. Two of 7 normal stomach tissues showed focal cytoplasmic reactivity of the surface epithelium. CP4, therefore, appears to react with an epitope with highly restricted expression in colorectal carcinoma. These studies demonstrate the complexities in dealing with an anti-peptide MAb with reactivity to an epitope which is accessible only under certain conditions.     

OVX1 and CEA in patients with colon carcinoma, colon polyps and benign colon disorders

The OVX1 tumor marker promises to complement CA125 for detection of early stage ovarian carcinoma. OVX1 has also been shown to be elevated in colon cancer patients. This study is designed to assess serum OVX1 levels in patients with specific stages of colon cancer, colon polyps or other GI disorders. Serum OVX1 and CEA were measured by radioimmunoassay or enzyme immunoassay for 206 patients at the time of colonoscopy or staging for colon carcinoma. In patients with stage I, II, III, or IV colon carcinoma, serum OVX1 was positive in 37%, 48%, 74% and 63%, respectively. Fifty-three percent of patients with colon polyps had elevated OVX1 levels, while OVX1 levels were positive in only 7% of healthy controls. If both OVX1 and CEA were considered, at least one of these markers was elevated in 36%, 60%, 79% or 89% of patients with stage I, II, III or IV colon carcinoma, respectively. The majority of patients with inflammatory bowel disease or diverticulosis also had elevated OVX1 levels. Both markers were positive in 27% of patients with colon carcinoma, and not in any patients with a normal colonoscopy or with a diagnosis of diverticulosis or hemorrhoids. In conclusion, serum OVX1 improves the sensitivity of CEA for detecting colon polyps and colon cancer; however, the use of OVX1 in this setting is hindered by its elevation in non-malignant colonic processes.