Material with carcinoembryonic antigen (CEA)-like achyity, isolated from colon lavages of healthy individuals, hasbeen chemically characterized. The CEA-like activity inthese lavages was purified by gel filtration on Sepharose 6Band Sephadex G-200 and by affinity chromatography onconcanavalin A linked to Sepharose. The purified materialmigrated in polyacrylamide-sodium dodecyl sulfate electrophoresis as a single diffuse band with mobility identical withthat of tumor CEA. The material with CEA activity fromtumor tissue and from normal colon lavages gave lines ofidentity in double diffusion experiments and had similarinhibition curves with essentially the same specific activitiesin a radioimmune assay for tumor CEA. The amino acid andcarbohydrate compositions were similar to that observedfor CEA tumor preparations. Lysine was the single aminoterminus. Methylation analysis demonstrated that the monosaccharide linkages were similar to those in CEA isolatedfrom tumor tissue.
INTRODUCTION
CEA4was demonstrated by Gold and Freedman (16, 17) insaline extracts of digestive tract tumors and embryonic guttissue. Antibody against the tumor extract, after absorptionwith normal tissueextract,retaineditsabilityto precipitatehumor extractsand fetaldigestivetractextractsbut nolonger precipitated extracts of normal tissue. The authorstherefore concluded that CEA was not present in nonmalignanhadulttissue.
Martin and Martin (26) subsequently found that it waspossible to inhibit the precipitation of CEA by anti-CEA withhigh concentrations of extracts of normal tissues. Their
I This research was supported by USPHS Grants CA 19163 and CA 17491
from the National Large Bowel Cancer Project and by Grants CA 16434 andCB 23854, all of the National Cancer Institute.
2 Present address: Department of Microbiology, University of Alabama in
Birmingham, University Station, Birmingham, Ala. 35294.3 To whom requests for reprints should be addressed, at Department of
Microbiology, University of Alabama in Birmingham, University Station, Birmingham, Ala. 35294.
4 The abbreviations used are: CEA. carcinoembryonic antigen; NCA, nor
mal cross-reacting antigen; Con A, concanavalin A; SDS, sodium dodecylsulfate.
Received March 2, 1977; accepted May 6, 1977.
findings raised the question of whether the requirement forlarge amounts of normal material in order to obtain inhibilion was caused by a concentration of CEA present in normal tissue several orders of magnitude lower than that intumor tissue on whether there was a substance in normaltissue that was weakly cross-reactive with CEA. The initialreport by Martin and Martin was followed by other reportsthat demonstrated Ihat extracts of normal tissue inhibited inradioimmune assays (3, 20, 23, 27, 28) and complementfixation assays (36) for CEA from tumor tissue.
However, in none of these laboratories was sufficientpurified material available to analyze structurally the substancefrom normaltissue.Somereportsincludedthe factthat the inhibition curves obtained from radioimmune assays of tumor and normal tissue extracts were parallel (4),although this evidence does not demonstrate chemicalidentity (12).
NCA [also called normal glycoprotein (25), CCEA2 (42),and CEX (7)] is a substance isolated from normal tissue thatcross-reacts with CEA from humor tissue but has a muchlowermolecularweight(43).NCAprobablyisalsoidenticalwith CCAIII (30) and with the fJEantigen of Orjasaeter (31,32). Immunodiffusion experiments have indicated that CEAand NCA have both unique and shared anhigenic determinants. The demonstration of NCA in normal tissue, whichcould be distinguished from humor CEA by its size, appeared for a time to resolve the controversy. However, somegroups subsequently reported the presence of material withCEA-like activity in normal serum and normal tissue extractsthat was similar hoCEA in eluhion from gel filtration columns(4, 21, 35). Unfortunately, the concentration of this substancein normaltissuewasso low that sufficientmaterialcould not be obtained for chemical characterization.
In 1974 Go et a!. (15) measured the secretion in variousportionsof the gastrointestinaltract of materialthat inhibihed in the assay for CEA. The colon secreted the largestquantity of the material. The inhibitory material eluled froma Sephadex G-200 column in the same position as CEA fromtumor tissue and well ahead of the usual elution position ofNCA. The presence of large quantities of material with CEAlike activity in the colon lavages of healthy individuals madeit possible ho isolate sufficient material to be characterizedchemically and directly compared with CEA. The isolationandcharacterizationof this materialis the subjectof thisreport.
CANCER RESEARCH VOL. 372638
Isolation and Immunochemical and Chemical Characterization ofCarcinoembryonic Antigen-like Substances in Colon Lavages ofHealthy Individuals1
Marianne L. 23 David G. Pritchard,2 Charles W. Todd, and Vay Liang W. Go
Divisionof Immunology,Cityof HopeNationalMedicalCenter,Duarte,California91010(M. L. E., D. G.P., C. W.TI, andGastroenterologyUnit,MayoClinicand Mayo Foundation, Rochester, Minnesota 55901 (V. L. G.J
CEA and AntI-CEA.CEAwas obtainedfrom livermetastases of colon adenocancinomas by treatment of a waterhomogenate of liver tissue with an equal volume of 2 Mperchlonicacid to precipitatethe proteins,followedbychromatography of the dialyzed supemnalant on Sephanose4B and Sephadex G-200 (5). A final purification on Con A-linked Sephanose was performed (33). The anhi-CEA serumused was obtained from a goat (Ace) producing antibodies 4years after an initial series of injections of 10 @gof CEA for 3months, followed by booster shots of 1 ho5 @gat 4, 11, 16,19, and 22 months (11). The bleedings used in the studywere taken on the 3rd (Ace 5) and 43rd (Ace 67 ho 70)months after initial immunization. These antisera had thesame cancer specificity when tested with clinical samplesas did other antisera available for clinical testing (W. L. Go,unpublished observations; Ref. 24).
Radioimmune Assay for CEA. CEA was measured by thetriple-isotope, double-antibody nadioimmune assay for CEA(8, 13). 57Cowas usedin placeof 22Naasa volumemarker(14).
Lavageof the Colonof HealthyIndividuals.The colonicsaline lavage was obtained from 14 healthy subjects (age 30to 56 years, 1 female and 13 males) whose sera CEA assayvalues were less than 2.5 ng/mI. Appropriate informed consent was obtained for all volunteers prior to study. Colonicsaline lavage was collected either by saline enema in 6subjects or by colonic perfusion technique (15) in 8 subjects. After the colon had been cleansed with 0.9% NaCIsolution, an additional 1000 to 1200 ml were obtained fromeach individual for the study. Care was taken to keep thesamples cold at all limes. Individual samples were centnifuged for 15 mm in a Sorvall GLC-2B centrifuge ho removeparticulate matter, filtered through a Millipore cellulosepad, and concentrated in an Amicon DC-2 concentratordialyzento200 ml.
Isolation of Material with CEA-Ilke Antigenic Determinants from Colon Lavages. The centrifuged and concentrahed lavage was treated at 4°with an equal volume of 2 Mperchlonic acid, centrifuged to remove the precipitated prohems, dialyzed against running deionized water, furtherdialyzed and concentrated using an Amicon PM 10 membrane (Amicon, Lexington, Mass.), and lyophilized. Thelyophilized samples from the 14 individuals were pooled. Atotal of 2.4 g of perchlonic acid extract was obtained. Thepool was chromatognaphed on a Sepharose 6B column(Phanmacia Fine Chemicals, Inc., Piscahaway, N. J.) thathad been equilibrated wihh 0.05 M phosphate buffer, pH 5.5,in 0.1 M NaCI. Fractions from the Sepharose 6B columncontaining the CEA activity as measured by radioimmuneassay were pooled, dialyzed, lyophilized, and applied hoSephadex G-200 columns equilibrated with the same phosphale buffer as above. Fractions containing CEA antigenicactivity were pooled, lyophilized, and chromahographed onCon A-Sephanose (Phanmacia).
Chromatographyon Con A-Sepharose.The samplewasdissolved in Con A buffer (1 M NaCI/0.1 M acetate, pH 5.5;and 10 mM concentrations each of MgCI2, MnCl2, and CaCl2)and applied to a column of Con A-Sepharose equilibrated at
40 with the same buffer. After elution of the nonbound
fraction with Con A buffer, the column temperature wasraised to 37°and the bound fraction was eluled with 20% a-methyl-D-mannoside (Grade Ill; Sigma Chemical Co. , SI.Louis, Mo.) in Con A buffer. This was dialyzed against 6additional changes of distilled waler, after no free a-methylD-mannosidehad been detected, and lyophilized.
SDS-PolyacrylamideElectrophoresis. Electrophoresiswas performed in 6% polyacrylamide gels containing 0.1%SDS in 0.1 M Tnis-glycine buffer, pH 8.1. Samples weresubjected to electrophoresis for 1.5 hr at 8 ma/gel. Gelswere stained with 0.5% Coomassie blue in 25% isopropylalcohol and 10% acetic acid and destained with 10% aceticacid.Duplicategelswere stainedforcarbohydrateby amodification (29) of the periodic acid-Schiff method of Segrest and Jackson (38). For determination of the position ofCEA activity, an unstained gel was cut into slices approximalely 1 mm thick. Each slice was shaken overnight in 0.5ml of a solution of 1 mg gelatin per ml in 0.075 M NaCIbuffered with 0.075 M sodium phosphate to pH 7.2. Analiquol of the supemnatant was tested in the madioimmuneassayforCEA.
Amino Terminal Analysis. Amino terminals were idenhified by a modified dansylation procedure of Hartley (19). Tothe sample (200 @g)dissolved in 50 @lof water were added50 @lof 0.2 M NaHCO3and 50 .d of 0.36% dansyl chloride inacetone (w/v). The sample was incubated for 1 hr at 37°,extracted 3 limes with loluene, dried under N2, and extraded with 300 @lof acetone/i N HCI (v/v). The dansylahedprotein was dissolved in 0.01 M acetic acid and applied ho aDowex 50, H@form column equilibrated with 0.01 N aceticacid. The column was washed with 5 to 10 ml of 0.1 N aceticacid, and the sample eluled with H20/acehone/concenInated NH4OH, 80/20/4, v/v/v. After evaporation ho drynessthe sample was hydrolyzed in 6 N HCI for 12 hr at 110°.TheHCI was evaporated off under N2, and the sample was dissolved in 100 @lof ethyl acetate saturated with water. Theethyl acetate extract was dried under N2and dissolved in 10
Amino Acid and Amino Sugar Composition.Sampleswere hydrolyzedundervacuum for24and 48 hrin0.5ml 3 Np-loluenesulfonicacid containing 0.2% 3-(2-aminoehhyl)indole (22). The amino sugars and basic amino acidswere eluhedfrom a PA35 (Beckman Instruments, Inc., Fullerton, Calif.) column using sodium citrate buffers (0.4 Nsodium citrate), pH 5.27 and 5.87. The acidic and neutralamino acids were eluled from an AA15 resin (Beckman) withsodium citrate buffers (0.2 N sodium citrate), pH 3.27 and4.55.
CarbohydrateAnalysis.Neutralsugarswereanalyzedbygas chromatography of the Inimethylsilyl derivatives of themelhyl glycosides according ho the procedure of Pritchardand Todd (34).
MethylatlonAnalysis. Sampleswere methylatedby themethod of Hakomori (18), in which the dimelhylsulfinyl anion was used ho generate the polysacchanide alkoxide before the addition of methyl iodide. The dimethylsulfinyl anion was prepared as described by Sandford and Conrad(37).
Aceto!ysis, hydrolysis, reduction, and acetylation of thepermethylated polysacchanides were performed using theprocedures described by SlelInemet a!. (39).
Identification of the partially methylaled aldilol acetaheswas carried out according to the method of Bjomndalet a!.(1) for the neutral sugar derivatives and according to themethod of Shellnen et a!. (39) for the amino sugar denivalives. A Vanian Model 2740 gas chromahograph equippedwith a flame ionization detector connected to a DupontModel 21-402B double-focusing mass spectrometer wasused for these analyses. A 6-foot column (2 mm insidediameter) of 3% ECNSS-M coated on Gas Chrom 0 (AppliedScience Laboratories, Inc. , Slate College, Pa.) was used hoseparate the aldilol acehates derived frOm the mehhylaledsugars. Isothermal nuns were made at 160°for the neutralsugars and at 180°for the amino sugars. Mass spectra of thecarbohydrate derivatives were taken over a mass mangeof35 to 600 atomic mass units using an ionizing potential of 70eV. Scans were taken every 8.47 sec, and the spectra wereprocessed using a Dupont 2i-094B disc-based data system.
MucopolysaccharideAnalysis.Electrophonesisof sampIes was carried out on a Beckman Model R-lOi MicrozoneCell as described previously (33). Mucopolysacchanideswere stained with 1% Alcian blue (Eastman Kodak Co.,Rochester, N. Y.) in 5% acetic acid.
RESULTS
Purification. After treatment with penchlonicacid to precipitatetheprotein,thesampleswere appliedhoa columnof Sephanose 6B (Chart 1). The material with CEA activity asmeasured by nadioimmune assay eluled from 42 to 62%column volume, in a pattern identical with that obtainedwith tumor extracts. Since the activity in the fractions eluting after 55% column volume represented a very small percentage of the lohal CEA-like activity, these fractions werenot further purified. The fractions from 45 to 55% of columnvolume were pooled for further purification on a SephadexG-200 column. The pattern was identical with that obtainedfrom tumor preparations (Chart 2).
Material from the G-200 column that eluted from 34 ho45% column volume was pooled, dialyzed, lyophilized, andapplied to a column of Con A linked to Sephamose. It haspreviously been demonstrated in preparations of tumor hissue that contaminating mucopolysacchanides and substances with blood group activity will not adsorb to Con Awhereas most of the material with CEA activity will adsorb.This activity can be eluted with 20% a-melhyl-D-mannopynanoside after the column temperature has been raised to 37°(33). Similar results were obtained with normal colon washings (Chart 3). The material that did not bind to Con Acontained mucopolysacchamides and a substance that inhibiled the agglutination of human 0 cells by H substance.The unbound fractions also contained a small amount ofCEA-like activity, but 1000-fold less than that which wasbound to Con A. The peak Ihat did not bind to Con A wasnot due to overloading of the column since incubation witha fresh batch of Con A did not absorb the activity.
Almost 100% of the material that inhibited in a nadioimmune assay for CEA was found in the Con A-adsorbed
% Column Volume
C)CD
0:3
Chart I . A perchloric acid extract of normal colon washing containing 2.4g of lyophilized material was dissolved in 33 ml of 0.05 N phosphate buffer,pH 5.5, in 0.1 N NaCI. The sample was divided into 3 portions which werechromatographed separately on a column of Sepharose 6B (161 x 2.5 cm)equilibrated with the same buffer. Identical patterns were obtained for all 3portions.
Sephadex G-200
A
I1A
)@‘A@4@ £ £
I
I
EN
UqLUCi
1.4
1.2
0
1.0 oC,(b
0.80
0.6@
0.4
0.2
1400
1200
1000
800
600
400
200
20% 40% 60% 80%
0/0 Column Volume
Chart 2. Pooled fractions from the Sepharose 6B column that containedCEA activity were dialyzed, Iyophilizad, and applied to a Sephadex G-200column (95 x 2.5 cm) equilibrated with 0.05 M phosphate buffer, pH 5.5, In0.1 M NaCI.
fraction. As is usually observed with CEA from tumor tissue,the CEA activity eluted as a sharp peak with a slight shoulden on the descending side and slightly ahead of the peak ofabsorbance at 220 and 280 nm. The material in Fractions148 to 160 was pooled and characterized chemically andimmunochemically.
Acrylamide Electrophoresis.The Con A-purifiedfraclions were analyzed by electrophonesis in 6% acnylamide/0.1% SOS. A diffuse, single band, 0.5 to 2 cm from theorigin, was obtained when the gels were stained with Coomassie blue or periodic acid-Schiff slain. The mobility of theband was identical with a standard of CEA prepared fromtumor tissue. The CEA activity measured by radioimmune
assay was located in the position in the gel that stained withCoomassie blue and periodic acid-Schiff stain.
Amino Acid and Amino Terminal Analysis. The aminoacid compositions of material with CEA-like activity fromtumor tissue and normal colon washings are very similar(Table 1). Aspartic acid (including asparagine)washhe mostabundant amino acid in both tumor and normal preparalions. Relatively high levels of senine, glulamic acid, proline, and threonine were found in material from bothsources. Lysine was found to be the single NH2-herminalamino acid in the material from normal colon washings.
Carbohydrate Compositionand MethylatlonAnalysis.Carbohydrate compositions of CEA samples isolated fromdifferent tumors vary slightly. The material isolated fromnormal colon washings has a carbohydrate compositionvery similar to that of CEA preparations (Table 2). All of the
carbohydrate structural units found in CEA were also foundin the material isolated from normal colon washings. Therelative amounts of the various linkages were very similar tothose observed in CEA (Table 2).
The relatively lower amount of total galactose in colonwashings may be related to the relatively higher percentageof terminal N-acehylglucosamine since previous studieshave suggested that galachose is linked to N-acetylglucosamine (6, 10).
ImmunochemicalCharacterization.The material fromnormal colon washings and tumor CEA gave lines of identityin double diffusion analysis against goat anti-CEA (Ace 5and Ace 67 ho 70). When tested in the radioimmune assaywith 125I-labeled CEA from tumor tissue, the sample fromnormal colon washings gave an inhibition curve that wassimilar to, although not absolutely identical with, the inhibilion curve wilh tumor CEA (Chart 4).
Table1
a Average values of CEA preparations from 3 different tumors.
DISCUSSION
The results reported here strongly support the view thatCEA is not restricted to fetal and neoplastic tissue but isalso present in healthy tissue. All volunteers who participaled in this study remain apparently healthy to date. It ispossible that the presence of a small quantity of CEA in thenormal adult is analogous to the small quantity of fetalhemoglobin that continues to be synthesized in the adult (2,45, 46).
The CEA-like material obtained from these normal volunteens had the same NH2-henminalamino acid residue as did
17 CEAfrom tumor tissue and a very similar amino acid composition (9, 41). The carbohydrate composition and the
15 linkage positions of the carbohydrate residues were also
very similar to those found in tumor CEA (6, 41). The elec
13 hrophoretic mobility of the material in SDS-polyacrylamide
@ eleclnophonesis was identical with that of tumor CEA. Fur11@ thenmore, the sequence of the 1st 24 amino-terminal nesi
@ dues was identical with that reported for tumor CEA prepa9@ rations (40).@
@1 Immunological results are consistent with the structural
7 @- identity of the substances from normal and humor tissue
I sincealineofidentitywasobtainedwiththe2substancesin5 double diffusion analysis. Although minor differences were
observed by radioimmune assay, the variation in the inhibi3 lion curve was no greater than has been observed by us (M.
L. Egan, unpublished observations) and others (44) for dif1 fenent preparations of CEA from tumor tissue on serum
samples.The collective chemical and immunological evidence ob
tamed in this study strongly suggests that the material isolaled from the colon of healthy individuals is very similar to,and perhaps identical with, the CEA of humor tissue.
S J, E. Shively, V. L. W. Go, C. W. Todd, and M. L. Egan. Amino Terminal
Sequence of a CEA-like Substance in the Colonic Lavages of Healthy Individuals, submitted for publication.
Chart 3. Material with CEA activity that had been purified by gel chromatography was applied to a water-jacketed column containing 100 ml ofCon A linked to Sepharose equilibrated with 1 M NaCI/0.1 N acetate, pH 5.5;and 10 mM concentrations each of MgCI2, MnCI2, and CaCI, (Con A buffer).The column was maintained at 4@until the nonabsorbad fractions eluted. Thecolumn temperature was then raised to 37@and the aluent was changed to20% a-methvl-D-mannoovranoside in Con A buffer.
a Subscripts 1 to 4 indicate CEA purified from different tumors. All tumors were liver
metastases that had originated from the colon.bNCW,normal colon washings; NA, not analyzed.
100
80
60
40
20
globin Restriction to a Few Erythrocytes (F cells) in Normal HumanAdults. Science, 188: 361-363, 1975.
3. Burtln, P., Martin, E., Sabine, M. C., and von Kleist, S. ImmunologIcalStudy of Polyps of the Colon. J. NatI. Cancer Inst., 48: 25—32,1972.
4. Chu, T. M., Raynoso, G., and Hansen, H. J. Demonstration of Cardnoembryonic Antigen in Normal Human Plasma. Nature, 238: 152-153,1972.
5. Coligan, J. E., Lautanschlegar, J. T., Egan, M. L., and Todd, C. W.Isolation and Characterization of Carcinoembryonic Antigen. Immunochemistry, 9: 377-386, 1972.
6. Coligan, J. E., Pritchard, D. G., Schnute, W. C., Jr., and Todd, C. W.Methylation Analysis of the Carbohydrate Portion of CarcinoembryonlcAntigen. Cancer Res., 36: 1915-1917, 1976.
7. Darcy, D. A., Tuberville, C., and James, R. Immunological Study ofCarcinoembryonic Antigen (CEA) and a Related Glycoprotein. BrIt. J.Cancer, 28: 147-160, 1973.
8. Egan, M. Carcinoembryonic Antigen. In: R. Nakamura (ad.), Immunepathology: Clinical Laboratory Concepts and Methods, pp. 564—580.Boston: Little, Brown and Co., 1974.
9. Egan, M. L., Coligan, J. E., Morris, J. E., Schnuta, W. C., Jr., and Todd,C. W. Antigenic Determinants on Carcinoembryonic Antigen: Chemicaland Immunological Studies. In: P. Bucalossi, V. Varonesi, and N. Cascinelli (eds.), Proceedings of the Eleventh International Cancer Congress,Florence, Italy, October 20 to 26, 1974, Vol. 1, pp. 244-2*8. Amsterdam:Excerpta Medics, 1975.
10.Egan,M. L.,Coligan,J.E.,Pritchard,D. G.,Schnute,W. C.,Jr.,andTodd, C. W. Physical Characterization and Structural Studies of theCarcinoembryonic Antigen. Cancer Res., 36: 3482-3485, 1976.
11. Egan,M. L., Collgan,J. E., and Todd, C. W. RadioimmuneAssayfortheDiagnosis of Human Cancer. Cancer, 34: 1504-1509, 1974.
12. Egan, M. L., Engvall, E., Ruoslahti, E. I., and Todd, C. W. Detection ofCirculating Tumor Antigens. Cancer, in press.
13. Egan, M. L., Lautenschlager, J. T., Coligan, J. E., and Todd, C. W.Radloimmune Assay of Carcinoembryonic Antigen. Immunochemistry,9: 289-299, 1972.
14. Egan, M. L., Todd, C. W.. and Knight, W. 5. “Ce:A Volume Marker forthe Triple Isotope Double Antibody Radioimmune Assay. Immunochemistry, in press.
15. Go, V. L. W., Ammon, H. V., Holtermuller, K. H., Krag, E., and Phillips, S.F. Quantification of Carcinoembryonic Antigen-like Activities in NormalHuman Gastrointestinal Secretions. Cancer, 36: 2346-2350, 1975.
16. Gold, P., and Freedman, S. 0. Demonstration of Tumor-SpecifIc Anti
C0
.0-CC
ng SampleAddedChart 4. Radioimmune assay inhibition curves obtained with “@I-IabeIed
CEAfrom tumor tissue,1:4000dilution of Ace(67to 70)goat anti-CEA,andCon A-purified CEA from tumor tissue (C) or from normal colon washings(0).
ACKNOWLEDGMENTS
We thank Ronald Card and Nancy Buker for their excellent tachnicalassistance. We thank William S. Schnute, Jr. , for determining the massspectra, and David Bills, for amino acid analysis.
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