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[C ANCER R ES EARCH 33, 313-322, F ebruary 1973]Carcinoembryonic Antigen and B lood Group Substances1D . A lastair R. Simmons2 and Peter Perlm annDep ar tm en t o f Immun ol og y, Wen ne r-Gr en I ns ti tu te , S to ck ho lm , Swe de n

SUMMARYImmunochemical studies support the view that carcino-em bry onic an tig en s from en to derm ally derived ad en oca rc ino -mata are incomplete blood group substances of the ABOsystem . T he d eficien t an tig en s h av e a tumo r-sp ecific a ctiv ity ,due to unmasking of a structural sequence that is cryptic inthe norm al situation. Two other serological activities of thetum or antigens are determ ined by ./V -acetylgalactosam inylpeptide and antigenic C om plex I sequences identical to thosefound in blood group substances. T he structural results im plythat the genetic changes in oncogenesis include deletion orrep ressio n o f the g en e(s) co ntro llin g th e tra nsferase(s) for th ete rm in al se qu en ce s o f c la ssic al ABO -a ctiv e g ly co pro te in s. T hedevelopm ent of specific s rodiagnostic tests for tum ors ofentoderm al origin will probably depend on the production ofsera w ith high activity for the tum or-specific determ inant andlittle or no activity for the norm al blood group sequences alsofo un d in c arc in oemb ry on ic a ntig en .

INTRODUCTIONStudies (26, 37) of antigenic variation in bacteria haveshown that the biosynthesis of cell-surface antigens iscontrolled by a coordinated sequence of regulating enzym esthat are coded in the nucleus of the cell. M oreover, the geneticchanges that follow spontaneous mutation, bacterial

conjugation, and lysogenic conversion m ay delete, repress, orotherw ise m odify the loci controlling the enzym es for norm alantigen synthesis so that the resulting new antigens arestructurally related to but serologically distinct from those ofth e p arent cell. In th ese circumstanc es, comp ara tiv e stru ctu ralanalyses of the parent and "mutant" antigens have not onlydefined the molecular basis of the change in antigenicspecificity but have also elucidated enzymic and geneticaspects of such antigenic variations. In this study we haveassumed that the genetic loci controlling normal humanantigen synthesis m ay be sim ilarly disturbed w hen the hum angenome is disrupted by carcinogenesis and that structuralan aly sis o f th e n ew tumo r an tig en s w ou ld clarify fu nd am en talaspects of the oncogenic event, provided that the normalantigen system had been carefully characterized forcomparison. Because blood group substances are human

1 Su pp ort ed b y a We ll com e/ Sw ed is h T ra ve lin g R es ea rc h F el lows hipf rom t he WeUc omeT r us t (Gr ea t B rit ai n) a nd b y G ra nt B 72 -1 6X -1 59 -0 8f rom the Swed is h Med ic al Re se ar ch Coun ci l.'P resent address: D epartm ent of B acteriology and Immunology,Un iv er si ty o f G la sg ow , West er n I nf irmary, G l as gow W. I .. Sc ot la nd .Re ce iv ed J ul y 1 7, 1 972ac ce pt ed Oc to be r 1 8, 1 972.

antigens for which the structural requirem ents of antigenicspecificity have been adequately defined (1, 23, 31, 43) andsince the loss of blood group specificity has been observed in anum ber of tum or types (3,4,18,28), we have investigated thestructural changes that occur in A BO substances followingoncogenesis to define the relationship of such modifiedsu bstan ces to tumor-sp ecific an tig en s. E arly in th ese stu dies w efound that our extracts from hum an gastrointestinal tum orscontained a "deficient" blood group substance that resem bledthe w ater-soluble, tum or-specific antigen first described byKorosteleva (20) and subsequently term ed CEA3 by Gold andF reedm an (11). T hus the original aim of this investigation w asrestricted more specifically to defining the structuralrelationship of CEA to blood group substances and toelucidating the general nature of the enzymic and geneticchanges involved in the production of this tum or antigen fromb lood g roup s ub sta nc es .M ATERIALS AND METHODSThere are several m ajor difficulties in studying m odifiedblo od g ro up su bsta nce s from p rim ary ad en ocarcin om ata o f th egastrointestinal tract. M ost im portantly, prim ary grow thscontain m ixed populations of norm al and tum or cells so thatextracts are necessarily mixtures of normal and tumorsubstances including com pletely synthesized blood groupglycoproteins that mask the presence of incomplete forms.

Furtherm ore, extracts of tum ors from the lower intestine arealso unsuitable for immunochem ical studies because they areh eav ily co ntam in ated w ith b acteria l an tig en s d eriv ed from theadherent slim e of organism s present on the m ucosal surfacee ve n a fte r c are fu l w as hin g. T he se p ro blems w ere c irc umve nte dby extracting some of the required antigens from largesecondary masses in a tissue that does not synthesizeABO -sp ecific su bstanc es. T he sp ecificity of such e xtrac ts w ascontrolled by extracting primary tumors to compare thebehavior of the primary and secondary antigens byquan ti ta ti ve serol og ical me thods .Source of Norm al Tissues and Tum ors. The norm al tissuesand tumors used in this study fell into 3 groups. Group 1co ntain ed th e fo llo wing 6 ad en ocarcin om ata th at o rig in ated in"blood group-producing" tissues of entodermal origin,namely: secondary hepatic masses (T2) from a primary ofpancreas; secondary hepatic masses (T8) from a primary ofcolon; a primary of ileocecal junction (Til); a primary ofstom ach (T14); secondary peritoneal m asses (T15) from theprimary of stomach (T14); and secondary masses in the

'T he abbreviations used are: C EA , carcinoem bryonic antigen;ELISA , e nzyme -l in ke d immuno so rb en t a ss ay .

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D . A lasta ir R . S immo ns a nd P eter P erlm an nbladder wall (T18) from a primary of colon. These 6 tumorsw ere the source of the specific antigens used in this study, andall were obtained at autopsy from patients of Blood G roup A.Group 2 contained 6 papillary carcinornata of the humanurinary bladder, w hich were included to determ ine whetherthe specific antigen from gastrointestinal tumors could bedetected in a different tumor type. These specimens wereobtained at operation from patients of different blood groups.Group 3 comprised 14 normal tissues, namely, 3 liverspecimens, 2 pancreas samples, 8 colon specimens, and 1stomach sample. These normal control specimens were allobtained at autopsy from patients of Blood G roup A.Extraction and Fractionation of Norm al Tissue and Tum orG ly co pro teins. In th e early stag es o f this in vestigatio n, some o fthe adenocarcinom a and norm al tissue antigens (G roups 1 and3) were extracted by the phenol method of M organ (32) withsubsequent purification by gel filtration on Sephadex G -200;these methods have been widely used in the preparation ofhuman ABO and related substances (9). When the 1stad eno carcin om a ex tracts w ere fou nd to co ntain a glyco pro teinfraction that resem bled C EA in its gel filtration properties andqualitative com position, the tissues w ere then extracted w ithperchloric acid as described by W inzler et al. (46), so that themethods used would be directly comparable with those usedby G old and Freedm an (10). H ow ever, the extracts continuedto be purified on Sephadex G-200, because this simpleprocedure gave good separation of the components. Thecolum n bed (110 x 5 cm ) w as eluted w ith phosphate-bufferedsaline containing 0.02% (w/v) sodium azide at a flow rate of25 ml/hr. Since fractions were collected every 20 min, thevolume of eluate per tube was approximately 8.3 ml. Thecolum n w as calibrated by running a series of m arker dextransof known molecular weight. In a few instances furtherp urific atio n w as a ttemp te d b y p re pa ra tiv e u ltra ce ntrifu ga tio naccording to the method of Kauffmann et al. (17). W ith the 6malignant papillomata of the bladder (Group 2 tissues),extraction was by the phenol:w ater m ethod of W estphal et al.(45) and/or w ith perchloric acid (46); but the yields obtainedwith these prim ary tum ors were usually insufficient to allowp urific atio n b y g el filtra tio n o r u ltra ce ntrifu ga tio n. A ll e lu ate sa nd e xtra cts w ere sto re d fre ez e-d rie d.H ydrolysis C onditions for A nalytical Studies. Sam ples foranalysis were hydrolyzed in sealed tubes in conditions thatwere appropriate to the constituent under test as follows:jV -acetylneuram inic acid (sialic acid), 80Qor 1 hr in 0.1 NHjSO4; hexoses and deoxyhexoses, 100 for 4 hr in l NH2SO4; hexosamines, 100 for 2 hr in 2 N HCU partialhydrolysis for the rem oval of acid-labile fucose, 100 for 20m in in 0 .1 N HC1 . A fter h ydro ly sis, sam ple s co ntain in g H 2S 04w ere neutralized with Am berlite IR-410 (HCO3~), whereasthose w ith HC1 w ere rapidly dried in a vacuum to remove theacid.Quantitative Analysis of Extracts. The constituents of theextracted antigens were first identified by descending paperchrom atography w ith the use of butan-l-ol:acetic acid:w ater(4:1:5, by volum e) (33) and ethyl acetate:acetic acid:w ater(3 :1 :3 , b y v olume) (1 6) a s so lv en ts . N eu tr al s ug ar c on stitu en tswere identified with silver nitrate reagent (40), and aminosugars and amino acids were identified w ith ninhydrin (2).

Q uantitative m icroanalysis of the sugar constituents w as thencarried out in tubes (7.5 x 1.0 cm ) w ith C arlsberg constrictionmicropipets (H. E . Pedersen, Copenhagen, Denmark) asfollow s: 7V -acetylneuram inic acid by the thiobarbituric acidmethod of Warren (42); glucose by the glucose oxidasem ethod (21); galactose by the galactose oxidase m ethod, w ithGalactostat reagents (W orthington Biochemical Corp.,Freehold, N.J.); fucose by the H2SO4 :cysteine method ofD isch e an d S hettles (5 ); total h exo sam in e by th e Morg an-E lso nreaction after jV -acetylation according to the method ofStrominger et al. (38); free glucosamine by the specificD-glucosam ine 6-phosphate W -acetylase m ethod (25); freegalactosamine by the method of Ludowieg and Benmaman(2 7); a nd TV -a ce ty lh ex os am in es b y th e mod ifie d Mo rg an -E lso nreaction of Reissig et al. (35). From the quantitative enzym eanalyses, all the sugars were present in the D form except forthe fucose, which is presumed to be in the L form. In theresults recorded below, each figure is the mean of at least 8determinations made within the following weight ranges:jV -acetylneuram inic acid, 5 to 30 nm oles; am inohexoses, 5 to50 nmoles; all other sugars, 10 to 100 nmoles. All readingswere m ade in a Hitachi M odel 124 spectrophotom eter fittedw ith s ta nd ar d m ic ro an aly tic al a cc es so rie s.Periodate Oxidation and Analysis of Periodate-treatedA ntigens. T he periodate reactions w ere set up at 0 by adding

80 n\ of 0.025 M NaIO4 to 0.25 mg of the test substance in2 50 J .f H2O. O xidation was allowed to proceed at 04nthe dark for 1 week. A t frequent intervals throughout thisperiod, the residual periodate was measured spectro-photometrically at 225 nm as described "by Rammlerand Rabinowitz (34). The extent of overoxidation and theam ount of periodate consum ed w ere determ ined graphicallyby the standard procedure of extrapolating the terminalportion of the periodate consum ption curve to zero tim e.The periodate-treated antigens were also recovered foranalysis of their sugar com ponents, as such studies often yieldva lu able stru ctu ral in fo rm atio n b y in dicatin g w hich resid ues, ifany, are protected from oxidation. T hese experim ents w ere setup as described above but with all the reactants scaled up8 -fo ld to p ro vid e su ffic ie nt o xid iz ed m ate ria l fo r re pu rific atio nand further study. In most cases the reaction was allowed torun for 18 to 24 hr by which time the primary reaction wasalmost complete and overoxidation was still m inimal.Oxidation was then stopped by adding 150 n\ of 0.01 M leadacetate to the reaction mixture. After 5 min at roomtem pe ra ture, the precipitate w as sep ara ted b y cen trifug ation a t2000 X g for 5 min, and a further 30 ilf 0.01 M lead acetatewere added to ensure that the precipitation of IO3" and IO4~was complete. After centrifugation at 2000 X g for 5 min, theclear neutral supernatant was dried in a vacuum . The productwas hydrolyzed, and its sugar content was analyzed asdesc ri bed above .Antisera. These were donated as follows: the eel serum todetect Blood G roup H specificity and the rabbit (R32) serumfor Forssm an activity, from Dr S. Ham marstrm ; the rabbitanti-L ea serum , w hich had been absorbed w ith ficin-treated OLea (a" b+) cells, and the rabbit anti-pneum ococcus type X IVserum, which was prepared in the laboratories of Dr. J. H.Hum phrey, both from Professor W . M . W atkins; the antigen

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CEA a nd B lo od G ro up S ubsta ncesC om plex I (M a) serum , w hich has been described in detail (8),from Dr. T . Feizi anH Dr. E . A . Kabat; and rabbit anti-CEAsera from D r H . 3G lcNAc (lacto -W -biose 1 )a nd 0Gal- (l - > 4 )- j3 -G lcNAc< 1 -6 )-3 -h ex enet etr ol( s) , wh ic h issp ecific in hib ito r o f th e an tig en ic C om plex I (M a) sero lo gicalsystem, from Dr. E . A. Kabat; j3G lcNAcl -* 30-Gall -2e ry th rito l, from D r. A . G au he.ELISA Studies. The assay procedure has been described indetail by Engvall et al. (6). The principle of the test may besum marized as follows. The antigen under study is coated onpolystyrene tubes and allowed to react w ith a specificantiserum prepared in the rabbit. After careful washing toremove free antiserum, the bound serum is treated withanti-rabbit IgG conjugated w ith alkaline phosphatase. A fterfu rth er c are fu l w as hin g to remove a ll e xc ess re ag en ts, b uffe re dp-nitrophenyl phosphate is added to measure the boundphosphatase and hence, indirectly, the extent of the prim aryantigen-antibody reaction. The tests are read spectro-photometrically at 400 nm after 30 min at roomtem perature or, alternatively, after an appropriate am ount of

color has developed. The test was used to assay a number ofd iffe re nt a ntig en -a ntib od y sy stems a s d es crib ed b elow.Serological Characterization of Tumor Extracts. Thes ero lo gic al p ro pe rtie s o f th e tumo r e xtra cts w ere c ha ra cte riz edin 2 series of experiments. In the 1st experiment, the tumorantigens were tested for blood group activity with the use ofhemagg lu tinatio n-in hib ition , prec ipitatio n, p re cip itation -in hib itio n, a nd ELISA te ch niq ue s a s fo llows .The A, B, and H specificities of the tumor fractions wereteste d b y in hib itin g A , B , an d H h om olog ou s h em ag glu tin ationsystems with 1 to 1000 ng of tumor substance per testacco rding to th e m etho d o f H ammarstr m et al. (1 5).L ea activity w as m easured by the degree of cross-reactionbetween the tumor antigens and Lea antiserum and alsobetween Lea substances and CEA antiserum with the ELISAmethod of Engvall et al. (6). In these tests the antigen wastitrated over the range of 0.25 to 16.0 /Jg against a 1:1000dilution of the appropriate antiserum . T he conjugates K 60 andK61 w ere used at dilutions of 1:300 and 1:500, respectively.Factor XIV and antigenic Complex I (Ma) activity weresought in the tumor extracts by testing them againstpneum ococcus type X IV and I (M a) antisera, respectively, bythe q uan titative p recip itatio n m eth od o f S ch iffm an et al. (36 ).T he results obtained in the Factor X IV system w ere confirm edby testing the 3 Factor XIV-active blood group precursors(Flinton and samples 484 and 502) against CEA antiserum inthe ELISA test (6). The results obtained in the antigenicCom plex I system were confirm ed by testing the inhibitoryeffect of 1 to 250 /ug o f CEA per test in a human O cell:anti-I(Ma) h emagg lu tin ati on s ys tem .Forssm an activity w as tested by m easuring the inhibitoryeffect of 4 to 1000 /jg of tumor antigen per test in theForssm an (sheep red cell:rabbit serum ) hem agglutinationsystem.In the 2nd series of experiments, an attempt was made toelucidate the structural sequences of the antigenicd ete rm in an ts o f CEA by in hib itin g h omolo go us CEA :a nti-CEAand heterologous CE A:anti-blood group system s w ith bloodgroup precursors and oligosaccharides of known m olecularstructure. These experim ents w ere m ade with the ELISA (6)a nd quanti ta tiv e p re cip ita tio n ( 36 ) te chnique s.

RESULTSFractionation of Extracts. The tumor and normal tissueextracts gave 4 main fractions (Chart 1) when purified onSephadex G -200 w ith continuous m onitoring of the eluate forprotein, nucleic acid, and sugar. These fractions weredesignated I, II, III, and IV in order of decreasing m olecular

weight.Fraction I (Tubes 60 to 80; 500 to 670-ml pool; M .W . >200,000) w as the exclusion peak. In the 3 norm al livers (C hartla) it consisted entirely of an a-(l ->) -l inked po lyg lucosanind istin guish ab le from live r glyco gen . N o b lo od g ro up or othe rfucose-containing polym ers could be detected chem ically orserologically. In the rem aining norm al tissue extracts (C hartl ), all o f w hich cam e from B lo od G ro up A -p ro du cin g o rg an s,the exclusion peak contained a fucose-rich polym er that was

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D . A la sta ir R . Simmo ns a nd P eter P erlm an n

ocoegN ormal LiverT9

gCM

OCO

I-O-08 -0-6-O-4-02 -l-O-i0-8-O 6-O4 -O-2-

Ib Normal PancreasT IO

Ca Panc rea s Li ve rT 2

Ocoa Co lonBladderT 18

4O 8O I2O I6OTube Number 20O 240

I H ID IVFraction NumberCha rt 1 . S ep had ex G -2 00 filtra tio n o f p erc hlo ric a cid ex trac ts fro mnorm al and tum or tissues, a, the gel filtration pattern of norm al liver(T 9) ex tra ct. T he p atte rn s o btain ed w ith th e a na log ou s ex tra cts o f theother norm al livers (T3 and T 4) w ere essentially the sam e, b, the gelfiltration pattern of norm al pancreas (T O )extract. T he patterns

obtained with all other normal tissue extracts including those ofstom ac h, c olo n, p an creas , a nd b lad der w ere also v ery sim ila r, c a nd d , 2rep res en ta tiv e g el filtra tio n p atte rn s o f e xtrac ts o f m etas ta tic m as sesfrom prim ary adenocarcinom ata of pancreas T 2 (c) and colon T 18 (d).In the T2 situation, there w as good separation of the Fraction I and IIregions. T he patterns obtained w ith the extracts from T 8 [carcinom a(C a) colon iver) and T15 (secondary peritoneal m asses) closelyrese mb led th at o f T 18 (c/), w hile th e T 14 (p rim ary o f sto mac h) e xtra ctgave a pattern (not shown) that was even m ore polydisperse in theFraction II region. There w as insufficient T il (prim ary of ileocecalju nc ti on ) e xt ra ct t o a tt empt p ur if ic ati on b y t his m eth od .indistinguishable from Blood Group A glycoprotein onq ua ntita tiv e a na ly sis a nd h em ag glu tin atio n-in hib itio n stu die s.In the tumor extracts (Chart 1, c and d), the excludedcomponent was also a fucose-containing polymer with thesam e qualitative sugar and am ino acid com position as bloodgroup substances; but it lacked A , B, and H specificity whente ste d in h emagg lu tin atio n- in hib itio n s ys tems.The Fraction II component (Tubes 81 to 115; 670- to960-ml pool; M .W ., 60,000 to 140,000) was present in theadenocarcinomata (Chart 1, c and d) but not in the normaltissue extracts (Chart 1, a and b}. It resembled the

glycoprotein of Tumor Fraction I in being of the samequalitative composition as blood group substances, but itpossessed none of the specificities of the latter. M oreover, asw ill be shown below, Fraction II has the same quantitativesugar com position as F raction I and, on a w eight-for-w eightbasis, they w ere serologically indistinguishable in the E LISAsystem. However, these findings do not establish thehom ogeneity or identity of these 2 fractions in which the onlydem on stra ted d ifferenc e is o ne of m olecu lar w eig ht.Fraction III (Tubes 116 to 160; 960- to 1330-ml pool)contained little except traces of contam inating substancesfrom adjacent peaks and Fraction IV (Tubes 161 to 240;1330- to 2000-ml pool) contained small amounts of nucleica cid only .Thus the tumors used in this study did not synthesizeABO-active glycoproteins although they originated in celltypes that normally do so. However, they did produce ananalogous glycoprotein w hich w as tum or specific in the sensethat it occurred in adenocarcinom ata of entoderm al origin butnot in norm al tissues or carcinom ata of the urinary bladder.T hese tum or glycoproteins, w hich are qualitatively sim ilar toblood group substances but serologically distinct from them ,were therefore studied more closely both chemically and

serologically to determ ine their relationship to A BO -actives ub sta nc es. T he re su lts w ere a s fo llows.Homogeneity of Tumor Glycoproteins. These fractionscontained no sim ple protein contam inants; their UV spectrashowed no absorption peak at 280 nm and tyrosine andtryptophan could not be dem onstrated am ong the am ino acidspresent after hydrolysis. S im ilarly, R NA contam ination w asnegligible, there being no absorption peak at 260 nm in theUV spectra and no ribose in the hydrolysis products.Furthermore, the tumor fractions contained no bloodgroup-active substances and, w ith the exception of the T 2 andT8 substances which will be described below , all were freefrom rese rv e p oly gluc osan s as sh ow n by th e ab sence o f g lu co seon hydrolysis. Thus the tumor glycoproteins are notapparently contam inated with other classes of com pounds.However, these results do not exclude heterogeneity withinthe glycoprotein class either as a mixture of biologicallyunrelated polym ers that happen to have the sam e qualitativesugar and amino acid composition or as a mixture ofbiologically related polymers that differ physically in thep ositio n, numb er, o r leng th o f sero lo gically active sid e c hains.T he 1st possibility w as investigated by quantitative chem icaland serological analysis of subfractions within each poolbefore bulking to detect inhom ogeneity across each peak. Nosuch inhomogeneity could be detected by these methods.However, by analogy with the blood group substances,physical heterogeneity due to variations in the number andlength of side chains probably also occurs in the tumorglycoproteins.Quantitative Analysis of Tumor Glycoproteins. Onqu an titativ e m icro an aly sis (T ab le 1 ), th e tumo r g ly co pro teinfraction s w ere in distin gu ish able ex cep t for th e T 18 su bstan cew hich w ill be described below . A ll contained sim ilar am ountso f f uc os e, g al ac to se , jV -a ce ty lg lu co sam in e, TV -a ce ty lg ala cto s-amine, and sialic acid; and all resembled blood groupsubstances in that their amino acid composition was

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CEA a nd B loo d G ro up Su bstan cesT able 1T he p er ce nt ag e o f s ug ar c ompo si ti on o f s ome r ep re se nt at iv e b lo od g ro up s ub sta nc es a nd t umor -s pe cif ic g ly co pr ot ein s (CEA 's )

Sug ar c omp os it io n (%)Sp ec if ic g ly co pr ot ei n o fesignationBlood

G roup Aubstance0BloodG ro up L eaubstance6CarcinomaofancreasCarcinomaofancreasCarcinomaofolonCarcinomaofolonCarcinomaoftomach0Carcinomaoftomach0CarcinomaoftomachCarcinomaoftomachCarcinomaofolonCarcinomaof colonT20T2T2T8T8T14T 14T15T15T18T18('ructionIIIIIIIIIIIIIIIITotal

jV-Acetylneur-yield (mg) aminiccid37.920.214.087.547.672.757.3269.539.235.956.72.15.07.98.55.33.97.17.47.37.42.73.7GalNAc20lose25.130.315.918.317.115.714.614.914.2sugarcontent%)82.374.6

a E xtrac ted fro m n orm al ad ult c olo n (T 20 ).l -'i gu re s a da pt ed fr om wor k o f L lo yd a nd K ab at ( 23 ).0 P rim ary tu mo r ex trac te d. T he re su lts fo r th e oth er p rim ary tu mo r (T l 1 ) a re n ot g iv en b ec au se th ere w as in su ffic ie nt m aterial fo r p urifica tio n.H ow ev er, th e m olar p ro portio ns of th e su gars in T l 1 w e re th e sa me a s fo r the o th er tum or g ly co protein s. T hu s, th e p rim ary a nd sec ond ary tu mora nt ig en s a pp ea r t o b e c hem ic al ly a nd s er ol og ic al ly i de nt ic al .

characterized by large amounts of serine and threonine butonly traces of tyrosine and tryptophan. On 1st isolation thespecific glycoproteins T2 and T8 resembled the CEAp rep aratio ns o f K ru pey et a l. (2 2), in asm uch as th ey co ntain edglucose and m annose in addition to the sugars and am ino acidscited above. H ow ever, on ultracentrifugation the glucose inthese preparations separated as a polyglucosan that proved tobe liver glycogen. Moreover, as 4 of the tumor-specificglycoproteins w ere m annose free and as this sugar w as presentin the analogous fractions from some normal tissues, it isalmost certainly not a constituent of the tumor-specificsu bstan ce. T hu s, as sh ow n in T ab le 1, the tum or glyco protein swere qualitatively indistinguishable from blood groupsubstances but quite distinct from them in 2 importantq uan tita tiv e re sp ects. F irst, th e po ly sacch aride comp on en ts ofthe tumor glycoproteins comprised 40 to 50% of thesepreparations, whereas in blood group substances thepolysaccharide m oiety accounts for 70 to 80% of the m olecule(23). Second, the fucose content of the tumor antigens wassignificantly lower than in blood group substances and, in 1ex ceptio na l tum or (T 18 ), th is co nstitu en t w as v irtu ally absen t.When the molar ratios of the sugar constituents werederived from the data in T able 1 w ith T V-acetylgalactosam inetaken as 2.0, the values for the other sugars in the tumorglycoproteins all fell w ithin the follow ing ranges: fucose, 0.8to 1.8 m oles; g ala cto se, 2.9 to 4.8 m oles; and . V -acety lg lu co s-amine , 1 .9 to 2 .5 m oles. T he extra cts o f the ade no carcin om ataof the stomach (T14 and T15) and colon (T18) all containedfucose , ga lac tose , TV-ace ty lg lucosamine , and TV-acet yl ga lact os -am ine in the proportions 1:3:2:2, respectively, except for theT 18 su bstan ce w hic h lack ed fu co se. O n qu an titativ e perio dateoxidation, one half of the W -acetylgalactosamine wasprotected w hereas the other half w as rapidly destroyed. T hesefindings suggest that there are 2 differently linked./V -acetylgalactosam ine residues in the tum or substance andthat the molar proportions given above may be the number ofsugar residues in the average tumor antigen side chain.

F urthe rm ore, b ecau se th e jV -acetylga lacto samine residu es inblood group substances also behaved sim ilarly on periodateoxidation (23), these residues are probably linked in the sam eway in both groups of polymers. From these analytical andperiodate oxidation studies, it would appear that the tum orglycoproteins may be incomplete blood group substanceslacking the end groups that determ ine ABO specificity. Thishypothesis is particularly attractive because it explains theobserved absence of blood group substances in the tumorsituation.S ero lo gica! C haracterizatio n o f C EA . A s n oted p rev io usly ,the tumor extracts used in these experiments have manyphysical and chemical features in common with CEA. Theirprobable identity with this antigen was therefore tested bytitrating them against specific CEA antiserum (2014) in theELISA system (6). As all the glycoproteins from theentod erm ally d eriv ed tumo rs (G ro up 1) reacted sp ecifica lly inthis system, they are presumed to be identical with CEA andshall now be referred to by that term .W hen the blood group specificities of CEA were tested asdescribed in "M aterials and M ethods," no A , B, H , L ea, F actorXIV, antigenic Complex I, or Forssman activity could bed em on strated w ith 1 e xce ption (T 18 ), w hich w ill be d escrib ed .T hus these determ inants are either com pletely m issing in C EAor present in a cryptic state. The molecular sequences thatd ete rm in e th ese sp ec ific itie s a re g iv en in C ha rt 3 a nd e sp ec ia llyin Chart 3a, which shows the detailed structure of a BloodG ro up A -spec ific sid e ch ain acco rd in g to F eizi et al. (8 ).W hen CE A w as characterized serologically, it w as found tocontain 3 specificities, w hich w ere arbitarily designated I, II,and III. These were distinguished as follows. CEA I was theimmu nod om in an t tumo r-sp ecific d eterm in an t as ju dg ed b y th eintensity and specificity of its reaction w ith C EA antiserum .CEA II activity, by contrast, was found to be common to allb lo od g ro up su bstan ces an d th eir precu rso rs, in asm uch as C EAcross-reacted weakly with all blood group antisera and allblood group substances cross-reacted w eakly w ith anti-C EA

FEBRUARY 1973 31 7


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D . A la sta ir R . S immo ns a nd P eter P erlm an nsera. W ith l exception (T18), CEA III was present in all CEAsamples as a cryptic determinant that was revealed whenterm inal acid-labile fucose and ./V -acetylneuram inic acidresidues were removed by partial hydrolysis as describedabove. As the fucose-deficient, jV-acetylneuraminicacid-containing T18 substances displayed CEA III activitywithout prior hydrolysis, the CEA III determinant is mostprobably m asked by fucose in the other CEA sam ples. These 3C EA sp ecific ities, I, II, an d III, w ere stu die d as fo llo ws.T he cry ptic C EA III d ete rm inan t w as in vestigated b y testin gpartially hydrolyzed CEA against blood group sera of knownspecificity. In these studies, which were carried out by thequantitative precipitation technique (36), the antigen (1 to1000 /jg) w as titrated against appropriate aliquots (50 jul) ofthe serum under test and was found to have Factor XIV andblood group antigenic Complex I (Ma) activity as shown inChart 2, a and b, respectively. As Factor XIV and antigenicC om plex I are d eterm in ed b y th e sequ en ces (3Ga ll -> -4GlcNAca nd (3Ga ll -> j3G lcN Acl -> 6, respectively, C EA III is alm ostc erta in ly id en tic al w ith th e s eq ue nc e Gall >-4 j3GlcNA cl - .

This conclusion was confirmed by inhibiting the CEA IIIreaction wi th 0-Gal -( l -4)-j3-GlcNAc-(l -> 6) -3-hexenetet rol (s )(Chart 2c). Because the fucose residue substituted on thissequence resembles the analogous fucose unit in ABOg ly co pro te in s in h av in g n o re co gn iz ab le se ro lo gic al sp ec ific ity ,it w as presum ed to be 3-O -substituted on the A '-acetylglucos-am ine residue as in norm al blood group substances (see C hart3b). However, the fucose-deficient CEA of T18 proved to beexceptional with respect to its CEA III activity and wasdesignated serotype 2 to distinguish it from the abovefucose-containing forms now designated serotype l. W hentested by the sam e serological m ethods, the serotype 2 antigenhad no A, B, H , Lea, or Forssman activity but, in contrast toserotype l, it displayed CEA III (Factor XIV and antigenicComplex I) activity without prior hydrolysis. From thesestudies and from its quantitative analysis in Table 1, CEA ofserotype 2 is probably a fucoseless form of serotype l (Chart3c).The CEA II determinant was common to all blood groupsubstances and their precursors when tested with CEAantiserum. These cross-reactions gave 5 to 15% of the colorintensity of the homologous CEA system in the ELISA test(Chart 2u) except in the case of Blood Group A where thereaction was about tw ice as strong. The CEA II determ inantw as studied by testing the inhibitory effect of the saccharidesof known structure in a standardized heterologous CEA:anti-Lea system with the ELISA m ethod (6). In these tests thestandardized system contained 2 /gof CEA; 1 to 500 /ag ofthe inhibitor contained in a 1:2000 dilution of L ea antiserum ;and conjugate K61 at a dilution of 1:500. As this system andall the cross-reactions between the ABO glycoproteins andCEA antiserum were inhibited by the GalNAc-peptidebackbone of the "degraded" blood group substance (Chart2e), CEA II is probably determined by this region of themole cule (C hart 3 , b an d c). T his fin din g offers an ex plan atio nfor the m ore m arked cross-reactivity of CEA antiserum withBlood Group A substance which has A-specific terminalimmunod om in an t jV -a ce ty lg ala cto samin e re sid ue s in a dd itio nto CEA II sp ec ific ity .Unlike CEA II and III which can be detected in normal

IDS

6. 42

Factor XIVAntiserum

IO8-6-

-2-

CEA B2O 4O 6O 8O IOOu q Ant ig en

A nti -1SerumCEA B2O 40 60 80 IOOp g A ntig en

IOOe 8O|| 6O- 4O

t* 2O -

2c I-O-r 0-8-CEA/Anti - O n *-I System O

ujO-4-O-2-nhibitor:, ->--(l*6)3hexenetetrol(s)

20 40 6O 80 IOOtig In hib ito rIOOc 8 0-]g|60-^40-_oo 20 - I nh ib it or :G alN A c - p ep tid e O-2-

1234p g A ntig en2fCE AAntiserum

32 64 125 25O 5OOp g In hib ito r 1234pg A ntigenChart 2. The serological characterization of CEA. a, partiallyh yd ro ly ze d CEA A h as F ac to r X IV a ctiv ity w here as un hy dro ly ze d CEAB has no such activity. The reaction with the hom ologous antigen(XIV) is also shown, b, partially hydrolyzed CEA A has antigenC om ple x I a ctiv ity w here as u nhy dro ly zed CEA B h as n o su ch ac tiv ity .c, th e I a ctivity o f CEA (CEA III d ete rm in an t) is sp ec ific ally in hib ite dby 0-G al-(l ->) -0 -G lcNAc- (l - )- 3- he xe ne te tr ol (s ). N o in hi bit io nwas noted with any of the other oligosaccharides tested, d, CEAa ntise ru m h as a n an tib od y c om pon en t th at rea cts w ith all b lo od g ro up

g ly co pr ot ei ns ( CEA I I d et ermi na nt ). A ll th e p re cu rs or s te st ed b eh av edquantitatively like the B and Lca substances shown, e, the cross-re ac tio n b etw een CEA a nd an ti-L ea sc rum (CEA II sy ste m) is in hib ite dby the G alN Ac peptide "backbone." A ll other C EA II cross-reactings ys tems w er e s im il ar ly i nh ib ite d. / , p art ia lly h yd ro ly ze d CF ,A A is 2 0 t o30% m ore active than unhydrolyzed C EA B and lactosam ine does notinhibit the C EA : anti-C EA (C EA I) system . N o inhibition w as notedw it h a ny o f th e o th er o li go sa cc ha ri de s te st ed .blood group substances and their precursors, thetumor-specific CEA I determinant has not yet beendemonstrated in any normal ABO-type glycoproteins. Anattem pt w as m ad e to in vestig ate its stru ctural n atu re b y testin gthe inhibiting effect of the saccharides of know n structure in ah om olo go us C EA :a nti-C EA sy stem , w hich had b een carefu llystandardized by the precise titration of all the reactants. Thetechnique differed from the usual ELISA method (6) in thatthe CEA antiserum (2014) was exposed to 1 to 250 /jg of theinhibitor under test for 15 min at room temperature. Thestan dardized sy stem co ntain ed the fo llo wing reac tan ts: 4 ju g ofCEA; 1 to 250 /-ig of the appropriate inhibitor contained in a1:1000 dilution of CEA antiserum; and conjugate K61 at adilution of 1:500. N o significant inhibition was produced byABO or related substances, and the oligosaccharides



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CEA a nd B lo od G ro up S ub sta nces

GalNAcFuc1'HFuc1241 Gal 13 GlcN

Chart 3aBlood group A substance"Gal I3 GlcNAc 13 Gal 3 GalNAc

i = pi type 2 chain |GalNAc 13 Gal 14 G lcNAci;uc Hmain chain

GalNAc

IIISenne or threonine

S enne or threonin eI

Chart 3bCEA serotype lV Gal I-3 GlcNAc l-*3 Gal

Gal 1 G lcNAc< XIV'I-CEA m 11Fu c

GalNAc

GalNAc

ISenne or threonineII

Senne or threonineCEA U I

on'S&I"5o.

Chart 3cCEA serotype 23 lcNAc 13 Gal 1 GalNAc -Senne or threonine

IGalNAc enne or threonine

CEA Ei-CEA m IChart 3. The structural relationship of Blood Group A substance and CEA's of serotypes 1 and 2. The structure of the Blood Group Aglycoprotein has been adapted from the studies of I-'eizi e t al. (8), but the "optional" secondary side chains that are present on som e prim ary

chains have been om itted in the interests of clarity. blood group and C EA determ inants that have been adequately characterized; - - - -,incom pletely characterized C EA I. A ll the fucosyl and jV -acetylgalactosam inyl residues are believed to be of the a-anom eric form and all theg al ac to sy l a nd jV -a ce ty lg lu co sami ny l r es id ue s w er e t ho ug ht t o b e o f t he o rm . Fuc , L -fu co se ; G al, D -g al ac to se ;/l , d et ermi na nt A ; / /= d et ermi na ntH ;L ea = d ete rm ina nt L ea ;A 7K , F ac to r X IV ;/, a ntig en ic C om ple x I(=CEA III).jSGa ll *G lcNA c, j3Ga ll *G lcNAc, and 0GlcNAcl ->3f3Gall - 2erythritol were likewise inactive in all concentrations tested. These results were confirmed by attempting to inhibit the titration of CEA against a 1:1000dilution of CEA antiserum with 250-A ig amounts of theinhibitor per test (Chart If). Thus the structure of CEA Iremains to be elucidated, but the finding that the activity ofCEA is increased by 20 to 30% on the removal of fucose(C ha rt 2 f) s ug ge sts th at th e A ^-a ce ty lg lu co samin e re sid ue o f th etype 2 chain or another unidentified cryptic structure isi nvolv ed i n t hi s d ete rm in an t.These studies of CEA I, II, and III also provide someindirect evidence about the nature of the C EA m ain chain. T hefind in g o f cry ptic an tig en ic C om plex I activ ity, an d th ereforeof type 2 chains in CEA, indicates that there is no defect inthe underlying acceptor main chain with the structure

0Gall - 3(3GlcNAcl *j3 Gall - 3GalNAc (C hart 3 a). T wofurther pieces of evidence support this view: (a) the molarproportions of the sugar residues in CEA are those expectedfor a substance consisting of normal main and type 2 chains;( > )h e q uan titativ e perio date o xida tio n stu dies sh ow th at, asin n orm al b lo od g rou p su bstance s, th e m ain ch ain con stitu en tsare all (1 ->)-linked as shown above. The absence of type 1chains was confirmed by testing blood group "precursor"substances O G and JS (Ist-stage periodate oxidation) againstCEA antiserum and by using these substances as inhibitors ofhom ologous CE A system s. A s these polym ers, w hich containcom plete an d incomp lete ty pe 1 ch ain s, respe ctiv ely (2 3,4 1),were inactive in these systems, CEA seems to be deficient intype 1 chains. Moreover, the association between CEA Ispecificity and the loss of the type 1 chains suggests that theterminal galactose of the main chain may also play a role in

FEBRUARY 1973 319


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the CEA I determinant as shown in Chart 3, b and c. Thus, thepreceding studies provide support for the view that CEA is anincomplete blood group substance lacking the peripheralsequences that determine A, B, H, and Lea specificity butretaining the m ore central sequences associated w ith G alN Acpeptide, Factor X IV , and antigenic C om plex I activity. F rom aconsideration of all the evidence available, the 2 CEAstructures may be as given in Chart 3, b and c. However, w iththe molecular sequence of the major determinant CEA I stillto be established, it should be stressed that the proposedstructures must be regarded as tentative until confirmed bys tr uc tu ra l anal ys is .DISCUSSIONIf CEA is assumed to be an incomplete blood groupsubstance, one of the characteristic changes induced by theoncogenic event is a failure to incorporate the type 1 chains(Chart 3, b and c). However, the isolation of serotype 2 CEAfrom T18 indicates that the genetic changes and relatedenzyme defects are not identical in every tumor. W hile it ispossible that CEA may ultimately prove to be a family of

related m olecules w ith common C EA I specificity, the presentstudy suggests that the m ajority of gastrointestinal tum orsprobably secrete C EA of serotype l.T he failure to incorporate the type 1 chains is probably dueto a defective transferase system though the precise nature ofthis defect is not understood at present. Confirm ation of thisview comes from the studies of Hakomori and Jeanloz (12),w ho show ed that there is an accum ulation of an Lea-specificpentasaccharide ceramide in gastrointestinal adenocar-cinomata. This substance, which proved to be a 0Gal 1 -30-GlcNAc l -/5Gal-G lc ce ram ide w ith an a-fuco syl re sid ue4 -0 -su bstituted o n th e A ^-ace ty lg lu cosam in e residu e (1 3), is atype 1 chain com plete w ith its L ea-specific fucose residue and"carrier" m oiety. Perhaps the sim plest interpretation of theseo bserv ation s is th at in ad eno carcin om ata o f en to derm al o rig inthere is a deletion or repression of a transferase(s) required forthe synthesis of blood group substance. The resultingincom plete blood group substance or CEA carries the new lyexposed CEA 1 determ inant and those blood group sequencesthat are proximal to the biosynthetic block. The unincorporated side chains, w ith those blood group specificitiesthat are distal to the block, continue to be synthesized butaccumulate in the form of the pentasaccharide ceramide ofHakomori. However, the A and H sequences associated withthe type 2 chains are also missing in all CEA preparationsstudied to date, which implies that most, if not all,gastrointestinal adenocarcinom ata m ay also have defects ino th er transferase system s fo r ABO su bstan ces. M ore rec ently ,th e a ccumu la tio n o f a la cto -A f- fu copenta os yl-O I I (- ce ram id e hasalso b een d escrib ed in some aden ocarcin om ata (4 7), in dicatin gthat in these instances there is a failure of incorporation oftype 2 chains. These results, taken together, confirm that thegenetic and related enzym e defects are not identical in everytumor.Antigenic variation in gastrointestinal tumors may becompared with smooth-to-rough mutation in bacteria andco ntrasted w ith ly so gen ic co nv ersion . In th e fo rm er a d efectiv e

CEA a nd B lo od G ro up S ub sta nc estran sferase also h alts the b io sy nth esis o f comp lete an tig en w iththe production of incom plete form s and the accum ulation ofthe unincorporated side chains. In this situation, w hen there isa loss of genetic inform ation, the structural sequences of theincom plete antigen and unattached side chains are unaltered.In ly so genic co nv ersio n, b y co ntrast, ne w ge netic in form atio nis carried into the cell by the bacteriophage so that newstructures appear in the antigens of such lysogenic strains. A sno new structural sequence (and therefore no new geneticinform ation) seem s to be required for CEA synthesis, there isno evidence that oncogenic viruses play a role in theproduction of tumor-associated antigens of this type.H ow ever, this possibility is not entirely excluded as one m ightpostulate an oncogenic virus w ith genetic effects on antigensynthesis that are restricted to repression of the ABOtransferases. In these circumstances no new structuralsequences would be expected in the resulting incompletet umor ant igens .It has been suggested that the biosynthesis of CEA isrepressed at the end of the 2nd trim ester of em bryonic life andthat its reappearance in tumors is the consequence ofd ere pre ss iv e d ed iffe re ntia tio n in itia te d b y th e o nc og en ic e ve nt(11). In our view the serological activity of CEA m ay sim plybe masked, because the biosynthesis of normal blood groupsub stan ces is comp leted b y the in corp oratio n o f th e p erip hera lA -, B-, H-, and Lea-specific sequences. M oreover, since theCEA antisera used in earlier studies have not beench aracteriz ed w ith resp ect to their C EA I, II, an d III activ ities,the nature of the determinant responsible for the cross-reactio n w ith fetal antig en h as n ot y et b een clarified .Since the pioneer studies of Thomson et ai. (39) in which itw as show n that C EA is a specific entoderm ally derived tum orsu bsta nce d etectab le in serum b y radioimmun oassay , a n um berof investigators (19, 24, 29, 30) have studied the level of thisantigen in the sera of patients with various diseases to assessth e reliab ility o f th is d ia gn ostic test in carcinoma o f th e co lo n.W hile there is general agreem ent that there is a high incidenceof circulating CEA in patients with entodermally derivedtumors, the position lias been somewhat complicated byreports (19, 24, 29, 30) of this antigen in many othercarcinom ata including those of lung, breast, ovary, bladder,and kidney and even in nonneoplastic conditions such assevere alcoholic liver disease, urem ia, ulcerative colitis, andovarian cysts. Som e of the confusion that has follow ed thesereports may be explained by the finding that CEA containsnot only a tum or-specific determ inant (C EA 1) b ut also norm alblood group sequences (CEA II and III). Thus, innonneoplastic conditions, the high CEA levels found may bedue to cross-reaction of CEA antiserum with normal bloodgroup substances secreted in large amounts in some of theseconditions. In the nonentoderm ally derived neoplasm s, it isnot known whether the high antigen levels found are due toreactions with CEA identical to that of entoderm al origin, tocro ss-reactio ns w ith ana lo go us C EA -lik e an tig en s, o r sim ply tothe presence of normal blood group substances. In one ofth ese stu dies (24 ), h ow ev er, th e ion -se nsitiv e de term in ant usedto m easure the C EA level w as probably not the tum or-specificdeterminant (CEA 1) but "a different antigenic site on theCEA m olecule," which m ay well be identical w ith our CEA 11.The success of future attem pts to elaborate s rodiagnostic



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CEA an d B lo od G rou p S ub sta ncestests for tum ors of entoderm al origin w ill probably depend onthe production of m onospecific CEA I antisera. O ur findingssuggest that this might be done either by absorption of theCEA II activity from CEA antisera or by the synthesis of anartificial C EA I an tigen for u se as an immu nizin g ag en t.ACKNOWLEDGMENTS

We thunk the follow ing to w hom w e are indebted for generous help:D r. B . A lm gard, D r. P . B iberfeld, D r. L . E rhardt, D r. G . H olm , D r. A . L .Mob erg, D r. G . Mobe rg er, an d D r. B . U nsg rdo f v ario us S to ck holmh os pit al s f or o bt ai nin g n orma l a nd c an ce ro us t is su es ; D r. H . E . C ar ls so n(Wenner-Gren Institute) for testing the Blood Group A and Bspecificity of the tumor tractions; D r. E . Engvall and Dr. S .Hammarstrom (W enner-Grcn Institute), Dr. T. Feizi and Dr. E. A.Rabat (Columbia University, New York, N . Y.). Dr. A . Gauhe(M ax-P lanck-Institut f r M edizinische F orschung, H eidelberg,Germany), D r. J. H . Humphrey (National Institute for M edicalR esearch, L ondon, E ngland), D r. H . 0rjasaeter (S tatens Institutt forFolkehelse, Oslo, N orway), and Professor W . M . W atkins (ListerI ns ti tu te , L on do n, E ng la nd ), f or r ea ge nt s a s in dic at ed i n th e t ex t.REFERENCES1 . C ep ellin i, R . P hy sio lo gic al G en etics of H um an B lo od F ac to rs. In:G . E. W . Wolstenholme and C. M . O 'Connor (eds.). Ciba

Symposium on the Biochemistry of Human Genetics, pp.242-263. L ondon: J. & A . C hurchill, 1959.2. Consden, R., Gordon, A. H., and M artin, A . J. P. QualitativeA naly sis o f P rotein s: a P artitio n C hromato grap hie M etho d U sin gP ap er . B io ch em . .,3 8: 2 24 -2 32 , 1 94 4.3. D avidsolm , I., K ovarik, S ., and L ee, C . L . A , B and O S ubstances inG as tr oin te st in al C ar cin oma. A rc h. P at ho l.,5 /.' 3 81 -3 90 , 1 96 6.4. D avidsohn, I., K ovarik, S ., and N i, L . Y . Isoantigens A . B and H inB enign and M alignant L esions of the C ervix. A rch. P athol., 87:3 06 -3 14 , 1 96 9.5. D ise e,Z ., and S hettles, L . A N ew S pectrophotom etric T est forthe D etection of M ethylpentosc. J. B iol. C hem ., 192: 579-582,

1951.6. Engvall, E ., Jonsson, K ., and Perlmann, P. Enzyme-linkedImmu no so rb en t A ss ay . I I. Q ua nti ta ti ve A ss ay o f P ro te in Ant ig en ,Imm uno glob unG, b y Mean s o f E nz ym e-L ab elled A ntig en an dAnt ib od y-Co ate d Tub es . B io ch im . B io ph ys . A cta , 2 51 : 4 27 4 34 ,1971.7. E ngvall, E ., and P erlm ann, P . E nzym e-L inked ImmunosorbentAssay (ELISA) Quantitative Assay of Immunoglobulin G .Immunochem is tr y, : 8 71 -8 74 , 1 971.8. F eizi, T ., K abat, E . A ., V icari, G ., A nderson, B ., and M arsh, W . L .Imm un och em ic al S tu die s o n B lo od G ro up s. X LIX . T he I A ntig enComp le x: D if fe re nc es amo ng Anti -I S er a R ev ea le d b y Qua nti ta tiv eP re cip itin S tu die s; P artial S tru ctu re o f th e I D eterm in an t S pecificfo r O ne A nti-I S eru m. J. Immu no l., 7 00 . 1 57 8-1 59 2, 1 97 1.9 . F io ri, A ., G iu sti, G . V ., P an ari, G ., an d P orce lli, G . G el F iltra tio n o fA BH S ubstances of H um an S aliva. J. C hrom atog., 55: 337-349,1971.10. G old, P ., and F reedm an, S . O . D em onstration of T um our S pecific-Antigens in Hum an Colonie Carcinomata by Im munologicalTolerance and Absorption Techniques. J. Exptl. M ed., 121:439-461, 1965.1 1. G ol d, P ., a nd F re edma n, S . O . S pe ci fi c C ar cin oemb ry on ic Ant ig en sof the H um an D igestive S ystem . J. E xptl. M ed., 722: 467-481,1965.12. Hakomori, S., and Jeanloz, R . W . Isolation of a Glycolipid

C ontaining F ucose, G alactose, G lucose and G lucosam ine fromH um an C an ce ro us T iss ue . J. B io l. C hem ., 23 9: 3 60 6-3 607 , 1 96 4.13. H akom ori, S., and Strycharz, G . D . Investigations on C ellularB lo od -G ro up Sub sta nc es . I . I so la tio n a nd Chemic al C ompo sit io n o fB lood-G roup A BH and Le Isoantigens of SphingoglycolipidN at ur e. B io ch em is try , 7 : 1 27 9- 12 86 , 1 96 8.1 4. H ammar st r m, S ., a nd K ab at, E . A . P ur if ic at io n a nd Cha ra ct er is ation of a B lood-G roup A R eactive H aem agglutinin from the S nailHelix p om atia a nd a Stu dy o f Its C om bin in g Site. B io chemis try , 8 :26 96 -2 70 5, 1 96 9.

1 5. H ammar strm , S ., L ag er cr an tz , R ., P er lm an n, P ., a nd Gus ta fs so n,B . E . Immunological S tudies in U lcerative C olitis. II. "C olon"Antigen and Human Blood Group A- and H-like Antigens inG erm free R ats. J . E xp tl. M ed ., /2 2: 1 07 5-10 86 , 1 96 5.16. Jermyn, M . A., and Isherwood, F. A . Im proved Separation ofSugars on the Paper Partition C hrom atogram . B iochem . J., 44:402-407, 1949.17. K auffm ann, F ., L deritz,O ., S tierlin, H ., and W estphal, O . Z urC hem ie der O -antigene von E nterobacteriaceae. I. A nalyse derZ uc he rb au st ei ne v on S almo ne lla O -a nti ge ne n. Z . B ak te ri ol . A bt. I .O rig., 1 78 : 4 42 -45 8, 1 960 .18. Kay, H. E. H., and W allace, D . M . A and B Antigens of TumoursArising from Urinary Epithelium . J. Nati. Cancer Inst., 26:1 349 -1 36 5, 1 96 1.1 9. K lcin ma n, M . S ., H arw ell, L ., an d T urn er, M . D . S tu dies o f C olo nieC arc inoma A ntig en s. G ut, 1 2: 1 -1 0, 19 71 .2 0. K oro st el ev a, V . S . S im ila rit ie s a nd V ar ia bil it y o f S pe cif ic A nt ig en sin H um an C an ce r. B ull. E xp t.. B io l. M ed ., 4 4: 8 7-9 3, 19 57 .2 1. K rug er. L ., Lde ritz,O ., S tro min ger, J. L .. an d Westp hal, O . Z urImmunchem ie der O -antigene von E nterobacteriaceae. V II. D ieZug eh ri gk ei t vo n H ex os en u nd 6 -D es ox yh ex os en i n S almo ne ll a-Lipopolysacchariden zur D -bzw . L -Reihe. B iochem . Z., 335:548-558, 1962.2 2. K ru pe y, J., G old, P ., a nd F ree dm an, S . O . P hy sico ch em ica l S tu dieso f th e C ar cin oemb ry on ic Anti ge ns o f t he Human D ig es ti ve Sys tem.J. E xptl. M ed., 128: 387-398, 1968.23. L loyd, K . O ., and K abat, E . A . Immunochem ical S tudies on B loodG ro up s. X LI. P ro po sed S tru ctu res for th e C arb oh yd rate P ortio nsof Blood Group A, B, H, Lewis3 and Lewisb Substances. Proc.N ati. A ca d. S ei. U . S ., 6 1: 14 70 -1 47 7, 1 96 8.24. Lo G erfo, P., K rupey, J., and H ansen, H . J. D em onstration of anA ntig en C ommon to S ev era l V arieties o f N eo pla sia. A ssa y U sin gZ irconyl P hosphate G el. N ew E ngl. J. M ed., 285: 138-141, 1971.2 5. Lde ri tz ,O ., S immons, D . A . R ., W estphal, O ., and S trom inger, J.L . A Spe cif ic M i cro de te rm in ati on o f G lu co samin e a nd t he Ana ly si sof Other Hexosamines in the Presence of Glucosamine. Anal.B io ch em ., 9 . 26 3-2 71 , 1 96 4.2 6. Lde ri tz ,O ., S ta ub , A . M ., a nd Westph al, O . Immu no ch em istry o fO a nd R A ntig en s o f S alm on ella an d R ela te d E nte ro ba cteriac ea e.B ac te rio l. R ev.,m - 19 2-2 55, 1 96 6.

2 7. L ud ow ie g, J ., a nd B enmaman , J . D . Col or im etr ie D iff er en ti ati on o fH ex os am in es . A na l. B io ch em ., 1 9: 8 0- 88 , 1 96 7.28. Masamune, H., Kawasaki, H ., Abe, S., and Oyama, 1.M olisch-positive M ucopolysaccharides of G astric C ancers asCompared with the Corresponding Components of GastricMuco sa e: F rac tio na tio n P ro ce dure o f G astric C anc er an d G astricMuc os a. T oh ok u J. E xp tl. M ed ., 6 8: 8 1-91 , 19 58 .29. M cNeil, C ., Ladle, J. N ., Helmick, W . M ., Trentelman, E., andW entz, M . W . A n A ntiserum to O varian M ucinous C yst F luid w ithCol on C an ce r S pe cif ic it y. C an ce r R es ., 2 9: 1 53 5- 15 40 , 1 96 9.30. M oore, T. L., Kupchik, H . Z., M arcon, N., and Zamcheck, N.Carcinoem bryonic Antigen Assay in Cancer of the Colon andPancreas and O ther D igestive Tract D isorders. A m. J. D igest.D ise ase s, 16 : 1 -7 , 19 71 .31. M organ, W . T . J. T he C roonian L ecture: A C ontribution to H um an

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Lacking A, B, H, Lea and Leb Specificities. Biochemistry, 9:3 41 4-3 42 1, 1 97 0.42. W arren, L. The Thiobarbituric Assay of Sialic A cids. J. B iol.Ch em ., 2 34 : 1 971- 1975 ,1 959.4 3. W atk ins , W . M . B lo od G ro up S ubs ta nc es . S cie nce , 7 52 . 17 2-1 81,1966.4 4. W atk ins , W . M . B io sy nthe sis o f B lo od G ro up S ub stan ces. C urre ntC oncepts and R esults. //;. D . A minoff (ed.). Blood and TissueA ntig en s, p p. 4 41 -4 59 . N ew Y ork: A cad em ic P re ss, In c., 1 970 .4 5. We st ph al , O ., Ld er it z,O ., a nd B ister, F . b erd ie E xtrak tien vo nB ak te ri en m it P he no l-Was se r. Z . N at ur fo rs ch ., 7 b: 1 48 -1 55 , 1 95 2.46. W inzler, R . J., D evor, A . W ., M ehl, J. W ., a nd Smyth, I. M . S tudieson the M ucoproteins of H um an Plasma. I. Determination andIso lation . J. C lin . In vest., 2 7. 60 9-6 16, 1 94 8.47. Y ang, H ., and H akom ori, S . A S phingoglycolipid H aving a N ovelT yp e o f C era mide an d L acto-TV-F uco pen tao se III. J. B io l. C hem.,246: 1192-1200, 1971.

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