Cell membrane, but not circulating, carcinoembryonic antigen is linked to a...

Vol. 155, No. 2, 1988

September 15, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 794-800

CELL MEMBRANE, BUT NOT CIRCULATING, CARCINOEMBRYONIC ANTIGEN IS LINKED

TO A PIIOSPHATIDYLINOSITOL-CONTAINING HYDROPHOBIC DOMAIN

Freder ic J e a n ' , Pascale Malapert +, Genev ieve Rougon + and Jacques Ba rbe t '

Immunotech S.A.', Lumlny, Case 915, 13288 Marsei l les Cedex 9, France

UA 202 CNRS +, I n s t i t u t de Chimie Biologlque, 13331 Marsei l les Cedex 3, France

Received July 20, 1988

S.UMMA~X.; Carc lnoembryonic a n t i g e n is p r e se n t in the cell membrane of most tumors of colorec ta l or igin and in the plasma of p a t i e n t s with colorecta l cancer and o ther mal ignanc ies . In th i s paper we demons t r a t e t h a t c a r c l n o - embryonic an t i gen can be re leased from HT-29 cells by p h o s p h a t i d y l l n o s i t o l specific phosphol ipase C. Tr i ton X-114 phase s e p a r a t i o n shows t h a t phospho - l ipase C conve r t s the a n t i g e n in to a water soluble pro te in . In addi t ion , plasma ca rc inoembryonlc a n t i g e n behaves as the c leaved a n t i g e n in phase s e p a r a t i o n exper iments . This s t rong ly sugges ts t h a t carc inoembryonlc a n t i g e n is a t t a ched to cell membranes by a g l y c o s y l - p h o s p h a t i d y l l n o s i t o l anchor and t h a t i t can be re leased in r i v e by enzymat ic c leavage of the hydrophobic ta i l . ~ 19~ Acaaem±c Press, Inc.

[NT!~QD.U.CTIQ~[; Carcinoembryonic a n t i g e n ( C E A ) , f i r s t descr ibed by Gold

and Freedman (1), is a 180-200 k i loda l ton (kDa) g lycoprote in , well e s t a b l i she d

as a tumor assoc ia ted an t igen . The leve l of c i r cu l a t i ng CEA de te rmined by

immunoassays is widely cons idered as a d iagnos t ic tool of g rea t i n t e r e s t in the

moni tor ing of colon carcinoma and o ther ma l ignanc ies (2). CEA is s t r u c t u r a l l y

r e l a t ed to a se r ies of a n t i g e n s (NCA), expressed by normal t i s sue s and p r e se n t

in the plasma of h e a l t h y Ind iv idua l s (3), t h a t c r o s s - r e a c t with seve ra l a n t i -

bodies a g a i n s t CEA.

Amino acid sequence a n a l y s i s (4) and cDNA clone c h a r a c t e r i z a t i o n (5)

have shown e x t e n s i v e homology be tween CEA and NCA and c lass i f ied them

wi th in the immunoglobul in s u p e r - f a m i l y (4). Fur thermore the repor ted nuc leo t ide

Abbrev i a t i ons : CEA, carc lnoembryonic an t igen ; NCA, normal c r o s s - r e a c - t ing an t igen ; G-PI, g l y c o - p h o s p h a t l d y l i n o s l t o l ; PI-PLC, P h o s p h a t i d y l i n o s i t o l - specific phosphol lpase C; CRD, c r o s s - r e a c t i n g de t e rminan t ; PBS, phospha te buffered sa l ine .

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved. 794

VOI. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

s e q u e n c e s exh ib i t a t t he C - t e r m i n a l domains some s im i l a r i t y wi th t h a t of T h y - 1

(6) and of o the r ce l l su r f ace g lycop ro t e ln s d e m o n s t r a t e d as anchored to p lasma

membrane by a complex g lycophospho l ip id con t a in ing p h o s p h a t i d y l i n o s i t o l (G-PI)

(7). This kind of p ro t e in can be r e l e a s e d from cel l membrane in a so luble form

by t r e a t m e n t wi th p h o s p h a t i d y l i n o s i t o l - s p e e i f i c phospho l ipase C (PI-PLC) (8).

In order to i n v e s t i g a t e t he n a t u r e of t he membrane anchor , we s tud ied the

r e l e a s e of CEA from human co lo r ec t a l carc inoma ce l l s HT-29 a f t e r PI-PLC t r e a t -

ment. In addi t ion , we examined w h e t h e r the hydrophobic t a i l is s t i l l p r e s e n t in

c i r c u l a t i n g CEA, and t r i ed to c h a r a c t e r i z e the s t r u c t u r e of the anchorage us ing

r abb i t an t i bod i e s d i r ec t ed a g a i n s t an ep l tope in the c a r b o h y d r a t e of the G-PI

ta i l , named cross r e a c t i n g d e t e r m i n a n t (CRD) (9).

MATERIAL AND METHODS: HT-29 ce l l s were grown in DMEM medium wi th 10% f e t a l bov ine serum.

A n t i - C E A monoclonal an t ibod ie s 35B and F023-C5 were pu rchased from YAMASA SHOYU Co., Ltd. ( Japan) and IMMUNOTECH (Marsei l les , France) r e s p e c t i - ve ly . Rabbit a n t l s e r a aga in s t CRD were k indly p rov ided by Dr. M. Fergusson (Oxford, England) and Dr. T. Bal tz (Bordeaux, France) .

Q u a n t i t a t i v e d e t e r m i n a t i o n of CEA was performed wi th an immunorad io - metr ic a s say k i t (ELSA-CEA, ORIS, France) .

PI-PLC was pur i f i ed in our l a b o r a t o r y from Baclllus thuringiensis c u l t u r e s as p r e v i o u s l y desc r ibed (10).

Live ce l l s were washed wi th p h o s p h a t e buf fe red sa l ine (PBS) pH 7.2, 0.05% bov ine serum albumin, t h e n i n c u b a t e d for 30 rain a t 37"C wi th or w i thou t 0.1 IU/ml PI-PLC in the same buffer ; s u p e r n a t a n t s were co l l ec ted for CEA q u a n t i t a - t i v e de t e rmina t i on . A l t e r n a t i v e l y , ce l l s (5. 108) were washed twice with DMEM and h a r v e s t e d in 25 mM EDTA in DMEM, to p repa re cel l homogena tes . Af t e r c e n - t r i f u g a t i o n the p e l l e t was homogenized in 600 lJl of 0.25 M sucrose , 0.5 mM p h e n y l m e t h y l s u l f o n y l f luor ide , 10 UI/ml apro t in in , 5 lJM p e p s t a t i n , 40 1JM l e u p e p - t in and 5 pg/ml a lpha 2 macrog lobul ln as p r o t e a s e s lnh lb i to r s . Af te r c e n t r i f u g a - t lon (30 mln, 100 000 g), the p e l l e t was r e suspended in 50 mM Tris-HC1 buf fe r pH 7.4, wi th p r o t e a s e s inh ib i to r s , a t a f ina l p ro t e in c o n c e n t r a t i o n of 11 mg/ml. Cell homogena tes were i n c u b a t e d for v a r i o u s t imes a t 37*C with or w i thou t 1 IU/ml PI-PLC, t h e n so luble and p a r t i c u l a t e m a t e r i a l s were s e p a r a t e d by c e n t r i - f uga t i on (100 000 g, 1 hr, 4"C).

Samples were sub jec ted to e l e c t r o p h o r e s i s us ing 7% SDS-po lyac ry lamlde gel according to the method of Laemmli (11), and e l e c t r i c a l l y t r a n s f e r r e d onto n i t r o c e l l u l o s e s h e e t a t 60 V for 5 hrs in 20 mM Tris-HC1 buf fe r pH 7.4 wi th 20% methano l (12). The n i t r o c e l l u l o s e s h e e t was f i r s t s t a i n e d with Ponceau red to r e v e a l p ro t e ins t h e n quenched for 2 hrs a t 37"C with 5% non f a t dry milk in PBS. I ncuba t i on wi th an t ibod ie s and a u t o r a d l o g r a p h y were conduc ted as desc r ibed (13). In some i n s t a n c e s s h e e t s were d e s a t u r a t e d accord ing to r e f e r e n c e 14 and reused for i n c u b a t i o n with a n o t h e r an t ibody .

Phase s e p a r a t i o n in T r i t on X - 1 1 4 was performed as desc r ibed by Bordier (15). Briefly, cel l homogena tes were i n c u b a t e d with or w i thou t PI-PLC as above; p lasma samples were d i lu ted t h r e e fold wi th water . One volume of 25 mM T r t s - ttC1 pH 7.4, 1513 mM NaCI, 4% Tr i ton X - 1 1 4 and 0.4 mM HgCI2 as PI-PLC i n h i b i - tor was added to each sample and kep t for 6 rain a t 4"C, t hen 6 rain a t 37°C and c e n t r i f u g a t e d for 6 rain, a t 37"C, 2500 g. Af t e r c e n t r i f u g a t i o n the upper aqueous phase was removed from the d e t e r g e n t phase a t t he bot tom of the tube .

RESULTS: Q u a n t i t a t i v e d e t e r m i n a t i o n of CEA in the s u p e r n a t a n t of l ive

HT-29 cel ls showed t h a t l m m u n o r e a c t i v l t y a s soc i a t ed to CEA could be r e l e a s e d

795

16-

14-

0.0

12-

IO-

B/T (%) 8-

6-

4-

2-

C) °

f -X~ not freafed

* ~i Xl I x I I I j X ~ 2,0 4.0 6.0 8.0 10.0 12.0 14.0 16.0

ng/ml or cells/ml x 10 -~ C

Vol. 155, No. 2, 1988 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

.Fl~ure 1: Quantitative determination of CEA released by PI-PLC treatment of HT-29 llve cells. Supernatants of HT-29 live cells, treated or not with PI- PLC, were subjected to CEA quantitative determination by Immunoassay. Results are plotted as bound to total radioactivity ratios versus sample dilutions. Standard CEA samples are included as positive controls.

Figure 2: Immunoblot analysis of pellets and supernatants of HT-29 cell membrane homogenates. The figure (left panel) shows immunoblots revealed with 35B antibody, and (right panel) Immunoblots revealed with F023-C5 antibody. Lanes - ENZ : control experiments without PI-PLC treatment, lanes + ENZ : experiments with PI-PLC treatment. SN corresponds to supernatants, P to pellets. Positions of molecular weight markers are shown in the figure.

by PI-PLC t r e a t m e n t , while v i r t u a l l y no CEA could be d e t e c t e d in the s u p e r n a -

t a n t when cel ls were no t t r e a t e d (Figure 1). Immunoblot expe r imen t s were p e r -

formed on pe l l e t s and s u p e r n a t a n t s of HT-29 cel l homogena tes , with or w i thou t

PI-PLC t r e a t m e n t . CEA d e t e c t i o n was done with two d i f f e r e n t an t i CEA m o n o -

c lonal an t ibod ies , 35B and F023-C5. Autorad iograms r e v e a l e d a broad band

migra t ing be tween 170 and 200 kDa cor respond ing to the r epor t ed molecu la r

weight of CEA (Figure 2). Without PI-PLC t r e a t m e n t , t he i n t e n s i t y of th i s band

was c l ea r ly more impor t an t in t he pe l l e t f r ac t ion , co r respond ing to cel l mem-

branes . By con t r a s t , the f r ac t ion of CEA r e c o v e r e d in the s u p e r n a t a n t i nc rea sed

d r ama t i ca l l y a f t e r PI-PLC t r e a t m e n t . I nc rea s ing i ncuba t i on t ime or enzyme

c o n c e n t r a t i o n did not allow comple te so lub l l l z a t i on of CEA: the maximum f r ac t i on

of CEA r e l e a s a b l e by PI-PLC t r e a t m e n t was 80% as shown by i n t e g r a t i o n of the

immunoreac t i ve bands (Table 1). This might r e f l e c t v a r i a t i o n in s u s c e p t i b i l i t y to

PI-PLC, as a l r e a d y r epo r t ed for o the r G-PI anchored p ro t e in s (7). Af t e r i n c u b a -

t ion wi thou t enzyme 10 to 20% of the CEA was found in the s u p e r n a t a n t ; t h i s

could be due to p r o t e o l y t l c c l e a v a g e or ac t lon of endogenous PI-PLC p r e s e n t in

cell homogena tes .

HT-29 cel l homogena tes t r e a t e d or not wi th PI-PLC and four serum

samples (2 n e g a t i v e and 2 CEA p o s i t i v e as measured by immunoradiomet r lc

796


Table I: Integration of CEA Immunoblot experlments a

PI-PLC treated Control Background

SN b pb SN P

cpm 46924 10662 4478 35966

percent of total 82 18 i0 90

445

a: Immunoblot experiments were conducted with antibody F023-C5 on pellets and supernatants of HT-29 cell membrane homogenates treated or not with PI-PLC. CEA Immunoreactlve bands were cut and counted for 125 1 radioactivity.

b: SN: supernatants, P: pellets.

assay) were submitted to phase separation in Triton X-II4 solution. Aqueous

phase and detergent phase were characterized by SDS electrophoresis and immu-

noblot. As shown In figure 3A, CEA from HT-29 cells behaved as expected for a

G-P! anchored protein: it was almost exclusively recovered in the detergent

phase in control membrane preparation, and, on the contrary in the aqueous

phase when treated wlth PI-PLC. Plasma CEA was found for the most part in the

aqueous phase in both CEA positive samples (Figure 3B), suggesting that clrcu-

lating CEA has lost its hydrophoblc tail. Quantitative determination of CEA by

immunoassay confirmed the Immunoblot experiments (Figure 4). Control plasma

samples (CEA nega t i ve ) did no t exh ib i t a ny s i g n i f i c a n t lmmunoreac t lv i ty .

In order to de te rmine whe ther c leaved CEA was express ing CRD d e t e r m i -

n a n t s , as repor ted for some G-PI anchored p ro te in (7), two r a b b i t a n t i s e r a , both

- E N Z +ENZ 1 2 I! A 3 4

Figure 3: Immunoblot analysis of Triton X-II4 phase separations. Immu- noblot experiments were conducted with F023-C5 as described In Material and Methods. Figure 3A shows results for HT-29 cell membrane homogenates treated (+ ENZ) or not (- ENZ) wlth PI-PLC and partitioned In Triton X-114. Figure 3B shows Immunoblots of serum samples after phase separation In Triton X-114 solution. Samples 1 and 2 are CEA negative, samples 3 and 4 CEA positive as determlnated by Immunoassay. SN corresponds to aqueous phases, P to detergent phases. Gels were run under non-reducing (A) and reducing (B) conditions, resulting In slight differences In CEA apparent molecular weight.

797


I [ ] Aqueous phase

[ ] Defergenf 100- phase

90-

80-

70-

60-

CEA (z) 50-

40-

30

20-

10-

0 •

Q

i

Cell Cell Plasma Plasma homogenafe homogenafe sample 3 sample 4 / ~ ' ~

PI-PLC nor freafed freafed

+ENZ

P ~SN TOP P jSN

i iii ;ii 2000

n6.5 . 9 2 5

6 6 . 2

' ~ 4 5 . 0

Figure 4: Q u a n t i t a t i v e de t e rmina t i on of CEA a f t e r phase s e p a r a t i o n in Tr i ton X-114 . Q u a n t i t a t i v e de t e rmina t i on of CEA was performed on aqueous and d e t e r g e n t phases , a f t e r phase s e p a r a t i o n in Tr i ton X-114. Resul ts for HT-29 cell membrane homogenates , t r e a t e d or not wi th PI-PLC, and serum samples 3 and 4 are p lo t t ed as the pe rcen tage of t o t a l CEA in each sample. The sum of CEA con ta ined in aqueous and d e t e r g e n t phases was 0.28 lag/rag p ro te in when membrane homogena tes were t r e a t e d with PI-PLC, and 0.26 lag/rag p ro te in In the control . This sum was 1.9 lag/ml for serum sample 3, and 5.6 pg/ml for serum sample 4 in accordance wi th va lues p rev ious ly de te rmined on whole samples before Tr i ton X - 1 1 4 phase s e p a r a t i o n (1.6 pg/ml and 5.3 lag/ml respec t ive ly ) .

Figure 5: Immunoblot ana ly s i s wi th an t i CRD an t ibodies . HT-29 cell mem- b rane homogena tes were submi t t ed to PI-PL0 t r e a t m e n t as descr ibed in Mater ia l and Methods. Ni t rocel lu lose s h e e t s were Incuba ted wi th F029-C5 an t ibody as pos i t ive cont ro l ( lef t) , or s equenc ia l ly wi th bo th ant i -CRD a n t l s e r a (r ight) .

r a i s e d a g a i n s t t h e s o l u b l e fo rm of t h e v a r i a n t s u r f a c e g l y c o p r o t e i n of

T r y p a n o s o m a b r u c e 1 a n d d i r e c t e d a g a i n s t CRD, w e r e u s e d t o d e t e c t CEA a f t e r P I -

PLC t r e a t m e n t , b y SDS e l e c t r o p h o r e s l s a n d b l o t t i n g o n t o n i t r o c e l l u l o s e . N o n e o f

t h e two a n t i s e r a t e s t e d was a b l e to r e v e a l t h e s o l u b l e fo rm o f CEA ( F i g u r e 5).

In c o n t r o l e x p e r i m e n t s u n d e r t h e s a m e c o n d i t i o n , P I - P L C - c l e a v e d h u m a n a l k a l i n e

p h o s p h a t a s e was l m m u n o r e a c t l v e w i t h b o t h a n t l s e r a ( n o t s h o w n ) ,

DISCUSSION: C o n v e n t i o n a l c e i l s u r f a c e p r o t e i n s a r e g e n e r a l l y a t t a c h e d to

p l a s m a m e m b r a n e b y a t r a n s m e m b r a n e h y d r o p h o b i c p o l y p e p t i d e c o n n e c t i n g t h e

l n t r a - a n d e x t r a - c e l l u l a r h y d r o p h l l l c d o m a i n s . H o w e v e r , a n u m b e r of p r o t e i n a r e

a t t a c h e d to c e l l m e m b r a n e i n a d i f f e r e n t way , u t i l i z i n g g l y c o s y l p h o s p h a t i d y l -

I n o s l t o l a s a m e m b r a n e a n c h o r . A m i n o a c i d s e q u e n c e s o f G - P I l i n k e d p r o t e i n s

p r e d i c t e d f rom cDNA s e q u e n c e s e x h i b i t a t t h e C - t e r m i n u s a s h o r t h y d r o p h o b l c

d o m a i n (15 to 20 r e s i d u e s ) w i t h o u t c y t o p I a s m l c d o m a i n (7) . Wi th t h i s r e s p e c t , C -

798


Table 2: Comparison of CEA C-terminal amino-acid sequence with other G- PI anchored proteins

Proteins C-terminal sequences ref.

CEA ..NNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI a 24 NCA ..NRTTVTMITV SGSAPVLSAVATVGITIGVLARVALI 5

Thy-i ..KLVKCGGISLLVQNTSWLLLLLLSLSFLQATDFISL 6 N-CAM 120 ..PTVIPATLGSPSTSSSFVSLLLSAVTLLLLC 25

a: The sequences have been tentat ively allgned at the N-terminal end of the hydrophobic domain (shown in bold characters) (see ref. 7).

t e rmina l s equences sugges t G-PI membrane anchorage for CEA and NCA as well

(Table 2). PI-PLC t r e a t m e n t and phase s epa ra t i on in Tr i ton X-114 provide

s t rong ev idence t h a t CEA is anchored on HT-29 cell membrane by a G-PI ta i l ,

and i t might be assumed to be l inked by the same way to plasma membrane of

o ther cell types . However, f u r t h e r i n v e s t i g a t i o n , such as e thano lamine and f a t t y

acid labe l l ing , are needed to d e f i n i t e l y conclude t h a t CEA is G-PI anchored.

Similarly, the a t t a c h m e n t of NCA to cell membranes should be s tud ied us ing

specific an t ibod ies .

After phase s e p a r a t i o n in Tr i ton X-114 of serum samples, c i r cu l a t i ng CEA

was recovered in the aqueous phase . This sugges ts t ha t , in yivo, CEA could be

re leased from the cell membrane in a so luble form by c leavage of the G-PI l ink,

poss ib ly th rough a c t i v a t i o n of an endogenous enzyme (16). ttowever, p h o s p h a t i -

d y l i n o s i t o l - s p e c i f l c phophol lpase D a c t i v i t y has been found in plasma t h a t

removes phospha t id lc acid from de t e rg e n t so lubi l lzed G - P I - b e a r i n g p ro t e in s (17).

Thus, a l t e r n a t i v e l y , i n t a c t CEA might be shed in plasma and s u b s e q u e n t l y

c leaved by the p lasma phosphol ipase ac t i v i t y . By con t r a s t , no CEA could be

de tec ted by immunoradtometr ic a s s ay in HT-29 cell cu l tu re medium, even a f t e r

long term cu l t u r e (not shown). Then d i sc repanc ies be tween c i r cu l a t i ng CEA leve l

and the p resence of CEA pos i t i ve tumor de tec ted by immunosc in t ig raphy (18),

could be a t t r i b u t e d to d i f f e r e n t i a l a c t i v a t i o n of the enzymat ic mechanisms r e s -

pons ib le for CEA re lease .

I n t e r e s t i n g l y , CEA, l ike o ther members of the immunoglobul in s u p e r - f a m i l y

anchored to plasma membrane by a G-PI l ink (Thy-1 (19), N-CAM 120 (20), LFA

3 (21)), did no t b ind ant l -CRD an t ibod ies . We can specu la t e t h a t th is f ea tu re

re f lec t s a specif ic s t r u c t u r e of the ca rbohydra te of the G-PI anchorage , as

repor ted for T h y - 1 (22), t h a t may prove c h a r a c t e r i s t i c of the members of the

s u p e r - family.

F u n c t i o n a l a d v a n t a g e s of the p h o s p h a t i d y l i n o s i t o l anchor compared with

c o n v e n t i o n n a l t r an smembrane po lypep t ide domain s t i l l remain unc lea r . However,

as a l r eady d i scussed (7, 23), i t is l ike ly t h a t G-PI anchored p ro te ins are more

mobile, and eas i ly r e l ea sab le from cell membranes by enzymat ic c leavage.

799


Al though the f u n c t i o n of CEA is no t known, I t might be i n v o l v e d in c e l l - c e l l

r ecogn i t i on p roce s se s . In t h a t case CEA r e l e a s e could o b v i o u s l y modu la t e i n t e r -

a c t i ons be tween ce l l s dur ing tumor growth and d i s s e m i n a t i o n .

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Cell membrane, but not circulating, carcinoembryonic antigen is linked to a...

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