(CANCER RESEARCH 48, 6906-6911. December 1, 1988]

A Mr 58,000 Glycoprotein Specifically Expressed in Developing Hamster Pancreasand Reexpressed in Hamster and Human Pancreatic Carcinomas1

A. Heredia2 and M. J. Escribano

E. K. 227 CNRS, Institut de Recherches Scientifiques sur le Cancer, B.P 8, 94802 Villejuif Cedex, France

ABSTRACT

A glycoprotein with a molecular weight of 58,000 specifically expressed in exocrine hamster fetal pancreas was characterized using amonoclonal antibody (Mab B4).

By immunoperoxidase, Mab B4 stained pancreatic tissue from the 1(1"'day of gestation (6 days before delivery) until the 10lh day after birth.

The maximal expression of the Mab B4-specific protein called fetalpancreatic (FP) protein was reached between delivery and the 5"' day of

postnatal life. Endocrine pancreas was negative at any developmentalstage. All adult pancreata examined were negative. Moreover, Mab B4was tested against a wide variety of fetal and adult tissues; only immaturepancreata were stained. Chemically induced pancreatic carcinomas werestrongly stained by this Mab. On the contrary, other tumors (liver andkidney) appearing simultaneously with pancreatic carcinomas were negative. Using a nitrocellulose immunofixation assay, FP protein was foundin all sera from hamsters bearing pancreatic tumors (23 cases tested).This protein was not detected in normal sera.

Mab B4 cross-reacted with a protein in human fetal pancreas extracts,that behaves similarly to the hamster FP protein: it is present exclusivelyin exocrine fetal pancreas and is reexpressed in pancreatic adenocarci-nomas.

The high tissue specificity of this protein, its oncofetal character, andrelease into the blood circulation make the FP protein a potential tumormarker of pancreatic cancer.

INTRODUCTION

The presence in the pancreas of components specific for fetalacinar cells was first evidenced in Syrian golden hamster usinga polyclonal antiserum raised against fetal pancreatic extracts(1,2). Important features of these antigens are their associationwith development and reexpression in pancreatic carcinomas(3, 4). During experimental carcinogenesis in a BOP3 hamstermodel (5-7), these antigens were already present in preneoplas-tic lesions (8, 3, 4). They first appeared in altered acini andprogressed through irregular hyperplasia to ductal carcinoma.This suggests an important role in transformation.

In Western blot, this antiserum recognized several antigensin fetal pancreatic extracts, in the relative molecular weightrange of 27,000 to 94,000 (1). Two main components of M,58,000 and 80,000 were identified as being two antigenicallyunrelated glycoproteins (2).

Recently, a family of proteins presenting many analogies withhamster fetal pancreatic proteins has been described in humanfetal pancreas. By Western blot, the main human componentshad a molecular weight of 110,000 and 60,000 (9). A monoclonal antibody exclusively recognizing the M, 110,000 corn-

Received12/28/87; revised4/12/88. 8/2/88; accepted8/24/88.The costs of publication of this article were defrayedin part by the payment

of page charges. This article must therefore be hereby marked advertisementinaccordancewith 18 U.S.C. Section 1734solelyto indicatethis fact.

1This work was partly financed by a grant (6394) from the Association for

Cancer Research, Villejuif, and Aid of Innovation (ANVAR), Paris. A. H. wassupported by a student stipend from the French government and from theAssociation for Cancer Research.

2To whom requests for reprints should be addressed.'The abbreviations used are: BOP, N-nitroso-bis(2-oxopropyl)amine; PBS,

phosphate-buffered 0.15 M NaCl, pH 7.4; Con A, concanavalin A; SDS, sodiumdodecyl sulfate; Mab, monoclonal antibody; NIF, nitrocellulose immunofixation;PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric-focusing. FAP protein, fetoacinar pancreatic protein; FP protein, fetal pancreatic protein.

ponent has been obtained (10, 11). It is a concanavalin A-reactive protein associated with the development of the pancreas and located in fetal acinar cells. It has been referred to asFAP (for fetoacinar pancreatic) protein. The expression of FAPprotein rose sharply in case of pancreatic carcinoma and wasfound to be present in the blood of most patients with thisneoplasia (12, 13).

In the present study, we prepared a murine monoclonalantibody (Mab B4) against the M, 58,000 hamster fetal protein(14). This protein belongs to the family of fetal pancreas-specificproteins already defined by the polyclonal antiserum, and shareswith FAP protein: pancreas specificity, association with development and reexpression in tumors. However the protein described here is located in acinar and ductular fetal cells whereasFAP-protein was described as acini-restricted (11). Therefore

the A/r 58,000 component has been referred to as FP proteinfor fetal pancreas protein.

This work describes the biochemical characteristics and tissuedistribution during development of the FP protein, its reexpres-sion in chemically induced pancreatic tumors and its presencein sera from hamsters with pancreatic carcinoma. The cross-reaction of Mab B4 with a human protein is also reported.

MATERIALS AND METHODS

Hamsters. Syrian golden hamsters maintained for many years in asyngeneic colony (15) were obtained from the IRSC facilities (Villejuif,France).

Normal Tissues. Whole embryos and fetuses of gestational ages 8, 9,10, 11, and 12 days were removed. Gestational age was assessed bymeans of the size and number of somites in embryos and fetuses.4

Pancreases were obtained from fetuses aged 13 and 15 days of gestation(the gestation period in hamsters is 16 days), from animals between 1day and 15 days after delivery and also from adult hamsters 2-4 monthsold. Other fetal and adult organs, including lung, heart, kidney, spleen,liver, and the whole gastrointestinal tract were taken from the sameanimals.

Tumoral Tissues. Primary pancreatic carcinomas were obtained in 2-month-old hamsters by treatment with BOP (1, 4). Secondary tumorswere raised by grafting pancreas from hamster treated with BOP intosyngeneic recipients as described previously (1, 8). Kidney and livertumors appearing simultaneously with pancreatic tumors after BOPinjection were also collected.

Human Material. Fetal pancreases were obtained after spontaneousor therapeutic abortion. Adult pancreases and other normal organscame from recently deceased donors of kidney grafts. Normal andpathological tissues were kindly donated by Dr. M. Gonzalez (HôpitalSaint Antoine, Paris, France) and Dr. M. Nap (Institute of HealthProvince of Friesland, Leeuwarden, The Netherlands).

Tissues for Histology. Immediately after excision, a piece of eachorgan was fixed in ethanol or formalin for 48 h and subjected toimmunohistological procedures.

Tissue Extraction. Pancreas from fetuses 13 to 15 days-old and fromnewborns were taken for tissue extraction since previous results fromour laboratory (1) showed that newborn and fetal hamster exhibit thesame antigens. At birth individual pancreas weight is around 0.15 mg.

' Carré-Llopis,A. Protéinesde différenciationdes acini pancréatiques.Leur

association avec le développementet la cancerisation du pancréas.Isolement etcaracterisation. Thesis in Sciences of Paris XI University, 1986.

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FETAL PANCREATIC PROTEIN IN CANCER PANCREAS

Immediately after removal, 100 pancreases were dipped into an anti-proteinase solution consisting of 0.4% E-amino-n-caproic acid w/v,aprotinin 40 units/ml and 10~5 M diisopropylfluorophosphate and

homogenized. The homogenates were centrifuged (20,000 rpm for 30min at 4°C)and the supernatants were immediately aliquoted andfrozen at —¿�80°C.Protein content in extracts evaluated by Lowry method

(16) was on the average 5 mg/ml. Normal and tumoral adult pancreaseswere processed similarly. Extracts of the other organs were prepared inthe same manner, with the exception that antiprotease solution contained only aprotinin.

Con A-reactive Fraction. This was prepared from extract of hamsterfetal pancreas as previously described (2). 5 ml of pancreatic extracts(5 mg protein/ml) were applied to a Sepharose-Con A column equilibrated with 0.02 MTris buffer, pH 7.4, containing 1 mM each of MnCl2,MgCl2, and CaClj. Elution was performed with 0.2 M methyl-a-D-mannopyrannoside. This Con A reactive (Con A+) fraction contained

the two major fetal proteins of M, 58,000 and 80,000 (2), as well asminor components including adult pancreas and normal serum proteinsas contaminants.

Production of Hybridomas. BALB/c mice, 8 weeks old, received 7s.c. monthly injections of the fraction purified on Con A affinitychromatography from fetal pancreas extracts (15 ^g total protein emulsified in complete Freund's adjuvant per injection). 3 days prior to

fusion the animals were given one i.v. booster injection of crude fetalpancreas. Spleen cells were fused with murine myeloma cells SP2/0according to the method of Buttin et al. (17). Fused cells were distributed in 24-well culture plates at a cell concentration of IO6 cells/ml.

Hybridomas were selected in ER medium containing hypoxanthine,aminopterin, and thymidine. Wells with 40% growth were selected forantibody-screening assays.

Antibody Screening. Since the fetal pancreas Con A+ fraction is

heterogeneous, monoclonal antibodies were selected on the basis oftheir restricted reaction with fetal pancreas. Therefore, culture supernatants were absorbed with adult pancreas, adult serum and fetal serum(7 mg, 20 pi, and 2 /il, respectively, per ml of hybridoma supernatant).After centrifugation at 20,000 rpm, the supernatants were tested bynitrocellulose immunofixation assay (NIF, modified dot blot), (18, 19)and immunohistology. Hybridomas strongly positive for fetal pancreaswere further tested by Western blot.

A total of 144 hybridomas were tested, and hybrid cells giving thestrongest reaction were cloned twice by limited dilution to obtain MabB4. Ascites were raised by injections of 5 x IO6cells into the peritoneal

cavity of BALB/c mice, pristane pretreated.Purification and Isotyping of Mab B4. Mab B4 was purified from

ascitic fluid using protein A-Sepharose CL4B (Pharmacia) affinitychromatography. Purification was controlled by electrophoresis oncellogel strips (Chemetron, Italy). The immunoglobulin subclass wasdetermined by double immunodiffusion analysis (20) using isotype-specific goat-antimouse antibodies (Sigma).

Polyacrylamide Gel Electrophoresis and Isoelectric Focusing. Iimiin-noblot Analysis. PAGE was performed in 10% acrylamide gels and inTris-glycine buffer pH 8.3 containing 0.1% SDS. Proteins electropho-retically separated were then transferred to nitrocellulose sheets(Schleicher and Schüll,FRG) according to the procedure of Towbin(21).

Isoelectric-focusing was done on polyacrylamide plates containing2.4% ampholine (LKB) with a pH range of 3.5-9.5. Migration wascarried out under 1500-V constant current for 1.5 h at room temperature. Transfer was done by uniform pressure of 2 g/cm2 for 1 h. In both

procedures (PAGE and IEF), nitrocellulose blots were then incubatedwith Mab B4 and peroxidase-labeled second antibody. The reactionwas visualized with 0.01% H2O2 and chloronapthol.

Enzyme Digestions. The fetal pancreas extracts were treated with (a)neuraminidase from Vibrio choleras (Behringwerke, Marburg, W. Germany) 0.02 U/mg of protein, in 50 mM sodium acetate buffer, pH 5.5,for 1.5 h at 37°C.The reaction was blocked by addition of l M K2HPO4,

pH 9, and (b) trypsin (Sigma) 450 U/mg of protein, in 40 mM Tris-HC1 containing 50 mM CaCl2, pH 8.1, for 2 h at 37°C.After that the

sample was mixed with type I-S trypsin inhibitor (Sigma) 2 Mginhibitor/

1 Mgtrypsin.

Controls containing all reagents except enzymes were made simultaneously in each case.

Periodate and /S-Mercaptoethanol Treatments. Periodate oxidationwas carried out for l h at room temperature. Periodic acid was used atconcentrations ranging from 0.01 to 0.08 M, the reaction was stoppedby NaOH.

Fetal pancreas extracts were treated with 0.2 M /3-mercaptoethanolin 300 mM Tris base buffer, pH 8.6, containing IO"3M of phenylmeth-ylsulfonyl fluoride and IO"1 M of /V-ethylmaleimide for 5 h at 4°C.

Then 0.4 M iodoacetamide was added to the sample and incubated for2 h at 4°Cin order to block the reaction. Controls in absence of periodic

acid and mercaptoethanol were performed under the same conditions.Immunohistology. Ethanol- or formalin-fixed tissues were embedded

in paraffin (22) and cut into 3-iim sections. Immunoreaction wasperformed by indirect ¡mmunoperoxidase. Sections were hydratedthrough graded alcohol and endogenous peroxidase activity was inhibited by incubation in 0.5% H2O2 in methanol for 30 min. Sections werethen incubated sequentially in (a) 2% egg albumin in PBS for l h toprevent nonspecific fixation, (b) Mab B4 at appropriate dilutions, 4 hat 4°C,and (c) peroxidase-labeled anti-mouse antibodies (Institut Pas

teur, France) diluted 1/100 for 30 min. Revelation of the peroxidaselabel was done with amino-ethylcarbazol (23). Sections were counter-stained with hematoxylin and mounted in PBS containing 50% glycerol.In all cases, controls with nonimmune mouse serum were done.

Serum Test. To investigate whether the FP protein is present in theblood the dot blot technique was used. Serum from hamsters bearingpancreatic tumors (2 p\) were deposited onto nitrocellulose paper, thenserially incubated in Mab B4 and peroxidase-labeled anti-mouse antibody. The reaction was visualized with H2O2 and chloronapthol. Allsera were tested in triplicate and normal sera were used as control.

RESULTS

Monoclonal Antibody Characterization

Three hybrid cells out of the 144 tested reacted in Westernblot with A/r 58,000 protein contained in fetal pancreas extracts.The hybridoma giving the strongest reaction was selected andcloned by limited dilution. After two cloning procedures, MabB4 was produced in the form of ascites. Ascitic fluid showedthe same specificity as culture supernatant.

After purification on protein A-Sepharose, antibody in asciticfluid was typed as IgGi, by immunodiffusion analysis (20); thisis in agreement with the fact that Mab B4 was fully eluted froma protein A column at pH 5.8 (24).

Antigen Characterization

In Western blot, Mab B4 in fetal pancreatic extract revealeda main component of M, 58,000 and a minor one of M, 54,000(Fig. 1, lane D). According to the total protein level Coomassie-stained M, 58,000 protein appears at relatively high concentrations as compared to other proteins in fetal extract (Fig. 1, laneB). In IEF immunoblots, a rather heterogeneous pattern exhibiting several bands in the 5.5-6.1 region was observed (data notshown). Adult pancreas extracts were negative in both techniques.

The binding of Mab B4 to FP protein was not altered afterantigen treatment with neuraminidase, periodic acid, and mercaptoethanol, whereas it was fully abolished after trypsin digestion.

Tissue Specificity

Mab B4 was tested by indirect immunoperoxidase in a largevariety of normal fetal and adult tissues namely: pancreas,kidney, heart, spleen, liver, stomach, duodenum, small intestine, and colon. Only fetal pancreas was stained. Tissue specificity was confirmed by the negative reaction of extracts from

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FETAL PANCREATIC PROTEIN IN CANCER PANCREAS

BMWxicr

200

97

68

43

15854

^—^J

18Fig. 1. Lane A, molecular weight markers (BRL). Lane B, hamster fetal

pancreas extracts, Coomassie-stained profile after SDS-PAGE. Immunoblotanalysis of hamster fetal pancreas extracts (Lane D) and adult Pa extracts (LaneO with Mab B4 (1/500 dilution) 4 h after transfer to a Schleicher & Schullmembrane. Peroxidase-labeled antibodies diluted 1/1000. Substrate 0.01% H2O2and chloronapthol. Total protein loaded was 80 i>nin Lanes B and /' and 200 ¡¡V.in Lane C.

these tissues by dot blot nitrocellulose membrane assay. Controls performed using normal mouse serum were negative.

Developmental Pattern

In the hamster the gestation period is 16 days. Embryogenesistakes place during about 8 days (22) and the pancreas is morphologically mature at Day 10 after birth (1).

Embryonic and Fetal Tissues. Sections from whole embryosand fetuses ranging from 8 to 12 days of gestation (five to sixspecimens each at 8, 9, 10, 11, and 12 days) and pancreasesobtained from fetuses aged 13 and 15 days of gestation wereexamined by immunohistology with Mab B4. The earliest staining was observed on the 10th day. This stage corresponds tothe beginning of pancreas organogénesis.A pancreatic rudimentembedded in a loose mesenchyme was seen near the hepaticprimordia, in all examined sections (Fig. 2). Moreover theundifferentiated cells forming this primitive structure were theonly tissue Mab B4 immunostained in the whole fetus. Duringthe following 24 h the epithelium of the developing pancreaticrudiment is arranged in branching tubules. Cells proliferate andbecome arranged into acinus-like structures with enlarged lumina. The stain with Mab B4 was located in the cytoplasm ofthese cells (Fig. 3A). The cytoplasmic compartment enlargedfrom this date and some morphologically distinguishable aciniwere seen at 13 days of gestation. Mab B4 immunostain regularly increased and was located in both still immature and newlyformed acini. This persisted until delivery.

Neonates until 15 Days after Birth. Forty-four pancreaseswere tested (N = 20 for newborns and three cases each at 1,3,5, 7, 9, 10, 12, and 15 days). In newborns, the Mab B4 reactionwas located in primitive ductular cells and acini (Fig. 3Ä).Atthis age staining intensity varied from one area to another,

Fig. 2. I. section of a hamster fetal pancreas at 10 day of gestation stainedwith hematoxylin & eosin. Note the pancreatic rudiment (arrow) near the hepaticprimordia (arrowhead), x 63. Bar, 10 ^m. B, greater magnification of primitivepancreatic structure (arrow), x 250. Bar, 10 /¿m.

probably due to the different developmental stage of cells.Maximal expression of FP protein, as judged by the intensityof immunoreaction, was observed around birth and persisteduntil the Sth day of postnatal life. During this period intralob-ular ducts were patent and appeared stained (Fig. 3Q, whereasinterlobular ducts observed were negative. After the 5th day,staining quickly decreased, so that at the 7th day some aciniremained faintly positive. Staining totally disappeared at the10th postnatal day (Fig. 3D). Thus, all sections examined atDays 10, 12, and 15 were negative.

Hamsters 2-4 Months Old. No stain at all was observed inall examined pancreas at this age.

FP Reexpression in Pancreatic Carcinoma

In our experimental conditions, BOP-treated hamsters developed pancreatic adenocarcinomas from 6 to 7 months afterthe first carcinogen injection. However, morphological alterations were observed from the 2nd month. Evolution in time ofpancreatic lesions were altered acinar cells [loss in cytoplasmicand zymogen contents and dilated lumina (26)], cystadenomas,and regular and irregular hyperplastic epithelium. Polyclonalanti-fetal pancreas serum showed immunoreactivity in preneo-plastic lesions, mainly in distorted acini and ducts with markedatypical epithelium (3, 4). By contrast, the Mab B4 stain wasonly observed in pancreatic carcinomas, either primary (Fig.4A) or produced by graft (Fig. 4B).

Reexpression of FP protein in pancreatic carcinomas was6908

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FETAL PANCREATIC PROTEIN IN CANCER PANCREAS

Fig. 3. .1. section of a hamster pancreas at12 days of gestation. Mah B4 staining is located in the cytoplasm of cells (arrows), x 400.Bar, 10 /im. B, section of a newborn hamsterpancreas showing a strong Mah B4 reaction inboth well-formed (arrows) and immature acini(arrowhead), x 400. Bar. 10 um. C, section of5-day old hamster pancreas. The immunoreac-tion is observed in the acini (arrows) and in theductular cells (arrowhead). A structure resembling islets (star) and vessels (curved arrow) arenegative, x 250. Bar. 10 >tm. D, pancreas section of 10-day old hamster showing duct (arrow) and acini Mab B4 negative, x 400. Bar,10 um. Immunohistology was performed byindirect immunoperoxidase. Substrate 0.01%H2O2 and aminoethylcarbazol. Hematoxylincounterstain.

;j>^

- rl V \ *» *" a^^ "-> •¿�'•̂* * •¿�** 7

» i * * 3C * -^ * - -*

confirmed by the dot blot technique: Mab B4 positivity wasseen in extracts from primary and grafted tumors.

Tumors of other organs which appeared simultaneously withpancreatic carcinomas after BOP treatment (liver, N = 10;kidney N = 4) were negative for Mab B4.

Serum Test

A qualitative test using the dot blot technique was performedin order to detect whether FP protein was present in the blood;23 sera from hamsters with pancreatic tumors were analyzed.All cases were positive for Mab B4. On the contrary, no reactionwas detected in normal sera. Its presence in other inducedtumors could not be tested, since they appeared simultaneouslywith pancreatic carcinoma in our experimental model. Therefore, the control group was limited to healthy animals.

Cross-Reaction of Mab B4 with Human Tissues

Mab B4 cross-reacted in dot blot technique with human fetalpancreas extracts. In Western blot, this monoclonal antibodyalso reacted with a component which migrated at the M, 58,000position (data not shown). Immunoperoxidase of all fetal pancreas sections analyzed (N = 10) showed a weak positive reac

tion in the cytoplasm of acinar cells. When Mab B4 was testedagainst other normal tissues summarized in Table 1, all caseswere negative. We also examined the presence of FP protein inpathological tissues: pancreatic adenocarcinomas were positive(seven out of 10 tested) whereas other tumors were negative(Table 2). In pancreatic tumors, a positivity was observed in themalignant ductular epithelium (Fig. 4Q. The immunoreactionwas located in the cytoplasm of tumoral cells.

Comparative Study of FP and FAP Proteins

In order to confirm whether FP and FAP ( 10,11 ) are differentproteins, a comparative study by Western blot and immunohis-tology was made. Thus Mab B4 and Mab J28 (specific for FAPprotein) (10, 11) were tested by Western blot against hamsterand human fetal pancreas extracts. Mab B4 recognized the M,58,000 protein in both, but Mab J28 reacted only with the M,110,000 FAP protein in human fetal pancreas extracts. Byimmunohistology, we tested the same pancreatic tumors withboth monoclonal antibodies (B4 and J28) and observed a verydifferent localization, i.e., Mab B4 reacted with the tumoralstructures themselves whereas as previously reported the stain-

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FETAL PANCREATIC PROTEIN [N CANCER PANCREAS

Table I Reactivity of monoclonal antibody B4 with human normal tissues

[

Tissues Number tested Staining

\

-'.

4CFig. 4. A, section of a priman pancreatic adenocarcinoma induced by BOP.

The Mab B4 stain is seen in the cytoplasm of atypical glandular structures(arrows), x 400. Bar, 10 >im.B. section of a well-differentiated adenocarcinomaof pancreas obtained by graft into syngeneic animals. Staining is seen in malignantductular cells. Note strong reaction on the basal portion of tumor cells (arrows).x 250. Bar. 10 /im. C, section of human pancreatic adenocarcinoma. Theimmunostaining is visible in the carcinomatous glands (arrows). Clusters of tumorcells are also stained (arrowhead). The reaction can be observed in the cytoplasmof malignant cells, x 250. Bar, 10 iim. Immunohistology was performed as inFig. 3.

Fetal pancreasAdult pancreasAdult Wirsung ductAdult duodenumAdult stomachAdult liverAdult colonAdult oesophagusAdult lungAdult kidney

10105567554

10" Indirect immunoperoxidase was applied in all instances. +, positive staining:

-. no reaction.

Table 2 Reactivity of monoclonal antibody B4 with human pathological tissues

TissuesPancreatitesPancreas

CA°Duodenum

CAGastricCABreastCAColonieadenomasColonieCAOvarian

tumorsNumber

tested310210105105Staining_+*—_———-

°CA, carcinomas.* Immunoreaction was performed by indirect immunoperoxidase procedure.

+, positive staining in 7 of 10 specimens; -, no reaction.

ing of J28 was mostly located in acinar cells surrounding thetumor (11, 12).

DISCUSSION

The FP protein reported in this study behaves, in SDS-PAGEimmunoblots, identically to the major antigen evidenced byBenedi et al. in hamster fetal pancreas extracts with the aid ofa polyclonal antiserum (1). The bands immunostained by MabB4 at the M, 58,000-54,000 position were comparable (sizeand shape) to those recognized by the polyclonal antibody. TheM, 54,000 component appears as a molecular variant of the M,58,000 protein. This antigen has been characterized as a gly-coprotein which binds to concanavalin A. Changes in glycosyl-ation can easily explain the double band pattern recognized byMab B4. In this regard, FP protein is similar to human FAPprotein (11) since in Western blot, Mab J28 (specific for FAPprotein) stained two bands at positions M, 110,000 and100,000. Results obtained after treatment of fetal pancreasextracts with enzymes or chemical reagents suggest that theepitope recognized by Mab B4 may be expressed on the peptidemoiety.

By immunohistology, we demonstrated that FP proteinexpression is development related: it is present from the earlieststage of pancreatic ontogenesis (10 days of gestation) when theonly pancreatic structure consists of a primitive duct-like structure embedded in a loose mesenchymal stroma. Later, thisprotein is expressed in exocrine fetal pancreas (cytoplasm ofacinar and ductular cells), but never in endocrine fetal pancreas.The synthesis of this protein as well as FAP protein starts at avery early stage of exocrine pancreas differentiation, and therefore these proteins can be regarded as organogenesis-specificproteins. Expression of FP protein slowly increased, reachingthe highest level between birth and 5 days after delivery. Thereafter, FP protein quickly decreased and totally disappearedwhen the definitive morphology of adult pancreas was acquired(on the 10th day of postnatal life).

As for the fetus, endocrine cells remained negative for MabB4 in neonates and adults in agreement with previous resultsobtained with polyclonal antiserum in both hamster (3, 4) and

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FETAL PANCREATIC PROTEIN IN CANCER PANCREAS

humans (9), suggesting the existence of independent pathwaysof differentiation for exocrine and endocrine pancreas.

The FP protein is not expressed in adult pancreas, but wasreexpressed in pancreatic carcinomas. FP protein was found inneoplastic stages and not in benign lesions observed duringcarcinogenesis, this suggests that not all oncofetal pancreaticantigens revealed using the polyclonal antiserum are simultaneously expressed (4).

The absence of FP protein in a wide variety of normal andpathological tissues, apart from immature pancreas and pancreatic tumors, demonstrates its high tissue specificity.

The serum test performed in order to verify the presence ofFP protein in the circulation showed clear positivity in 100%of the sera from hamsters with pancreatic tumors. This findingindicates that FP protein is released into the blood in thismalignancy.

Mab B4 cross-reacted with human tissues: in immunohistol-ogy, fetal pancreas and pancreatic carcinomas were stained withthe same localization as in hamster. In Western blot, Mab B4reacted in human fetal pancreas extracts with a band that alsomigrated at the M, 58,000 position, whereas the M, 110,000FAP protein was not recognized. Conversely, the Mab J28which possesses high affinity for FAP protein, did not reactwith the M, 58,000 FP protein. The fact that Mab B4 recognizesa protein with 58,000 molecular weight in hamsters and humans, and that Mab J28 reacts only with the human M, 110,000protein, led us to infer that the A/r 58,000 protein (FP) isprobably a highly conserved molecule present in both speciesand that the M, 110,000 protein (FAP) is a molecule with anonconserved region in the epitope recognized by Mab J28,found only in humans and not in hamsters.

Therefore, M, 58,000 and 110,000 represent two antigeni-cally unrelated proteins, associated with development of humanfetal pancreas and implicated in cancer of the pancreas in man.Nevertheless, the localization of these proteins in human pancreatic carcinomas is different: FAP protein is seen mainly inperitumoral acini and rarely in transformed cells. In contrast,FP protein is present in tumor structures.

FAP and FP proteins have some characteristics in common,i.e., developmental-related expression. Con A binding ability,organ-tissue specificity, and reexpression in pancreatic tumors.However, the FP protein possesses its own properties likely tobe expressed in the tumor itself which allow us to consider itas a valuable protein. Moreover, as this protein passes into theblood in hamsters bearing pancreatic tumors it could be anadditional tumor marker for serum diagnosis of pancreaticcancer and therefore a means of enhancing the diagnosticpotential and survival rate in this malignancy.

ACKNOWLEDGMENTS

The authors thank Dr. P. Burtin for helpful discussion and criticalreading of the manuscript, Dr. M. Gonzalez (Hôpital Saint Antoine,Paris) and Dr. M. Nap (Institute of Health Province of Friesland,Leeuwarden) for the kind gift of normal and pathological humantissues. They also wish to thank Dr. B. Loridon-Rosa for the histológica! examinations and C. Cuadrado for excellent technical assistancewith histology and J. Urani and J. Shimotake for English corrections.

REFERENCES

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2. Carre-Llopis, A., and Escribano, M. J. Isolation and characterization of twooncofetal glycoproteins from hamster pancreas using concanavalin A andpreparative electrophoresis. Biochim. Biophys. Acta, 880: 101-107, 1986.

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9. Escribano, M. J., Cordier, J., Nap, M., Ten Kate, F., and Burtin, P. Differentiation antigens in fetal human pancreas. Reexpression in cancer. Int. J.Cancer, J«:155-160, 1986.

10. Escribano, M. J., and Albers, G. H. R. A monoclonal antibody, recognizinga concanavalin A reactive, fetoacinar and cancer associated human pancreaticprotein. Tumor Biol., 7: 253, 1986.

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1988;48:6906-6911. Cancer Res   A. Heredia and M. J. Escribano  Pancreatic CarcinomasHamster Pancreas and Reexpressed in Hamster and Human

58,000 Glycoprotein Specifically Expressed in DevelopingrMA

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