Retinoic Acid Binding Protein in Human and Experimental Pancreatic Carcinomas in Hamsters*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 13, No. 5 Copyright 1983, Institute for Clinical Science, Inc. Retinoic Acid Binding Protein in Human and Experimental Pancreatic Carcinomas in Hamsters* MARTIN BRANDES, B.A.*, MARK S. GESELL, PhD.1\ HISAKO UEDA, M.S c.t, JUAN C. MILLAN,.D.f, ROBERT M. QUINLAN, M.D.$, CALVIN ERNST,.D 4, W ILLIAM F. SINDELAR, M.D., CAROLE C. KURMAN, B.S., SANDRA HARRIS, B.A.t, and DAVID BRANDES, M.D.f f Departments of Pathology, Baltimore City Hospitals and Johns Hopkins University School of Medicine, Baltimore, MD td epartm ent of Surgery, Baltimore City Hospitals, Baltimore, MD Surgery Branch, National Cancer Institute, National Institutes o f Health, Bethesda, MD ABSTRACT Cytoplasmic retinoic acid binding protein (crabp) is present in human fetal pancreas and becom es nondetectable in the norm al adult pancreas. The binding protein for retinoic acid becomes apparent again in pancreatic cancer. Similar fluctuations in the content of crabp occur in the hamster. The binding protein is undetectable in the normal adult ham ster pancreas, while it was detected in several transplantable adenocarcinomas in the Syrian golden hamster. Introduction Vitamin A com pounds are essential for m aintenance of visual function, epithelial cell differentiation, and reproductive capacity Retinoic acid is capable of m aintaining th e differentiated state of normal epithelial structures and may in * Supported in part by NIH Biomedical Research Support Grant SO 7-RR-556, and Pathology Restricted Fund (CPPA). duce differentiation of various kinds of anaplastic tum or cells1216'36'42 43 and rev erse h y p erp lastic and p rem alig n an t changes induced by chem ical carcinog e n s.3'4041 A cytoplasm ic p ro te in th at binds retinoic acid specifically, retinoic acid binding protein (crabp), has been d etected in various tissues, adult and fetal8'914 26'27 and in cultured cells.21-22'23'24' It has been suggested that the effects of retinoic acid may be m ediated by this binding protein ' /83/ $01.20 Institute for Clinical Science, Inc.

2 Most p ertinent to this study is the fact that crabp has been detected in various hum an tum ors, including breast, lung, cervix, colon, and prostate cancer5' ,30,33 anc] jn animal tum ors; however, the binding protein for retinoic acid was either undetectable or present in lower concentrations in th e norm al adult tissues from which the tum ors originated. In anim al tissu es, th e o ccu rren ce of crabp is far more widespread in fetal organs than in th e ad u lt,8-26 suggesting that the expression of this binding protein is lost during post-natal developm ent. Changes are reported by us in the content of crabp during post-natal developm ent and in the course of malignant transformation in fetal, adult, and carcinomatous pancreatic tissues, both in humans and in the Syrian golden hamster. Methods and M aterials H u m a n P a n c r e a t ic T is s u e H um an norm al pancreas and pancreatic carcinom as w ere obtained from discarded portions of four surgical resection specimens. Fetal pancreatic tissue was procured from discarded portions of five cases of spontaneous abortions and was pooled to obtain a sample sufficient for assay. Samples w ere cooled to 4 and used immediately or frozen at 70 C and stored. In no case was the tim e betw een specim en rem oval and cooling greater than 30 minutes. H a m s t e r P a n c r e a t ic T is s u e s Normal pancreas was obtained from six inbred Syrian ham sters 20 to 30 weeks old. T hree established nitrosam ine-induced pancreatic carcinom as in inbred Syrian ham sters '39 were assayed for the presence of retinoic acid binding protein. The Scarpelli tum or35 arose in a fem ale in b re d Syrian ham ster, in jected weekly for 10 weeks, with 10 mg per Kg N -nitrosobis (2-oxopropyl) am ine. The PA N C R EA TIC R E T IN O IC A C ID B IN D IN G P R O T E IN 4 01 Pour SG pancreatic carcinoma31-32 was induced in Syrian golden hamsters by subcutaneous injection of 2,2'-dihydroxy-di- N -propylnitrosam ine at 125 to 500 mg p e r Kg body w eig h t dose range. T he CBP pancreatic carcinoma38 originated in an in b re d Syrian golden h am ster, by w eekly inoculation of 250 mg p e r Kg body w eight of N -nitrobis-(2-hydroxypropyl) amine. The tumors were propagated by serial subcutaneous transplant in in b re d Syrian golden h am sters and harvested when they reached an approxim ate diam eter of 2 cm. All tissues w ere collected on ice and rapidly frozen in liquid nitrogen and kept at -70 C until used. Histological controls for each of the tissues used in the study w ere prepared by frozen sections and perm anent paraffin sections of form alin-fixed sam ples. S ections w ere stain ed by ro u tin e hem atoxylin-eosin methods. C h e m i c a l C o m p o u n d s A ll-trans-(ll,12-3h) retinoic acid (1.45 Ci mmol) was a gift. * All other chemicals w ere p u rch ased elsew h ere, f R etinoic acid was dissolved in absolute ethanol containing 1 mg ml dl-a-tocopherol and stored at 0 to 4. [3H] R e t i n o i c A c i d A ssays The methods of assay for cellular retinoic acid binding protein were m odified14 from procedures published by Ong, Page, and Chytil29 and Sani and Corbett.34 Tissues w ere hom ogenized in five volumes (wgt/vol) 50 m M tris-h C l p H 7.5 and cen trifu g ed one h o u r at 100,000 x g- Five hundred m icroliters of the resulting supernatant cytosol w ere incubated with saturating am ount (200 pmols) of [3H] retinoic acid alone or w ith 200-fold excess * Hoffman-LaRoche, Nutley, NJ. t Sigma, St. Louis, MO.

3 4 02 B R A N D E S, G E S E L L, U E D A, M IL L A N, E T A L unlabeled retinoic acid, eith er com pound contained in 5 xl solvent. The samples w ere incubated 18 hours in the dark, after which free ligand was separated from that bound to macromolecules by treatm ent with 100 xl dextran (0.05 percent)-coated charcoal (0.5 percent).19 Charcoal was rem oved by centrifugation for 15 m inutes at 1000 X g and 200 xl of the supernatant was layered onto 5 to 20 p ercen t linear sucrose gradients. G rad ien ts w ere cen trifu g ed two hours at 369,400 X g under gradient reorienting conditions in a Sorval TV 865 rotor and Sorval OTD 65 centrifuge. Two hundred jjli sam ples w ere collected from the gradients and the amount of radioactivity in each fraction was measured in a Tri-carb liquid scintillation counter. $ As standards, ovalbumin (4S) and whale myoglobin (2S) w ere run in parallel gradients. The amounts of protein w ere m easured by the m ethod of Lowry, et al.25 To estim ate the quantity of cellular retinoic acid binding protein present in the samples, the am ount of radioactivity present in the 2S region of th e g rad ien t was co n v erted to fem tomoles of retinoic acid bound. The [3H] retinoic acid bound in the presence of a 200-fold excess of nonradioactive retinoic acid subtracted from the total [3H] retinoic acid bound yields the retinoic acid specifically bound in th e sam ple. This quantity, divided by the amount of protein in the sample, yields the am ount of retinoic acid specifically bound p er mg of cytosol protein and is expressed as fmol per mg. A pooled sample of the Dunning R3327 tumor, previously shown to contain crabp,14 was used as a control for every assay reported here. Analysis of the binding data on the repeated assays of the pooled sample yielded a coefficient of variation (C.V.) of 16 percent for the assay and quantitation procedures. Results Sucrose density gradient centrifugation of cytosol prepared from the pancreas of the Syrian golden hamster revealed a peak of radioactivity in the 2S region, which could be abolished by excess unlabelled retinoic acid indicative of specific binding of [3H] retinoic acid. The 2S peak was not observed in the normal adult ham ster pancreas, whereas it was present in three adenocarcinomas of the ham ster pancreas: the CBP1 (figure 1), the Pour SG, and the Scarpelli (table I). In the hum an, crabp was detected in the fetal pancreas and in pancreatic carcinom a, w hile n o rm al adult pancreas tissue did not contain detectable amounts of the binding protein (table I). In every sample, binding of [3H] retinoic acid was observed in the 4S region FRACTION NUMBER F i g u r e 1. Representative sucrose density gradient profile of cytosol containing crabp, as seen in adenocarcinoma of the hamster pancreas, CBP. Similar profiles were seen in the Pour SG, Scarpelli LHC hamster tumors, in human fetal pancreas and in human pancreatic carcinoma (table I). O O [3H] RA: Cytosol extract incubated with tritium labeled retinoic acid only. [3H] RA x RA: Cytosol extract incubated with tritium labeled retihoic acid in the presence of 200 fold concentration of unlabeled retinoic acid.

4 TABLE I Binding of (%) Retinoic Acid by Cytoplasmic Retinoic Acid Binding Protein in Human and Hamster Pancreatic Tissues Hamster P a n c r e a s Normal (n = 6) CBPI (n = 6) Pour SG Scarpelli Human P a n c r e a s Normal adult (n = 2) Normal fetal (pooled sample) Cancer Case #1 Case #2 Case #3 Case #4 PA N C R E A T IC R E T IN O IC A C ID B IN D IN G P R O T E IN 4 03 fm o l/m g Not detectable 7805 ± fm o l/m g Not detectable Data are presented as the mean ± standard error of the mean where n was sufficient. Other data represent determinations made on individual cases or pooled samples * CBp38,39; P oursg31'32' Sarpelli35: Transplantable pancreatic duct carcinomas in Syrian Golden hamsters. of the sucrose density gradient profiles of which (figure 1) is representative. This binding was not saturable and was not abolished by excess unlabeled retinoic acid indicating the presence of a high capacity, low specificity class of bin d er. W hen the binding of [3H] retinoic acid in the 2S peak is converted to fmol [3H] retinoic acid bound per mg cytosol protein (fmol p er mg) it can be seen that the value is generally higher and less variable in the ham ster pancreatic cancers (e.g., CBP1: C.V. among samples = 45 percent) than in the hum an cancers (C.V. among samples = 79 percent) (table I). The greater variability in the contents of crabp in human pancreatic tissues is attributed to wide variations in connective tissue, the presence of areas of necrosis, and th e ad m ix tu re o f cr A B P-negative normal pancreatic acini in the samples of carcinoma. In the non-neoplastic tissue, crabp was detectable in the hum an fetal pancreas (1180 fmol per mg) whereas in both th e norm al a d u lt h am ster and hum an p an creas it was u n d e te c ta b le (table I). Assays for crabp in fetal ham ster pancreas could not be performed because of the impossibility of obtaining the amount of tissue required for the assay. Histologically, the ham ster experimental tum ors w ere strikingly similar to human pancreatic cancer, and showed an admixture of well differentiated and anaplastic components (figures 2 and 3). Discussion This study has shown that whereas demonstrable levels of crabp are present in hum an pancreatic carcinom a and in human fetal pancreas, this binding protein cannot be detected in normal adult pancreatic tissue. This may indicate for the first tim e in the hum an a correlation among developm ental stage, malignant transform ation and fluctuations in this binding protein in the pancreas. The expression of this binding protein in malignant tissues, after becoming und e te c ta b le in p o stn atal life, has b een com pared to the reappearance of some fetal proteins in some hum an malignant tum ors.8-29 These studies have also shown that dem onstrable levels of crabp are present in nitrosam ine-induced tran s plantable pancreatic carcinomas, in contrast to the absence of this binding protein in normal adult ham ster pancreas. It is of interest to note that the reappearance of crabp occurred in tum ors of similar histology w hether spontaneously arising, as in the hum an, or induced by carcinogen, as in the hamster. The expression of crapb in hum an pancreatic cancer may have implications in the possible endogenous modulation of tumor growth, in view of the postulated role of retinoids in the control of cell proliferation and in cell differentiation (see ref. 22 for review), apparently mediated, in some cases, through the interaction of retinoic acid with its binding

5 404 B RA N D ES, G E S E L L, U ED A, M ILLA N, E T A L protein (crabp)7'8-9-16'22' in a fashion similar to the interaction between steroid hormones and their receptors.817 It may be postulated that such ligandreceptor interactions could induce differ entiation of the anaplastic fraction of the cell population ordinarily present in pan creatic carcinoma, as well as the redifferentiation of the in-situ dysplastic ducts so frequently detected in this neoplasia. This retinoic acid mediated effect might result in a m ore hom ogenous, w ell-differen tiated tumor, presumably of less aggres sive behavior. In a similar fashion, the inhibitory effects of retinoic acid on DNA synthesis3746, also mediated through its binding protein, may contribute to slowing the rate of tumor progression. Previous studies from this and other laboratories on anim al and hum an tu mors have dem onstrated that pretreat ment with retinoids may potentiate the effects of conventional agents such as cy totoxic drugs and x-irradiation.1'2'4'61118 W hether or not retinoids may also en hance the effects of chem otherapeutic drugs and x-irradiation currently used in treating hum an pancreatic cancer re mains to be demonstrated. Acknowledgments The authors thank Dr. W. E. Scott of Hoffman-LaRoche, Inc., Nutley, New Jersey, for generously pro viding various retinoids used in this study, and the Gerontology Research Center (NIA) for space and facilities. Thanks are also due to Dr. Parvis Pour, Department of Pathology, University of Nebraska and to Dr. Dante Scarpelli, Department of Pathology, Northwestern University for making available exper imental tumor lines. F ig u r e 3. Human pancreatic carcinoma. This is a moderately differentiated area similar in histologic appearance to areas present in the experimental ad enocarcinomas of the hamster pancreas. X380. Representative photomicrographs revealing histo logical similarities between hamster and human pan F ig u r e 2. Transplantable adenocarcinoma of the creatic tumors are illsutrated in figures 2 and 3. The hamster pancreas (CBP). This shows an area of mod histologic composition of human and experimental erately differentiated carcinoma, as frequently ob hamster tumors is usually characterized by the pres served in many human pancreatic tumors. X380. ence of well differentiated, moderately differen tiated, and anaplastic areas.

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