[CANCER RESEARCH 50. 5829-583X September 15. 1990] Lorglumide and Loxiglumide Inhibit Gastrin-stimulated DNA Synthesis in a Rat Tumoral Acinar Pancreatic Cell Line (AR42J)1
Catherine Seva,2 Jean Luc Scemama, Marie JoséBastie, Lucien Pradayrol, and Nicole Vaysse InstituÃNational de la Sanie et de la Recherche Medicale. L'nité151,C.H. l '. Rangueil, Bat. 1.3, 31054 Toulouse Cedex, France
ABSTRACT Another class of potent cholecystokinin and gastrin receptor antagonists has been developed from a benzodiazepine analogue Many reports emphasized the role of gastrin as growth factor on isolated from Aspergillus alliaceus, asperlicin (18). In this fam normal gastrointestinal mucosa and pancreas. In the present study, we ily, L364.718 (19) has been found to be the most potent analyzed the proliferate effects of cholecystokinin (CCK) and gastrin antagonist available until now on the pancreatic CCK1 receptor peptides on a rat tumoral pancreatic cell line, AR42J, which possesses both CCKAand CCKB receptor subtypes. with the same affinity as the synthetic peptide, CCKg. The results showed a good correlation between the binding of gastrin L364,718, used at micromolar concentrations, is able to inhibit to CCKBreceptor |A., 1.125 ±0.3(SD) n\i| and its ability to either induce gastrin binding and to antagonize biological events induced by ornithine decarboxylase activity |50% effective concentration, 0.6 ±0.3 gastrin peptides such as gastric smooth muscle contractions n\i| and |'H|-thymidine incorporation [50% effective concentration, 2 ± (20). 0.4 IIM|.Furthermore, the ability of different cholecystokinin and gastrin Pancreatic acinar cells from dog and guinea pig have been antagonists such as proglumide and asperlicin derivatives (respectively, shown to possess two classes of CCK receptors (21, 22). The CR1409, CR1505, and L364.718) were tested. We found that all antag onists displaced '"I-labeled gastrin binding, with the following order of CCKA receptor, having a high affinity for CCK and a low affinity for gastrin peptides, is involved in pancreatic enzymatic potencies: L364.718 > CR1409 > CR1505 > proglumide. Furthermore, secretion. By contrast, the CCKB receptor does not discriminate the 50% inhibitory concentration of CRI409 and CR1505 to inhibit gastrin stimulated ornithine decarboxylase activity (an early event in between CCK and gastrin peptides, and biological events related volved in cell proliferation) and |'H|thymidine incorporation were in to this pancreatic CCKB subtype are still poorly documented. agreement with their constants of inhibition (A,) on gastrin binding. The We have shown previously that a rat tumoral pancreatic cell 1.364.7IX compound, at a concentration which fully occupied the CCKA line (AR42J) possesses both CCKA and CCKB receptor subtypes without affecting the CCKB, had no effect on gastrin stimulated ornithine (23) as described on dog (21) and guinea pig (22) pancreas. We decarboxylase activity and |'I l|th>miiliiu- incorporation. In addition, this have also demonstrated that occupancy of CCKB receptor by compound appeared to be a full agonist on CCKBreceptor. gastrin stimulates early events associated with cell proliferation These results confirm the implication of the CCKB receptor in the such as ornithine decarboxylase activity (24) and activation of proliferativi response of AR42J cells to gastrin. the Na+/H+ antiporter (25). In the present study we analyze the effects of gastrin on the proliferation of AR42J cells, measuring ODC activity modula INTRODUCTION tion and ['Hjthymidine incorporation. We also examine the Gastrin and its structural analogue pcntagastrin have been abilities of CR 1505, CR 1409, and L364.718 to antagonize these shown to modulate the growth of normal gastrointestinal mu gastrin-induced effects. cosa (1) and pancreas (2-4) through stimulation of protein and DNA synthesis. Recent studies have pointed out that these MATERIALS AND METHODS peptides act also as growth factors on gastrointestinal cancers. Gastrin and pentagastrin stimulate the growth of colon carci Chemicals. 1!SI-BH[A'-succinimidyl-3-(4-hydroxy-[5-125I]iodophenyl) propionate) (specific activity, 2000 Ci/mmol) and [5'-'H]thymidinc nomas in vivo (5) and transplanted into the nude mice (6). In addition, they induce proliferation of colon and gastric cancer (specific activity. 18 Ci/mol) were obtained from Amersham, France. The COOH-terminal nonapeptide (Thru, Nle,7)CCK,,.3, (CCK,) and cell lines (7, 8). human gastrin:_|7ds were purchased from Bachern, Switzerland. Bovine Proglumide is a weak although competitive antagonist of serum albumin, soybean trypsin inhibitor, and bacitracin were from gastrin receptors (9), requiring large doses in vivo as well as in Sigma, France. Lorglumide (CR1409) and loxiglumide (CRI505) were vitro. This antagonist has been found to inhibit gastrin-stimu- kindly provided by Dr. L. Rovatti (Rotta Research Laboratory. Milan. lated acid secretion (10). Recently, it has been reported to Italy). L364.7I8 was a gift from Dr. P. S. Anderson (Merck, Sharp and prevent gastrin-induced trophic effects on normal gastrointes Dohme Research Laboratories, West Point, PA). tinal mucosa (11) and colorectal carcinomas (12-14). By con Cell Culture. AR42J cells, originally obtained by Jessop and Hay trast, some recent reports have shown either a stimulating effect (26) from a rat exocrine pancreatic tumor (azaserine induced), were a gift from Dr. C. Logsdon (San Francisco, CA). These cells were of proglumide when used at high concentrations (15) or a routinely grown in Dulbecco's modified Eagle's medium (glucose, 1 g/ nonspecific growth-restricting effect on a fibroblast cell line liter), supplemented with 10% fetal calf serum. Cells were plated at 5 which does not possess gastrin receptors (16). These findings x IO5cells/ml and the medium was changed every 2 days. have pointed out the necessity for development of more potent For incorporation assay of ['Hjthymidine. cells were plated into 35- derivatives like lorglumide (CR1409) and loxiglumide mm-well dishes, allowed to attach overnight, and then cultured in (CR1505) (17). medium containing 1% fetal calf serum during 18 h to increase the number of cells in the G0-G, phase, prior to serum or hormone addition. Received .1/6/90: accepted 6/4/90. The costs of publication of this article were defrayed in part by the payment Ligand Binding Studies. Binding assays were carried out on isolated of page charges. This article must therefore he hereby marked advertisement in cells as described previously (24). Briefly 1.2 and 2.4 x 10' cells were accordance with 18 I'.S.C. Section 1734 solely to indicate this fact. 1The present work was supported by grants from the Association pour la 3The abbreviations used are: CCK. cholecystokinin: ODC. ornithine decar Recherche sur le Cancer (6414). EEC (ST¡JOOI-.1F). and RégionMidi-Pyrénéesboxylase; I2'I-BH. '"l-labeled Bollónand Hunter reagent [,V-succinimidyl-.1-(4- (8700777). hydroxy[5-'"l|iodophenyl)propionate]; G2_i7ds. 16desulfatcd human gastrinj 17; 2To whom requests for reprints should be addressed, at INSERIVI U 151. ICso, concentration of drug that inhibited by 50rr: ECM. concentration of drug C.H.U. Rangueil. Bat. L.I. .11054 Toulouse. France. that induced 50r; of the maximal response. 5829
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respectively incubated with 60 pmol of i:i5I-Bolton & Hunter-CCK,, and 100 120 pmol of I2il-Bolton & Hunter-Gj.nds and various concentrations of analogues or antagonists in a Krebs-4-(2-hydroxyethyl)-l-piperazine- E ethanesulfonic acid buffer in a total volume of 0.5 ml at 25°Cuntil 3 equilibrium (30 min). Specific binding was defined as the excess binding "x over that in blanks containing lO^*1Munlabeled peptide. 10 75 Ornilhine Decarboxylase Assay. ODC activity was measured on E cytosolic extracts from AR42J cells as described in Ref. 24 with minor modifications. Briefly, cells were lysed by liquid N2 freezing and thawing followed by a 10-min centrifugation at 100,000 x g. Supernatants were incubated in the presence of pyridoxal 5-phosphate (50 MM).i.-['4C]- ornithinc (0.25 /jCi) and L-ornithine (0.2 mxi). An aliquot was stored 50 for protein measurement according to the method of Bradford (27). I'HjThymidine Incorporation Assay. DNA synthesis was estimated by measurement of ['H]thymidine incorporation into the trichloroacetic O O acid-precipitable material. The cells were cultured as described previ o ously and treated with agents twice a day. I'HjThymidine (0.1 /jOi/ml) 25 was added during the last hour of a 24-h treatment period. Cells were then washed with serum-free medium to remove unincorporated |'HJ- thymidine. and the DNA was precipitated with 5% trichloroacetic acid at 4°Cfor 15 min. Precipitates »erewashed twice with 95% ethanol and dissolved in 1 ml of 0.1 N NaOH. Radioactivity was measured by 00 liquid scintillation counting. o -11 -10 -9 -8 -7 -6 Gastrin concentrations (LOG M) RESULTS loglXVX - 1) Binding Assays. In Fig. 1, proglumide and its derivatives B (CR1409 and CR1505) were compared with G^,7ds for their ability to inhibit I25I-BH-G2 nds binding. All these compounds decreased radioligand binding in a dose-dependent manner. CR1409 was the most potent of the proglumide derivatives with a 1C»,of3 ±0.2(SD) /IMwhereas CR1505 and proglumide inhibited i:sI-BH-Gv ,7ds binding with lower potencies (respec tively, ICio 13 ±0.2 /iM and >18 m\i). Compared with L364J18 (1C,,, 0.35 ±0.1 MM)CR1409 was 10-fold less effi cient to displace I25I-BH-G: ,7ds binding on AR42J cells.
100»
X (0
o 654 m Antagonist concentration (-log M) Fig. 2. A, ability of CF1409 antagonist (o inhibit gastrin-stimulatcd ODC i activity in AR42J cells. AR42J cells cultured in serum-free medium for 18 h were (N incubated with rising concentrations of gastrin (O) alone or in the presence of O different concentrations of CRI409 (I, x; 10. D; 100 0M. V) for 2 h. Results are the means of 4 different experiments each performed in duplicate. Basal and I maximal ODC' activity averaged, respectively. 2.2 ±0.1 and 5.5 ±0.2 nmol CO2/ m h/mg protein. B, Schild plot analysis of the inhibition curves with CR 1409 from A. The values have been calculated by loglconcentrations ratio - I) versus log antagonist.
Hofstee plot (28) representations of the data were found to O -11 -10 -9 -8 -7 -6 -5 -4 -2 be linear, demonstrating the interaction with a single class of binding site (data not shown). Concentration (LOG M) ODC Activity. As shown previously, Gi-ivds increased ODC Fig. 1. Displacement of '"I-BH-Gj ]7ds binding on AR42J cells by antago nists. '!'l-BH-G2_,,ds (120 pM) was incubated with AR42J cells (2.4 X 10') for 1 activity in a dose-dependent manner. The gastrin EC5(>was 0.6 h at 25°Cin the presence of various doses of the different molecules tested ±0.3 niM(Figs. 2 and 3). The maximal effect reaching 5.5 ± (Gj.„,•:I..164.718. X; CRI409. D; CRI505. V; proglumide. O). Each point 0.2 nmol CO^/h/ mg protein was obtained with 10 n\i gastrin. represents the means of 5 independent experiments each performed in duplicate. Total binding averaged 2.9 ±0.8'V of total radioactivity and nonspecific binding We next examined the effects of CR1409 and CR1505 on averaged 0.2 ±0.01%. gastrin-stimulated ODC activity. CR1409 (Fig. 2A) produced a 5830
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100 CR1505 (slope, 1.2). The comparison of the two PA2 values obtained with CR1409 and CR1505 (respectively, 6.5 ±0.4 and 5.25 ±0.2) indicated, like binding studies, that CR1409 was the more potent of these two antagonists. Fig. 4 shows the remaining gastrin-stimulated ODC activity in percentage of maximum obtained with CR1409 and CR1505. In these experiments, gastrin was used at a concentration which produces the maximal response (10 nM). In presence of 10 ^M CR1409 or CR1505, respectively, 72 ±2% and 33 ±1% inhibitions were obtained. A dose of 100 ¿IMCRI409 caused a 96 ±5% inhibition of gastrin-stimulated ODC activity, while CR 1505 at the same concentration causes a 67 ±6%inhibition. At a concentration of 1 n\i, L364.718 occupies the CCKA receptor without affecting the CCKB (24) and does not prevent gastrin-stimulated ODC activity (Fig. 4). When used alone at 1 nM, this antagonist had no effect on basal ODC activity but higher concentrations caused a dose-dependent stimulation (Fig. 5). A maximum of 5.3 ±0.1 nmol COi/h/mg protein was obtained with 10 ^M L364.718, showing that this compound o & O -11 -10 -9 -8 -7 acts as an agonist on CCKB receptor-stimulating ODC activity. On the other hand, when used alone, even at the highest Gastrin concentrations (LOG M) concentration tested (100 >¿M),neither of the two proglumide derivatives had a significant effect on ODC activity (Fig. 5). loglXVX - 1) [•'H|ThymidineIncorporation. On the basis of prior studies in B which we observed that serum deprivation increases the ratio of cells in the Go-G, phase to 80% (23), we used similar conditions to test gastrin effects on AR42J cell proliferation by ['Hjthymidine measurement. The optimal peptide stimulation period was found to be 24 h before ['Hjthymidine addition in the culture medium. As shown in Fig. 6, gastrin stimulated pHjthymidinc incor poration in a dose-dependent manner. A 10 nM gastrin concen Slope : 1.2 tration induced a maximal stimulation of 145 ±5% of control. Half-maximal stimulation (EC50) was produced by 2 ±0.4 nM. Concentrations lower than 1 nM had low or no significant effects on ['H]thymidine incorporation. Surprisingly, CCK9 tested in the 0.1-100 nM concentration range did not stimulate ['Hjthymidine incorporation (Fig. 6). We analyzed the effects of the antagonists on gastrin-stimu-
PA2 •5.25
543 Antagonist Concentration (-log M) 100 Fig. 3. A. ability of CR1505 antagonist to inhibit gastrin-stimulated ODC activity in AR42J cells. The cells cultured in serum-free medium for 18 h were x incubated with rising concentrations of gastrin (O) alone or in the presence of CO different concentrations of CFI505 (10, G; 100 nM. V) for 2 h. Results are the 75 m means of 4 different experiments each performed in duplicate. Basal and maximal ODC activity averaged, respectively. 2.2 ±0.1 and 5.5 ±0.2 nmol CO,/h/mg protein. B. Schild plot analysis of the inhibition curves by CR1505 from A. The values have been calculated by log(concentrations ratio —I) versus log antagonist. 8 so parallel rightward shift in the initial gastrin dose response, with no change in the maximal response. Furthermore, the magni tude of the shift was proportional to the concentration of the 25 antagonist. At concentrations of 1, 10, and 100 ¿¡Mtheshift of the gastrin dose response was respectively about 5, 50, or 500. A Schild plot (29) of the data (Fig. 2B) gave a slope of 0.9, indicating a competitive antagonism. Similar studies with CR1505, at 10 and 100 »M(Fig. 3/1), Fig. 4. Remaining gastrin-stimulatcd ODC activity obtained with antagonists. also gave a parallel shift to the right of the gastrin dose response, AR42J cells were cultured as described in Figs. 2 and 3 and treated for 2 h with proportional to the concentration of this antagonist (respec 10 n\i gastrin (D) alone or in the presence of various concentrations of the different antagonists (L364.718, 1 nM, D; CR1505. 10 and 100 „M.QCRI409. tively, shifts about 3 and 30). A Schild plot of the data (Fig. 10 and 100 JIM. •).Values are means of 3 separate experiments performed in 3Ä) was in agreement with a competitive antagonism of duplicate. 5831
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studies have used the nonpeptidic antagonist proglumide to prevent the gastrin-promoting growth effects and to demon strate the mediation via a gastrin receptor. We have found previously that AR42J cells possess both O a CCKA and CCKB receptor subtypes and that CCKB receptor is D) coupled to an early event involved in cell proliferation/stimu lation of ODC activity (24). In the present study, we were able to demonstrate that occu CM pation of CCKB receptor subtype in AR42J cells led to an Oa increase in cell proliferation as measured by ['Hjthymidine incorporation. o E For the first time, we also compare the ability of different e antagonists available to inhibit either gastrin binding on CCKB receptor subtype or gastrin-stimulated ODC activity and cell proliferation. In the first part of this work, using i:5I-BH-G2_17ds as radi- oligand. we compared the ability of proglumide and its deriva tives to occupy gastrin receptors on AR42J cells. The relative order of potencies was CR1409 > CR1505 » proglumide (respective IC50 3 ftM > 13 pM » 18 IHM).The same order of potencies was observed when we displaced I25I-BH-CCK9 bind Concentration (LOG M) ing by these compounds (data not shown). IC50s were, respec Fig. 5. Antagonist effects on basal ODC activity in AR42J cells. Cells were tively, 15 fiM > 25 ftM » 4.8 HIM.As shown by these results, cultured as described in Figs. 2 and 3 and treated with \arious concentrations of the different antagonists for 2 h (control.«; L364.718. •:CR1505. D; CR1409. these antagonists display a low selectivity between CCKA and D). Values are means of .1separate experiments performed in duplicate. CCKH receptors on AR42J cell line. In our previous studies, L364.718, a derivative of asperlicin, 150 presented a high degree of selectivity toward the two receptors on AR42J cells (24). Indeed, the binding affinity of this antag onist for the CCKA receptor (IC5I>0.2 ±0.1 n\i) was about 2000 times higher compared to its affinity for the CCKB (IC50 0.35 ±0.1 /UM)and had the same affinity as CCK,, on its own receptor. These data are in agreement with those obtained by- Jensen et al. (30) on acini from guinea pig pancreas. In their studies, L364.718 discriminates in the same range of potencies between CCK\ and CCKB receptor subtypes. CR1409 (IC50 3 ±0.2 f
Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. INHIBITION OF GASTRIN-STIMULATED DNA SYNTHESIS BY ANTAGONISTS stimulates the terminal event of cell proliferation/DNA synthe 13. Bcauchamp. R. D., Townsend, C. M.. Singh. P.. Glass, E. J., and Thompson, sis as shown by ['HJthymidine incorporation. Preliminary ex J. C. Proglumidc. a gastrin receptor antagonist, inhibits growth of colon cancer and enhances survival in mice. Annu. Surg., 202: 303-309, 1985. periments have shown that the maximal mitotic effect of gastrin 14. Smith. J. P., and Solomon, T. E. Effects of gastrin, proglumide and soma- was observed after 24 h of treatment in serum-deprived me tostatin on growth of human colon cancer. Gastroenterology, 95: 1541-1 548. 1988. dium. Treatment twice a day was necessary to see a growth- 15. Vamaguchi, T.. Tabata. K.. and Johnson. L. R. Effect of proglumide on rat promoting effect of the peptide on AR42J cells. In these con pancreatic growth. Am. J. Physiol., 249: G294-G298. 1985. ditions, gastrin stimulates [1H)tbymidine incorporation with an 16. Imdahl. A., St. Eggstein, C., Crone, C., and l-arthmann, E. H. Growth of colorectal carcinoma cells: regulation in vitro by gastrin pentagastrin and the EC50 of 2 ±0.4 n\i. The results presented here show a good gastrin-receptor antagonist proglumide. J. Cancer Res. Clin. Oneol.. 115: correlation between the binding of gastrin on CCKB receptor 388-392. 1989. 17. Rovati, L. C., Ban!. M., Makovec. F., Revel, L., and Setnikar. I. Lorglumide (Kd 1.125 ±0.3 nM) and its ability to induce ODC activity and loxiglumide: two potent and specific antagonists of peripheral CCK. In: (EC50 0.6 ±0.3 nM) and DNA synthesis (EC,,, 2 ±0.4 nivi). J. P. Bali and J. Martinez (eds.). Gastrin and CCK, Chemistry. Physiology and Pharmacology, pp. 45-48. Amsterdam: Elsevier. Science Publishers Furthermore, we show the potency of CR1409 and CR1505 B.V., 1988. to inhibit gastrin-stimulated proliferation in vitro. These data 18. Chang. R. L. S.. Lotti, V. J.. Monaghan. R. L., Birnbaum. J., Stapley, E. O., fit well with their abilities to occupy the CCKB receptor subtype Goet/, M. A., Albers-Schonberg. G., Patchett. A. A., Liesch. J.. llensens, O. and to block gastrin-stimulated ODC activity. These results D.. and Springer. J. P. A potent non peptidic cholecystokinin antagonist selective for peripheral tissues isolated from Aspergillus aUiaceus. Science confirm the important role played by the CCKB receptor sub (Washington DC). 230: 177-179. 1985. type in the mediation of the proliferative events induced by 19. Bene. E., Bock. M. G.. Rittle. K. E., Dipardo. R. M., Whitter. W. L., Veber, D. F., Anderson. P. S., and Freidingcr. R. M. Design of potent orally effective gastrin on this cell line. non peptidal antagonists of the peptidic hormone cholecystokinin. Proc. Nati. Acad. Sci. USA. «.-4918-4922, 1986. 20. Huang. S. C.. Zhang, L.. Chiang. H. C. V.. Wank. S. A.. Matón. P. N.. REFERENCES Gardner, J. D.. and Jensen. R. T. Benzodia/epine analogues L365.260 and L364.7I8 as gastrin and pancreatic CCK receptor antagonists. Am. J. Phys iol.. 257.-G169-G174. 1989. 1. Johnson. L. R. Gastrointestinal hormones and their functions. Annu. Rev. 21. Fourmy, D., /ah¡di. A.. Fahre. R.. Pradayrol. L... and Ribet. A. Receptors Physiol.. 39: 135-158. 1977. 2. Folsch. U. R. Regulation of pancreatic growth. Clin. Gastroenterol.. 13:679- for cholecystokinin and gastrin peptides display specific binding properties and are structurally different in guinea pig and dog. Eur. J. Biochem., 765: 699, 1984. 683-692. 1987. 3. Solomon. T. E.. Morisse!. J.. Wood, J. G.. and Bussjacger, L. J. Additive 22. Yu, D. H.. Noguchi. M.. Zhou. C.. Villanueva. M. L.. Jensen. R. T.. and interaction of pentagastrin and secretin on pancreatic growth in rats. Gastro- Gardner. J. D. 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Role of a rat pancreatic cell line. Am. J. Physiol.. 256: G846-G850. 1989. gastrin and gastrin receptors on the growth of a transplanted mouse colon 25. Bastie, M. J.. Delvaux. M.. Dufresne. M.. Saunier-Blanche. J. S., Vaysse, carcinoma (MC 26) in BALB/c mice. Cancer Res.. 46: 1612-1616. 1986. N., and Ribet, A. Distinct activation of Na*/H* exchange by gastrin and 7. Watson, S. A.. Durrani. L. G.. Crosbie. J. D.. and Morris. D. L. The in vitro CCK peptides in acini from guinea pig. Am. J. Physiol., 254: G25-G32, growth response of primary human colorectal and gastric cancer cells to 1988. gastrin. Int. J. Cancer, 43: 692-696. 1989. 26. Jessop. H. W.. and Hay. R. J. Characteristics of two rat pancreatic exocrine 8. Watson, S.. Durrani. L.. and Morris. D. Gastrin: growth enhancing effects cell lines derived from transplantable tumors. In Vitro (Rockville), 16: 212. on human gastric and colonie tumours cells. Br. J. Cancer, 59: 554-558. 1980. 1989. 27. Bradford, M. M. A rapid and sensitive method for the quantitation of 9. Rovati. A. L. The relationships between chemical structure of a new dicar- microgram quantities of protein utilizing the principle of dye binding. Anal. boxylic amino acid derivative and antigastrin activity in the rat. Br. J. Biochem., 72: 248-254. 1976. Pharmacol.. 34: 677-682. 1968. 28. Hofstee. B. H. J. An evaluation of constants I'm and Km in enzyme reaction. 10. Magous. R., and Bali. J. P. Evidence that proglumide and benzotript antag Science (Washington DC). 7/6:329-331. 1952. onize sccretagogue stimulation of isolated gastric parietal cells. Regul. Pep- 29. Schild. H. O. PAx and competitive drug antagonism. Br. J. Pharmacol., 4: tides. 7:233-241, 1983. 277-280, 1949. 11. Johnson, L. R.. and Guthrie. P. D. Proglumide inhibition of trophic action 30. Jensen, R. T., Von Schrenck, T., Yu. D.. Wank. S. A., and Gardner. J. D. of pentagastrin. Am. J. Physiol.. 246: G62-G66. 1984. 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Catherine Seva, Jean Luc Scemama, Marie José Bastié, et al.
Cancer Res 1990;50:5829-5833.
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