Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6696-6700, July 1993 Biochemistry Tumors in hepatobiliary tract and pancreatic islet tissues of transgenic mice harboring gastrin simian virus 40 large tumor antigen fusion gene (developmental regulation/gene expression) ANTHONY G. MONTAG*, TATSUZO OKAt, KWANG HEE BAEKt, CHEL SOON CHOIt, GILBERT JAYt, AND KAN AGARWALt§ Departments of *Pathology and tBiochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637; and tLaboratory of Virology, Jerome H. Holland Laboratory, American Red Cross, Rockville, MD 20855 Communicated by Donald F. Steiner, April 19, 1993

ABSTRACT Gastrin is expressed in the gastric antrum A GPl.5Tag and in fetal pancreatic islets but not in adult islets. We have now identified the hepatobiliary tract as another, previously un- EcoRl Pvu II TATA PstI AUG Pvu II BamHI known, potential site of gastrin gene expression. Two human I I lI I I gastrin simian virus 40 large tumor antigen (SV40 T antigen) ______s* poly(A) Human gastrin (1.5 Kbp) fusion genes containing 1.5 kb of 5' flanking sequence and 10.5 T antigen (2.7 Kbp) kb that included 5.5 kb upstream, 1.5 kb downstream, and the entire transcribed region were used to generate transgenic B GP1O.5Tag mice. Analysis of several transgenic lines, derived from both * 13.5 Kpb - fusion genes, revealed development of transmissible hepatobil- EcoRl TATA EcoRl HindIli XhoI iary tract tumors and pancreatic islet cell tumors. Analysis of I I oma each of the tumor cells demonstrates expression of SV40 T antigen but no expression of gastrin. Of the two fusion genes, 5.5 Kpb 1.5 Kbp only the 10.5-kb sequence induces hyperplasia of gastrin- Bgeln ( Apal producing cells in the antrum. Analysis of these cells demon- T antigen (3.0 Kbp) strates expression of SV40 T antigen and gastrin, suggesting FIG. 1. Structures of the human gastrin gene regions fused with that the 10.5-kb sequence is sufficient for gastrin cell hyper- SV40 T antigen. (A) Schematic of the human gastrin gene 5' flanking plasia in the antrum. These data raise the possibility that 1.5-kb region fused with SV40 Tag gene. This DNA construct is gastrin is transiently expressed in the hepatobiliary tract. designated as GP1.5 Tag. The dark box represents exon 1 (8). The position ofthe poly(A) site is indicated. (B) Map ofthe human gastrin The gastrointestinal peptide gastrin regulates gastric acid gene fused with SV40 Tag gene. The site of the Tag gene insertion is secretion and stimulates gastric mucosal as well as pancreatic also indicated. The AUG codon of the human gastrin gene was growth (1, 2). In the stomach, gastrin is expressed in G cells mutated to GUG codon. This DNA construct is designated as GP10.5 localized in the antral mucosa, beginning during the weaning Tag. period and continuing throughout adult life (3, 4). In addition, kb upstream, 1.5 kb downstream, and the entire transcribed expression ofgastrin in developing fetal islets of Langerhans region, in transgenic mice. has been observed in some species, such as rat, but is subsequently repressed in the adult tissue (5). This selective MATERIALS AND METHODS expression in fetal islets and subsequent repression in adult islets may indicate a regulatory role of gastrin in terminal Construction of Fusion Genes and Production of Transgenic differentiation of the pancreatic islets (3, 5). Gastrin- Mice. All DNA manipulations were carried out as described producing islet cell tumors occur in humans, suggesting that (7). The human gastrin gene 1.5-kb 5' flanking region was loss of gastrin repression can accompany neoplastic trans- excised from plasmid p235 (8) by digestion with EcoRI and formation ofislet cells; however, such tumors rarely produce Pst I and then subcloned into the same sites of pUC9. The insulin, in keeping with their embryonal character. recombinant plasmid was digested with Pst I, blunt ended by Transcriptional regulatory elements in the human gastrin Klenow fragment, and then ligated to SV40 Tag sequences gene have recently been defined by transfection ofpermanent contained in a 2.9-kb Bgl I-Apa I DNA fragment (whose ends gastrin-expressing islet cell lines with a fusion gene contain- had also been made blunt by the T4 DNA polymerase). The ing elements from the human gastrin gene promoter fused to recombinant plasmid, designated GP1.5 Tag, consists of 1.43 a reporter gene. Positive and negative elements that control kb from the 5' flanking region and 62 nucleotides from the islet cell-specific transcription are located between nucleo- untranslated region of the human gastrin gene linked to the tides -108 and -76 (6). Whether the same elements specify SV40 genome encoding Tag and small tumor antigen (Fig. gastrin transcription in antral mucosa is not known inasmuch 1A). as G-cell-derived cell lines are not available. To overcome The construction of GP10.5 Tag involved inactivation of this problem, we have analyzed the expression of two fused two HindIII sites located in the untranslated region of P1042 genes, one containing the simian virus 40 large tumor antigen DNA (8) and alteration of the initiator methionine codon of (SV40 Tag) gene fused to the gastrin gene 1.5-kb 5' flanking human gastrin to a valine codon by site-directed mutagenesis. sequence and the other to a 10.5-kb region that includes 5.5 The EcoRI-EcoRI fragment from p235 DNA was excised and

The publication costs of this article were defrayed in part by page charge Abbreviations: SV40, simian virus 40; T, large tumor antigen; H&E, payment. This article must therefore be hereby marked "advertisement" hematoxylin/eosin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 6696 Downloaded by guest on September 24, 2021 Biochemistry: Montag et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6697 ligated to the EcoRP site of the mutated p1042 DNA. Thus, DNAs from GP1.5 Tag and GP10.5 Tag mice were digested the pUC8 plasmid contained the 10.5-kb gastrin gene in which by Pvu II to generate an =2.0-kb fragment and by EcoRI plus two of the three HindIII sites were inactivated by site- BamHI to generate a 4.3-kb fragment (Fig. 1). Southern blots directed mutagenesis (7). The internal EcoRI located in large were probed with an EcoRI-BamHI fusion gene fragment intron (8) was also inactivated by mutagenesis. This plasmid, labeled by nick-translation to a specific activity of =2 x 108 designated pGP10.5, was then digested with HindIII, blunt- cpm/,ug of DNA. ended by Klenow, and then ligated to the same blunt-ended Total RNA was extracted from deep-frozen tissues after 2.9-kb Bgl I-Apa I DNA fragment as described above. This homogenization in a 4 M guanidine thiocyanate solution and GP10.5 Tag construct contained the entire gastrin gene pelleting through a cushion of CsCl, as described (10). Total including flanking regions and the Tag coding region inserted RNA was analyzed using an S1 nuclease protection assay at the HindlIl site ofexon 2 (Fig. 1B; see ref. 8). Replacement employing single-stranded cDNAs specific for Tag, mouse of the gastrin gene methionine codon by valine should allow gastrin, and mouse insulin II mRNAs. The Tag single-stranded translation initiation at first AUG codon of the SV40 T probe consisted of Stu I-Stu I fragment representing map antigen open reading frame (Fig. 1B). positions 5190-5230 of SV40 and -15 to +61 of gastrin Both plasmids, GP1.5 Tag and GP10.5 Tag, were digested promoter. This 120-nucleotide fragment was subcloned in with EcoRI, BamHI, Xho I, and FnuDII, and the fragments M13mpl8. Single-stranded labeled probe complementary to containing fusion genes were purified by sucrose gradient mRNA was prepared by the primer extension-restriction centrifugation. The DNA fragments, free from plasmid se- enzyme digestion method, as described (11). Extension of quences, were used for microinjection of CD1 oocytes (9). primer in primer-phage DNA complex by Klenow in the DNA and RNA Isolation and Detection. DNA was extracted presence of [y-32P]dATP resulted in double-stranded DNA from tail biopsies and analyzed by Southern blotting (7). Tail that was cleaved by BamHI. The single-stranded probe was

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FIG. 2. Histological and immunocytochemical analysis of pancreas, hepatobiliary tract, and gastric antral mucosa. (A-C) Pancreas from GP1.5 Tag mouse showing islet cell tumor and islet hyperplasia. (A) H&E stain. Islet tumor and islet hyperplasia are indicated by straight and curved arrows, respectively. (B) Insulin staining. (C) T antigen immunostaining. (D-F) Pancreas from GP10.5 Tag mouse showing islet tumor immunostaining. (D) H&E staining showing islet tumor (straight arrow) and nonhyperplastic islets (curved arrow). (E) Insulin immunostaining. (F) T antigen immunostaining showing nuclear staining in islet tumor (straight arrow) but not in adjacent normal islet (curved arrow). (G and H) Bile duct lesions from GP1.5 Tag mouse. (G) H&E stain of bile duct carcinoma. (H) T antigen immunostaining of hyperplastic bile ducts. (J and K) Hepatic lesions in GP10.5 Tag mouse. (J) H&E stain of hepatoceliular carcinoma (arrow) and adjacent dysplastic liver. (K) T antigen immunostaining showing reactivity in hepatocellular carcinoma (straight arrow) and bile duct cyst (curved arrow). (Iand L) Stomachs from GP1.5 Tag mouse and GP10.5 Tag mouse, respectively. (I) Gastrin immunostaining showing scattered gastrin-positive cells without hyperplasia. (L) Gastrin immunostaining showing microscopic neuroendocrine tumors. Downloaded by guest on September 24, 2021 6698 Biochemistry: Montag et al. Proc. Natl. Acad Sci. USA 90 (1993) isolated by 8 M urea/PAGE as a 196-nucleotide fragment, of mouse eggs. The GP1.5 Tag and GP10.5 Tag DNA constructs which the 108-nucleotide region corresponded to Tag-human- resulted in six and five founder animals, respectively. These gastrin mRNA hybrid (designated as Tag probe). The mouse animals contained 5-106 copies of the newly integrated gastrin probe was prepared by subcloning the HindIII-Nco I sequences. Several lines from each construct were propa- region (447 bp; T.O. and K.A., unpublished results) into gated (data not shown). Analysis of several generations of HindIII-Xba I sites of M13mpl8. Primer extension was per- offspring revealed =50% transmission with crosses to non- formed from the phage DNA as described above and the DNA transgenic CD1 animals. was then digested by Nco I. The 244-nucleotide probe con- Development of Tumors in Hepatobiliary Tract. Animals tained 182 nucleotides complementary to the mouse gastrin harboring GP1.5 Tag DNA were clinically normal at a young HindIII-Nco I gene region (designated as gastrin probe). age but developed symptoms of weakness by 75-90 days of The mouse insulin II probe was prepared by subcloning the age. Necropsy, at 90-100 days of age, revealed numerous Sma I-Nde I region (200 bp; ref. 12) into the Sma I site of solid white tumor nodules in the liver. Histologic examination M13mp18. Primer extension was performed from the phage of these tumor tissues characterized them as bile duct car- DNA as described above and the DNA was digested by Sma cinomas composed of glandular acini with marked nuclear I. Of the 240-nucleotide single-stranded probe, 170 nucleo- pleomorphism (Fig. 2G), usually associated with hyperplasia tides were complementary to the mouse insulin II mRNA of bile ducts within portal tracts (Fig. 2H). In addition, all (designated as insulin II probe). Each of the single-stranded animals had some degree of hepatocellular dysplasia as probes (2-4 x 105 cpm) was hybridized in 15 pl with the evidenced by large bizarre hepatocytes (data not shown). T indicated amount of total RNA as described (13). These antigen was expressed in dysplastic bile duct epithelium and samples were treated with 200 units of S1 nuclease in 200 ul biliary tumors (Fig. 2H) as well as in occasional dysplastic at 37°C for 30 min. After processing of the reaction mixtures, hepatocytes as evidenced by Tag-positive staining. Interest- the samples were analyzed by 8 M urea/8% PAGE gels. ingly, however, no gastrin expression in hepatic tumors or in Immunohistochemistry. Tissues were fixed in either forma- the native parenchyma was revealed by immunohistochem- lin or Bouins' fixative. Paraffin-embedded tissues were sec- istry (data not shown). These results are in complete agree- tioned at 5 ,um. Sections were stained with hematoxylin/eosin ment with the results of Tag and gastrin mRNA studies. Tag (H&E) or were incubated with antisera to T antigen (gift from mRNA was expressed in the tumor tissue (Fig. 3A), whereas D. Hanahan) and neuroendocrine peptides (rabbit anti-gastrin, mouse gastrin mRNA was not detectable (Fig. 3B), by S1 glucagon, vasoactive intestinal peptide, and cholecystokinin, nuclease analysis. DAKO; rabbit anti-somatostatin, Immunoclear; guinea pig The animals carrying GP10.5 Tag DNA also showed biliary antiserum to insulin, DAKO). Antigen-antibody complexes duct changes, with biliary duct cysts and dysplasia, but were visualized with the avidin-biotin-peroxidase technique withoutfully developed bile duct tumors ofthe kind seen in the (14). Briefly, following a 1-hr incubation with the primary GP1.5 Tag animals. GP10.5 Tag animals developed hepato- antibody at 25°C, sections were incubated for 30 min with cellular dysplasia and hepatocellular carcinoma, often multi- biotinylated goat anti-rabbit or anti-guinea pig IgG (Zymed focally throughout the liver (Fig. 2J). T antigen was present in Laboratories; 1:40 diluted, with 10o mouse serum). A final tumor nodules, dysplastic hepatocytes, and bile duct cysts, 45-min incubation was performed with ABC complex (Vector but not uniformly in normal hepatocytes or in bile duct Laboratories) using diaminobenzidine as substrate. epithelium (Fig. 2H). No gastrin was detected in the hepato- Transplantation of Islet Cell Tumor Tissue in Nude Mice. biliary tract. These results suggest that GP1.5 Tag and GP10.5 Athymic nu/nu nude mice were obtained from Charles River Tag genes contain regulatory regions that direct the expression Breeding Laboratories and housed in a clean laminar flow of T antigen in the hepatobiliary tract. Since gastrin has not room. Transplantation oftumor to nude mice was carried out been found to be expressed in this tissue of adult animals, by sterilely removing tumor tissue from surrounding normal these data raise the possibility that gastrin is transiently pancreatic or liver parenchyma, dissecting away any fibrous expressed at some stage in hepatobiliary tract development. pseudocapsule, and mincing 0.1-0.3 g of the resulting mate- Development of Islet Cell Tumors Associated with Hypogly- rial in Hanks' balanced salt solution. Following several cemia. The GP1.5 Tag transgenic mice lost considerable passages through an 18-gauge needle, 0.2-0.4 ml of washed weight at 75-90 days of age and developed symptoms of cell suspension was injected into the subcutaneous tissues of weakness. Administration of glucose through drinking water the infrascapular area. temporarily ameliorated the symptoms, indicating deficiency Glucose Assays. Glucose determinations were carried out of blood glucose. Estimation of blood glucose levels showed on whole blood collected from tail veins using the glucose marked decrease in glucose levels from 160 mg/dl at 60 days oxidase method in kit form (Sigma). of age to 45 mg/dl at 90 days of age. Indeed, most animals in this pedigree (generations one to six) died from hypoglycemic RESULTS shock between the average ages of 80 and 100 days. In Generation of Transgenic Mice. Nucleotides -150 to +62 contrast, 20%o of GP10.5 Tag animals developed islet cell of the human gastrin gene are sufficient to support the tumors and tended to live for >200 days. Death appeared to expression of a DNA sequence lacking guanine nucleotide be more frequently due to massive tumor burden than from (G-less cassette) in transiently transfected HeLa and CV-1 islet cell tumors. cells (K.H.B. and K.A., unpublished data). Contrary to these Gross examination of the moribund mice carrying GP1.5 observations is the fact that expression of the intact endoge- Tag and GP10.5 Tag revealed tumors in their pancreas that neous gene is limited to gastrin-producing enteroendocrine were histologically composed of ribbons and nests of poly- cells located in the antrum ofthe stomach and fetal pancreatic gonal cells surrounded by a fine vascular network (Fig. 2 A islets, suggesting that the intact animal may be required to and D). Additionally, the GP1.5 Tag animals displayed dif- identify the gastrin gene sequences specifically directing its fuse islet hyperplasia and focal ductal hyperplasia. The tumor expression to these tissues. Two constructs were generated. tissues of both transgenic lines strongly stained for insulin (i) The 5' flanking 1.5-kb human gastrin region was linked to and Tag (Fig. 2 B, C, E, and F), whereas other neuroendo- the coding sequence of SV40 Tag and small tumor antigen crine peptides, including gastrin, cholecystokinin, glucagon, (GP1.5 Tag, Fig. 1A). (ii) A second construct included 10.5 somatostatin, and vasoactive intestinal peptide, were absent kb of the human gastrin gene consisting of 5' flanking, 3' by immunohistochemistry (data not shown). Pancreatic duc- flanking, and exon and intron regions (Fig. 1B). The recom- tal hyperplasia in GP1.5 Tag animals was also associated with binant DNAs were injected into the pronucleus of fertilized focal insulin expression in ductal cells. Downloaded by guest on September 24, 2021 Biochemistry: Montag et al. Proc. Natl. Acad. Sci. USA 90 (1993) 6699

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1 2 3 4 5 6 7 8 9

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FIG. 3. Si nuclease mapping of RNAs in tissues from GPl.5 Tag transgenic mice. Tissues examined from the same line of transgenic mice are indicated. (A) Analysis of the RNA for T antigen expression. Total RNA (30 ,ug) from the indicated tissues was hybridized with uniformly labeled 196-nucleotide Tag-gastrin fusion probe and the resulting hybrids were digested with S1 nuclease (13). Lane 1, undigested probe. (B) Analysis of the RNA for mouse gastrin expression. The 244-nucleotide probe was hybridized to each total RNA (55 Ag) and digested with Si nuclease. Lane 1, undigested probe; lane 8, protected probe. (C) Analysis of the RNA for T antigen and mouse insulin II in tissues from islet tumor and normal pancreas. Total RNA from normal pancreas (50 Mg) and from islet tumor (5 Mg) was hybridized in combination with T antigen and mouse insulin II probes. Lane 1, T antigen probe; lane 2, islet tumor RNA plus T antigen probe; lane 3, insulin II probe; lane 4, islet tumor RNA plus T antigen probe plus insulin II probe; lane 5, normal pancreatic RNA plus Tag probe plus insulin II probe.

Analysis of total RNAs from tumor tissues obtained from ng/dl) for a week but subsequently declined rapidly over a GP1.5 Tag mice for mouse insulin II and Tag mRNAs by S1 period of 2-3 weeks (5-10 ng/dl). Development of hypoglyce- nuclease supported the immunohistochemical data. Mouse mic coma in nude mice was more dramatic in comparison to insulin II and Tag mRNAs were expressed in the tumor tissues transgenic animals, but both animals had a 15- to 20-fold drop (Fig. 3C). Expression of 10-fold higher level of insulin II in their glucose levels at the time oftheir death. Islet cell tumor mRNA in the islet tumor relative to normal pancreatic tissue tissue from GP10.5 Tag animals was less potent in producing from a littermate suggests that hypoglycemia is associated hypoglycemia in nude as well as transgenic mice. These mice with increased level ofinsulin II expression (Fig. 3C, lane 4 vs. survived twice as long as GP1.5 Tag mice. This may be due, in lane 5). Insulin I mRNA levels were not determined because part, to the slower growth rate of the GP10.5 Tag islet tumors of its low-level expression relative to insulin II mRNA. in comparison to GP1.5 Tag tumors, thus resulting in a smaller Based upon the previously documented finding that gastrin islet tumor burden. Histologically, the transplanted tumors gene is transiently expressed in the fetal islets during a time of from both sets ofanimals were characteristic ofislet cell tumors rapid islet cell development (5, 15, 16), the data raise the and stained for insulin and T antigen. possibility that coexpression of Tag with gastrin causes neo- Development of Gastric Antral G-Cell Hyperplasia in plastic transformation of islet cells. Interestingly, however, GPO1.5 Tag Animals and Not in GP1.5 Tag Animals. Com- suppression of the endogeneous gastrin gene is not accompa- parison ofgastric antral mucosa from GP1.5 Tag animals and nied by suppression of exogenously introduced Tag-gastrin their littermates revealed no significant increase in cells fusion gene. It is possible that the continued expression ofthe immunoreactive for gastrin even though a low level of T fusion gene may reflect Tag-mediated cellular changes that antigen reactivity was observed in the antral mucosa (Fig. selectively affect fusion gene expression. 21). Analysis of GP10.5 Tag animals revealed a significant Transplantation of Islet Ceil Tumor Tissues in Nude Mice increase in antral mucosal cells immunoreactive for gastrin Induces Hypoglycemia. To assess whether bile ductal, hepa- and T antigen. Frequently these cells were located in clusters tocellular, or islet cell tumors caused hypoglycemia, each of and small tumors (Fig. 2L). Transplantation of GP10.5 Tag the tissues was transplanted in nude mice and their devel- antral mucosal tissue in nude mice resulted in slow growth of opment of hypoglycemia was followed. The GP1.5 Tag bile a solid tumor. Cytoplasmic staining for gastrin and nuclear ductal and GP10.5 Tag hepatocellular tissues grew rapidly in staining for T antigen of the tumor tissues were similar to the these animals but neither of them significantly altered the original tumor tissues (data not shown). normal blood glucose levels (data not shown). Analysis of bile ductal tissue showed a ductal pattern of growth that is DISCUSSION characteristic of adenocarcinoma (data not shown). The Studies of gastrin promoter activity by transfection of neuro- hepatocellular tissue produced a tumor with a sinusoidal blastoma and GH4 cells revealed that nucleotides -50 to +62 growth pattern characteristic of hepatocellular carcinoma. were sufficient to support basal levels oftranscription (17, 18). Islet cell tumor tissue, collected from GP1.5 Tag series Similar studies involving a gastrin-producing islet cell line animals of different lines and generations, induced hypoglyce- demonstrated a requirement for a cis-regulatory domain lo- mic coma within 21-28 days of transplantation to nude mice. cated between -108 and -76 in the gastrin gene (6), but the The blood glucose levels remained at preinjection levels (80-100 same region did not regulate transcription in GH4 cells (18). Downloaded by guest on September 24, 2021 6700 Biochemistry: Montag et al. Proc. Natl. Acad Sci. USA 90 (1993) Since gastrin is expressed in a number ofneuroendocrine cells, suggesting that the expression is not dependent on the site of including pituitary cells (19), vagal neurons (2), and pancreatic insertion but is instead regulated by the fusion gene itself. islets and antral G cells (5, 20), regulation of its expression in These preliminary dataraise the possibility thatfusion genes these sites could involve complex sets of cis-regulatory ele- may be transiently expressed in conjunction with gastrin in the ments. To confirm expression of gastrin in the previously affected tissues during development, transforming the anlage identified tissues and to reveal as yet unidentified sites, we and leading to hyperplasia and tumor formation in postnatal have employed the transgenic mouse technology, which offers life. Most of the phenotypic manifestations of both lines the distinct advantage over the cell transfection procedure in reported here may involve derivatives of the embryonic fore- that the expression ofthe transgene can be surveyed in all cell gut. Gastrin expression in rodents is evident in the 8 cells of types. These studies demonstrated that the human gastrin Tag pancreatic islets between 15 and 19 days of gestation (5, 6), tapering offas gastric antral production begins in the postnatal fusion genes GP1.5 Tag and GP10.5 Tag are accurately tran- period. Pancreatic gastrin mRNA is identical to that of antral scribed in the hepatobiliary tract and in the islets of pancreas gastrin, and they share the same transcriptional initiation site. and consequently induce tumors in these tissues. Expression Differential regulation of promoter or suppressor sequences of Tag in the pancreatic islet cells from both fusion genes that modify expression must be responsible for the differential indicates location of islet cell-specific regulatory elements pattern of gastrin gene expression in the hepatobiliary tree, within the 1.5-kb region. This finding is consistent with the pancreas, and antrum. The gastrin SV40 T antigen transgenic previous demonstration that the 5' flanking 200-bp region mice studies described here offer a unique system ofneoplasia contained islet cell-specific regulatory elements (6, 21). The of foregut derivatives. Studies of embryologic expression of unexpected finding is the expression of Tag in the hepatobil- these and other related transgenes will be necessary to delin- iary tract from both fusion genes because gastrin is not known eate the underlying cause of the aberrant tumor formation. be in this to expressed tissue. The elements specific for islet We are grateful to Lucy Ho for expert technical assistance, Dr. Jeff cell and hepatobiliary tract are localized within the 1.5-kb Gordon for critical reading of the manuscript, Hoeon Kim for sequence, whereas for effective antral expression additional preparation of the figures, and Janie Booker for preparation of the sequences localized within the 10.5-kb gastrin gene region are manuscript. This research was generously supported by National needed. Interestingly, the fusion genes do not appear to be Institutes of Health Grant DK21901. expressed in the pituitary cells and vagal neurons, as demon- 1. Walsh, J. H. & Grossman, M. I. (1975) N. Engl. J. Med. 292, strated by histological analysis of these tissues; previously 1324-1377. these tissues were thought to be sites of gastrin expression. 2. Walsh, J. H. (1981) in Physiology ofthe Gastrointestinal Tract, Expression of GP1.5 Tag and GP1O.5 Tag genes resulted in ed. Johnson, L. R. (Raven, New York), Chap. 3, p. 59. distinct islet proliferation. In GP1.5 Tag there was uniform 3. Larsson, L. I. (1980) Am. J. Physiol. 239, 237-246. 4. Johnson, L. R. (1980) in Gastrointestinal Hormones, ed. Glass, hyperplasia with tumor formation, whereas in the GP1O.5 Tag J. (Raven, New York), Chap. 22, p. 507. animals only sporadic tumor formation was seen. These find- 5. Brand, S. J. & Fuller, P. J. (1988) J. Biol. Chem. 263, 5341- ings raise the possibility of high-level transformation of the 5347. 3-cell population in the GP1.5 Tag animals and low-level 6. Wang, T. C. & Brand, S. J. (1990) J. Biol. Chem. 265, 8908- transformation in the GP10.5 Tag animals. This distinct phe- 8914. notype may be due, in part, to transient coexpression offusion 7. Sambrook, J., Fritsch, F. & Maniatis, T. (1989) in Molecular with in the fetal islets a islet Cloning: A Laboratory Manual (Cold Spring Harbor Lab. genes gastrin during time of rapid Press, Plainview, NY). cell development with failure of repression of the GP1.5 Tag 8. Ito, R., Sato, K., Helmer, T., Jay, G. & Agarwal, K. (1984) gene but only partial repression of the GP10.5 Tag gene. Proc. Natl. Acad. Sci. USA 81, 4662-4666. Similar observations were reported in the study ofinsulin Tag 9. Hogan, B., Constantini, F. & Lacy, E. (1986) Manipulating the fusion gene expression in transgenic animals (22, 25). Mouse Embryo: A Laboratory Manual (Cold Spring Harbor The T antigen expression and neoplastic transformation in Lab. Press, Plainview, NY), pp. 1633-1641. the hepatobiliary tree was an unexpected finding in both 10. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, lines. Not all bile duct epithelium in the GP1.5 Tag animals, W. J. (1979) Biochemistry 18, 5294-5299. nor all hepatocytes in the GP10.5 Tag animals, expressed T 11. Sato, K., Ito, R., Baek, K. H. & Agarwal, K. (1984) Mol. Cell. in Biol. 6, 1032-1043. antigen. Tumor formation both lines was sporadic, without 12. Wentworth, B. M., Schaefer, I. M., Villa-Komaroff, L. & complete transformation of an entire cell type. Embryolog- Chirgwin, J. M. (1986) J. Mol. Evol. 23, 305-312. ically, the liver and biliary tract form from the same foregut 13. Berk, A. J. & Sharp, P. A. (1978) Cell 12, 721-732. outpouchings as the pancreas. It is possible that gastrin plays 14. Hsu, S. M., Rainel, L. & Fanger, H. (1981) J. Histochem. a trophic role in the developing liver, similar to its role in the Cytochem. 29, 577-580. developing pancreas. 15. Larsson, L., Rehfeld, J. F., Sundler, F. & Hakanson, R. (1976) Focal expression of T antigen was seen in nuclei of antral Nature (London) 262, 609-610. cells in GP1.5 Tag animals, but gastrin-immunoreactive cells 16. Brand, S. J., Anderson, B. M. & Rehfeld, J. F. (1984) Nature were not increased. In contrast, GP10.5 animals had in- (London) 309, 456-458. 17. Thiell, L. E., Wiborg, 0. & Vuust, J. (1987) Mol. Cell. Biol. 7, creased numbers of gastrin-producing cells, with occasional 4329-4336. microscopic tumors. The short life-span of the GP1.5 Tag 18. Godley, J. M. & Brand, S. J. (1989) Proc. Natl. Acad. Sci. animals may have precluded the development of tumors in a USA 86, 3036-3040. cell type with an inherently low rate ofproliferation, whereas 19. Powell, C. T., Ney, C., Aran, P. & Agarwal, K. (1985) Nucleic the GP10.5 animals had a longer life-span to develop gastrin Acids Res. 13, 7299-7305. cell hyperplasia. 20. Noyes, B. E., Mevarech, M., Stein, R. & Agarwal, K. (1979) The SV40 T antigen, dnrven by its own promoter, produces Proc. Natl. Acad. Sci. USA 76, 1770-1774. choroid plexus tumors (23) but has not been shown to produce 21. Merchant, J. L., Demediuk, B. & Brand, S. J. (1991) Mol. Cell. the findings reported here. The chromosomal site oftransgene Biol. 11, 2686-2696. 22. Hanahan, D. (1985) Nature (London) 315, 115-122. insertion may also determine the phenotype, particularly if it 23. Van Dyke, T. A., Finlay, C., Miller, D., Marks, J., Lozano, G. interrupts a somatic gene (24). These mutations are usually & Levine, A. J. (1987) J. Virol. 61, 2029-2032. manifested as recessive lethal traits, whereas the animals 24. Cuthbertson, R. A. & Klintworth, G. K. (1988) Lab. Invest. 58, reported here were heterozygous. In addition, the phenotype 484-502. in our studies was consistent with different founder lines, 25. Hanahan, D. (1989) Science 246, 1265-1275. Downloaded by guest on September 24, 2021