American Journal ofPathology, Vol. 140, No. 5, May 1992 Copyight © Amerian Association ofPathologists Transgenic Mice That Develop Pituitary Tumors A Model For Cushing's Disease Are Helseth,*t Gene P. Siegal, Egil Haug,11 The hypothalamus, pituitary gland and adrenal cortex and Victoria L. Bautch* constitute an endocrine axis. A hypothalamic peptide, From the Department ofBiology,* University ofNorth corticotropin-releasing hormone (CRH),1 stimulates the Carolina at Chapel Hill, Chapel Hill, North Carolina; the pituitary to secrete adrenocorticotropic hormone (ACTH); Department of Tumor Biology,t Institutefor Cancer ACTH stimulates the adrenal cortex to produce and se- Research, The Norwegian Radium Hospital, Oslo, Norway; crete glucocorticosteroids. Chronic ACTH overstimula- the Departments ofPathology, Cell Biology and Surgery, tion of the adrenal cortex induces the steroidogenic path- University ofAlabama at Birmingham, Birmingham, way by enhancing gene transcription of the involved en- Alabama; and the Hormone Aker Laboratory,II Hospital, Glucocorticoids are Oslo, Norway zymes.2 normally elevated in response to stress and fasting. In fasting, corticosteroids maintain blood glucose by stimulating gluconeogenesis. Transgenic mice that developed adrenocorticotropic Feeding in the presence of high concentrations of gluco- hormone (ACTH)-producing pituitary tumors were corticoids may cause hyperglycemia, leading to hyper- generated with the polyoma early region promotor insulinism that in turn results in a central deposition of fat.3 linked to a cDNA encoding polyoma large T antigen Cushing's syndrome is the clinical result of chronic (PyLT). Light microscopic examination of the pitu- overexposure to endogenous cortisol (e.g., an adrenal itaries showed normal morphology at 4 months of cortical adenoma) or prolonged administration of syn- age, either unremarkable morphology or microade- thetic glucocorticoids in humans. The latter is seen in nomaformation at9 months ofage, and up to 5 mm large groups of patients treated for chronic autoimmune large adenomas in clinically ill transgenic mice at diseases, patients with serious allergies such as asthma, 13-16 months of age. At age 9 months, transgenic and certain groups of cancer patients. The side effects of mice weighed significantly more than corresponding glucocorticoid therapy often limit the therapeutic useful- control mice, but they began wasting at approxi- ness of the drugs. Excessive pituitary secretion of ACTH mately I year of age. The adrenal glands of these is called Cushing's disease and most often results from a older PyLT-1 mice showed a weight increase and ex- pituitary tumor, usually designated as a microadenoma. hibited a medullary hyperplasia Subcutaneous The more common symptoms of the disease are truncal transplants of transgenic pituitary tumors to non- obesity, hypertension, hyperglycemia, muscular weak- transgenic, immunocompetent mice resulted in tu- ness, and dystrophic skin changes. Measurement of mors with a morphology and ACTH immunoreactiv- ACTH levels in plasma from the inferior petrosal sinuses ity similar to the primary tumor. The effects of hy- can distinguish ACTH-secreting pituitary tumors from percorticotropism were more enhanced and occurred other causes of hyperadrenocorticotropism.4 Microade- with a shorter latency in the mice carrying transgene nomas can be removed selectively through a transsphe- pituitary transplants than in the PyLT-1 transgenic noidal resection, leaving normal gland in situ. For the few mice themselves. Moreover, these transplanted mice patients with a vessel plexus surrounding the pituitary showed a weight increase with an axial deposition gland5 and for patients with macroadenomas,6 trans- pattern and hypertrophy of the adrenal cortex that sphenoidal surgery may be impossible or insufficient. resembled the findings in human Cushing's disease. Plasma ACTH levels were significantly increased in Supported by grants from The Norwegian Cancer Society (AH), and from clinically ill transgenic mice and even higher levels the NIH (CA45727) (GPS) and (HL43174) (VLB). Accepted for December were found in the transplant mice. Thus both mu- publication 13,1991. Address reprint requests to Dr. Victoria L. Bautch, rine models should Department of be useful for studying Cushing's Biology, CB#3280, University of North Carolina at Chapel Hill, Chapel Hill, disease. (AmJPathol 1992, 140:1071-1080) NC 27599. 1071 1072 Helseth et al AJP May 1992, Vol. 140, No. 5 Adjunctive therapies with radiation and drugs that de- sex-matched controls at 4 and 9 months of age in addi- crease ACTH secretion or inhibit corticosteroid synthesis tion to sick mice. Each group contained 4-5 experimen- are available, but they have major shortcomings in terms tal mice and 3-6 control mice. of efficiency and side effects.7 Therefore, animal models for Cushing's syndrome and Cushing's disease are relevant for investigating in Transplant Mice detail the systemic effects of ACTH and glucocorticoids Pituitary tumors were carefully excised from the trans- and new treatment modalities. We have generated trans- genic animal (PyLT-1), minced with fine scissors and genic mice with a polyoma large T-antigen cDNA trans- rinsed in sterile saline. Tumor pieces were further minced gene that heritably form pituitary tumors with detectable with sterile scalpel blades and forced through a 1 9G hy- symptoms at about 1 year of age. The pituitary tumor and podermic needle before subcutaneous injection of 0.1 ml the testes express the PyLT transgene at the mRNA level tumor slurry into 6-10 week B6D2F1 nontransgenic fe- (VLB unpublished results). We have shown both at the male mice (first passage). Tumors were passaged four light- and electron microscopic level that these tumors times using this technique, using 3-4 week old B6D2F1 produce ACTH (R. Holm, unpublished results). We report mice in passages 3 and 4. Between 4 and 15 test ani- that the PyLT-1 transgenic mice and immunocompetent mals were transplanted in each experiment and accom- mice carrying transplanted transgenic pituitary tumors panied by 3-5 controls. All transplant mice were females show changes in total body weight, adrenal weight, and except for the third passage in which both genders were histology of several organs. In addition, the transplant used. The transplant mice were weighed every 2-4 mice have an axial fat deposition pattern and adrenal weeks, and the implant site was both inspected and pal- cortex hypertrophy. The circulating levels of ACTH are pated. Five months after transplantation all the third pas- markedly increased in both transgenic and transplant sage animals were anesthetized, and tumor diameter mice. These mice may therefore be useful models for was measured. A group of six randomly chosen female research related to Cushing's syndrome and Cushing's transplant mice were autopsied at this point. The remain- disease. ing nine females and eight males were followed for an additional 3 months. Material and Methods Pathology Transgenic Mice After lethal anesthesia, blood samples were obtained Transgenic mice were generated as described8 by mi- and the mice were subjected to complete autopsy. The croinjection of the polyoma (Py) early region promotor organs were quickly removed, the weights were re- linked to the cDNA encoding Py large T (PyLT) into the corded after excess fat was trimmed, the specimens male pronuclei of fertilized eggs. The cDNA has a dele- were fixed in 10% buffered formalin (Tissue-Fixx, Lerner tion in the late region and at the origin of replication that Laboratories, PA), and they were processed for paraffin prevents autonomous replication. Transgenic pups in a embedding or fixed and processed for ultrastructural litter were identified by the presence of PyLT in their ge- analyses (R. Holm, unpublished results). The paraffin nomic DNA. The DNA was isolated from tail biopsies sections were stained in batches with H&E. Tumor spec- taken at 2 weeks of age, digested with Hindlll, transferred imens from five representative cases (two PyLT-1 mice to nitrocellulose and hybridized with a 32P-labelled probe and three mice receiving subcutaneous transplants) against the Py sequences.8 Nontransgenic mice were were processed for immunohistochemical demonstration used as control animals in the study. The mice were kept of the anterior pituitary hormones using standardized in a room with humidified and filtered air together with techniques as previously described.910 The antibodies other transgenic lineages. The facility is approved by the were used in the commercially available prediluted American Association for Accreditation for Laboratory forms: ACTH and PRL (both from Lipshaw, Detroit, Ml), Animal Care (AAALAC) and is under the care of Dr. J. TSH, FSH, LH, and GH (all from Biomedica, Foster City, Pick. The mice had food and water ad libitum and were CA). inspected on a daily basis. Mice were anesthetized with 0.65 ml 2.5% avertin per 30 g animal weight (100% aver- ACTH Measurement tin is a weight/volume formulation composed of 5g 2,2,2 tribromoethanol (Aldrich Chemical Company Inc., WI) in Blood was obtained by heart puncture, transferred to 10 ml 2-methyl-2-butanol (Fluka Chemical Corp., NY)). chilled microfuge tubes, centrifuged and heparin plasma The time course of weights and morphology were ana- was stored at - 70 C until assayed. ACTH was measured lyzed by killing transgenic mice together with age- and using a double-antibody radioimmunoassay (RIA) as de- Transgenic Model