Pituitary Hyperplasia in Glycoprotein Hormone Alpha Subunit-, p18INK4C-, and p27kip-1-Null Mice
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64936-x
ISSN1525-2191
AutoresRicardo V. Lloyd, Katharina H. Ruebel, Shuya Zhang, Long Jin,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoMost spontaneously developing hyperplastic and neoplastic lesions of the pituitary occur in the anterior pituitary. Targeted disruption of various cell-cycle proteins, including Rb, p27kip1 (p27), and p18INK4c (p18), is associated with intermediate lobe pituitary hyperplasia. To develop a model of anterior pituitary proliferation to study the pathogenesis of pituitary tumors, we crossed the glycoprotein hormone α-subunit (αSU)-null mice that develop thyroid-stimulating hormone (TSH) cell hyperplasia with p18-null mice. The resulting offsprings developed accelerated enlargement of the anterior lobe with predominantly TSH cell hyperplasia. Immunohistochemical and histological analyses of these mice along with p27/p18 double-null mice, p18-null mice, and p27-null mice showed evidence of TSH, adrenocorticotropic hormone, prolactin, and luteinizing hormone hyperplasia. To determine whether there were alterations of p27 and the target proteins implicated in the ubiquitin degradation of p27 and other cyclin-dependent kinase inhibitors, we examined expression of SKP 2, Grb 2, and Jab 1 in the pituitaries of null mice. In the αSU-null mice there were decreased levels of SKP 2 and elevated levels of Grb 2 expression by Western blot analysis. Immunohistochemical analysis of the pituitary showed elevated Grb 2 in αSU-null and p18/αSU double-null mice. Jab 1 levels were not different from controls in the pituitary. These results show that 1) the p18/αSU double-null mice represent a good model to study the rapid development of anterior pituitary hyperplasia, and 2) various proteins important in p27 and other cyclin-dependent kinase inhibitor protein degradation are altered in the pituitary of αSU-null and p18/αSU double-null mouse models. Most spontaneously developing hyperplastic and neoplastic lesions of the pituitary occur in the anterior pituitary. Targeted disruption of various cell-cycle proteins, including Rb, p27kip1 (p27), and p18INK4c (p18), is associated with intermediate lobe pituitary hyperplasia. To develop a model of anterior pituitary proliferation to study the pathogenesis of pituitary tumors, we crossed the glycoprotein hormone α-subunit (αSU)-null mice that develop thyroid-stimulating hormone (TSH) cell hyperplasia with p18-null mice. The resulting offsprings developed accelerated enlargement of the anterior lobe with predominantly TSH cell hyperplasia. Immunohistochemical and histological analyses of these mice along with p27/p18 double-null mice, p18-null mice, and p27-null mice showed evidence of TSH, adrenocorticotropic hormone, prolactin, and luteinizing hormone hyperplasia. To determine whether there were alterations of p27 and the target proteins implicated in the ubiquitin degradation of p27 and other cyclin-dependent kinase inhibitors, we examined expression of SKP 2, Grb 2, and Jab 1 in the pituitaries of null mice. In the αSU-null mice there were decreased levels of SKP 2 and elevated levels of Grb 2 expression by Western blot analysis. Immunohistochemical analysis of the pituitary showed elevated Grb 2 in αSU-null and p18/αSU double-null mice. Jab 1 levels were not different from controls in the pituitary. These results show that 1) the p18/αSU double-null mice represent a good model to study the rapid development of anterior pituitary hyperplasia, and 2) various proteins important in p27 and other cyclin-dependent kinase inhibitor protein degradation are altered in the pituitary of αSU-null and p18/αSU double-null mouse models. The molecular changes leading to the pathogenesis of anterior pituitary tumors are primarily unknown. Recent studies with targeted disruption of cell-cycle genes such as retinoblastoma (Rb), p27kip1 (p27), and 18INK4C (p18) have provided some insights into the role of cell-cycle proteins in the development of pituitary tumors.1Jacks T Fazeli A Schmitt EM Bronson RT Goodell MA Weingberg RA Effects of Rb mutation in the mouse.Nature. 1992; 359: 295-300Crossref PubMed Scopus (1521) Google Scholar, 2Hu N Gutsman A Herbert DC Bradley A Lee WH Lee EY Heterozygous Rb-1 delta 20/+ mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance.Oncogene. 1994; 9: 1021-1027PubMed Google Scholar, 3Harvey M Vogel H Lee EY Bradley A Donehower LA Mice deficient in both p53 and Rb develop tumors primarily of endocrine origin.Cancer Res. 1955; 55: 1146-1151Google Scholar, 4Nakayama K Ishida N Shirane M Inomata A Inoue T Shishido N Horii I Loh DY Nakayama KI Mice lacking p27kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia and pituitary tumors.Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar, 5Kiyokawa H Kineman RD Manova-Todorova KO Soares VC Hoffman ES Ono M Khanam D Hayday AC Frohman LA Koff A Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27 (kip1).Cell. 1996; 85: 721-732Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar, 6Fero ML Rivkin M Tasch M Porter P Carow CE Firpo E Polyak K Tsai LH Broudy V Perlmutter RM Kaushansky K Roberts JM A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis and female sterility in p27kip1-deficient mice.Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1343) Google Scholar, 7Franklin DS Godfrey VL Lee H Kovalev GI Schoonhoven R Chen-Kiang S Su L Xiong Y CKD inhibitors p18INK4C and p27kip1 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis.Genes Dev. 1998; 12: 2899-2911Crossref PubMed Scopus (340) Google Scholar, 8Franklin DS Godfrey VL O'Brien DA Deng C Xiong Y Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity.Mol Cell Biol. 2000; 20: 6147-6158Crossref PubMed Scopus (248) Google Scholar Most of these hyperplastic pituitaries in Rb-, p27-, and p18-null mice develop in the intermediate lobe, so these are not good models to study anterior pituitary tumor development, which is where most of these tumors develop spontaneously in rodents and humans. Single knockout of the α-subunit gene9Stahl JH Kendall SK Brinkmeier ML Greco TL Watkins-Chow DE Campos-Barros A Lloyd RV Camper SA Thyroid hormone is essential for pituitary somatotropes and lactotropes.Endocrinology. 1999; 140: 1884-1892Crossref PubMed Scopus (45) Google Scholar and transgenic mice expressing the growth hormone-releasing hormone with a metalloproteinase-driven promotor crossed with p27-null mice10Teixeira LT Kiyokawa H Peng XD Christov KT Frohman LA Kineman RD p27kip1-deficient mice exhibit accelerated growth hormone-releasing hormone (GHRH)-induced somatotrope proliferation and adenoma formation.Oncogene. 2000; 19: 1875-1884Crossref PubMed Scopus (29) Google Scholar have also been used to study anterior pituitary hyperplasia.9Stahl JH Kendall SK Brinkmeier ML Greco TL Watkins-Chow DE Campos-Barros A Lloyd RV Camper SA Thyroid hormone is essential for pituitary somatotropes and lactotropes.Endocrinology. 1999; 140: 1884-1892Crossref PubMed Scopus (45) Google Scholar, 10Teixeira LT Kiyokawa H Peng XD Christov KT Frohman LA Kineman RD p27kip1-deficient mice exhibit accelerated growth hormone-releasing hormone (GHRH)-induced somatotrope proliferation and adenoma formation.Oncogene. 2000; 19: 1875-1884Crossref PubMed Scopus (29) Google Scholar The levels of p27 proteins are decreased in many human cancers compared to normal tissues and have prognostic significance, suggesting that p27 may be a tumor suppressor gene. However, there are few mutations in the p27 gene and the mRNA levels are relatively unchanged compared to the decreased levels of p27 protein in tumors.11Lloyd RV Erickson LA Jin L Kulig E Qian X Cheville JC Scheithauer BW p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers.Am J Pathol. 1999; 154: 313-323Abstract Full Text Full Text PDF PubMed Scopus (549) Google Scholar, 12Desdouets C Bréchot C p27: a pleiotropic regulator of cellular phenotype and a target for cell cycle dysregulation in cancer.Pathol Biol. 2000; 48: 203-210PubMed Google Scholar These observations suggest that the proteins regulating posttranslational degradation of p27 may be potential targets to explain the mechanism of down-regulation of p27 and other cyclin-dependent kinase inhibitory (CDKI) cell-cycle genes during tumor development. Although it has been shown that the ubiquitin-proteasome system13Krek W Proteolysis and the G1-S transition: the SCF connection.Curr Opin Genet Dev. 1998; 8: 36-42Crossref PubMed Scopus (143) Google Scholar, 14Ciechanover A The ubiquitin-proteasome proteolytic pathway.Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar regulated short-lived CKDI proteins such as p27, the role of various proteins in the degradation are currently being investigated.15Montagnoli A Fiore F Eyton E Carrano AC Draetta GF Hershiko A Pagono M Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.Genes Dev. 1999; 13: 1181-1189Crossref PubMed Scopus (512) Google Scholar In studies with p27, the jun-activated protein Jab 1,16Tomoda K Kubota Y Kato J Degradation of the cyclin-dependent kinase inhibitors. p27kip1 is instigated by Jab 1.Nature. 1999; 398: 160-165Crossref PubMed Scopus (551) Google Scholar various F-box proteins including SKP 217Gstaiger M Jordan R Lim M Catzavelos C Mestan J Slingerland J Krek W Skp2 is oncogenic and overexpressed in human cancers.Proc Natl Acad Sci USA. 2001; 98: 5043-5048Crossref PubMed Scopus (441) Google Scholar, 18Carrano AC Eytan E Hershko A Pagano M SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27.Nat Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1341) Google Scholar, 19Chiaur DS Murthy S Cenciarelli C Parks W Loda M Inghirami G Demetrick D Pagano M Five human genes encoding F-box proteins: chromosomal mapping and analysis in human tumors.Cytogenet Cell Genet. 2000; 88: 255-258Crossref PubMed Scopus (25) Google Scholar, 20Nakayama K Nagahama H Minamishima YA Matsumoto M Nakamichi I Kitagawa K Shirane M Tsunematsu R Tsukiyama T Ishida N Kitigawa M Nakayama K Hatakeyama S Targeted disruption of SKP2 results in accumulation of cyclin E and p27kip1, polyploidy and centrosome overduplication.EMBO J. 2000; 19: 2069-2081Crossref PubMed Scopus (634) Google Scholar and the signal-transducing adaptor protein Grb 221Sugiyama Y Tomoda K Tanaka T Arata Y Yoneda-Kato N Kato J Direct binding of the signal-transducing adaptor Grb2 facilitates down-regulation of the cyclin-dependent kinase inhibitor p27kip1.J Biol Chem. 2001; 276: 12084-12090Crossref PubMed Scopus (33) Google Scholar, 22Birge RB Knudsen BS Besser D Hanafusa H SH2- and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction.Genes Cells. 1996; 276: 595-613Crossref Scopus (122) Google Scholar have been shown to have a regulatory roles in p27 degradation. In this study, we targeted pituitary hyperplasia to the anterior pituitary of p18-deficient mice by creating double-null animals with loss of the p18 and α-subunit genes. These mice as well as p27-null and p18/p27 double-null mice were used to examine expression of some of the major proteins that play a role in p27 and other CDKI ubiquitin-mediated degradation of CDKIs. The p27 mice with a C57BL/6 background (a gift from Dr. M. L. Fero and J. L. Roberts, Fred Hutchinson Cancer Center, Seattle, WA), the p18 mice with a C57BL/6 background (a gift from Drs. D. S. Franklin and Y. Xiang, University of North Carolina, Chapel Hill, NC), and the α-subunit of glycoprotein hormones (αSU) mice had a background of C57BL/6J (a gift from Dr. S. A. Camper, University of Michigan, Ann Arbor, MI) were all maintained in a specialized mouse barrier facility at the Mayo Clinic. F2 mice heterozygous for p27, p18, or αSU were generated from F1 mice in each group. Mice were genotyped and the resulting F1 mice were intercrossed to created double-null animals. The p18/αSU double-null mice were derived from crossing p18-null mice with αSU heterozygous mice. The p27/p18 double-null mice were derived from crossing p27 and p18 heterozygous mice. All animals were mainly B6 in their genetic background. Genotyping was done by polymerase chain reaction (PCR). All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Tail snips were taken from all mice at 4 weeks of age and genomic DNA extracted for genotyping. The PCR reactions contained 1.25 U of Taq polymerase, 1× PCR buffer, 1.5 of mmol/L magnesium chloride (Promega, Madison, WI), 100 ng of each primer, and 1 [μ]l of genomic DNA in a total volume of 25 [μ]l unless otherwise specified. All PCR products were resolved on a 2% agarose gel stained with ethidium bromide. Two sets of primers were used to amplify the intact α-subunit wild-type allele and the disrupted nonfunctional allele. The wild-type sense primer (5′-GCA TAT CCC ACT CCC GCC AGG) located within exon 3 was used in conjunction with an antisense primer (5′-CAA TTA AAG AGG ATA AAT GCA GGT GTC GCC) within intron 3 resulting in a 220-bp product. Detection of the disrupted or null allele used a sense primer (5′-TGC CTT TTG TAT TAT CAG GGT ACC TAG ACT) and an antisense primer located within the neo gene (5′-CGC CTT CTA TCG CCT TCT TGA CGA GTT CTT) resulting in a 1.1-kb product.23Kendall SK Samuelson LC Saunders TL Wood RI Camper SA Targeted disruption of the pituitary glycoprotein hormone α-subunit produces hypogonadal and hypothyroid mice.Genes Dev. 1995; 9: 2007-2019Crossref PubMed Scopus (216) Google Scholar Twice the amount of Taq polymerase was used for the null allele PCR (2.5 U). The α-subunit-null mice and the p18/αSU double-null mice required genetic sexing because of hypogonadism and hypothyroidism. Males were identified through PCR detection of the sry gene on the Y chromosome.24Gubbay J Collignon J Koopman P Capel B Economou A Munsterberg A Vivian N Goodfellow P Lovell-Badge R A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes.Nature. 1990; 346: 245-250Crossref PubMed Scopus (1354) Google Scholar The sense primer (5′-GTC TAG AGA GCA TGG ACGG GCC) and antisense primer (5′-ACA GGT GTG CAG CTC TAC TCC) amplified a 381-bp product in males only.25Kulig E Camper SA Kuecker S Jin L Lloyd RV Remodeling of hyperplastic pituitaries in hypothyroid α-subunit knockout mice after thyroxine and 17 β-estradiol treatment: role of apoptosis.Endocr Pathol. 1998; 9: 261-274Crossref PubMed Scopus (21) Google Scholar PCR for the wild-type and null alleles of the α-subunit gene as well as the sex-determining PCR shared the same PCR profile: 95°C for 5 minutes; 30 cycles at 94°C for 1 minute, 58°C for 1 minute, and 72°C for 2 minutes followed by 10 minutes of final extension at 72°C. Detection of p27 wild-type allele and p27-null allele used one common sense primer (5′-TGG AAC CCT GTG CCA TCT CTA T). The antisense primers used were specific for the p27 wild-type allele (5′-GAG CAG ACG CCC AAG AAG C) and the neo-disrupted allele (5′-CCT TCT ATG GCC TTC TTG ACG). PCR was performed for 40 cycles with an annealing temperature of 57°C. The wild-type allele resulted in a 1300-bp product and the null allele in a 600-bp product.6Fero ML Rivkin M Tasch M Porter P Carow CE Firpo E Polyak K Tsai LH Broudy V Perlmutter RM Kaushansky K Roberts JM A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis and female sterility in p27kip1-deficient mice.Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1343) Google Scholar A sense primer for the p18 gene (5′-AGC CAT CAA ATT TAT TCA TGT TGC AGG) was used in combination with the wild-type antisense primer (5′-CCT CCA TCA GGC TAA TGA CC) and null antisense primer (5′-CCA GCC TCT GAG CCC AGA AAG CGA AGG). PCR amplification was performed at an annealing temperature of 60°C for 35 cycles and produced a 600-bp wild-type amplicon or a 400-bp null amplicon.7Franklin DS Godfrey VL Lee H Kovalev GI Schoonhoven R Chen-Kiang S Su L Xiong Y CKD inhibitors p18INK4C and p27kip1 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis.Genes Dev. 1998; 12: 2899-2911Crossref PubMed Scopus (340) Google Scholar Animals were monitored daily and sacrificed at preplanned intervals or when they showed signs of morbidity. Body weight and specific organ weights were obtained at the time of sacrifice and genotyping was rechecked in some cases. Portions of specific tissues, including pituitaries and liver, were frozen in liquid nitrogen, stored at −70°C, and used for protein analysis and other studies. For histological analysis tissues were fixed in 10% buffered formalin, dehydrated in increasing concentrations of ethanol, cleared in xylene, and embedded in paraffin blocks. Hematoxylin and eosin (H&E) stains were performed on all blocks and other sections were used for immunohistochemical analysis. Mitotic figures in the pituitary were counted in 50 fields at a final magnification of ×400. Antibodies for prolactin (PRL) (used at 1/1000), growth hormone (GH) (used at 1/1000), thyroid stimulating hormone (TSH) (used at 1/500), and luteinizing hormone (LH) (used at 1/500) were obtained from the National Pituitary Distribution Agency, Bethesda, MD. Adrenocorticotropic hormone (ACTH) (used at 1/500) was obtained from DAKO (Santa Barbara, CA). Antibodies to p27 (1/1000; Transduction Laboratories, Lexington, KY), SKP 2 (1/100; Santa Cruz Inc., Santa Cruz, CA), Jab 1 (1/500, Santa Cruz, Inc.), and Grb 2 (1/500; Santa Cruz, Inc.) were used for immunohistochemistry. Immunohistochemical staining was done as previously reported23Kendall SK Samuelson LC Saunders TL Wood RI Camper SA Targeted disruption of the pituitary glycoprotein hormone α-subunit produces hypogonadal and hypothyroid mice.Genes Dev. 1995; 9: 2007-2019Crossref PubMed Scopus (216) Google Scholar, 24Gubbay J Collignon J Koopman P Capel B Economou A Munsterberg A Vivian N Goodfellow P Lovell-Badge R A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes.Nature. 1990; 346: 245-250Crossref PubMed Scopus (1354) Google Scholar with antigen retrieval used for p27, SKP 2, Jab 1, and Grb 2. Antigen retrieval was performed in an 800-W microwave oven using 0.1 mol/L of citrate buffer, pH 6.0, for 5 minutes followed by 20 minutes of cooling at room temperature. Immunohistochemical staining was done by incubation with the primary antibody overnight and the reaction product was developed with diaminobenzidine.26Jin L Tsumanuma I Ruebel KH Bayliss JM Lloyd RV Analysis of homogeneous populations of anterior pituitary folliculostellate cells by laser capture microdissection and reverse transcription-polymerase chain reaction.Endocrinology. 2001; 142: 1703-1709Crossref PubMed Scopus (66) Google Scholar, 27Jin L Kulig E Qian X Scheithauer BW Eberhardt NL Lloyd RV A human pituitary cell adenoma line proliferates and maintains some differentiated functions following expression of SV40 large T antigen.Endocr Pathol. 1998; 9: 169-184Crossref Scopus (41) Google Scholar The avidin-biotin complex peroxidase kits were obtained from Vector Laboratories (Burlingame, CA). Semiquantitation of immunohistochemical staining was done by grading the nuclear immunoreactivity as 0, negative; +, rare or <5% cells positive; ++, focal or 25% of cells positive. Four to five pituitaries were analyzed for each group. Western blots were done as previously reported.27Jin L Kulig E Qian X Scheithauer BW Eberhardt NL Lloyd RV A human pituitary cell adenoma line proliferates and maintains some differentiated functions following expression of SV40 large T antigen.Endocr Pathol. 1998; 9: 169-184Crossref Scopus (41) Google Scholar, 28Qian X Jin L Grande JP Lloyd RV Transforming growth factor-β and p27 expression in pituitary cells.Endocrinology. 1996; 137: 3051-3060Crossref PubMed Scopus (100) Google Scholar Briefly, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was done and the electrophoresed proteins subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA) and subjected to immunoblot analysis for p27 (1/1000 dilution), SKP 2 (1/400), Jab 1 (1/500), Grb 2 (1/250), and β-actin (1/1000; Sigma Chemical Co., St. Louis, MO). The reaction product was detected with enhanced chemiluminescence (Amersham, Arlington Heights, IL). β-Actin was used to normalize for equal loading of the gels. Densitometric analysis of the bands was done with a Fluor-S multimager (Bio-Rad, Hercules, CA). Results were expressed as relative densitometry units, and all data were normalized with β-actin. A linear response was obtained using different amounts of proteins on the gel. Each experiment was repeated two to three times. Results were expressed as the mean ± SEM. Statistical analysis was done with the Wilcoxon rank sum test. Genotype analyses by PCR were used to characterize the mice resulting from the various crosses (Figure 1). Null mice that were deficient in the αSU gene developed anterior pituitary hyperplasia in a time-dependent manner as previously reported.25Kulig E Camper SA Kuecker S Jin L Lloyd RV Remodeling of hyperplastic pituitaries in hypothyroid α-subunit knockout mice after thyroxine and 17 β-estradiol treatment: role of apoptosis.Endocr Pathol. 1998; 9: 261-274Crossref PubMed Scopus (21) Google Scholar, 29Stahl JH Kendall SK Brinkmeier ML Greco TL Watkins-Chow DE Campos-Barros A Lloyd RV Camper SA Thyroid hormone is essential for pituitary somatotropes and lactotropes.Endocrinology. 1999; 140: 1884-1892Crossref PubMed Google Scholar The p18- and p27-null mice developed enlarged intermediate lobes in a time-dependent manner. Animals sacrificed at 1 year of age from the αSU-null group had pituitaries weighing 73 ± 20 mg. The average weights of p18- and p27-null mice pituitaries were 6.1 ± 1.5 mg and 12.8 ± 5.4 mg, respectively. The p18/αSU double-null mice had a more rapid growth of their anterior pituitaries compared to either the p18-null or αSU-null animals (Figure 2). When the mice were sacrificed by 5 months of age, the pituitaries were sevenfold larger than the αSU-null mice sacrificed at the same time (P < 0.05) and fivefold larger than the p18-null mice sacrificed at 11 months of age on average (Figure 2). Mice that were p18 heterozygous and αSU-null sacrificed at 5 months of age had pituitaries that were slightly larger than those of the αSU-null mice (Figure 2). The p27/p18 double-null mice developed large pituitaries by 2 to 3 months of age and animals became moribund at this age because of the enlarged pituitaries, unlike their control littermates. Examination of the pituitaries showed that at 2 to 3 months of age, they were sevenfold larger than the p18-null mice at 11 months of age and nearly twofold larger than the p27-null mice at 11 months of age (Figure 3). The pituitaries of p27 heterozygous/p18-null mice were slightly larger than the control mice at the same age (Figure 3). Histological analysis revealed diffuse and nodular or adenomatous hyperplasia of the pituitary (Figure 4; A to F). For p18- and p27-null mice, this was mainly intermediate lobe ACTH cell hyperplasia (Table 1 and Figure 4, A and B), although one PRL-positive hyperplastic gland was present in 1 of 10 p18-null mice examined by immunohistochemistry (Figure 4E). The αSU-null mice had TSH cell hyperplasia in the anterior lobe of the pituitary. The p18/αSU double-null mice had mainly anterior lobe TSH cell hyperplasia when sacrificed at 5 months of age with less developed intermediate lobe ACTH cell hyperplasia at this age (Figure 4, C and D).Table 1Immunohistochemical Analysis of Hyperplastic PituitariesIntermediate lobeAnterior LobeGroupnACTHPRLGHTSHLHACTHp27-null1212—————p18-null1091————αSU-null5———500p27/p18—————double-null55p18/αSU————double-null949n = number of pituitaries immunostained in each group.Ten wild-type pituitaries were analyzed as controls.Hyperplastic LH nodules were identified in 2 of 6 p27 heterozygous/p18 null mouse pituitaries. Open table in a new tab n = number of pituitaries immunostained in each group. Ten wild-type pituitaries were analyzed as controls. Hyperplastic LH nodules were identified in 2 of 6 p27 heterozygous/p18 null mouse pituitaries. The p27/p18 double-null mice had markedly enlarged pituitaries with intermediate lobe ACTH cell hyperplasia (Table 1, Figure 4B) at the time of sacrificing at 2 to 3 months of age. Nodular LH cell hyperplasia was present in two of six pituitaries from p27 heterozygous/p18-null mice (Table 1, Figure 4F) along with intermediate lobe ACTH cell hyperplasia. Analysis of mitotic activity showed between one to five mitoses per 50 high-power fields in the p27-null, p18-null, αSU-null, as well as the double-null mice whereas none to one mitoses per 50 high-power fields were present in the anterior pituitary of control mice. When pituitary tissues for p27-null, p18-null, and αSU-null mice were analyzed by Western blots for p27, SKP 2, the highest levels of Grb 2 and Jab 1, the αSU-null mice had the lower levels of SKP 2 and Grb 2 protein that were 3.5-fold higher than the control wild-type liver. The p27-null mice pituitary tissue had Grb 2 levels that were 2.3-fold higher than the control mice (Figure 5, A and B). The levels of Jab 1 were not significantly different in any of the pituitary tissues from p27-, p18-, or αSU-null mice by Western blot analysis (Figure 5). Analysis of immunohistochemical staining showed mainly nuclear, but some cytoplasmic, staining (Figure 6). Only nuclear staining was evaluated. Staining of the pituitaries showed the highest levels of Grb 2 in the αSU- and p18/αSU double-null mice (Table 2). p27 immunoreactivity was not present in the pituitaries of the p27-null and p27/p18 double-null mice. Jab 1 levels were similar to wild type in the pituitary by Western blotting and by immunohistochemistry.Table 2Immunohistochemical Analysis of Pituitary Tissues in Wild-Type and Null MiceGroupp27SKP 2Jab 1Grb 2Wild type++++++++p27-null0+++++++p18-null+++++++++++p27/p18double-null0++++++++αSU-null++++++++++++p18/αSUdouble-null++++++++++++Analysis of staining: 0, negative; +, rare or <5% cells positive; ++, focal or 25% of cells positive.Between four to five pituitaries per group were analyzed. Open table in a new tab Analysis of staining: 0, negative; +, rare or <5% cells positive; ++, focal or 25% of cells positive. Between four to five pituitaries per group were analyzed. Pituitary hyperplasia and tumor development is most commonly seen in the anterior pituitary of rodents and humans. These lesions often develop spontaneously,30Lloyd RV Tumors of the Pituitary.in: Stinson SF Schuller HM Reznik G Atlas of Tumor Pathology of the Fischer Rat. CRC Press, Boca Raton1990: 265-289Google Scholar, 31Horvath E Kovacs K Fine structural cytology of the adenohypophysis in rat and man.J Electr Micro Tech. 1998; 8: 401-432Crossref Scopus (94) Google Scholar although they can also be induced by chemicals such as hormonal treatment.32Lloyd RV Estrogen-induced hyperplasia and neoplasia in the rat anterior pituitary gland. An immunohistochemical study.Am J Pathol. 1983; 113: 198-206PubMed Google Scholar Mice with targeted disruption of the Rb, p27, and p18 genes often develop pituitary lesions in the intermediate lobe.1Jacks T Fazeli A Schmitt EM Bronson RT Goodell MA Weingberg RA Effects of Rb mutation in the mouse.Nature. 1992; 359: 295-300Crossref PubMed Scopus (1521) Google Scholar, 2Hu N Gutsman A Herbert DC Bradley A Lee WH Lee EY Heterozygous Rb-1 delta 20/+ mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance.Oncogene. 1994; 9: 1021-1027PubMed Google Scholar, 3Harvey M Vogel H Lee EY Bradley A Donehower LA Mice deficient in both p53 and Rb develop tumors primarily of endocrine origin.Cancer Res. 1955; 55: 1146-1151Google Scholar, 4Nakayama K Ishida N Shirane M Inomata A Inoue T Shishido N Horii I Loh DY Nakayama KI Mice lacking p27kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia and pituitary tumors.Cell. 1996; 85: 707-720Abstract Full Text Full Text PDF PubMed Scopus (1478) Google Scholar, 5Kiyokawa H Kineman RD Manova-Todorova KO Soares VC Hoffman ES Ono M Khanam D Hayday AC Frohman LA Koff A Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27 (kip1).Cell. 1996; 85: 721-732Abstract Full Text Full Text PDF PubMed Scopus (1150) Google Scholar, 6Fero ML Rivkin M Tasch M Porter P Carow CE Firpo E Polyak K Tsai LH Broudy V Perlmutter RM Kaushansky K Roberts JM A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis and female sterility in p27kip1-deficient mice.Cell. 1996; 85: 733-744Abstract Full Text Full Text PDF PubMed Scopus (1343) Google Scholar, 7Franklin DS Godfrey VL Lee H Kovalev GI Schoonhoven R Chen-Kiang S Su L Xiong Y CKD inhibitors p18INK4C and p27kip1 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis.Genes Dev. 1998; 12: 2899-2911Crossref PubMed Scopus (340) Google Scholar, 8Franklin DS Godfrey VL O'Brien DA Deng C Xiong Y Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity.Mol Cell Biol. 2000; 20: 6147-6158Crossref PubMed Scopus (248) Google Scholar, 33Park MS Rosai J Nguyen HT Capodieci P Cor
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