Morphological, Histochemical, Immunohistochemical, and Ultrastructural Characterization of Tumors and Dysplastic and Non-Neoplastic Lesions Arising in BK Virus/tat Transgenic Mice
1999; Elsevier BV; Volume: 154; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)65375-8
ISSN1525-2191
AutoresGiuseppe Altavilla, Cecilia Trabanelli, Michela Merlin, Antonella Caputo, Massimo Lanfredi, G. Barbanti‐Brodano, Alfredo Corallini,
Tópico(s)Virus-based gene therapy research
ResumoTo study the role in AIDS pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Tat protein, a transactivator of viral and cellular genes, we generated transgenic mice with a recombinant DNA containing BK virus (BKV) early region and the HIV-1 tat gene, directed by its own promoter-enhancer. DNA hybridization revealed that the transgene is stably maintained in all organs of transgenic mice as a tandem insertion in a number of copies ranging from 5 to 20 per cell. In addition, tat and BKV RNA were expressed in all tissues. Transgenic mice developed three types of lesions: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. Tumors of different histotypes, such as lymphomas, adenocarcinomas of skin glands, leiomyosarcomas, skin squamous cell carcinomas, hepatomas, hepatocarcinomas, and cavernous liver hemangiomas, developed in 29% of transgenic animals. The majority of tumors were malignant, invasive, and producing metastases. Conversely, tumors of only two histotypes (lymphomas and adenocarcinomas of skin glands) appeared in control mice. Hyperplastic and dysplastic lesions were more frequent in transgenic than in control mice and involved the skin or its adnexes, the liver and the rectum, indicating multiple targets for the activity of the transgene. Pyelonephritis, frequently complicated with hydronephrosis, inflammatory eye lesions, and amyloid depositions represented the most frequent non-neoplastic lesions detected in transgenic mice. Many of the pathological findings observed in this animal model are comparable to similar lesions appearing in AIDS patients, suggesting a relevant role for Tat in the pathogenesis of such lesions during the course of AIDS. To study the role in AIDS pathogenesis of the human immunodeficiency virus type 1 (HIV-1) Tat protein, a transactivator of viral and cellular genes, we generated transgenic mice with a recombinant DNA containing BK virus (BKV) early region and the HIV-1 tat gene, directed by its own promoter-enhancer. DNA hybridization revealed that the transgene is stably maintained in all organs of transgenic mice as a tandem insertion in a number of copies ranging from 5 to 20 per cell. In addition, tat and BKV RNA were expressed in all tissues. Transgenic mice developed three types of lesions: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. Tumors of different histotypes, such as lymphomas, adenocarcinomas of skin glands, leiomyosarcomas, skin squamous cell carcinomas, hepatomas, hepatocarcinomas, and cavernous liver hemangiomas, developed in 29% of transgenic animals. The majority of tumors were malignant, invasive, and producing metastases. Conversely, tumors of only two histotypes (lymphomas and adenocarcinomas of skin glands) appeared in control mice. Hyperplastic and dysplastic lesions were more frequent in transgenic than in control mice and involved the skin or its adnexes, the liver and the rectum, indicating multiple targets for the activity of the transgene. Pyelonephritis, frequently complicated with hydronephrosis, inflammatory eye lesions, and amyloid depositions represented the most frequent non-neoplastic lesions detected in transgenic mice. Many of the pathological findings observed in this animal model are comparable to similar lesions appearing in AIDS patients, suggesting a relevant role for Tat in the pathogenesis of such lesions during the course of AIDS. Several types of tumors are associated to human immunodeficiency virus type 1 (HIV-1) infection and AIDS.1Beral V Jaffe H Weiss R Cancer, HIV and AIDS.Cancer Surv. 1991; 10: 1-3PubMed Google Scholar, 2Biggar RJ Cancer in the acquired immunodeficiency syndrome: an epidemiological assessment.Semin Oncol. 1990; 17: 251-260PubMed Google Scholar, 3Cremer KJ Spring SB Gruber J Role of human immunodeficiency virus type 1 and other viruses in malignancies associated with acquired immunodeficiency syndrome.J Natl Cancer Inst. 1990; 82: 1016-1025Crossref PubMed Scopus (52) Google Scholar Epidemiological investigations established that non-Hodgkin's B-cell lymphoma (NHL) and Kaposi's sarcoma (KS) significantly increase in HIV-1-infected patients.4Beral V Epidemiology of Kaposi's sarcoma.Cancer Surv. 1991; 10: 5-22PubMed Google Scholar, 5Harnly ME Swan SH Holly EA Kelter A Padian N Temporal trends in the incidence of non-Hodgkin's lymphoma and selected malignancies in a population with a high incidence of acquired immunodeficiency syndrome (AIDS).Am J Epidemiol. 1988; 128: 261-267PubMed Google Scholar, 6Obrams GI Grufferman S Epidemiology of HIV associated non-Hodgkin lymphoma.Cancer Surv. 1991; 10: 91-102PubMed Google Scholar Moreover, other tumors, such as basal cell and squamous cell skin carcinoma, malignant melanoma, and carcinoma of the rectum and of the uterine cervix as well as hepatocellular carcinoma are frequently present as a complication of AIDS.7Rabkin C Blattner WA HIV infection and cancers other than non-Hodgkin lymphoma and Kaposi's sarcoma.Cancer Surv. 1991; 10: 151-160PubMed Google Scholar The lack of immune surveillance that causes a state of severe immunodeficiency in HIV-1 infection and AIDS plays a major role in the induction of these tumors. However, reactivation of latent viral infections may participate in AIDS-associated oncogenesis. Indeed, Epstein-Barr virus is frequently reactivated in NHL in AIDS patients,8Luxton JC Thomas JA Crawford DH Aetiology and pathogenesis of non-Hodgkin lymphoma in AIDS.Cancer Surv. 1991; 10: 103-119PubMed Google Scholar and KS has been associated to some viruses such as cytomegalovirus,9Giraldo G Beth E Kourilsky FM Henle W Miké V Huraux JM Andersen HK Gharbi NR Kyalwazi SK Puissant A Antibody patterns to herpes-viruses in Kaposi's sarcoma: serological association of European Kaposi's sarcoma with cytomegalovirus.Int J Cancer. 1975; 15: 839-848Crossref PubMed Scopus (115) Google Scholar, 10Giraldo G Beth E Henle W Henle G Miké V Safai B Huraux JM McHardy J de-Thé G Antibody patterns to herpes viruses in Kaposi's sarcoma. II. 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AIDS and Associated Cancers in Africa.in: Giraldo G Beth-Giraldo E Clumeck N Gharbi Md-R Kyalwazi SK de Thé G S Karger, Basel1988: 175-181Google Scholar, 18Negrini M Rimessi P Mantovani C Sabbioni S Corallini A Gerosa A Barbanti-Brodano G Characterization of BK virus variants rescued from human tumours and tumour cell lines.J Gen Virol. 1990; 71: 2731-2736Crossref PubMed Scopus (39) Google Scholar HHV-6,19Bovenzi P Mirandola P Secchiero P Strumia R Cassai E Di Luca D Human herpesvirus 6 (variant A) in Kaposi's sarcoma.Lancet. 1993; 341: 1288-1289Abstract PubMed Scopus (57) Google Scholar and HHV-8.20Chang Y Cesarman E Pessin MS Lee F Culpepper J Knowles DM Moore PS Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (5030) Google Scholar Moreover, the Tat protein of HIV-1 is involved in tumor pathogenesis in HIV-1-infected patients. 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67: 277-287Crossref PubMed Google Scholar and is taken up by uninfected cells.25Frankel AD Pabo CO Cellular uptake of the Tat protein from human immunodeficiency virus.Cell. 1988; 55: 1189-1193Abstract Full Text PDF PubMed Scopus (2369) Google Scholar, 26Helland DE Welles JL Caputo A Haseltine WA Transcellular transactivation by the human immunodeficiency virus type 1 Tat protein.J Virol. 1991; 65: 4547-4549Crossref PubMed Google Scholar Tat induces growth, adhesion, migration, and invasion of KS spindle cells27Barillari G Gendelman R Gallo RC Ensoli B The tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi's sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.Proc Natl Acad Sci USA. 1993; 90: 7941-7945Crossref PubMed Scopus (342) Google Scholar, 28Albini A Fontanini G Masiello L Tacchetti C Bigini D Luzzi P Noonan DM Stetler-Stevenson WG Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2.AIDS. 1994; 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60: 577-630Crossref PubMed Scopus (281) Google Scholar, 32Buonaguro L Barillari G Chang HK Bohan CA Kao V Morgan R Gallo RC Ensoli B Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines.J Virol. 1992; 66: 7159-7167Crossref PubMed Google Scholar, 33Scala G Ruocco MR Ambrosino C Mallardo M Giordano V Baldassarre F Dragonetti E Quinto I Venuta S The expression of interleukin 6 gene is induced by the human immunodeficiency virus type 1 Tat protein.J Exp Med. 1994; 179: 961-971Crossref PubMed Scopus (233) Google Scholar Tat is highly angiogenic in vivo,30Albini A Benelli R Presta M Rusnati M Ziche M Rubartelli A Paglialunga G Bussolino F Noonan D HIV-tat protein is a heparin-binding angiogenic growth factor.Oncogene. 1996; 12: 289-297PubMed Google Scholar, 34Ensoli B Gendelman R Markham P Fiorelli V Colombini S Raffeld M Cafaro A Chang HK Brady J Gallo RC Synergy between basic fibroblast growth factor and HIV-1 Tat protein in induction of Kaposi's sarcoma.Nature. 1994; 371: 674-680Crossref PubMed Scopus (543) Google Scholar and transgenic mice expressing the tat gene develop skin lesions closely resembling the early phases of KS.35Vogel J Hinrichs SH Reynolds RK Luciw PA Jay G The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice.Nature. 1988; 355: 606-611Crossref Scopus (464) Google Scholar, 36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar In a previous study, transgenic mice were generated by means of a recombinant DNA (pRPU3R-Tat) containing BKV early region and the HIV-1 tat gene, directed by the HIV-1 long terminal repeat (LTR). BKV early region encodes the viral T antigen (TAg), which is a potent transcriptional activator of HIV-1 LTR, thus inducing expression of Tat in all organs and tissues of transgenic mice. BKV/tat transgenic animals developed vascular lesions in the dermis, skin tumors, lymphomas, liver cell dysplasia, and hepatocellular carcinoma.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar As an extension of our previous findings, in this study we have analyzed in detail the morphological, histochemical, immunohistochemical, and ultrastructural characteristics of tumors, of hyperplastic and dysplastic lesions, and of non-neoplastic lesions arising in BKV/tat transgenic mice. The plasmid used to generate transgenic mice is pRPU3tat.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar This recombinant DNA contains tat cDNA directed by its own promoter-enhancer, the HIV-1 LTR. In addition, it contains the complete BKV early region, consisting of the coding sequences for large T antigen and small t antigen as well as the early promoter and enhancer, the replication origin, and poly A sequences. This construct produces three mRNAs, one transcribed from tat cDNA and two transcribed from BKV early region, expressing large T antigen and small t antigen, respectively. The generation of BDF transgenic mice carrying BKV/tat sequences has been previously described.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar Seven founder mice were identified. Each founder gave rise to a heterozygous transgenic mouse line by crossing initially with normal BDF mice and then with heterozygous transgenic mice of the same lineage to obtain animals homozygous for the transgene. Homozygous transgenic mice were later crossed with outbred CD1 mice, a strain particularly sensitive to the effects of tat.35Vogel J Hinrichs SH Reynolds RK Luciw PA Jay G The HIV tat gene induces dermal lesions resembling Kaposi's sarcoma in transgenic mice.Nature. 1988; 355: 606-611Crossref Scopus (464) Google Scholar Control animals were normal BDF mice and BDF crossed with CD1 mice. Animals were routinely examined twice a week for the appearance of symptoms, new phenotypes, and tumors. All mice either died of natural causes or were sacrificed for advanced symptoms or lesions and were subjected to autopsy. Organs were taken for histological and histochemical examination and for molecular studies. Tissue samples taken at autopsy were fixed in 10% formalin in phosphate-buffered saline (PBS) for 12 to 24 hours and embedded in paraffin. A fragment of each sample was also cryopreserved at −80°C. Paraffin-embedded sections (3 to 5 μm) were stained with hematoxylin and eosin (H&E) and treated by periodic acid-Shiff (PAS) reaction. Warthin-Starry silver stain37Bancroft JD Stevens A Theory and Practice of Histological Techniques. Churchill Livingstone, Edinburgh1982Google Scholar was carried out on ulcerative skin lesions. The avidin-biotin-peroxidase complex (ABC) technique was used for the immunohistochemical studies performed on paraffin sections. The panel of antibodies used in this study included AE-1 and Cam 5.2 (anti-low molecular weight cytokeratins; Dako, Glostrup, Denmark), S-100 (Dako), anti-α-actin (Dako), HHF 35 (anti-actins), anti-desmin (Dako), anti-vimentin (Dako), anti-light and -heavy immunoglobulin chains (Dako), and anti-CD19, -CD20, -CD21, -CD45R, and -CD45RO (Dako). Briefly, after removal of paraffin and rehydration, endogenous peroxidases were blocked with 0.3% H2O2 in methanol. Then the samples were incubated with mouse or rabbit primary antibodies for 10 to 12 hours at 4°C. Biotinylated anti-mouse and anti-rabbit immunoglobulins (Dako) were used as secondary antibodies followed by incubation with avidin-biotin-peroxidase conjugates and development in diaminobenzidine (Sigma Chemical Co., St. Louis, MO). Morphological characterization of lymphomas was carried out according to the Standard Murine Lymphoma Classification.38Pattengale PK Tumours of the lymphohaematopoietic system.in: Turusov VS Mohr U World Health Organization Pathology of Tumours in Laboratory Animals, vol 2: Tumors of the Mouse. ed 2. IARC, Lyon1994: 651-670Google Scholar BKV TAg was detected on sections of frozen tissues by indirect immunofluorescence using a first serum to TAg derived from hamsters bearing tumors induced by BKV-transformed cells and a second rabbit serum to hamster IgG conjugated with fluorescein isothiocyanate. For ultrastructural investigations, tumors were fixed with glutaraldehyde in PBS (0.2 mol/L, pH 7.2) for 8 hours, post-fixed in 1% OsO4 in PBS for 4 hours, dehydrated, and embedded in Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate and then examined in a Hitachi H-7000 electron microscope. Southern blot hybridization was carried out according to standard techniques,39Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar using BKV/tat (pRPU3Rtat) recombinant DNA as a probe labeled with 32P by nick translation to a specific activity of 1 × 109 to 6 × 109 cpm/μg, as previously reported.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar RT-PCR was used for amplification of BKV early region and tat, as previously described.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar Reverse transcription of total cytoplasmic RNA was carried out with the commercial kit provided by Invitrogen (San Diego, CA), according to the manufacturer's instructions. Two 23-mer oligonucleotides (5′-TAGGTGCCAACCTATGGAACAGA-3′ and 5′-GAAAGTCTTTAGGGTCTTCTACC-3′) were used to amplify a 180-bp sequence of BKV early region, whereas a 21-mer oligonucleotide (5′-GAAGCATCCAGGAAGTCAGCC-3′) and a 24-mer oligonucleotide (5′-ACCTTCTTCTTCTATTCCTTCGGG-3′) were used to amplify a 239-bp sequence of tat cDNA. The amplified products were hybridized with an internal BKV DNA oligonucleotide probe (5′-AATCTTCATCCCATTTTTCA-3′) or the complete tat cDNA labeled with 32P by polynucleotide kinase.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar To detect the transgene, DNA extracted from tails of the seven founder mice was doubly digested with BamHI and EcoRI, which produce four fragments on pRPU3Rtat, the transgenic plasmid containing BKV early region and tat DNA.36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar The restricted DNAs were then subjected to Southern blot hybridization. All founder mice showed the presence of the four BamHI/EcoRI fragments and of additional hybridization bands related to integration of pRPU3Rtat into flanking host DNA sequences (data not shown). The molar ratio of the four restriction fragments, measured by densitometric analysis, indicated a number of pRPU3Rtat molecules ranging from 10 to 50 copies/cell. Moreover, the results were suggestive of a single integration even of multiple copies of pRPU3Rtat arranged in a tandem array. All generations of transgenic mice obtained by mating animals belonging to the same transgenic line stably maintained this hybridization pattern. All tissues and organs in each transgenic line showed the presence of the pRPU3Rtat DNA as tandem insertions in a variable number of copies (5 to 20) per cell (Figure 1). tat and BKV RNA were detected in all tissues and in primary tumors of the transgenic mice by RT-PCR (Figure 2, A and B) with the exception of lung, which was negative for tat RNA. tat and BKV DNA and RNA were never detected in normal control mice, as expected, because mice, contrary to humans, do not bear a BKV latent infection. In addition, polyoma virus, which produces a latent infection in mice, is not homologous to BKV sequences. As previously reported,36Corallini A Altavilla G Pozzi L Bignozzi F Negrini M Rimessi P Gualandi F Barbanti-Brodano G Systemic expression of HIV-1 tat gene in transgenic mice induces endothelial proliferation and tumors of different histotypes.Cancer Res. 1993; 53: 5569-5575PubMed Google Scholar BKV TAg was constantly detected by immunofluorescence as a nuclear granular staining in tumors and organs (liver, lung, kidney, brain, and skin) of transgenic mice (data not shown).Figure 2Analysis of the expression of BKV early region and tat by RT-PCR. RNAs were extracted from tissues and transcribed into cDNA by reverse transcription. The cDNAs were amplified by PCR, migrated on 2% agarose gel, and hybridized to a 32P-labeled oligonucleotide probe internal to BKV early region (A) or to a complete 32P-labeled tat cDNA (B). A: Lane l, BKV early region amplified as a control by RT-PCR from the T53 cell line, constitutively expressing BKV TAg and tat36; lane 2, kidney; lane 3, skin; lane 4, lung; lane 5, brain; lane 6, liver. B: lane 1, tat amplified as a control by RT-PCR from the T53 cell line; lane 2, kidney; lane 3, lung; lane 4, liver; lane 5, skin; lane 6, RT-PCR solution without RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A number of phenotypes were observed in homozygous and heterozygous transgenic animals with no differences between the two groups. A total of 171 transgenic mice (135 BDF homozygotes and 36 BDF × CD1 hybrids) and 400 control animals were subjected to autopsy and histological examination of all organs. All transgenic animals were affected by lesions, whereas in BDF and BDF × CD1 control mice, only 44 of 400 animals (11%) developed lesions. A total of 282 and 68 lesions arose in transgenic and control animals, respectively (Table 1).Table 1All Types of Lesions Observed in BKV/tat Transgenic and Control MiceAnimalsTransgenic mice with lesionsControl mice with lesionsBDF135/135 (100%)38/316 (12.0%) P < 10−38BDF× CD136/36 (100%)6/84 (7.1%) P = 10−25Total171/171 (100%)44/400 (11.0%) P < 10−38P = probability by Fisher's exact test. Open table in a new tab P = probability by Fisher's exact test. The lesions observed in transgenic and in control mice were grouped in three morphological categories: 1) tumors, 2) hyperplastic and dysplastic lesions, and 3) non-neoplastic lesions. This last group included two subtypes: 1) specific lesions and 2) secondary lesions. Specific lesions were observed almost exclusively in transgenic animals, whereas secondary lesions were caused by other illnesses such as chronic infections and tumors or were unrelated to the experimental conditions (spontaneous lesions). Fifty tumors (29.2%) were observed in transgenic mice (Table 2), and more than one type of tumor was frequently present in the same animal. The majority of the neoplasms were malignant, invasive, and producing metastases, except for benign hemangiomas of the liver. In control animals, 15 tumors (3.7%) of only two histotypes were found: adenocarcinomas of the skin glands and lymphomas (Table 2). In control mice, lymphomas were invariably detected in the oldest animals. In transgenic and control animals, tumors appeared both in males and females.Table 2Tumors Observed in BKV/tat Transgenic and Control MiceTransgenic miceControl miceTumor histotypeNumberPercentage*The percentage is related to the total of transgenic mice (171) and control mice (400), respectively.P, probability by Fisher's exact test.NumberPercentage*The percentage is related to the total of transgenic mice (171) and control mice (400), respectively.P, probability by Fisher's exact test.Skin adenocarcinoma137.661.5 P = 10−4Skin squamous cell carcinoma31.7Leiomyosarcoma105.8Hepatoma/hepatocarcinoma52.9Liver hemangioma42.3Lymphoma137.692.2 P = 10−3Lung carcinoma10.6Lipoma10.6Total5029.2153.7 P = 10−17* The percentage is related to the total of transgenic mice (171) and control mice (400), respectively.P, probability by Fisher's exact test. Open table in a new tab In transgenic mice, the skin with the adnexes, the eyes, the liver, the rectum, and the lymphoid system were the most frequently involved organs. These tissues also showed hyperplastic and dysplastic alterations associated with tumors, and tumors manifested an early onset in younger animals compared with control mice. These results are consistent with a greater frequency and a wider range of phenotypic alterations in transgenic mice than in control mice. These tumors showed a nodular growth pattern with great invasiveness in the adjacent tissues. The tumor cells appeared undifferentiated with scarce basophilic cytoplasm and enlarged hyperchromic nucleus (basaloid cells, Figure 3A). Chromatin granules were adherent to the nuclear membrane, and nucleoli were often evident. PAS reaction for cytoplasmic mucins was constantly negative. More differentiated zones consisted of 1) tubular or glandular structures lined by epithelial cells with apocrine secretion features (cytoplasmic blebs of secretion) and differentiation and 2) large cystic spaces resulting from the confluence of glandular structures and sometimes containing prominent papillae lined by epithelial apocrine cells. The ratio between differentiated and undifferentiated zones was variable with slight prevalence of the undifferentiated ones. Immunohistochemical reactions confirmed the histological diagnosis showing strong reactivity to CAM 5.2, AE-1, and S-100 antibodies in the cytoplasm of the neoplastic cells. Ultrastructural observations demonstrated numerous lumens lined by microvilli. The neoplastic cells showed blebbing apical cytoplasm and tight junctions of the membrane and contained rough endoplasmic reticulum, mitochondria, and large electron-dense secretory granules, 300 to 800 nm in size, that resembled lysosomes (Figure 3B). Skin adenocarcinomas in control mice were very similar to those observed in transgenic mice, except that they appeared in a significantly lower proportion of animals (Table 2). Three tumors of the skin observed in transgenic mice belonged to this group; two were deeply located nodular tumors, and one had a verrucous esophitic appearance. Neoplastic cells situated in the periphery had scarce basophilic cytoplasm and large hyperchromatic nuclei like basal-type epidermal cells, tending to a progressive maturation toward the center of the tumor nodule (Figure 3C). In the center of the tumor, where the differentiation was completed, squamous cells, horny cysts, and keratin scales were observed. One of the tumors
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