Development of Hyperplasias, Preneoplasias, and Mammary Tumors in MMTV-c-erbB-2 and MMTV-TGFα Transgenic Rats
1999; Elsevier BV; Volume: 155; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)65124-3
ISSN1525-2191
AutoresBarry R. Davies, Angela Platt‐Higgins, Günter Schmidt, Philip S. Rudland,
Tópico(s)T-cell and Retrovirus Studies
ResumoHuman cDNAs corresponding to two epidermal growth factor-related products that are overexpressed in human breast cancers, that for c-erbB-2 (HER-2) and for transforming growth factor α (TGFα), have been cloned downstream of the mouse mammary tumor virus (MMTV) long terminal repeat promoter and injected into the pronucleus of fertilized oocytes of Sprague-Dawley rats to produce transgenic offspring. Expression of the transgenic mRNAs is not detectable in mammary tissue from virgin transgenic rats but is detected in mammary tissue from certain lines of mid-pregnant transgenic rats. When two such lines of either type of transgenic rat are subjected to repeated cycles of pregnancy and lactation, they produce, primarily in the mammary glands, extensive pathologies, whereas virgin transgenic rats produce no such abnormalities. Multiparous transgenic female offspring from c-erbB-2-expressing lines develop a variety of focal hyperplastic and benign lesions that resemble lesions commonly found in human breasts. These lesions include lobular and ductal hyperplasia, fibroadenoma, cystic expansions, and papillary adenomas. More malignant lesions, including ductal carcinoma in situ and carcinoma, also develop stochastically at low frequency. The mammary glands of transgenic females invariably fail to involute fully after lactation. Similar phenotypes are observed in female MMTV-TGFα transgenic rats. In addition, multiparous TGFα-expressing female transgenics frequently develop severe pregnancy-dependent lactating hyperplasias as well as residual lobules of hyperplastic secretory epithelium and genuine lactating adenomas after weaning. These transgenic rat models confirm the conclusions reached in transgenic mice that overexpression of the c-erbB-2 and TGFα genes predisposes the mammary gland to stochastic tumor development. Human cDNAs corresponding to two epidermal growth factor-related products that are overexpressed in human breast cancers, that for c-erbB-2 (HER-2) and for transforming growth factor α (TGFα), have been cloned downstream of the mouse mammary tumor virus (MMTV) long terminal repeat promoter and injected into the pronucleus of fertilized oocytes of Sprague-Dawley rats to produce transgenic offspring. Expression of the transgenic mRNAs is not detectable in mammary tissue from virgin transgenic rats but is detected in mammary tissue from certain lines of mid-pregnant transgenic rats. When two such lines of either type of transgenic rat are subjected to repeated cycles of pregnancy and lactation, they produce, primarily in the mammary glands, extensive pathologies, whereas virgin transgenic rats produce no such abnormalities. Multiparous transgenic female offspring from c-erbB-2-expressing lines develop a variety of focal hyperplastic and benign lesions that resemble lesions commonly found in human breasts. These lesions include lobular and ductal hyperplasia, fibroadenoma, cystic expansions, and papillary adenomas. More malignant lesions, including ductal carcinoma in situ and carcinoma, also develop stochastically at low frequency. The mammary glands of transgenic females invariably fail to involute fully after lactation. Similar phenotypes are observed in female MMTV-TGFα transgenic rats. In addition, multiparous TGFα-expressing female transgenics frequently develop severe pregnancy-dependent lactating hyperplasias as well as residual lobules of hyperplastic secretory epithelium and genuine lactating adenomas after weaning. These transgenic rat models confirm the conclusions reached in transgenic mice that overexpression of the c-erbB-2 and TGFα genes predisposes the mammary gland to stochastic tumor development. Transgenic techniques have been used to create many models of mammary neoplasia in the mouse1Amundadottir LT Merlino G Dickson RB Transgenic mouse models of breast cancer.Breast Cancer Res Treat. 1996; 39: 119-135Crossref PubMed Scopus (51) Google Scholar, 2Cardiff RD The biology of mammary transgenes: five rules.J Mammary Gland Biol Neoplasia. 1996; 1: 61-74Crossref PubMed Scopus (36) Google Scholar and, together with models of tissue reconstitution,3Edwards PAW Abram CA Bradbury JM Genetic manipulation of mammary epithelium by transplantation.J Mammary Gland Biol Neoplasia. 1996; 1: 75-90Crossref PubMed Scopus (29) Google Scholar have provided an insight into the function of various oncogenes in the development of breast cancer. 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Two of the most consistently expressed dominantly acting oncogenes in human breast cancer are those for transforming growth factor α (TGFα) and for c-erbB-2 (HER-2). 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Thus, expression of neu in the mammary glands of transgenic mice has been reported to result in the rapid development of multifocal mammary tumors that metastasize with high frequency,27Muller WJ Sinn E Pattengale PK Wallace R Leder P Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene.Cell. 1988; 54: 105-115Abstract Full Text PDF PubMed Scopus (1001) Google Scholar, 28Guy CT Webster MA Scholler M Parsons T Cardiff RD Muller WJ Expression of the neu proto-oncogene in the mammary epithelium of transgenic mice induces metastatic disease.Proc Natl Acad Sci USA. 1992; 89: 10578-10582Crossref PubMed Scopus (658) Google Scholar, 29Luccini F Sacco MG Hu N Villa A Brown J Cesano L Mangiarini L Rindi G Kindl S Sessa F Vezzoni P Clerici L Early and multifocal tumors in breast, salivary, Harderian and epididymal tissues developed in MMTV-neu transgenic mice.Cancer Lett. 1992; 64: 203-209Abstract Full Text PDF PubMed Scopus (113) Google Scholar whereas other laboratories have reported only the stochastic development of mammary tumors with little evidence of metastasis.30Bouchard L Lamarre L Tremblay PJ Jolicoeur P Stochastic appearance of mammary tumors in transgenic mice carrying the MMTV c-neu proto-oncogene.Cell. 1989; 57: 931-936Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 31Suda Y Aizawa S Furuta Y Yagi T Ikawa Y Saitoh K Yamada Y Toyoshima K Yamamoto T Induction of a variety of tumors by c-erb-B2 and clonal nature of lymphomas even with the mutated gene (Val 659 to Glu 659).EMBO J. 1990; 9: 181-190Crossref PubMed Scopus (74) Google Scholar Expression of the non-mutated form of the rat c-erb-B2 gene in transgenic mice also resulted in the stochastic development of mammary tumors, many of which metastasized.32Guy CT Webster MA Schaller M Parson TJ Cardiff RD Muller WJ Expression of the neu proto-oncogene in the mammary epithelium of transgenic mice induces metastatic disease.Proc Natl Acad Sci USA. 1992; 89: 10578-10582Crossref PubMed Scopus (1032) Google Scholar The reasons for these discrepancies are unclear but may depend on novel additional somatic activating mutations within neu/c-erb-B2.33Siegel PM Dankort DL Hardy WR Muller WJ Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.Mol Cell Biol. 1994; 14: 7068-7077Crossref PubMed Scopus (190) Google Scholar In view of the potential advantages of the rat as a model for human breast cancer and in view of the discrepancies between the mouse transgenic models, we have developed a method to produce transgenic rats using the same MMTV promoter linked to the Rous sarcoma virus (RSV) long terminal repeat (LTR) enhancer to drive expression in the mammary glands of either the human TGFα or the wild-type human c-erbB-2 genes. This approach has produced a variety of mammary hyperplasias, preneoplastic lesions, and tumors in the rat. In this communication, we report an analysis of the generation and phenotypes of these animals to the second (F2) generation. For the c-erbB-2 transgene, a 4.4-kb HindIII fragment containing the normal, unmutated human c-erbB-2 cDNA16Yamamoto T Ikawa S Akiyama T Semba K Nomura N Miyajima N Saito T Toyoshima K Similarity of protein encoded by the human c-erb-B2 gene to epidermal growth factor receptor.Nature. 1986; 319: 230-234Crossref PubMed Scopus (1140) Google Scholar was cut out of the plasmid pSV2-erb-B2 and subcloned into the cloning vector pPolyIII-I,34Lathe R Vilotte JL Clark AJ Plasmid and bacteriophage vectors for excision of intact inserts.Gene. 1987; 57: 193-201Crossref PubMed Scopus (118) Google Scholar to generate the plasmid pPolyIII-erb. A 1548-bp EcoRI-SmaI fragment containing the RSV-LTR enhancer linked to the MMTV-LTR promoter was excised from the plasmid pMam-neo (Clontech, Palo Alto, CA) and cloned into the EcoRI and EcoRV sites of pPolyIII-I to generate pIII-MMTV. The c-erbB-2 cDNA was then transferred to pIII-MMTV using the XbaI-SalI sites, to generate the plasmid pIII-MMTV-erb. A 667-bp SmaI-XbaI fragment containing a splice and polyadenylation signal from the 3′ end of the human growth hormone (hGH) gene was excised from the plasmid pBShGH (a gift from Dr. J. Gordon, Washington University, St. Louis, MO) and subcloned into the SmaI-XbaI sites of pBluescriptKS− to generate the plasmid pBlue-hGH3′. To complete the transgenic construct, the hGH3′ sequence was transferred from pBlue-hGH3′ to pIII-MMTV-erb by digesting with SalI (Figure 1a). For the MMTV-TGFα transgene, a 925-bp EcoRI fragment containing the unprocessed 917-bp TGFα cDNA sequence was cut out of the plasmid phTGF1–10-925 (a gift from Dr G. Bell, University of Chicago, Chicago, IL) and cloned into pPolyIII-I to generate the plasmid pPolyIII-TGFα. The same 667-bp SmaI- XbaI fragment containing the splice and polyadenylation signals as above was cloned into the SmaI-XbaI sites of pPolyIII-TGFα to generate the plasmid pPolyIII-TGFα-hGH3′. The same 1548-bp EcoRI-SmaI fragment containing the enhancer and MMTV-promoter as above was then cloned into the EcoRI and SmaI sites of pBluescriptKS− to generate pBlue-MMTV. To complete the transgenic construct, a BamHI-XbaI fragment from pPolyIII-TGFα-hGH3′ was cloned into the BamHI and XbaI sites of pBlue-MMTV to generate pBlueMMTV-TGFα-hGH3′ (Figure 1b). The identities of both plasmids were confirmed by restriction endonuclease mapping, and the correct nature of the ligation points was confirmed by dideoxy chain termination DNA sequencing. The completed transgenes MMTV-erb-hGH3′ and MMTV-TGFα-hGH3′ were released from their parental plasmids before being microinjected by digestion with NotI and SalI, respectively (Figure 1). All animals were maintained and procedures were performed in accordance with the British Home Office Animals (Scientific Procedures. Act 1986, under Project License 80/00733. Transgenic rats were generated by pronuclear injection of linearized DNA into fertilized eggs of Sprague-Dawley rats (Charles River Laboratories, Kent, UK), as previously described for transgenic mice35Hogan B Constantini F Lacy E Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar with the following modification. The female rats were superovulated at 30 days of age by continuous infusion rather than an injection of purified porcine pituitary follicle-stimulating hormone (FSH; Vetrepharm, London, Ontario, Canada) via Alzet miniosmotic pumps (Alzet model 2001, Alza Scientific Products, Palo Alto, CA)36Armstrong DT Opavsky MA Superovulation of immature rats by continuous infusion of FSH.Biol Reprod. 1988; 39: 511-518Crossref PubMed Scopus (67) Google Scholar. Each pump was filled with 200 μl of FSH diluted in sterile saline and was inserted intraperitoneally into pentobarbital-anesthetized animals 2 days before mating. Synchronization of ovulation was induced 48 to 52 hours later by an intraperitoneal injection of 100 ng of luteinizing-hormone-releasing hormone analogue (des-gly10(d-ala)-LHRH-ethylamide, Sigma Chemical Co., Poole, UK). After being mated overnight, the females with vaginal plugs were sacrificed by cervical dislocation. The pumps were transferred to a second set of animals, and embryos were collected in Dulbecco's phosphate-buffered saline (PBS) from the oviducts of plugged females. Embryos were rinsed free of cumulus cells in 0.1% (w/v) hyaluronidase and transferred to modified M2 medium for microinjection or modified M16 medium (280 mOsm) for culture at 38.5°C in 5% (v/v. CO2 until pronuclei became distinguishable.35Hogan B Constantini F Lacy E Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar Pronuclear injections were performed on a Nikon inverted microscope equipped with Narishige micromanipulators and Normarski optics. The excised DNA constructs were injected at approximately 2 ng/μl in 10 mmol/L Tris/HCl, 0.1 mmol/L EDTA, pH 7.4.37Brinster RL Chen HY Trumbauer ME Yagle MK Palmiter RD Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.Proc Natl Acad Sci USA. 1985; 82: 4438-4442Crossref PubMed Scopus (829) Google Scholar After injection of one pronucleus in each embryo (as evidenced by pronuclear expansion), all embryos were incubated in modified M16 until transfer. Epinephrine at 0.1% (w/v) was applied to the ovarian bursa of pseudopregnant recipients, and the bursa was torn to allow access to the infundibulum. The embryos were then transferred bilaterally to the oviduct using a finely drawn glass pipette38Warren J Blakemore S Theriogenology. 1993; 9: 337Abstract Full Text PDF Google Scholar either into day 1 pseudopregnant recipients (synchronous) or, after overnight culture, at the early two-cell stage into day 1 (asynchronous) or day 2 (synchronous) recipients. Transgenic founder rats and subsequent transgenic offspring were identified by Southern blot analysis of genomic DNA from tails of F1 litters at 10 days of age.35Hogan B Constantini F Lacy E Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1986Google Scholar Ten micrograms of appropriate restriction-enzyme-digested tail tip DNA was fractionated on 0.8% (w/v) agarose gels, transferred to Hybond N+ membranes (Amersham International, Little Chalfont, UK. by blotting in 0.4 mol/L NaOH and hybridized with the human c-erbB-2 or human TGFα cDNA probe labeled by random incorporation of [32P]dCTP (random primed DNA labeling kit, Boehringer, Mannheim, Germany) to a specific activity of 0.5 × 109 to 1 × 109 dpm/μg39Sambrook J Fitsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar and used to screen for transgene-positive rats. Transgene copy number was estimated by comparison with copy number controls. Total RNA was isolated from tissues and tumors using the guanidinium isothiocyanate/cesium chloride method.39Sambrook J Fitsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar Poly(A)-containing RNA was isolated using the PolyAtract mRNA isolation system (Promega, Madison, WI). For RNA hybridizations, 10-μg samples of poly(A)-containing RNA were subjected to denaturing-gel electrophoresis using formaldehyde39Sambrook J Fitsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar and transferred to nylon filters (Hybond N+). Hybridization and washing conditions were carried out according to the manufacturer's instructions (Amersham). Filters were subsequently hybridized using the above cDNA probes under the same conditions to a cloned cDNA corresponding to non-muscle actin to ensure consistency of loading between lanes. The radioactively hybridized filters were exposed to x-ray film, and those lanes containing different mRNA preparations were scored positive if a band corresponding to the correct sized mRNAs for c-erb-B2 or TGFα was detected. A preparation of mRNA from MCF-7 cells40Soule HD Vazquez A Long A Albert S Brennan MA A human cell line from a pleural effusion derived from a breast carcinoma.J Natl Cancer Inst. 1973; 51: 1409-1413Crossref PubMed Scopus (2156) Google Scholar was used as a positive control. Mammary glands were dissected from the skin, mounted on glass slides, fixed overnight in Methacarn (60% (v/v) methanol, 30% (v/v) 1,1,1-trichloroethane, 10% (v/v) glacial acetic acid) and then stained with carmine.41Banergee MR Wood BG Lin FK Crump LR Tissue Culture Association Manual, vol 2.in: Sanford KK TCA, Gaithersburg, MD1976: 457-462Google Scholar Macroscopically identifiable tumors were cut out and processed for histology separately. Glands were cleared by immersing in methyl salicylate. Whole mounts were photographed using Ilford Technical Pan film. Areas of interest and suspect lesions were cut out of the whole mount and processed for histology. Samples of mammary glands and any suspect lesions, including tumors, were fixed overnight in Methacarn, dehydrated in 70% (v/v. ethanol, and embedded in paraffin wax at 60°C on a Tissue Tek III embedding center (Miles, Slough, UK). Precooled blocks at −20°C were sectioned at room temperature on an Anglia AS 300 rotary microtome (Raymond A. Lamb, London, UK) using stainless steel disposable blades (Raymond A. Lamb). Sections 2 μm thick were transferred onto glass microscope slides that had been pretreated with glycerin albumin (Raymond A. Lamb) and incubated for at least 1 hour at 60°C. Sections were stained with hematoxylin and eosin (H&E). Details were recorded from at least two sections of each mammary gland/lesion. Photographs were recorded on Ilford Pan F film. Slides were examined by two independent observers (B.R. Davies and P.S. Rudland), and pathology was diagnosed as defined by the UK Royal College of Pathologists Working Group.42Royal College of Pathologists Working Group Pathology reporting in breast cancer screening.J Clin Pathol. 1991; 44: 710-725Crossref PubMed Scopus (55) Google Scholar Immunocytochemical staining of tissue sections was carried out using an antibody complex/horseradish peroxidase method (Dako, High Wycombe, UK).43Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 29:577–580Google Scholar Sections were rehydrated, treated with 0.5% (v/v) H2O2 in methanol to remove endogenous peroxidase and then with 0.5% (w/v) saponin for 30 minutes, and washed in water and then in PBS. They were then incubated with either a mouse monoclonal antibody to human TGFα (Oncogene Science, Cambridge, UK) or a rabbit polyclonal antibody to a human c-erbB-2 peptide (Dako), for 1 hour at room temperature. Sections were subsequently incubated with second antibody (biotinylated rabbit anti-mouse IgG or goat anti-rabbit IgG; Dako) and then with the streptavidin-biotin horseradish peroxidase complex, and finally, the color was developed with diaminobenzidine in H2O2. Selected sections were also stained with rabbit polyclonal antibodies to human callus keratin, chick smooth muscle actin, and mouse laminin, as previously described.44Warburton MJ Mitchell D Ormerod EJ Rudland PS Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating and involuting rat mammary gland.J Histochem Cytochem. 1982; 30: 667-676Crossref PubMed Scopus (170) Google Scholar Sections were counterstained using Mayers hemalum. The percentage of immunocytochemically stained cells was verified from five randomly chosen fields from at least two sections of each of the mammary lesions by two independent observers. The specificity of staining for each antibody was checked by the complete abolition of immunocytochemical staining without the first antibody and by previous incubation of the first antibody with preparations of the requisite antigen.44Warburton MJ Mitchell D Ormerod EJ Rudland PS Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating and involuting rat mammary gland.J Histochem Cytochem. 1982; 30: 667-676Crossref PubMed Scopus (170) Google Scholar Photographs were recorded in a Reichert-Polyvar microscope on Ilford Pan F film. Hybridizations of the human c-erbB-2 and TGFα cDNAs to tail-tip DNAs indicated that 7/105 (7%) and 6/42 (14%) of the offspring resulting from oviduct transfers of MMTV-c-erbB-2. and MMTV-TGFα-microinjected embryos, respectively, were transgenic. The founder animals contained from less than 1 up to 50 copies of the integrated transgene per haploid genome (Table 1) and, where successfully detected, transmitted the transgene either in Mendelian or mosaic fashion to their F1 generation. Thereafter, transmission, if it occurred, was exclusively Mendelian (Table 1). The results were confirmed by hybridizing a probe coding for the MMTV promoter to the same tail-tip DNA (not shown).Table 1Generation of MMTV-c-erbB-2 and MMTV-TGFα Transgenic RatsFounderSexCopies of transgeneInheritanceTransgene mRNAMMTV-c-erbB-2 ERB/1F10–20Mendelian+ ERB/2F1–5Mendelian+ ERB/3F∼50Mendelian+ ERB/4M1 and >20*Two independent integration sites.Mendelian− ERB/5F<1Unknown†Sub-fertile, all the rats from the single litter were negative for the transgene.− ERB/6F5–10Mosaic− ERB/7M1Sex-linked‡Transgene on Y chromosome, no female transgenic offspring produced.−MMTV-TGFα TGF/1F5–10Mendelian+ TGF/2F1Mosaic+ TGF/3M10Mosaic− TGF/4M10Mosaic− TGF/5M5–10No transmission− TGF/6M1–5Unknown§Could not be successfully mated.Founder indicates transgenic rat produced from either the MMTV-erb-hGH3′ or MMTV-TGFα-hGH3′ constructs. Copies of transgene were determined by Southern hybridizations to tail-tip DNA in relation to 1, 10, and 100 copy DNA controls (Materials and Methods). Inheritance was either Mendelian or mosaic occurring in less than 50% of the F1 offspring, but thereafter inheritance was Mendelian in the F2 generation. Transgene mRNA was determined by Northern hybridizations in mamm
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