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American Journal of Pathology, Vol. 138, No. 5, May 1991 Copyright © Ameffcan Association of Pathologits
Neuronal and Glial Properties of a Murine Transgenic Retinoblastoma Model Tero Kivela,* Ismo Virtanen,t Dennis M. Marcus,t Joan M. O'Brien, James L. Carpenter,11 Edgard Brauner, Ahti Tarkkanen,* and Daniel M. Albertt From the Departments of Ophthalmology and Anatomy, t University of Helsinki Helsinki, Finland; the David G. Cogan Eye Pathology Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, and the Department of
Pathology and Medicine,11 Angell Memorial Hospital, Boston, Massachusetts
Antigenic properties of a murine transgenic model for hereditary retinoblastoma induced by a chimeric gene coding for Simian virus 40 large T antigen, an oncogene that inactivates the retinoblastoma susceptibility gene product, were studied by immunohistochemistry. All transgenic mice develop bilateral intraocular retinal tumors in the inner nuclear layer with Homer Wright-like rosettes, and one quarter develop midbrain tumors resembling trilateral retinoblastoma Cell lines TE-I and TM-I were established from intraocular and metastatic tumors, respectively. Intraocular tumors reacted with antibodies to neuron-specific enolase and synaptophysirn while vimentint glial fibrillary acidic, and S-100 proteins were detected only in reactive glia derived from adjacent retina The midbrain tumors showed weak reactivity to synaptophysin, and they blended with reactive astrocytes positive for glial markers. The tumors were negative for cytokeratins. Finally both derived cell lines expressed synaptophysin and individual neurofilament trnpletproteins in immunofluorescence and Western blotting, supporting their essentially neuronal nature. The antigenic profile resembles human retinoblastoma, but differences in morphology and antigen distribution suggest a more close relationship to neurons of the inner nuclear layer than to photoreceptor cells (AmJPathol 1991, 138:1135-1148)
Retinoblastoma is a unique cancer with several unusual characteristics. It generally affects only children aged less than 8 years.' About one third of retinoblastomas are
hereditary and these cases are typically multifocal and bilateral.1 Patients with hereditary retinoblastoma may develop a concurrent pineal or midbrain tumor in addition to bilateral ocular neoplasms,2- and they are prone to have secondary unrelated malignant tumors later in life.56 A normal dominant antioncogenic allele in the retinoblastoma locus is required for controlled cell proliferation and, consequently, for orderly development of the retina.7-11 Mutational inactivation of both gene copies results in retinoblastoma, which will be hereditary if the first mutation occurred in a germline cell.11 Although retinoblastoma does not occur spontaneously in any animal species, a corresponding murine gene has been identified.12'13 In addition to inactivation due to mutation, certain viral oncoproteins can directly bind to and functionally inactivate the normal product of the retinoblastoma gene.1417 Indeed, during attempts to induce transgenic chromophobe adenomas in mice with one such oncogene, the Simian virus 40 large T antigen, one strain accidentally emerged, which develops hereditary retinal and midbrain tumors reminiscent of human retinoblastoma.1-20 In this paper we characterize further murine retinal and midbrain tumors and derived cell lines as to their neuronal and glial properties, and compare them to normal murine retina and to results previously obtained with human retinoblastoma in our laboratory.2145
Materials and Methods
Transgenic Mice Generation and propagation of the transgenic mice was described previously.18'19 Briefly, a chimeric gene consisting of the luteinizing hormone p-subunit promoter region and the Simian virus 40 (SV40) early region, which includes the protein-coding region for large T (T-Ag) and small t antigens, was purified and microinjected into ferSupported by grants from the Eye and Tissue Bank Foundation, Finnish Cancer Research Fund, Finnish Medical Research Council, Massachusetts Lions Eye Research Fund, and NIH grant EY01917. Accepted for publication January 7, 1991. Address reprint requests to Tero KivelA, MD, Ophthalmic Pathology Laboratory, Department of Ophthalmology, Helsinki University Central Hospital, Haartmaninkatu 4 C, SF-00290 Helsinki, Finland.
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tilized single cell murine ovocytes.18 Transgen-bearing offspring was selected by slot-blot analysis of tail DNA using a SV40 T-Ag-specific probe, and the animals were mated with CB6F1/J male and female breeder mice.18'19 One male transgenic mouse and all of its offspring developed bilateral retinal neoplasms, which were first seen microscopically at the age of about 2 months and filled the vitreous cavity by the age of 5 to 6 months.18'19 In addition, about one quarter of the offspring developed focal midbrain tumors, which apparently originated in the subependymal matrix layer of the mesencephalon, inferior to the cerebral aqueduct.2' The affected mice die due to widespread metastatic disease or to the midbrain tumor.18--0 For immunohistochemical studies, seven eyeglobes from six transgenic mice with retinal neoplasms (ages 2, 3, 4.5, and 6 months) and brains from four mice with midbrain tumors (ages 1.5,4, and 4.5 months) were fixed in 10% neutral-buffered formalin and embedded in paraffin. Before embedding, the brain specimens that included the cranial vault were decalcified in 5% nitric acid for 24 hours. Frozen specimens of intraocular tumors were obtained from three transgenic mice (ages 3,4, and 5 months) and sections from midbrain lesions from two mice (ages 3.5 and 4 months). For control purposes, formalin-fixed and paraffin-embedded eyeglobes from two mice (ages 2 and 5 months), and frozen sections from one mouse (age 6 months) that did not bear the transgene also were examined using identical methodology.
Cell Culture Several cell cultures were initiated from murine intraocular tumors and their cervical lymph node metastases. Tumor tissue (2 x 107 cells) was injected subcutaneously into nude mice. The subcutaneous tumors were transferred into tissue culture and grown in Dulbecco's Minimum Essential Medium (DMEM) containing 5% fetal calf serum, 15% horse serum, penicillin, and streptomycin. Although most cultures failed to proliferate, cell lines TE-1 and TM-1 could be established from intraocular and metastatic retinal transgenic tumors, respectively. For immunohistochemical and Western blotting analysis, the two established cell lines were maintained in suspension culture in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal calf serum (Gibco, Paisley, UK) and antibiotics. They were also cultured in plastic culture dishes with glass coverslips in RPMI 1640 medium supplemented with 1.0 mmol/ (millimolar) sodium butyrate (Fluka Ag, Buchs, FRG), as previously described.25
Immunohistochemical Stainings Immunoperoxidase stainings of formalin-fixed and paraffin-embedded material were carried out using commercial versions (Vectastain ABC Elite Kits, Vector Laboratories, Burlingame, CA) of the avidin-biotinylated peroxidase complex (ABC) method, as has been described.21 25 Frozen sections, spread cells on coverslips, and cytocentrifuge specimens were fixed in methanol for 10 minutes at -200C and studied by indirect immunofluorescence microscopy. In these experiments, secondary goat anti-mouse and goat anti-rabbit IgG antisera conjugated to fluorescein and rhodamine isothiocyanate (Boehringer Mannheim, Mannheim, FRG; 1:50), respectively, were used as has been described.22'24'25 The primary anti-neuron-specific enolase (lot 084; dilution 1:1000), ant4S-100 protein (lot 026; 1:2000), and anti-myelin basic protein (lot 045B; 1:200) rabbit antisera were purchased from Dakopatts a/s (Glostrup, Denmark). Murine monoclonal antibodies to synaptophysin (clone SY38; lot 11618322-02; 2 ,ug/ml; Boehringer Mannheim),26 HNK-1 epitope (ant-Leu-7; lot J01 19; 1:25; Becton Dickinson Monoclonal Center, Mountain View, CA), cytokeratin 18 (clone CK5; lot 37F-4958; Sigma Chemical Co., St. Louis, MO), cytokeratins 8,18, and 19 (clone CAM 5.2; lot L0102; undiluted; Becton Dickinson), and a conformational epitope common to most cytokeratins (clone lu-5; lot 10968521-01; undiluted; Boehringer Mannheim)27 were obtained commercially. Mouse monoclonal anti-glial fibrillary acidic protein (GFAP), anti-200 kd, and anti-1 60 kd neurofilament triplet protein antibodies (clones 1 A3 and 1 3AA8),28 as well as polyclonal rabbit antisera against vimentin, GFAP, and 68 kd neurofilament triplet protein-9 have been previously described. Murine monoclonal IgM antibodies against vimentin were prepared as described.' When antibodies to synaptophysin and intermediate filaments were used, the sections were pretreated with 0.4% pepsin (2500 FIP-U/g; E. Merck, Darmstadt, FRG) in 0.01 N hydrochloric acid for 15 minutes at 37°C to reduce background and to enhance the intensity of specific staining.2224 In control series, the primary antibody was omitted or substituted by normal rabbit serum or unrelated IgG and IgM monoclonal antibodies. The secondary anti-mouse anti-sera bound unspecifically to blood vessels, areas of necrosis and hemorrhage due to the presence of mouse immunoglobulins.
Polyacrylamide Ge/ Electrophoresis and Immunoblotting For polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the cells were harvested by
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centrifugation and boiled in electrophoresis sample buffer.31 Electrophoresis was carried out in 8% slab gels using the method of Laemmli.31 The polypeptides then were transferred onto nitrocellulose sheets and immunostaining was done as has been described.25'32 In immunoblotting experiments, rabbit antisera to GFAP and the three neurofilament triplet proteins provided by Dr. D. Dahl (Harvard University, Boston, MA) also were used.
Results
Light Microscopy of Autopsy Specimens The intraocular and midbrain tumors examined spanned a spectrum from incipient to advanced necrotic lesions. The earliest retinal tumors consisted of small groups of neoplastic cells with pleomorphic nuclei in the inner plexiform layer, most often near the ora serrata region (Figure 1A, B). Scattered neoplastic cells often were also seen elsewhere in the inner nuclear layer, mainly close to the outer plexiform layer (Figure 1C). In older mice, the tumors were typically multicentric and infiltrated adjacent retinal layers. The tumors consisted of polygonal neuroblastic cells, which had relatively large nuclei and scanty cytoplasm (Figure 1 D). The nuclei contained multiple small nucleoli and chromatin clumps, they were often lobulated, and many bizarre giant forms were seen. Mitotic figures, many of which were polyploid or otherwise aberrant, were seen very frequently (Figure 1 D). Scattered necrotic cells and confluent areas of necrosis were both common. The neoplastic cells frequently had thick cytoplasmic processes that formed variable fibrillary areas between clusters of tumor cells. Many of these resembled Homer Wright rosettes sometimes seen in human retinoblastoma (Figure 1 E). Such rosettes were present both in incipient and in advanced tumors (Figure 1 A). Small tumor cell groups with cytoplasmic processes sometimes could simulate fleurettes of differentiated human retinoblastoma (Figure 1 F). Most fibrillary areas were of haphazard shape, however, and bore close resemblance to plexiform layers of normal murine retina (Figure 1 G). FlexnerWintersteiner-like rosettes, which have a central lumen and are pathognomonic for human retinoblastoma, were not observed in the sections studied by immunohistochemistry. The advanced tumors filled the vitreous cavity and led to total detachment and destruction of the remaining retina. These tumors also invaded the retinal pigment epithelium, choroid, optic nerve, and anterior chamber. They contained frequent necrotic foci and caused subretinal and anterior chamber hemorrhage. Extraocular growth
was not observed. The morphology of tumor cells did not differ from early lesions and many rosettes were still present. The tumors had no stroma but were supplied by many small capillaries derived from destroyed retina and choroid. The morphology of the associated midbrain tumors already was described in detail.20 These primitive subependymal midline neoplasms, which consist of clusters of anaplastic tumor cells with pleomorphic nuclei and abundant mitoses, typically arise close to the cerebral aqueduct. The morphology of the tumor cells resembles that of retinal lesions (Figures 2A, B), but cytoplasmic processes are ill defined and complete Homer Wrightlike rosettes usually are not found in midbrain tumors.
Immunohistochemistry of Autopsy Specimens Rabbit antiserum to neuron-specific enolase (NSE) reacted with perikarya and cell processes in all layers of normal murine retina with the exception of outer and inner segments of photoreceptors, which were only weakly labeled (Figure 3A). Most tumor cells, both in early and in advanced cases, were not labeled, and the neoplastic foci were seen easily as negative areas within an otherwise positively reacting inner nuclear layer (Figure 3B). However, in all studied specimens, a population of neoplastic cells exhibited a moderate cytoplasmic positivity for NSE, and fibrillary areas, including the centers of Homer Wright-like rosettes, reacted positively (Figure 3B, C). No unequivocal positive reaction could be detected in any of the midbrain tumors studied, although normal cerebral neurons and their processes were labeled strongly (Figure 4A, B). The monoclonal antibody SY38 reacted exclusively with the plexiform layers of normal murine retina (Figure 3D). It reacted strongly with all Homer Wright-like rosettes and other fibrillary areas, but perinuclear reaction was seen very rarely either in cells that formed them or in undifferentiated tumor cells in formalin-fixed paraffin sections (Figure 3E). However, in frozen sections studied by indirect immunofluorescence, faint granular perinuclear reaction was seen in a population of neoplastic cells (Figure 3F). The brain tumors were all negative for synaptophysin in formalin-fixed and paraffin-embedded sections, but some tumor cells contained a few positive granules in frozen specimens (Figure 4C). The surrounding neuropil displayed a granular positivity in all specimens regardless of tissue processing. The polyclonal rabbit antiserum and all monoclonal antibodies to individual neurofilament triplet proteins labeled the nerve fiber layer of normal murine retina (Figure 3G). The antiserum to the low-molecular-weight triplet
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Murine Transgenic Retinoblastoma 1139 AJP May 1991, Vol. 138, No. 5
Figure 1. Morphology of the transgenic murine retinoblastomalike tumors (hematoxylin and eosin). A: Moderately advanced intraretinal tumor, which hasformed several Homer Wright-like rosettes (r), replaces the normal retina between the inner plexiform (ipl) and outer (onl) nuclear layers. 7he tumor has destrcyed the entire inner nuclear and most of the outerplexiform layer (x 190; age, 4.5 months). B: An early tumor focus close to the ora serrata in the inner nuclear layer (X300; age, 3 months). C: 7Te inner nuclear layer contains scattered pathologic cells (within circle) at the border of the outer plexiform layer (X300; age, 2 months). D: An advanced tumor, which has totally destroyed the retina, is composed of neoplastic cells with very pleomorphic nuclei Several mitotic figures (within circles), some of which are polyploid (m), as well as giant nuclei (gn), are present. Many plexiformlike areas (pl) of variable size and shape are found among the tumor cells (X375; age, 4.5 months). E: Frequent Homer Wright-like rosettes are formedfrom intertwined cytoplasmic processes of neoplastic cells (x600; age, 4.5 months). F: Small groups of tumor cells sometimes form fleurettelike bundles of cytoplasmic processes (within box) in addition to rosettes (x 600; age, 4.5 months). G: In many cases, the processes of tumor cellsform largerfibrillaiy areas, which closely resemble plexiform layers of normal murine retina (x 600; age, 4.5 months). In this and subsequentfigures: nfl, nervefiber; gcl, ganglion cell; ipl, inner plexiform; inl, inner nuclear; opl, outer plexiform; onl, outer nuclear; and prl, photoreceptor cell layer. 4
protein in addition revealed perinuclear fibrillary reaction in ganglion cell bodies and in their processes in the inner plexiform layer (Figure 3G), and labeled the horizontal cells in some specimens. The frozen sections, in which labeling of horizontal cells was constant, revealed positive fibrils among tumor cells in eary retinal lesions (Figure 3H). In more advanced tumors, only nerve fibers from destroyed retina were detected in most specimens (Figure 31). Centers of Homer Wright-like rosettes and fibrillary areas always were negative. No positive tumor cells were seen in any midbrain tumor studied, even though positively reacting nerve fibers radiating from adjacent brain always were present (Figure 4D). Both polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) reacted with astrocytes of the retina but did not label radial glia of MOller, except in the ora serrata region (Figure 5A). However, even in eyes with very early lesions, where only single neoplastic cells were present in the inner nuclear layer, radial fibers throughout the retina reacted positively for GFAP up to the outer plexiform layer, and near the tumorous foci they
had thick processes that reached the outer limiting membrane (Figure 5B). All neoplastic cells were completely negative for GFAP. In advanced lesions, only perivascular and scattered reactive glial cells that derived from the infiltrated retina and optic nerve were seen (Figure 50). In midbrain tumors, an extensive meshwork of positive glial processes, which belonged to large stellate astrocytes with small vesicular nuclei (Figure 2B), intimately surrounded unlabeled tumor cells (Figure 4E, F). The extent of this reactive gliosis varied from specimen to specimen, but positive transitional cells resembling neoplastic cells were never seen. The polyclonal antiserum and monoclonal antibodies to vimentin reacted with astrocytes, radial glial fibers, and horizontal cell processes throughout the normal murine retina (Figure 5D). Murine retinal and midbrain tumors were entirely negative for vimentin, although positive reaction was seen in reactive and entrapped glial cells within these tumors (Figure 5E). In very advanced cases, fibrous ingrowth from infiltrated choroid reacted positively.
Figure 2. Morphology of the transgenic murine midbrain tumors (hematoxylin and eosin stain). A: Undifferentiated tumor cells without obvious rosettesgrow in a diffusepattern and typically show little invasion of the adjacent normal brain (br) tissue (X375; age, 1.5 months). B: Individual tumor cells have pleomorphic and bypercbromatic nuclei with indented nuclear membranes. Note the presence of cells with oval, vesicular nuclei with single, well-defined nucleoli (within circles), corresponding to reactive astrocytes, among more typical tumor cell nuclei (X375; age, 4 months).
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AJP May 1991, Vol. 138, No. 5 Figure 3. Neuronalproperties of the munne retinoblastomalike tumors(F and H, indirect immunofluorescence; others, immunoperoxidase staining). A: Antiserum to neuron-specific enolase (NSE) reacts with all neurons of normal murine retina. Note posite reaction in the inner nuclear (lnl) layer (X300; age, 2 months). B: Strong positive reaction is seen in neuronal cells originatingJfrom the destroyed inner nuclear layer (arrowheads), while the tumor (tum) that replaces this layer contains neoplastic cells that react moderately for NSE (x275; age, 4.5 months). C: Several neoplastic cells react with antiserum to NSE in an advanced tumor (X335; age, 6 months). D: In normal murine retina, synaptophysin is detected in both plexiform layers (ipl and opl), as well as in some processes ofbipolar cells in the inner nuclear layer (mnl) (X300; age, 5 months). E: Monoclonal antibody to synaptopbysin reacts intensely with all Homer Wright-like rosettes andplexiform areas (pi). Many tumor cells have positive cytoplasmic processes (arrowheads), but perinuclear reaction (within circle) is seldom observed (x425; age, 4.5 months). F: Some granular cytoplasmic reactivity for synaptophysin is seen in an early tumor (tunm) that replaces the retina between the inner and outer plexiform layers (x400; age, 3 months). G: Polyclonal antiserum to the 68-kd neurofilamentprotein reactsfaintly with ganglion cells and strongly with their processes in the nerve fiber (nfl) and inner plexiform layers. 7he honzontal cells in the outer plexiform layer are negative in this retina (X335; age, 2 months). H: Some positive fibers are seen with the same antibody in early retinal tumor close to the positive horizontal cell processes in the outer plexiform layer (x400; age, 3 months). I: In an advanced tumor, only scattered nerve processes originating from infiltrated retina react positively (x300; age, 6 months).
Rabbit antiserum to S-100 protein reacted with retinal astrocytes and labeled radial glial processes, especially in the region of the outer nuclear and plexiform layers (Figure 5F). Neoplastic cells in all specimens were negative and there was no obvious increase in its expression in reactive glial cells in any specimen (Figure 5G). The number of labeled cells in retinal tumors always was smaller than that of GFAP-positive cells. In midbrain tumors, a similar distribution of reactive glial cells was seen as with antibodies to GFAP, although these were not as clearly delineated due to the more diffuse cytoplasmic reaction (Figure 4G). Ependymal cells also were positive for S-100 protein. The polyclonal antiserum to myelin basic protein labeled myelin sheaths of the optic nerve and myelinated white matter in the murine brain (Figure 4H), but it did not react with murine retina or with intraocular and midbrain tumor cells. The HNK-1 antibody did not react with murine tissues. The monoclonal antibody to an evolutionally conserved conformation-dependent epitope common to most cytokeratins gave a moderately positive reaction with murine ciliary epithelium, retinal pigment epithelium, and choroid plexus cells. It did not label intraocular or midbrain tumor cells in any specimen studied. The other two antibodies to simple epithelial cytokeratins did not react with murine tissues.
Cell Culture Experiments Both tumor cell lines grew normally in suspension as floating aggregates without adhering to the culture flask (Figure 6A). They could be induced to spread on coverslips when exposed to sodium butyrate, after which the cells acquired cytoplasmic processes (Figure 6B). As judged by indirect immunofluorescence using rabbit antisera and monoclonal antibodies, both cell lines were negative for vimentin and GFAP. In contrast, the cells exhibited a clear fibrllary perinuclear reaction with all polyclonal and monoclonal antibodies to neurofilament triplet polypeptides used (Figure 6C). Furthermore a distinct perinuclear
granular positive reaction was seen in both cell lines with the SY38 antibody (Figure 6D). These results were identical with suspension-cultured and spread cells. Immunoblotting of homogenized cell preparations confirmed the presence of a 200-kd band corresponding to the high-molecular-weight neurofilament triplet protein with the monoclonal 1 3AA8 antibody (Figure 7, lane 2), a 68-kd low-molecular-weight neurofilament triplet protein with the polyclonal rabbit antiserum (Figure 7, lane 3), and a 38-kd polypeptide compatible with synaptophysin with the SY38 antibody (Figure 7, lane 4). Neither GFAP nor vimentin could be detected by immunoblotting in either cell line with any of the antibodies used (Figure 7, lanes 5 and 6).
Discussion The transgenic murine strain presently investigated is the first animal model of human hereditary retinoblastoma.1>-' Its close clinical and histopathologic resemblance to human retinoblastoma probably is explained by the fact that both develop due to inactivation of the normal retinoblastoma gene product.11'19 In the transgenic mice, messenger RNA corresponding to Simian virus 40 large tumor antigen is detected in tumor tissue, and antibodies to this oncoprotein coprecipitate the protein encoded by the murine retinoblastoma gene, providing evidence for its functional inactivation in these cells. 12,15,18,19 The hereditary transgenic tumors and human retinoblastomas affect newborns, are multifocal and bilateral, and invade adjacent structures in an identical fashion.18&0 Quite strikingly, one quarter of the transgenebearing mice develop midbrain neoplasms analogous to human trilateral retinoblastoma.24'0 Under the light microscope, most tumor cells are undifferentiated, often they are arranged to rosette-like formations, and the tumors easily outgrow their blood supply and become necrotic.1' Finally microtubules, dense-core neurosecretory granules, cilia without a central tubule, nuclear triple-membrane structures, and evidence of photore-
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