Retinoblastoma protein in microphthalmic mice

Exp Toxic PathoI2000; 52: 17-22 URBAN & FISCHER http://www.urbanfischer.de/journals/exptoxpath

Institute of Anatomy, University of Leipzig, Germany

Retinoblastoma protein in microphthalmic mice JAN RICHTER, ELKE BRYLLA, CLAUDIA LENK, JAN ERNSTBERGER, and HEIDEGARD HILBIG With 5 figures Received: September 25, 1998; Accepted: October 19, 1998 Address for correspondence: H. HILBIG, Institut fUr Anatomie, Universitat Leipzig, Liebigstr. 13, D - 04103 Leipzig, Germany.

Key words: Retinoblastoma; Microphthalmic mice.

Summary A microphthalmic strain of mice was used to study immunoresponse of the retinoblastoma protein. Comparing wild-type, heterozygote and homozygote microphthalmic eyes, we found an increasing labelling of phosphorylated retinoblastoma protein (pRb) in the retinal pigment epithelium. Additionally, microphthalmic eyes expressed pRb in the neuroepithelium. Especially rosettes were strongly labelled.

Introduction Retinoblastoma, the most important primary tumor of the retina, has also provided a model of oncogenesis with exciting insights into the specific genetic alterations required to transform normal cells into cancerous cells. Flexner (1881) and Wintersteiner (1897) described and illustrated the characteristic and almost pathognomic rosettes, which have been given their name. Many investigators have favoured the concept that these are not glial tumors but neuroblastic neoplasms and that the formation of rosettes is an attempt to produce photoreceptor cells. Some electron microscopic observations support this concept (Tso 1980). In man, it is impossible to study the development of rosettes. It seems worthy to reveal retinal rosettes in an animal model. We used the microphthalmic strain of mice (mi). The homozygote microphthalmic littermates of this strain (mi/mi) show a lack of pigmentation and bilateral colobomatous microphthalmia. Heterozygotes of the strain (+/mi) appear normal with grey pigmentation and normal eyes (HERTWIG 1942). The wild-type littermates (+/+) can be used as controls. In the inner layer of the microphthalmic eye cup, there is an overproliferation of cells in comparison with the outer layer. Rosettes are developed. The typical persisting coloboma consists of many rosettes of neurons. These rosettes have two or three layers of neurons. The rosettes reach the chiasma of the optic nerve.

In the heterocygotes, the amount of pigmentation of the eye cup differs from nearly unpigmented to normally pigmented eyes. Light microscopically, the reduced amount of pigment in the retinal pigment epithelium (RPE) seems to lead to micromalformations in the morphology of the retinas (HILBIG et al. 1996). The retinal rosette seems to be the endpoint of the aberrant development. Comparing these features with those of the cells of origin in retinoblastoma (Tso 1980) we find similarities. In man, current research efforts are focussing on the identification of novel-tumor associated genes and gene loci and the molecular differentiation of morphologically homogenous tumor entities. The retinoblastoma gene (13qI4) is well known (FRANCOIS et al. 1975; FRANCOIS 1977; PASSARGE 1994). The retinoblastoma gene (Rb) had been cloned and characterized by FRIEND et al. (1986), FUNG et al. (1987) and LEE et al. (l987a, b). The DNA of the Rb has 4757 nucleotides and codes a protein developed by 928 amino acids (RILEY et al. 1994). The molecular weight of the Rb-protein is 105 kD. This Rb gen seems to be the main tumor suppressor during ontogenetic development. The ability of suppression depends on phosphorylation of the Rb-protein. Unphosphorylated Rb-protein suppresses the S-phase of the cell cycle (GOODRICH et al. 1991). The similarities of the morphology of rosettes both in man and in the microphthalmic mouse raised up the question whether there are causal similarities. Therefore, we compared Rb-protein antibody binding structures of +/+, +/mi and milmi mice. Since the Rb gene encodes a phosphoprotein ubiquitously expressed in normal cells (RILEY et al. 1994), we attempted to separate the blood proteins using gel electrophoresis.

Material and methods A total of 21 mice was used, i.e. 5 +/+, 5 +/mi, 5 mi/mi mice (at the postnatal day 7) for immunohistochemical stu0940-2993/00/52/01-017 $ 12.00/0

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min (BSA, Serva) and applied at 4 °C for 48 hours. Thereafter, the sections were treated with ZAM-Cy 3 [F(ab')2Fragment IgG, DIANOVA) as fluorochrome-linked secondary antibody diluted I :50 in TBS-BSA. Thorough buffer rinses with TBS were carried out after each incubation step. Air-dried sections were mounted in entellan (Merck). Using the DAB-method, the sections were washed in 0.1 M TBS (pH 7.4) and endogenous peroxidase was blocked with 3 % HP2 for 30 min. They were then again rinsed in TBS and incubated with the Rb antibody diluted I :30 in TBS and applied at 4 °C for 24 hours. After four rinses in TBS, the sections were incubated with biotinylated secondary antibody (Vectastain) at a final dilution of I :200 for 24 hours at 4°C. After rinses they were treated with the Avidin-Biotin-Complex (Vectastain) for two hours, rinsed again and then treated with Diaminobenzidine (DAB, Sigma) for light microscopic visualization. For electron microscopy, animals were deeply anaesthetized with ether. Intracardiac perfusion was performed using paraformaldehyde (4 %) and glutaraldehyde (5 %) in 0.1 M phosphate buffer. Eyes and blocks of the optic nerve were excised, embedded in Micropal and sectioned for electron microscopy. To test whether there could be differences in the composition of plasma proteins we carried out gel electrophoresis using 7 % acrylamide gel. We investigated blood of +/+, +/mi and mi/mi mice at postnatal days 6, 7, 8 and 9. Electrophoresis was carried out during 45-48 min with voltage of 200 V. For visualization the gels were incubated in Coomasie Brilliant Blue G 250 (Fluka) for five minutes and thereafter rinsed and stored in acetic acid/methanol solution.

A

Results Fig. 1. Comparison of pRb immunoresponse in the retinas of +/+ (A) and +/mi mice (B): No immunoresponse in +/+ but in +/mi. Bar: 10 /lm.

dies and two animals of each group (at postnatal day 15) for electron microscopical studies. All animals were housed under a natural light-darkness schedule and given free access to food and water. The maintenance of the animals was approved by the University Animal Care Committee according to the Declaration of Helsinki. The rules of laboratory animal care (NIH publication No. 86-23) and the German law for animal protection were followed. The animals were deeply anaesthetized with ether, decapitated, and the heads immediately frozen in liquid nitrogen. Seven /lm sections were cut using a cryostat, mounted on glass slides and fixed with acetone. For the detection of the Rb-protein we used a monoclonal antibody (SA-188, BIOMOL, Hamburg, Germany) which recognizes underphosphorylated and highly phosphorylated Rb 110-114 kDa protein of humans, and to lesser extend mouse. The epitope is human Rb (300-380). We visualized the reaction product using both the DAB method and fluorescence immunohistochemistry. Unspecific binding was blocked with 5 % normal goat serum (DAKO) for two hours at room temperature. Then the sections were incubated with the Rb antibody which had been diluted I :30 in 0.05 M Tris buffer solution (TBS, pH 7.6) with 5 % bovine serum albu18

Exp Toxic Pathol 52 (2000) I

Immunoresponse of the investigated phosphorylated Rb protein (pRb) was found in the retinas of +/mi and mi/mi mice, never in those of the +/+ controllittermates (fig. I). In the heterozygote mice, the expression of pRb seemes to depend on the amount of pigment and the morphology of the RPE. Figure 2 shows two examples of those comparing the light microscopic features of pigment and the fluorescent features of pRb expression. A strong pRb immunoresponse was restricted to the RPE and choroidea. Weak immunoresponse reached from the RPE towards the neuroepithelium. In the mi/mi mice, the unstructured or folded outer nuclear layer (ONL) of the neuroepithelium showed labelled cell surfaces (fig. 3A, B). Rosettes were strongly pRb immunopositive (fig. 3C). Electron microscopic photographs revealed photoreceptor cells forming the rosettes (fig. 3D). Photoreceptors were characterized by cell nuclei with oval shape and clustered chromatin. Summarizing, the expression of pRb depends on the amount of pigment in the RPE. Lacking pigment could cause the expression of pRb in the neuroepithelium. There is some evidence that pRb was expressed by photoreceptor cells. Expansion of RPE cells into the neuroepithelium could revealed in +/mi mice exclusively. Figure 4 gives a schematic drawing of the localizations in which pRb positive structures were found.

Fig. 2. Examples for the correlation of the morphology of the RPE cells visualized by their pigment (B, D) and the pRb immunoresponse visualized by the fluorochrome Cy 3 (A, C). Bar for A-D: 10 Jlm.

From our immunohistochemical results we conclude that there could be a gradation of phosphorylation of the suppressor protein. Such a gradation of the amount of ubiquitous pRb was revealed by gel electrophoresis of blood plasma (fig. 5). Comparing the three groups of animals, differences in the globuline fraction of blood plasma proteins are visible (arrows in fig. 5). That preliminary result could be only a first step to further investigations using electrophoresis. Nevertheless it reveals ubiquitous differences between the littermates of the microphthalmic strain of mice.

Discussion There is accumulating evidence that the induction of cancer may in some cases involve inactivation of negative cell growth regulators. The human retinoblastoma gene,

Rb 1, may be an example of this type of gene action. The gene encodes a nuclear phosphoprotein ubiquitously expressed in normal cells. Mutations of the Rb 1 gene are found in cells of many human cancers, and insertion of the wild-type gene will often suppress the neoplastic properties of these cells. A cDNA clone for the murine homolog of Rb I reveals a high degree of conservation with the human gene. In the mouse embryo, the expression of Rb 1 gene was ubiquitous (BERNARDS et al. 1989). The mouse Rb 1 gene was shown to suppress the transcription of the rat neu oncogene (Yu et al. 1992). Several research groups have created disrupted alleles of the Rb 1 and report that heterozygotes for the mutant show no predisposition to retinoblastoma. But the homozygotes die in utero with neuronal and hematopoietic system abnormalities (CLARKE et al. 1992; JACKS et al. 1992; LEE et al. 1992). Transfer of human RbI mini-trangene into the mutant mice corrects the defects (LEE et al. 1992). Exp Toxic Pathol 52 (2000) 1

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Fig. 3. A, B: Various labellings of pRb positive structures in mi/mi retinas; C: Strong immunoresponse in a rosette of mi/mi. Bar for A-C: 10 11m. D: Electron microscopic micrograph of a retinal rosette developed by photoreceptor cells. 20

Exp Toxic Pathol 52 (2000) 1

Our results show many similarities in the morphology of microphthalmic eyes to that of retinoblastoma in man. The light and electron microscopic features support the idea that the microphthalmic strain of mice could be a model for retinoblastoma. Our results corrobarate with the concept that these are not glial tumors but a local overproliferation of precursor cells both of possible photoreceptor cells and Muller glial cells in the retina. A differentiation of the cells stops the tumor development (MESSMER et al. 1984). It was revealed that the pRb was related to the amount of pigment in the RPE. The mi gene appears essential for pigment cell development and survival (HEMESA TH et al. 1994). It is locus for which 17 mutant alleles have been described (13-18). Here, we found an overlapping with the Rb 1. The mi gene was cloned and shown to predict a basic-helix-loop-helix-leucine zipper factor (HODGKINSON et al. 1994). Members of this class of proteins contain a basic domain through which they bind DNA. They dimerize through the other domain mentioned above. Structurally and functionally similar domains are also found in other transcprition factors. All these proteins form specific homo- and heterodimers and they have wide-ranging roles in regulation gene expression, cell proliferation and cell differentiation (TACHIBANA et al. 1994). All of those facts lead to the hypothesis that the microphthalmia associated genes and retinoblastoma genes could be linked. Evidence for that was given by YAVUZER et al. (1995). In their paper entitled "The microphthalmia gene product interacts with the retinoblastoma protein in vitro and is a target for deregulation of melanocyte-specific transcription" they found that the repression of melanocyte-specific TRP-l by the adenovirus E 1A correlated with the binding of the adenovirus to pi 05Rb. It was revealed that the basic-helix-loop-helix-leucin zipper pro-

tein was a transcription factor which can interact with the retinoblastoma gene product. The expression of the mi gene was reduced around 50-fold in the nonpigmented cells compared to that of the pigmented cells. Our results using gel electrophoresis corroborate the findings that the immunoresponse of phosphorylated Rb protein depends on the amount of pigment in the RPE and that ubiquitous differences of specific proteins (which were not specified in this paper) could depend on the amount of pigment in the whole body. It seems unlikely that the pRb could be shown in the gel electrophoresis. May be, there are more pRb bounded or associated proteins. About 30 cell proteins were found which bind to the pRb: transcription factors (FATTAEY et al. 1993; HELIN et al. 1992; KIM et al. 1992), growth factors (RUSTGI et al. 1991; QUIAN et al. 1993), protein kinases (Hu et al. 1992; KATO et al. 1993;

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Fig. 4. Schematic drawing of the pRb positive structures in mi/mi retinas.

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Fig. 5. Results of the gel electrophoresis of blood plasma. Arrows indicate different amounts and labellings.

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mi/mi

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mi/mi

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LIN et al. 1991), phosphatases (DURFEE et al. 1993) and nuclear matrix proteins (MANCINI et al. 1994). Little is known ofthe molecular mechanisms underlying the interaction of these proteins and the differentiation or overproliferation of cells. Our results corroborate indications to parallels between suppression of overproliferation by the Rb protein and the reduced expression of Mi protein which is required for pigment cell differentiation by lacking pigment.

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