The genomes of various tumour cells contain mutant oncogenes that act dominantly, in that their effects can be observed when they are introduced into non-malignant cells. There is evidence for another class of oncogenes, in which tumour-predisposing mutations are recessive to wild-type alleles. Retinoblastoma is a prototype biological model for the study of such recessive oncogenes. This malignant tumour, which arises in the eyes of children, can be explained as the result of two distinct genetic changes, each causing loss of function of one of the two homologous copies at a single genetic locus, Rb, assigned to the q14 band of human chromosome 13. Mutations affecting this locus may be inherited from a parent, may arise during gametogenesis or may occur somatically. Those who inherit a mutant allele at this locus have a high incidence of non-ocular, second tumours, almost half of which are osteosarcomas believed to be caused by the same mutation. Here we describe the isolation of a complementary DNA segment that detects a chromosomal segment having the properties of the gene at this locus. The gene is expressed in many tumour types, but no RNA transcript has been found in retinoblastomas and osteosarcomas. The cDNA fragment detects a locus spanning at least 70 kilobases (kb) in human chromosome band 13q14, all or part of which is frequently deleted in retinoblastomas and osteosarcomas.

/pdf/a-human-dna-segment-with-properties-of-the-gene-that-kc0q4f65ya.pdf

A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma

Recent evidence indicates the existence of a genetic locus in chromosome region 13q14 that confers susceptibility to retinoblastoma, a cancer of the eye in children. A gene encoding a messenger RNA (mRNA) of 4.6 kilobases (kb), located in the proximity of esterase D, was identified as the retinoblastoma susceptibility (RB) gene on the basis of chromosomal location, homozygous deletion, and tumor-specific alterations in expression. Transcription of this gene was abnormal in six of six retinoblastomas examined: in two tumors, RB mRNA was not detectable, while four others expressed variable quantities of RB mRNA with decreased molecular size of about 4.0 kb. In contrast, full-length RB mRNA was present in human fetal retina and placenta, and in other tumors such as neuroblastoma and medulloblastoma. DNA from retinoblastoma cells had a homozygous gene deletion in one case and hemizygous deletion in another case, while the remainder were not grossly different from normal human control DNA. The gene contains at least 12 exons distributed in a region of over 100 kb. Sequence analysis of complementary DNA clones yielded a single long open reading frame that could encode a hypothetical protein of 816 amino acids. A computer-assisted search of a protein sequence database revealed no closely related proteins. Features of the predicted amino acid sequence include potential metal-binding domains similar to those found in nucleic acid-binding proteins. These results provide a framework for further study of recessive genetic mechanisms in human cancers.

https://science.sciencemag.org/content/sci/235/4794/1394.full.pdf

Human retinoblastoma susceptibility gene: cloning, identification, and sequence

The first well-documented observation of retinoblastoma (Rb) in several members of one family was published by Lerche (1821). Four of seven siblings, children of healthy parents, were affected. At that time, the tumor almost invariably led to death during infancy or early childhood. The first pedigrees with transmission through two or more generations were observed only after enucleation of the affected eye had been introduced by von Graefe. More recently, the mode of inheritance has been identified as autosomal dominant with incomplete penetrance; Fig. 13.1 shows a typical pedigree. Unilateral as well as bilateral cases may occur in the same family, and the tumor may even skip one generation. Statistical analysis of many families (see Vogel 1979) led to the conclusion that the penetrance of the dominant gene causing Rb is about 90%; i.e., 90% of heterozygotes will be affected.

Genetics of retinoblastoma

Retinal cell culture has been a useful tool for ocular research. Although it does not replace the intact eye, retinal cell cultures provide convenient experimental systems for the assessment of numerous retinal processes. As with any in vitro systems, cell culture offers great advantages, but not without potential limitations. Advantages include controllable conditions that allow for the assessment of isolated cellular functions, a more affordable system when compared with the more costly animal research, and time course flexibility. Potential limitations include loss of native tissue architecture, lack of functional feedback from other retinal cell types, and a questionable correlation between in vitro and in vivo findings. However, for a great number of research applications, the advantages offered by in vitro systems outweigh the limitations.

Retinal cell culture can be routinely used to determine the cell specificity of promoter sequences,1 the effect of mutations on the structure and function of retinal proteins,2 or the role of multiple domains on the function of retinal proteins.3 Furthermore, retinal cell culture has been applied in studies of cell growth, death, differentiation, and cytotoxicity (for review, see Ref. 4).

Photoreceptor cells are terminally differentiated, specialized neuronal cells with a limited capacity for cell division. Therefore, to establish a line of photoreceptor cells, it is essential to transform them, possibly with a virus. Immortalized cell lines of several ocular cell types currently exist, including Muller,5 ganglion,6 corneal endothelial,7 and RPE cells.8 A cell line expressing retina-specific genes, including interphotoreceptor retinol-binding protein (IRBP) and cone transducin, has been isolated from a mouse ocular tumor.9 In addition, Y-7910 and WERI-Rb11 are immortalized human retinoblastoma cell lines available for the study of photoreceptors. Initially, it was thought that the Y-79 cells were of cone cell origin,12 but more recently these cells have been shown to express rod-specific antigens, such as opsin, transducin, phosphodiesterase, and recoverin.13,14 Primary retinal cultures have been created from several vertebrate retinas, including those of humans.15 These types of cultures, in addition to being tedious to prepare, are not adequate for some types of studies, because of their heterogeneity, limited cell division, and special conditions for growth as monolayers. Thus, there is a need for additional photoreceptor cell models that are homogeneous, passageable, and easily grown as a monolayer by using standard tissue culture techniques.

Herein, we describe a mouse photoreceptor-derived cell line (661W) immortalized by the expression of simian virus (SV)40 T antigen (T-ag) under control of the human IRBP promoter.16 Cellular, and molecular analyses show that these cells express cone but not rod photoreceptor markers, which suggests that the cells arise from a cone photoreceptor lineage. For this reason, the 661W cell line should contribute significantly to the study of cone photoreceptor cell function and of diseases affecting cone photoreceptor cells, including mechanisms of photoreceptor cell death in various retinal dystrophies.

/pdf/expression-of-cone-photoreceptor-specific-antigens-in-a-cell-5g2ay6um6n.pdf

Expression of Cone-Photoreceptor–Specific Antigens in a Cell Line Derived from Retinal Tumors in Transgenic Mice

Tumor cells can be induced to differentiate in vitro by biochemical manipulation of their culture environment. In the studies described here, the effects of medium conditioned by human retinal pigmented epithelial (RPE) cells on Y79 human retinoblastoma cells have been examined. RPE-conditioned medium in conjunction with laminin and a poly-D-lysine substratum is observed to induce neuronal differentiation of Y79 cells. The cells extend long cellular processes and exhibit immunologically detectable neurotypic properties. In contrast, control Y79 cells not exposed to medium conditioned by RPE cells exhibit only infrequent neuronal phenotypes. This response of Y79 cells to RPE-conditioned medium indicates that factors secreted by RPE cells can act as inducers of neuronal differentiation in retinoblastoma cells and suggest that such factors may be of importance in the development and differentiation of the neural retina.

Janice L. Wilcox

Papers

Characteristics of an established cell line of retinoblastoma.