Retinoblastoma is a malignant childhood tumor which can occur in the hereditary or non-hereditary forms. The genetic changes that are suspected to be the underlying cause of the hereditary predisposition and the subsequent development of the disease have been proposed to be a recessive mutation of a gene in the chromosomal region 13q14.1 We have recently isolated the cDNA clone of a gene encoded in this region and provided definitive structural evidences that it has all the properties of the putative retinoblastoma (Rb) gene. Approximately forty percent of the retinoblastoma examined with our Rb gene probe had structural changes including, in some cases, homozygous internal deletion with corresponding truncated transcripts. While these data tend to imply that the complete inactivation of the Rb gene is related to the development of retinoblastoma, virtually nothing is known about what the normal role of Rb gene is and how is its inactivation related to tumorigenesis. The main objective of this proposal is to study the function of the Rb gene and to under- stand how it is regulated. Specifically we will first gain an understanding of the spatial and temporal expression of the Rb gene. We will examine the RNA expression of the Rb gene in different tissues. Since the Rb gene is conserved in human and in mouse, we will examine the RNA expression of the RB gene in different mouse tissues at different stages of development and/or differentiation of various tissues. Secondly, we will examine the location of the Rb gene in the cell by using antibodies raised against the gene product. These experiments should provide a clue as to the site of action of the Rb gene product. Finally, since the inactivation of both alleles of the Rb gene is associated with the development of retinoblastoma, we will attempt to a) suppress tumorigenecity of retinoblastoma by reintroduction of the Rb gene (under an appropriate promotor) and test these transfected cells for tumorigenicity in nude mice; and b) induce tumorigenicity in the fibroblasts from patients with known structural aberrations at the Rb gene locus. This will be done by disrupting the remaining intact Rb allele with Rb genomic fragments via homologous recombination. The resulting cells will be tested for tumorigenicity in nude mice.
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