Temperature-sensitive RB mutations linked to incomplete penetrance of familial retinoblastoma in 12 families

Abstract

The tumor-suppressor activity of the retinoblastoma protein (RB) is encoded within a protein-binding ("pocket") domain that is targeted for mutations in all cases of familial retinoblastoma and in many common adult cancers. Although familial retinoblastoma is a paradigm for a highly penetrant, recessive model of tumorigenesis, the molecular basis for the phenotype of incomplete penetrance of familial retinoblastoma is undefined. We studied the RB pocket-binding properties of three independent, mutant RB alleles that are present in the germline of 12 kindreds with the phenotype of incomplete penetrance of familial retinoblastoma. Each arises from alterations of single codons within the RB pocket domain (designated "delta 480," "661W," or "712R"). Under the same conditions, we studied the properties of wild-type (WT) RB, an RB point mutant isolated from a lung carcinoma sample (706F) and an adjacent, in vitro-generated point mutant (707W). The delta 480, 661W, and 712R mutants lack pocket protein-binding activity in vitro but retain the WT ability to undergo cyclin-mediated phosphorylation in vivo. Each of the low-penetrant RB mutants exhibits marked enhancement of pocket protein binding when the cells are grown at reduced temperature. In contrast, in this temperature range, no change in binding activity is seen with WT RB, the 706F mutant, or the 707W mutant. We have demonstrated that many families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles with temperature-sensitive pocket protein-binding activity. The variable frequency for tumor development in these families may result from reversible fluctuations in a threshold level of RB pocket-binding activity.

Figure 1

A, In vivo phosphorylation analysis. Plasmids encoding WT RB (lanes 1–4), 706F mutant RB (lanes 5–8), and the incomplete-penetrant mutant 712R (lanes 9–12) were cotransfected with the indicated cyclin plasmids (D2, D3, and E) into the RB (−/−) tumor cell line H2009 and were subjected to α-RB immunoblotting. Phosphorylation is indicated by the slight retardation in migration of the RB protein signal (ppRB). Protein-size marker is shown on the left. B, In vitro E2F1 binding assay. cDNA plasmids encoding WT or the 706F and 712R mutants were in-vitro translated with [³⁵S]-methionine and were subjected to SDS-PAGE analysis before (1 μl of lysate; lanes 1, 4, and 7) or after precipitation with either GST alone (10 μl of lysate; lanes 2, 5, and 8) or GST-E2F1 fusion proteins (10 μl of lysates; lanes 3, 6, and 9).

Figure 2

A, Schematic representation of the RB protein. The black rectangles represent the A and B domains of the RB pocket, which is required for large T binding and for tumor suppression. The approximate locations of the missense mutations tested in this study are indicated. B, Activities of phenotypes. Null refers to the inactivating RB mutations that characterize familial retinoblastoma and selected common adult cancers. The DER is calculated as the sum of the number of eyes clinically involved with retinoblastoma, divided by the number of obligate carriers for each kindred, as described in the Subjects, Material, and Methods section. Clinical data sufficient to determine the mean DER score shown were available from only seven families (661W) or one family (712R). References for these kindreds are indicated in the Subjects, Material, and Methods section.

Figure 3

A, β-Galactosidase filter analysis for WT and for the 706F and 712R mutants, assayed after growth of cells at either 24°C or 30°C, as indicated. Two independent transformants are shown for each RB cDNA. B, β-Galactosidase filter analysis of WT or the five different RB point mutants, as indicated. Two independent transformants are shown for each RB cDNA, and assays were performed after growth of cells at either 37°C, 30°C, or 24°C. The yellow-green signal indicates the streaked yeast colonies transferred to the nitrocellulose filter. The purple-blue signal represents β-galactosidase activity as a reporter for a protein-binding interaction between the RB clones and large T antigen.