The existence of tumor suppressor genes, or 'anti-oncogenes', was suggested by previous experiments in which the fusion of a normal cell with a malignant counterpart was noted to suppress the malignant phenotype of the latter, as well as from studies demonstrating non-random loss of chromosomal material from a variety of tumors. One such gene, RB 1, was recently cloned and sequenced. Its product, Rb is a nuclear phosphoprotein which has properties of a cell-cycle regulatory element. Rb is bound (?inactivated) by three DNA viral oncoproteins (ElA, SV40 large T, and HPV E7) which each contain a short, homologous, colinear sequence implicated both in Rb binding and in their ability to transform cells. As these viruses are otherwise unrelated, it seems likely that the segment of their genomes encoding this sequence is derived from a host gene encoding a protein which forms a complex with Rb in a similar manner. This hypothesis is strengthened by the observation that those naturally occurring Rb mutants which have been tested to date are defective in El A/T/E7 binding and by preliminary data which suggest that a large region of Rb is necessary for this binding to occur. The identification of this gene and its protein product might potentially provide an important clue as to how Rb suppresses cell growth. An in vitro binding assay will be used to map the minimum region of Rb necessary for binding ElA/T/E7. Expression of Rb mutants which bind to ElA/T/E7 can then be examined for their ability to suppress cell growth in Rb-/- cell lines as well as for their ability to induce biological effects in Rb+/+ cells through competition with endogenous Rb for this putative cellular factor. A panel of anti-Rb antibodies will be used to look for proteins which co-immunoprecipitate with Rb and/or with Rb mutants. El A/T/E7 binding Rb mutants will also be overproduced and used in an attempt to biochemically isolate Rb binding proteins.
| Flemington, E K; Speck, S H; Kaelin Jr, W G (1993) E2F-1-mediated transactivation is inhibited by complex formation with the retinoblastoma susceptibility gene product. Proc Natl Acad Sci U S A 90:6914-8 |
