Cloning and Early Characterization of Rb

Rb is a prototypical tumor suppressor gene. Retinoblastoma, a childhood tumor of the eye, is found in either an inherited or a sporadic pattern, and it was hypothesized that this observation could be accounted for by a model requiring two mutations in a tumor suppressor gene (Knudson, 1971). In the inherited form, the existence of one germline mutation would predispose to multiple tumors because only a single additional mutation in the remaining wild type allele would need to occur in the initial tumor cell. In contrast, in sporadic cases only single tumors were found because each tumor required two independent mutational events in the same precursor cell. Analysis of chromosomal deletions in inherited cases of retinoblastoma allowed the chromosomal position of Rb to be determined, and the Rb gene was then identified by chromosomal walking to find genes that were deleted, mis-expressed or contained point mutations in either hereditary or sporadic cases of retinoblastoma but not in normal tissues or other tumor types (Dunn et al., 1988; Friend et al., 1986; Lee et al., 1987). Although Rb was shown to be a nuclear protein that could associate with DNA, the sequence itself did not reveal a biological function of Rb that could underlie its tumor suppressor function.

Insight into this tumor suppressor function was initially obtained from studies of the viral oncogene El a, an adenovirus immediate early protein. Expression of El a alone had been shown to immortalize tissue culture cells, while expression of Ela and either Elb or Ras together could transform cells. To determine the mechanism by which Ela immortalized cells, cellular proteins that co-precipitated with Ela were analyzed. Among the major proteins was a 105 KD band that was identified as the tumor suppressor, Rb (Whyte et al., 1988). Mutations of Ela that abolished binding to Rb also eliminated its ability to transform cells in conjunction with Ras. Deletion mapping showed that the region of Ela required to induce DNA synthesis and to immortalize cells was also required to bind to both Rb and pi07, another Ela associated protein later found to be a

Corresponding Author: Wei Du, Tel: (773) 834-1949, Fax: (773) 702-4394, E-mail: [email protected]

member of the Rb family (Whyte et al., 1989). These studies connected the ability of viral oncogenes to transform cells with their ability to interact with a cellular tumor suppressor protein and raised the possibility that the tumor suppressor function of Rb could be mediated by its interaction with other cellular targets.

Subsequent experiments led to the identification of numerous cellular targets of Rb over the years. Among these, the first and the best studied are the E2F transcription factors, which were originally identified among the cellular factors that bound to the adenovirus E2 promoter. In uninfected cells, a large complex was found bound to the E2F binding site, while in cells expressing Ela a smaller, "free" E2F was present. Ela was shown to be capable of dissociating the larger E2F complex, and this depended on a conserved domain showing homology to other viral oncoproteins (Bagchi et al., 1990). Given Ela's interaction with Rb and its ability to release "free" E2F, it was hypothesized that Rb might be part of the larger E2F complex, and this was confirmed by purification of that complex (Bagchi et al., 1991). Furthermore, it was shown that E2F only interacted with a hypophosphorylated form of Rb (the active form) and that Rb was part of the complex dissociated by Ela (Chellappan et al., 1991).

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