FUNCTION OF THE RETINOBLASTOMA PROTEIN

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The retinoblastoma gene is the prototype of a class of cancer genes known as tumor suppressor genes. Tumor suppressor genes are defined by two key characteristics: (1) mutation of these genes promotes tumorigenesis (2) the selective advantage conferred by mutation of these genes is due to the loss of gene function. We are investigating the function of the retinoblastoma gene product (pRB). The best understood function of pRB is as a regulator of transcription factors. During the last grant cycle we characterized the ability of pRB and related proteins to regulate the E2F transcription factor and to control the expression E2F-target genes. The activation of E2F promotes cell cycle progression and, in normal cells, pRB acts to repress of E2F-dependent transcription. A great deal is known about the biochemistry of protein complexes formed between E2F and pRB-family proteins but the in vivo functions of this regulatory system are poorly understood. In the last two years we have established two experimental systems to examine the functions of these proteins. First, using homologous recombination we generated mutant alleles of E2F-1 in the mouse and studied the effects of eliminating E2F-1 function. Previous work had shown that E2F-1 behaved as an oncogene when over-expressed in tissue culture. However mice lacking E2F-1 developed a broad range of tumors revealing that E2F-1 acts to suppress tumor formation in vivo. In the next grant cycle we propose to continue this investigation of E2F function. We will concentrate on the characterization of the phenotype of E2F-1 null cells, on the phenotype of cells lacking DP-1 (the heterodimeric partner of E2F-1), and on the phenotype of cells lacking multiple pRB-regulated E2F s. To test role of the pRB/E2F-1 complex in pRB and E2F-1 functions we will characterize mouse strains carrying specific mutant alleles of E2F-1 that eliminate this interaction. A second area of functional studies that we initiated in the last grant cycle has also given unexpected findings. pRB is one member of a family of proteins, including p107 and p130, which share strong structual homologies. During the characterization of 3T3 cell lines prepared from pRB, p107 or p30 nullizygous mouse embryos, we observed that pRB, and p107 or p130 have specific and opposite effects on adipocyte differentiation. Whereas pRB promotes differentiation, p107 and p130 block adipocyte differentiation. We have finished the initial characterization of these differences and in the next round of this grant will investigate the functional differences between pRB p107 and p130 in adipogenesis and in other defined differentiation systems.