Several proteins that associate with cellular promoters have recently been found to have an essential role in the control of cellular proliferation and neoplastic transformation. The importance of one of these, the retinoblastoma protein (Rb), was recognized when it was found to be expressed in every normal tissue, but mutated or deleted in tumors such as retinoblastoma, osteosarcoma, cervical cancer, small cell lung cancer, and bladder cancer. When wild-type Rb is reintroduced into these tumors, they lose their malignant characteristics, therefore, Rb has been called a tumor suppressor. We have demonstrated that Rb is a transcriptional repressor during G-O/G-1 and that is likely to constrain cell division because it represses the expression of a set of genes that is required for DNA synthesis. In support of such a role for Rb, we have found that in Rb(-) cells DNA synthesis persists in G-O/G-1-arrested cells. It is understandable then how the loss of Rb function could lead to a loss of cell cycle control. In quiescent cells (G-O/G-1) Rb is tethered to the promoters of genes by E2F, a protein that binds to a specific site in the promoters. In proliferating cells, Rb is released from E2F in late G-1 and it is replaced on E2F by a complex consisting of a 107 kDa protein (plO7), cyclin A, and cdk2 kinase. We have demonstrated that this S phase complex is a transcriptional activator, suggesting that it mediates the S phase- specific activation of the genes for DNA synthesis enzymes. These results suggest that E2F sites alternate between transcriptional inhibitors and activators during the cell cycle and that this activity is responsible for preventing expression of genes for DNA synthesis enzymes in G-0/G-1 and activating these genes in S phase. We will examine the functions of these proteins that associate with E2F. To analyze the function of Rb, we have constructed a fusion gene where Rb is linked to the DNA binding region of the yeast protein Gal-4. The Gal-4 DNA binding domain has been widely used to tether transcriptionally active proteins to promoters as a tool to examine their activities. In transfection assays, Rb-Gal-4 inhibited transcription from promoters containing Gal-4 binding sites, suggesting that Rb is a transcriptional repressor and that E2F only serves to tether Rb to promoters. Mutations and deletions in Rb will be made and fused to Gal-4. The activity of the resulting fusion proteins will be analyzed in transfection assays to identify the repressor domain of Rb. Similar technology will be used to identify and characterize the transcriptional activator in the S phase-specific E2F complex. Additionally, the role of E2F will be examined in vivo. We will functionally inactivate E2F sites by sequestering cellular E2F. This will be done by transfection of a competitor plasmid containing E2F binding sites. We anticipate that E2F is required for cell proliferation, and these experiments should provide the first direct evidence that this is so. The proposed studies should lead to a further understanding of cell cycle control and the mechanisms underlying the loss of this control that occurs in tumorogenesis.
| Dean, D C; Weintraub, S J; Holtzman, M J (1994) A molecular switch between lung development and cancer: is the tumor suppressor Rb turned on during bronchial epithelial cell differentiation and turned off in small cell lung cancer? Am J Respir Cell Mol Biol 11:249-50 |
