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Query: UNIPROT:P06889 (Mol)
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The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.
Mol Cell Biol 1993 Dec
PMID:Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes. 824 49

Cyclin D1 is a G1-specific cyclin that has been linked to lymphoid, parathyroid, and breast tumors. Recent studies suggested that high protein levels of cyclin D1 are not always produced when cyclin D1 mRNA is overexpressed in transfected cells, suggesting that posttranscriptional events may be important in cyclin D1 regulation. The mRNA cap-binding protein (eukaryotic initiation factor 4E [eIF-4E]) is a potential regulatory of several posttranscriptional events, and it can itself induce neoplastic transformation. Consequently, we examined eIF-4E as a potential regulator of cyclin D1. Overexpression of cyclin D1 mRNA in NIH 3T3 cells did not increase cyclin D1 protein. In contrast, overexpression of eIF-4E markedly increased the amount of cyclin D1 protein in NIH 3T3 cells. This increase was specific to cyclin D1 in comparison with the retinoblastoma gene product, c-Myc, actin, and eukaryotic initiation factor 2 alpha. We also examined cyclin D1 protein in cells expressing an estrogen receptor-Myc fusion protein because we previously found that eIF-4E increases after induction of c-myc function. In these cells, increased levels of eIF-4E protein were closely followed by increases in levels of cyclin D1 protein, but the level of cyclin D1 mRNA was not increased. We conclude that increases in cyclin D1 levels may result from increased expression of eIF-4E, and this regulation may be one determinant of cyclin D1 levels in the cell.
Mol Cell Biol 1993 Dec
PMID:Elevated levels of cyclin D1 protein in response to increased expression of eukaryotic initiation factor 4E. 824 56

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.
Mol Cell Biol 1993 Dec
PMID:Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F. 824 95

E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action of a family of proteins.
Mol Cell Biol 1993 Dec
PMID:The retinoblastoma protein binds to a family of E2F transcription factors. 824 96

The retinoblastoma (RB) gene is the prototype tumor suppressor gene. Mutations in this gene are often associated with the occurrence of various tumors. Several mutations have been found in the promoter region of the gene, suggesting that inappropriate transcriptional regulation of the RB gene contributes to tumorigenesis. Sequence analysis of the RB promoter has revealed a potential E2F recognition site within a region critical for RB gene transcription. By using the cloned E2F-1 gene, here we report that (i) RB expression is negatively regulated by its own gene product, (ii) E2F-1 binds specifically to an E2F recognition sequence in the RB promoter and transactivates the RB promoter, (iii) overexpression of RB suppresses E2F-1-mediated stimulation of RB promoter activity, and (iv) the expression of the RB gene is paralleled by the expression of the E2F-1 gene during cell cycle progression. These results demonstrate that expression of RB is negatively autoregulated through E2F-1.
Mol Cell Biol 1994 Jan
PMID:The transcription factor E2F-1 mediates the autoregulation of RB gene expression. 826 96

mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformed cells, suggesting that the promoter region of thrombospondin was less efficiently transcribed in Ni-transformed cells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumor suppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of an expression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondin promoter such that the expression was higher in normal cells than in transformed cells.
Mol Cell Biol 1994 Jan
PMID:Loss of thrombospondin transcriptional activity in nickel-transformed cells. 826 52

Abnormalities of the retinoblastoma (Rb) gene have been reported in some human cancers, including hepatocellular carcinomas (HCCs). We examined by Southern blotting the status of the Rb gene in HCCs induced in rats in four experimental models. A low frequency of Rb gene alterations, detected as novel hybridizing bands unique to each tumor, was observed. Expression of the Rb protein product was examined in the HCCs and in seven established rat hepatoma cell lines studied. It appears, therefore, that alterations in the structure or expression of the Rb gene do occur but probably do not contribute in a major way to hepatocarcinogenesis in the rat.
Mol Carcinog 1993
PMID:Low frequency of retinoblastoma gene alterations in rat hepatocellular carcinomas. 828 Mar 71

Esophageal carcinomas from 24 patients, most of whom were smokers and consumed alcoholic beverages daily, were analyzed for mutations in exons 5-8 of the p53 tumor suppressor gene. Mutations were identified by polymerase chain reaction amplification and direct sequencing in 12 of 24 (50%) of the samples; almost half of the mutations were at A:T base pairs. Nuclear accumulation of p53 protein, determined by immunohistochemistry with the CM-1 polyclonal antibody, was observed in all cases in which a missense mutation in the p53 gene was detected. None of the 24 carcinomas had amplification of the mdm2 gene, an alternate pathway to p53 loss of function. Alterations involving three other cancer-related genes associated with human esophageal carcinogenesis, c-erbB-1/epidermal growth factor receptor (EGFR), c-myc, and retinoblastoma (Rb), were examined by Southern blot or immunohistochemical analysis in the same sample set to explore the possibility of a link between oncogene activation and loss of tumor suppressor function. While no associations were observed between amplification of the c-myc or EGFR genes and p53 abnormalities, a significant correlation (P < 0.01) was seen between the presence of p53 mutation and EGFR overexpression. Absence of Rb protein, measured immunohistochemically, was observed in four tumors, none of which had aberrations of the p53 gene.
Mol Carcinog 1993
PMID:Correlation of p53 mutations with epidermal growth factor receptor overexpression and absence of mdm2 amplification in human esophageal carcinomas. 828 Mar 79

The transcription factor E2F activates the expression of multiple genes involved in cell proliferation, such as c-myc and the dihydrofolate reductase gene. Regulation of E2F involves its interactions with other cellular proteins, including the retinoblastoma protein (Rb), the Rb-related protein p107, cyclin A, and cdk2. We undertook a detailed analysis of E2F DNA-binding activities and their cell cycle behavior in primary human T cells. Three E2F DNA-binding activities were identified in resting (G0) T cells with mobilities in gel shift assays distinct from those of previously defined E2F complexes. One of these activities was found to be a novel, less abundant, Rb-E2F complex. The most prominent E2F activity in resting T cells (termed complex X) was abundant in both G0 and G1 but disappeared as cells entered S phase, suggesting a possible role in negatively regulating E2F function. Complex X could be dissociated by adenovirus E1A with a requirement for an intact E1A conserved region 2. However, X failed to react with a variety of antibodies against Rb or p107, implicating the involvement of an E1A-binding protein other than Rb or p107. In addition to these novel E2F complexes, three distinct forms of unbound (free) E2F were resolved in gel shift experiments. These species showed different cell cycle kinetics. UV cross-linking experiments suggested that a distinct E2F DNA-binding protein is uniquely associated with the S-phase p107 complex and is not associated with Rb. Together, these results suggest that E2F consists of multiple, biochemically distinct DNA-binding proteins which function at different points in the cell cycle.
Mol Cell Biol 1993 Jul
PMID:Cell cycle analysis of E2F in primary human T cells reveals novel E2F complexes and biochemically distinct forms of free E2F. 832 Dec 4

Cell transformation by adenovirus-E1A proteins is mediated by binding to cellular proteins whose functions are thereby inactivated or altered. The various properties of the E1A proteins are reviewed in relation to their binding to cellular proteins. A number of the cellular proteins which associate to E1A have been identified: the retinoblastoma-susceptibility protein (Rb), the p107 protein, cyclin A and the p33cdk2 kinase. Recent data have shown that those proteins are also able to bind to transcription factor E2F. Binding of Rb to E2F represses the transcription-activating potential of E2F. E1A can sequester the regulatory proteins, like Rb, and thereby release free, active E2F. The domains in E1A that are essential for this transcriptional regulation are also required for the transforming properties of E1A.
Mol Biol Rep 1993 Apr
PMID:Adenovirus-E1A proteins transform cells by sequestering regulatory proteins. 832 55


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