ABSTRACT
To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.
Subject(s)
Adenocarcinoma/genetics , Breast Neoplasms/genetics , Cell Cycle Proteins/physiology , Cellular Senescence/genetics , Chromosomes, Human, Pair 17/genetics , DNA-Binding Proteins , Gene Amplification , Gene Expression Regulation, Neoplastic , Neoplasm Proteins/physiology , Protein Biosynthesis , Repressor Proteins/physiology , T-Box Domain Proteins/physiology , Adenocarcinoma/metabolism , Animals , Breast Neoplasms/metabolism , COS Cells , Carrier Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/isolation & purification , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16 , E2F Transcription Factors , E2F1 Transcription Factor , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Deletion , Genes, BRCA1 , Humans , Mice , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasm Proteins/isolation & purification , Neoplastic Syndromes, Hereditary/genetics , Nuclear Proteins/genetics , Oncogenes , Polycomb Repressive Complex 1 , Promoter Regions, Genetic , Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Retinoblastoma-Binding Protein 1 , T-Box Domain Proteins/genetics , T-Box Domain Proteins/isolation & purification , Transcription Factor DP1 , Transcription Factors/antagonists & inhibitors , Transfection , Tumor Cells, Cultured , Tumor Suppressor Protein p14ARFABSTRACT
The E2F transcription factor family participates in growth control presumably through transcriptional activation of genes that promote entry into S phase. E2F activity is believed to be controlled across the cell cycle by association with various cellular proteins, including the product of the retinoblastoma gene (pRB). We find that E2F-1 proteins are heterogeneously phosphorylated in insect cells, as a result of which they migrate as a doublet on SDS-polyacrylamide gels. This electrophoretic shift is shown to be dependent upon specific phosphorylation of E2F-1 on serine-375 (S375), near the pRB-binding site. Phosphorylation on S375 also occurs in human cells. E2F-1 was most efficiently phosphorylated on this residue by cyclin A/cdk2 kinase, and to a lesser extent by cyclin A/cdk2, irrespective of the presence of the pRB-related p107 protein. Phosphorylation of E2F-1 on S375 greatly enhanced its affinity of pRB in vitro. These results suggest a novel way of regulating E2F-1 activity, namely by cell-cycle-dependent phosphorylation of this transcription factor.
Subject(s)
Carrier Proteins , Cell Cycle Proteins , Cyclin-Dependent Kinases/metabolism , DNA-Binding Proteins , Retinoblastoma Protein/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Cells, Cultured , E2F Transcription Factors , E2F1 Transcription Factor , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Phosphorylation , Protein Binding , Retinoblastoma-Binding Protein 1 , Spodoptera , Transcription Factor DP1 , Transcription Factors/chemistryABSTRACT
Expression of the gene encoding the cell cycle regulator cyclin D1 is strongly repressed in adenovirus type 5 E1 (Ad5E1)-transformed cells. Since cyclin D1 is a regulator of cell proliferation, modulation of its abundance may affect cell cycle control. Therefore, we studied the importance of cyclin D1 repression for cell transformation by Ad5E1. We found that forced expression of cyclin D1 does not affect the transforming potential of Ad5E1. Similarly, cyclin D1 overexpression did not affect the efficiency of colony formation, the proliferation rate, or the cell cycle distribution of Ad5E1-transformed cell lines, whereas the colony formation of untransformed cell lines was strongly inhibited. Thus, repression of cyclin D1 expression is not required for initiation or maintenance of cell transformation by Ad5E1. In addition, we show that the growth-suppressive effect of cyclin D1 correlates with cyclin D1 binding to cdk4 rather than to proliferating cell nuclear antigen PCNA.
Subject(s)
Adenovirus E1 Proteins/genetics , Cyclins/metabolism , Oncogene Proteins/metabolism , Transformation, Genetic , Animals , Cell Division , Cell Line , Cell Line, Transformed , Chlorocebus aethiops , Clone Cells , Cyclin D1 , Cyclins/genetics , Gene Expression , Humans , Oncogene Proteins/genetics , PhenotypeABSTRACT
The cdk-inhibitor p21(CIP1/WAF1) inhibits the activities of cyclin-dependent kinases and proliferating cell nuclear antigen, thereby repressing cell-cycle progression and DNA replication. Transforming oncogenes, such as E1A of human adenovirus 5 (Ad5), may interfere with such growth-inhibitory proteins. In this study, we show that in various Ad5E1-transformed cells, p21(CIP1/WAF1) is expressed and that, in general, expression is not downregulated. In addition, colony-formation assays show that in Ad5E1-transformed cells highly overexpressed p21(CIP1/WAF1) can still cause growth inhibition. FACS experiments indicate, however, that a G1 arrest induced by moderate overexpression of p21(CIP1/WAF1) can be overcome by E1A. The E1A proteins may interfere with the function of p21(CIP1/WAF1) by binding. Indeed, p21(CIP1/WAF1) binds with its cyclin/cdk-binding N terminus to the transforming N-terminal and CR1 region of the E1A proteins. Together, these results lend support to the model that E1A can interfere directly with p21(CIP1/WAF1) function and thereby stimulates cell growth.