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ABSTRACT: Genomic instability is thought to be critical for the development of cancer. Among its causes microsatellite instability (MIN) and chromosomal instability (CIN) have attracted the most attention. Cell cycle checkpoints and DNA repair mechanisms are the first line of defense against DNA damage. Among the most dangerous DNA lesions are double-strand breaks. The response to DNA double strand breaks is regulated mainly by the serine/threonine kinases ATM and Chk2 and their downstream target the tumor suppressor p53, which in turn stimulates the expression of growth-inhibitory genes like p21 or pro-apoptotic genes like Bax. The balance between these gene products determines the fate of a cell. EAPP is a nuclear phosphoprotein that is frequently upregulated in human tumors. We have recently shown that EAPP levels are critical for cellular homeostasis. DNA damage elevates EAPP levels and its overexpression results in G1 arrest and impairs apoptosis in a p21-dependent manner. EAPP binds to the p21 promoter, stimulates its activity and seems to be essential for transcription initiation. In the present work we show that EAPP also regulates the phosphorylation status and thus the activity of Chk2. EAPP binding seems to trigger the dephosphorylation of P-Chk2 resulting in its inactivation. A newly described function of Chk2 in mitosis that secures genomic integrity might also be affected by EAPP overexpression. This might explain the abundance of EAPP in aneuploid tumor cells.
Cell cycle (Georgetown, Tex.) 07/2011; 10(13):2077-82. · 5.36 Impact Factor
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ABSTRACT: The E2F-associated phosphoprotein (EAPP) is a ubiquitous nuclear protein that interacts with the activating members of the E2F family of transcription factors and increases the activity of several cell-cycle regulated promoters in an E2F-dependent manner. Our previous studies also showed that EAPP levels are elevated in most transformed human cells. To examine the molecular basis of this increase of EAPP we isolated and studied the nucleotide sequence at the 5' end of the EAPP gene. In silico analysis revealed a TATA-less promoter with several putative binding sites for transcription factors, the most probable ones being Sp1, Sp3 and Egr-1. We could confirm the binding of these factors in vitro by electrophoretic mobility shift assays, supershift experiments and competition assays. Additionally we could validate the binding in vivo by chromatin-immunoprecipitation assays. To analyse the function of these transcription factors in the expression of EAPP, we performed reporter-assays with the promoter and truncations thereof. We found that Sp1 and Egr-1 stimulate the EAPP promoter, whereas Sp3 acts as a repressor that could even overcome the positive effect of the activators. Increasing the amounts of Sp3 also caused a strong reduction of EAPP, but the overexpression of Sp1 or Egr-1 resulted in only marginally higher EAPP levels. Our results suggest that the elevated EAPP levels in transformed cells can be caused by reduced Sp3 activity, but higher Sp1 activity might also play a role.
The International Journal of Biochemistry & Cell Biology 07/2008; 40(12):2845-53. · 4.63 Impact Factor
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ABSTRACT: E2F transcription factors play an essential role in cell proliferation and apoptosis and their activity is frequently deregulated in human cancers. In a yeast two-hybrid screen we identified a novel E2F-binding protein. Due to its strong phosphorylation we named it EAPP (e2F-associated phosphoprotein). EAPP is localized in the nucleus and interacts with E2F-1, E2F-2, and E2F-3, but not with E2F-4. Examination of a number of human cell lines revealed that EAPP levels are elevated in most transformed cells. Moreover, EAPP mRNA was detected in all investigated human tissues in varying amounts. EAPP is present throughout the cell cycle but disappears during mitosis. In transfection assays with reporters controlled by either an artificial E2F-dependent promoter or the murine thymidine kinase promoter, EAPP increased the activation caused by E2F-1 but not by E2F-4. Surprisingly, the promoter of the p14(ARF) gene, which was also activated by E2F-1, became repressed by EAPP. Overexpression of EAPP in U2OS cells resulted in a significant increase of cells in S-phase, whereas RNAi-mediated knock down of EAPP reduced the fraction of cells in S-phase. Taken together, these data suggest that EAPP modulates E2F-regulated transcription, stimulates proliferation, and may be involved in the malignant transformation of cells.
Molecular Biology of the Cell 06/2005; 16(5):2181-90. · 4.94 Impact Factor
