Regulation of the E2F-associated phosphoprotein promoter by GC-box binding proteins.

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.

EAPP, a novel E2F binding protein that modulates E2F-dependent transcription.

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.

The tumor suppressor p53 and histone deacetylase 1 are antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 gene.

The cyclin-dependent kinase inhibitor p21/WAF1/CIP1 is an important regulator of cell cycle progression, senescence, and differentiation. Genotoxic stress leads to activation of the tumor suppressor p53 and subsequently to induction of p21 expression. Here we show that the tumor suppressor p53 cooperates with the transcription factor Sp1 in the activation of the p21 promoter, whereas histone deacetylase 1 (HDAC1) counteracts p53-induced transcription from the p21 gene. The p53 protein binds directly to the C terminus of Sp1, a domain which was previously shown to be required for the interaction with HDAC1. Induction of p53 in response to DNA-damaging agents resulted in the formation of p53-Sp1 complexes and simultaneous dissociation of HDAC1 from the C terminus of Sp1. Chromatin immunoprecipitation experiments demonstrated the association of HDAC1 with the p21 gene in proliferating cells. Genotoxic stress led to recruitment of p53, reduced binding of HDAC1, and hyperacetylation of core histones at the p21 promoter. Our findings show that the deacetylase HDAC1 acts as an antagonist of the tumor suppressor p53 in the regulation of the cyclin-dependent kinase inhibitor p21 and provide a basis for understanding the function of histone deacetylase inhibitors as antitumor drugs.

Transcriptional regulation of the human chorionic gonadotropin beta gene during villous trophoblast differentiation.

Expression of the trophoblast-specific subunit of human chorionic gonadotropin, CGbeta, is associated with fusion of cytotrophoblasts into a multinuclear syncytium. Here, we studied regulation of the CGbeta5 gene in trophoblasts undergoing in vitro syncytialization. Transfection of luciferase reporters harboring different lengths of the CGbeta5 upstream sequence revealed that the proximal promoter region (-345 to +114) is sufficient to govern differentiation-dependent induction. Mutational analyses suggested that two selective promoter factor (Sp) and three activating protein 2 (AP-2) recognition sequences are necessary for full activity of the promoter. During syncytialization these elements interacted with increasing amounts of the transcription factors Sp1, Sp3, and AP-2alpha in electrophoretic mobility shift assay, but only AP-2alpha binding rose upon elevation of cAMP levels with forskolin. Increasing expression of different isoforms of Sp1 and Sp3 could also be detected by Western blot analyses. Sp1/Sp3 localized to syncytial nuclei both in differentiated cultures and in term placental tissue, suggesting assembly of functional transcriptional complexes. Costaining of the transcription factors with E-cadherin on term placental sections revealed that 47 and 33% of cytotrophoblast nuclei were negative for Sp1 and Sp3, respectively. In contrast, immunohistochemistry of early tissue demonstrated expression of Sp1 in the majority of cyto- and syncytiotrophoblasts, whereas Sp3 was absent from the syncytium. Sp1 and Sp3 induced wild-type/mutant promoter constructs upon transfection in Sp-deficient SL-2 cells, indicating that the Sp elements function as activating sequences. The data suggest that increasing concentrations of Sp1, Sp3, and AP-2alpha enhance transcription of CGbeta in differentiating term trophoblasts, whereas a different combination of factors may control expression in early placentas.

Expression of the human Hand1 gene in trophoblastic cells is transcriptionally regulated by activating and repressing specificity protein (Sp)-elements.

The tissue-specific basic helix-loop-helix protein Hand1 is essential for the formation of trophoblast giant cells of the murine placenta. In humans, Hand1 is detectable in trophoblastic tumour cells suggesting an equivalent role in trophoblast differentiation. To understand its mode of expression we have cloned and characterized the human Hand1 gene promoter. Primer extension analyses suggest that transcription initiates 19 nucleotides downstream of the TATA element of the proximal 5' flanking region. Expression of luciferase reporter constructs harboring deletions of the 9.5 kb Hand1 5' flanking sequence defines a promoter region within 274 bp upstream of the transcriptional start site. Compared to a reporter bearing only the TATA box, the proximal promoter activates transcription up to 30-fold. However, transcriptional activity of the region was observed in both Hand1-expressing and non-expressing cell lines. Sequencing, DNAseI footprint analyses and electrophoretic mobility shift assays reveal the presence of four GC-rich sequences, which show different affinities to the endogenous specificity proteins (Sp), and a CCAAT box. In vitro, the Sp-elements mainly interact with Sp1 and Sp3 while the CCAAT element is recognized by the alpha CAAT binding factor protein. Mutant luciferase reporters bearing single active or inactive recognition sites demonstrate that two of the four Sp-binding sites (I and IV) contribute little to the overall transcription rate. The two other Sp-cognate sequences, II and III, downregulate and activate reporter expression 2.3- and 2.6-fold, respectively. Co-transfections of Sp1/Sp3 expression vectors and mutated reporter constructs in Sp-deficient SL2 cells indicate that the Sp-binding site II and III indeed function as repressing and activating enhancer sequences. In summary, the data suggest that constitutive expression of the Hand1 gene in cultured cells is regulated by a complex interplay of Sp-proteins interacting with activator and repressor elements.

Regulation of the MDR1 promoter by E2F1 and EAPP.

Multidrug resistance (MDR), one of the main reasons for diminishing efficacy of prolonged chemotherapy, is frequently caused by the elevated expression of the ABCB1/MDR1 gene encoding PGP (P-glycoprotein). EAPP (E2F Associated PhosphoProtein) is a frequently overexpressed protein in human tumor cells. It inhibits apoptosis in a p21-dependent manner. We show here that EAPP stimulates the MDR1 promoter resulting in higher PGP levels. Independently of EAPP, E2F1 also increases the activity of the MDR1 promoter. Co-expression of pRb inhibits E2F1-, but not EAPP-dependent promoter activation. The upregulation of PGP might contribute to the survival of tumor cells during chemotherapy and worsen the prognosis for the patient.

Novel inhibitors of cyclin-dependent kinases combat hepatocellular carcinoma without inducing chemoresistance.

Treatment options for hepatocellular carcinoma using chemotherapeutics at intermediate and advanced stages of disease are limited as patients most rapidly escape from therapy and succumb to disease progression. Mechanisms of the hepatic xenobiotic metabolism are mostly involved in providing chemoresistance to therapeutic compounds. Given the fact that the aberrant activation of cyclin-dependent kinases (CDK) is frequently observed in hepatocellular carcinomas, we focused on the efficacy of the novel compounds BA-12 and BP-14 that antagonize CDK1/2/5/7 and CDK9. Inhibition of those CDKs in human hepatocellular carcinoma cell lines reduced the clonogenicity by arresting cells in S-G2 and G2-M phase of the cell cycle and inducing apoptosis. In contrast, primary human hepatocytes failed to show cytotoxicity and apoptosis. No loss of chemosensitivity was observed in hepatocellular carcinoma cells after long-term exposure to inhibitors. In vivo, treatment of xenografted human hepatocellular carcinomas with BA-12 or BP-14 effectively repressed tumor formation. Moreover, BA-12 or BP-14 significantly diminished diethylnitrosamine (DEN)-induced hepatoma development in mice. These data show that BA-12 or BP-14 exhibit strong antitumorigenic effects in the absence of chemoresistance, resulting in a superior efficacy compared with currently used chemotherapeutics in hepatocellular carcinomas.

EAPP modulates the activity of p21 and Chk2.

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.