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The transcription factor YBX1 can act as a mediator of signals transmitted via the EGFR-RAS-MAPK axis. YBX1 expression has been associated with tumor progression and prognosis in multiple types of cancer. Immunohistochemical studies have revealed dependency between YBX1 expression and individual EGFR family members. We analyzed YBX1 and EGFR family proteins in a colorectal cancer (CRC) cohort and provide functional analyses of YBX1 in the context of EGFR-RAS-MAPK signaling. Immunohistochemistry for YBX1 and EGFR family receptors with two antibodies for YBX1 and EGFR were performed and related to clinicopathological data. We employed Caco2 cells expressing an inducible KRASV12 gene to determine effects on localization and levels of YBX1. Mouse xenografts of Caco2-KRASV12 cells were used to determine YBX1 dynamics in a tissue context. The two different antibodies against YBX1 showed discordant immunohistochemical stainings in cell culture and clinical specimens. Expression of YBX1 and EGFR family members were not correlated in CRC. Analysis of Caco2 xenografts displayed again heterogeneity of YBX1 staining with both antibodies. Our results suggest that YBX1 is controlled via complex regulatory mechanisms involving tumor stroma interaction and signal transduction processes. Our study highlights that YBX1 antibodies have different specificities, advocating their use in a combined manner.

Low molecular weight heparin (LMWH) is routinely used for antithrombotic treatment of cancer patients. Preclinical- and clinical data suggest that LMWH has beneficial effects for cancer patients beyond the prevention of thrombosis, i.e. by inhibiting metastasis. It is, however, unclear whether heparin has an impact on the efficiency of chemotherapy in cancer patients. Here we show that a therapeutic dosage of LMWH tinzaparin reverses cisplatin resistance of A2780cis human ovarian cancer cells to the level of sensitive cells. This novel activity of tinzaparin is associated with intense transcriptional reprogramming. Our gene expression profiling experiments revealed that 3776 genes responded to tinzaparin treatment. For this reason tinzaparin has a complex impact on diverse biological processes. We discovered that tinzaparin inhibits the expression of genes that mediate cisplatin resistance of A2780cis cells. In contrast tinzaparin induced the expression of genes that antagonize drug resistance. This activity of tinzaparin is mediated by cell surface proteoglycans, since enzymatic cleavage of heparan sulfates prevented the reversal of cisplatin resistance. These data indicate that cell surface heparan sulfate proteoglycans play an important role for chemotherapy resistance. The results of this study shed a new light on LMWH application in cancer therapy and suggest tinzaparin as promising treatment option of ovarian cancer patients in combination with anticancer drugs. Future clinical trials are needed to validate these findings., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The WT1 gene encodes a zinc finger transcription factor important for normal kidney development. WT1 is a suppressor for Wilms tumour development and an oncogene for diverse malignant tumours. We recently established cell lines from primary Wilms tumours with different WT1 mutations. To investigate the function of mutant WT1 proteins, we performed WT1 knockdown experiments in cell lines with a frameshift/extension (p.V432fsX87 = Wilms3) and a stop mutation (p.P362X = Wilms2) of WT1, followed by genome-wide gene expression analysis. We also expressed wild-type and mutant WT1 proteins in human mesenchymal stem cells and established gene expression profiles. A detailed analysis of gene expression data enabled us to classify the WT1 mutations as gain-of-function mutations. The mutant WT1(Wilms2) and WT1(Wilms3) proteins acquired an ability to modulate the expression of a highly significant number of genes from the G2/M phase of the cell cycle, and WT1 knockdown experiments showed that they are required for Wilms tumour cell proliferation. p53 negatively regulates the activity of a large number of these genes that are also part of a core proliferation cluster in diverse human cancers. Our data strongly suggest that mutant WT1 proteins facilitate expression of these cell cycle genes by antagonizing transcriptional repression mediated by p53. We show that mutant WT1 can physically interact with p53. Together the findings show for the first time that mutant WT1 proteins have a gain-of-function and act as oncogenes for Wilms tumour development by regulating Wilms tumour cell proliferation., (© The Author 2014. Published by Oxford University Press.)

The frequent development of cellular resistance to cisplatin in cancer patients is a serious limitation for clinical drug therapy. However, cisplatin resistance is incompletely understood. We have shown that cisplatin-resistant A2780 ovarian cancer cells (A2780cis) can efficiently be eliminated by liposomal cisplatin, which displayed similar cytotoxicity towards both A2780 and A2780cis cells. This may, at least in part, be related to a higher intracellular accumulation of the drug within the resistant cells after liposomal entry. However, the superior cytotoxicity of the liposomal drug was not reflected by DNA platination. This suggests a more complex mode of action of liposomal cisplatin, most likely affecting different signaling pathways. To gain insight into the resistance gene signature, a whole-genome gene expression analysis was performed for A2780cis cells, untreated or treated with half-minimal inhibitory concentration (IC50) of free and liposomal cisplatin. Strong differences in the functional networks affected by free and liposomal cisplatin became evident. p53 was identified as a key factor directing differences in the apoptotic processes. While free cisplatin induced the intrinsic pathway of apoptosis, liposomal cisplatin induced expression of genes of DNA damage pathways and of the extrinsic pathway of apoptosis. These predictions from gene expression data were confirmed at the protein and function level. This sheds new light on liposomal drug carrier approaches in cancer and suggests liposomal cisplatin as a promising strategy for the treatment of cisplatin-resistant ovarian carcinoma.

Background: The Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s)., Results: To address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter., Conclusion: Compartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.

Previously we reported that liposomal cisplatin (CDDP) overcomes CDDP resistance of ovarian A2780cis cancer cells (Krieger et al., Int. J. Pharm. 389, 2010, 10-17). Here we find that the cytotoxic activity of liposomal CDDP is not associated with detectable DNA platination in resistant ovarian cancer cells. This suggests that the mode of action of liposomal CDDP is different from the free drug. To gain insight into mechanisms of liposomal CDDP activity, we performed a transcriptome analysis of untreated A2780cis cells, and A2780cis cells in response to exposure with IC50 values of free or liposomal CDDP. A process network analysis of upregulated genes showed that liposomal CDDP induced a highly different gene expression profile in comparison to the free drug. p53 was identified as a key player directing transcriptional responses to free or liposomal CDDP. The free drug induced expression of essential genes of the intrinsic (mitochondrial) apoptosis pathway (BAX, BID, CASP9) most likely through p38MAPK activation. In contrast, liposomal CDDP induced expression of genes from DNA damage pathways and several genes of the extrinsic pathway of apoptosis (TNFRSF10B-DR5, CD70-TNFSF7). It thus appears that liposomal CDDP overcomes CDDP resistance by inducing DNA damage and in consequence programmed cell death by the extrinsic pathway. Predictions from gene expression data with respect to apoptosis activation were confirmed at the protein level by an apoptosis antibody array. This sheds new light on liposomal drug carrier approaches in cancer and suggests liposomal CDDP as promising strategy for the treatment of CDDP resistant ovarian carcinomas., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Purpose: We studied the response of human embryonic stem cells (hESC) to the β-emitter (131)I, which affects the entire cell and to the Auger electron emitter (125)I-deoxyuridine ((125)I-dU), primarily affecting the deoxyribonuleic acid (DNA). The effects were also studied in keratinocytes as a prototype for somatic cells., Methods: HESC (H1) and human keratinocytes (HaCaT, human) were exposed to (125)I-dU (5 × 10(-5) - 5 MBq/ml) and (131)I-iodide (5 × 10(-5) - 12.5 MBq/ml) and apoptosis was measured by DNA-fragmentation. Cell morphology was studied by light microscopy and electron microscopy. Transcriptional profiling was done on the Agilent oligonucleotide microarray platform., Results: Auger-process induced no apoptosis but a strong transcriptional response in hESC. In contrast, HaCaT cells showed a pronounced induction of apoptosis but only a moderate transcriptional response. Transcriptional response of hESC was similar after (125)I-dU and (131)I treatments, whereas HaCaT cells expressed a much more pronounced response to (125)I-dU than to (131)I. A striking radiation-induced down-regulation of pluripotency genes was observed in hESC whereas in keratinocytes the enriched gene annotations were related primarily to apoptosis, cell division and proliferation., Conclusions: Human embryonic stem cells respond to ionizing radiation by (125)I-dU and (131)I in a different way compared to keratinocytes. Transcriptional response and gene expression appear to facilitate an escape from programmed cell death by striking a new path which probably leads to cell differentiation.

Unlabelled: Analysis of gene expression profiles is no longer exclusively a task for bioinformatic experts. However, gaining statistically significant results is challenging and requires both biological knowledge and computational know-how. Here we present a novel, user-friendly microarray reporting tool called maRt. The software provides access to bioinformatic resources, like gene ontology terms and biological pathways by use of the DAVID and the BioMart web-service. Results are summarized in structured HTML reports, each presenting a different layer of information. In these report, contents of diverse sources are integrated and interlinked. To speed up processing, maRt takes advantage of the multi-core technology of modern desktop computers by using parallel processing. Since the software is built upon a RCP infrastructure it might be an outset for developers aiming to integrate novel R based applications., Availability: Installer, documentation and various kinds of tutorials are available under LGPL license at the website of our institute http://www.pharma.uni-bonn.de/www/mart. This software is free for academic use., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties., Competing Interests: The authors have declared that no competing interests exist.

Wilms tumors (WTs) are genetically heterogeneous kidney tumors whose cells of origin are unknown. Tumors with WT1 mutations and concomitant loss of the wild-type allele represent a distinct subgroup, frequently associated with mutations in CTNNB1. Here, we describe the establishment and characterization of long-term cell cultures derived from five individual WTs with WT1 mutations. Three of these tumor cell lines also had CTNNB1 mutations and an activated canonical Wnt signaling pathway as measured by beta-catenin/T cell-specific transcription factor (TCF) transcriptional activity. Four of the five Wilms cell lines had a stable normal karyotype for at least 25 passages, and four lines showed loss of heterozygosity of chromosome 11p due to mitotic recombination in 11p11. Gene expression profiling revealed that the WT cell lines are highly similar to human mesenchymal stem cells (MSCs) and FACS analysis demonstrated the expression of MSC-specific surface proteins CD105, CD90 and CD73. The stem cell like nature of the WT cells is further supported by their adipogenic, chondrogenic, osteogenic and myogenic differentiation potentials. By generating multipotent mesenchymal precursors from paraxial mesoderm (PAM) in tissue culture using embryonal stem cells, gene expression profiles of PAM and MSCs were described. Using these published gene sets, we found coexpression of a large number of genes in WT cell lines, PAM and MSCs. Lineage plasticity is indicated by the simultaneous expression of genes from the mesendodermal and neuroectodermal lineages. We conclude that WTs with WT1 mutations have specific traits of PAM, which is the source of kidney stromal cells.

The clinical application of cisplatin to treat solid tumours is often limited by the development of tumour cell resistance against this cytostatic agent. Although liposomal carriers of cisplatin are currently in clinical development, approaches to functionally overcome cisplatin resistance by liposomes have hardly been reported. We prepared PEGylated cisplatin-containing liposomes with diameters of about 110 nm and targetability to transferrin receptors (TfR) to correlate cisplatin cell uptake with cytotoxicity in sensitive and cisplatin resistant ovarian cancer cells A2780 compared to the free drug. Whereas the cell entry of free cisplatin was reduced by factor 4 after 24h in resistant cells, liposomal uptake was similar in both cell lines and not affected by resistance. Cytotoxicity was clearly related to intracellular platinum levels, which were even higher for liposomal vs. free cisplatin in the resistant cells after 24, 48, and 72 h and slightly lower in the sensitive cells. However, TfR targeting was of less impact on activity in comparison to non-targeted liposomes. Detection of cellular ATP levels within 24h allowed postulations on the intracellular fate of the liposomes. Altogether, this study strongly supports approaches to overcome cisplatin resistance by a liposomal application of the drug., (Copyright 2010 Elsevier B.V. All rights reserved.)

Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1(S102) stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1-transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1(S102D) enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1(S102) expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44(High)/CD24(Low) cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f.

The G protein-coupled P2Y(11) receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y(11) receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y(11) agonist NF546 [4,4'-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-alpha,alpha'-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC(50) of 6.27 and is relatively selective for P2Y(11) over P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(12), P2X(1), P2X(2), and P2X(2)-X(3). Adenosine-5'-O-(3-thio)triphosphate (ATPgammaS), a nonhydrolyzable analog of the physiological P2Y(11) agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4'-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA(2) of NF340 was 8.02 against ATPgammaS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y(11)-mediated effects in monocyte-derived dendritic cells. Similarly to ATPgammaS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPgammaS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y(11) receptors in both recombinant and physiological expression systems and could show a P2Y(11)-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y(11) receptors.

Purpose: To investigate the potential of Y-box-binding protein YB-1, a multifunctional protein linked to tumor aggressiveness and multidrug resistance, to identify patients with breast cancer likely to benefit from dose-intensified chemotherapy regimens., Patients and Methods: YB-1 was immunohistochemically determined in 211 primary tumors from the prospective, randomized West German Study Group WSG-AM-01 trial in high-risk (> or = 10 involved lymph-nodes) breast cancer (HRBC). Predictive impact of YB-1 was assessed by multivariate survival analysis, including time-varying factor-therapy interactions., Results: At median follow-up of 61.7 months, patients receiving rapidly cycled tandem high-dose therapy (HD; two cycles [2x] epirubicin 90 mg/m(2) and cyclophosphamide 600 mg/m(2) every 14 days, followed by 2x epirubicin 90 mg/m(2), cyclophosphamide 3,000 mg/m(2), and thiotepa 400 mg/m(2) every 21 days) had better disease-free survival (DFS; hazard ratio [HR] = 0.62; 95% CI, 0.44 to 0.89) and overall survival (OS; HR = 0.59; 95% CI, 0.4 to 0.89) than those receiving conventional dose-dense chemotherapy (DD; 4x epirubicin 90 mg/m(2) and cyclophosphamide 600 mg/m(2), followed by 3x cyclophosphamide 600 mg/m(2), methotrexate 40 mg/m(2), and fluorouracil 600 mg/m(2) every 14 days). High YB-1 was associated with aggressive tumor phenotype (negative steroid hormone receptor status, positive human epidermal growth factor receptor 2 and p53 status, high MIB-1, unfavorable tumor grade) and poor OS (median 78 v 97 months; P = .01). In patients with high YB-1, HD yielded a 63-month median DFS (P = .001) and a 46-month median OS advantage (P = .002) versus DD. In multivariate models, patients with high B-1 receiving HD (v DD) had one third the hazard rate after 20 months for DFS and one sixth after 40 months for OS., Conclusion: In a randomized prospective cancer therapy trial, for the first time, a strong predictive impact of YB-1 on survival has been demonstrated: enhanced benefit from HD (v DD) therapy occurs in HRBC with high YB-1. Future trials could therefore address optimal chemotherapeutic strategies,taking YB-1 into account.

Platinum plays a central role in the therapy of ovarian cancer, and the emergence of platinum resistance is a major obstacle for clinical management of the disease. We treated A2780 ovarian cancer cells by weekly cycles of cisplatin over a period of 6 months and unveiled that enhanced insulin-like growth factor I receptor (IGF-IR) expression and autocrine IGF-I are associated with hyperactivation of the IGF-IR and phosphatidylinositol-3-OH kinase (PI3K) pathways in cisplatin-resistant cells. IGF-IR expression levels increased during treatment cycles and correlated with cisplatin resistance. Purified IGF-I induced cisplatin resistance in diverse ovarian cancer cell lines, and small molecule inhibitors proved that IGF-IR and PI3K are essential for cisplatin resistance. Similar results were obtained with BG-1 ovarian cancer cells. Cytogenetic and array comparative genomic hybridization analyses revealed selection and de novo formation of chromosomal alterations during resistance development. An analysis of gene expression profiles of primary ovarian carcinomas identified the regulatory subunit PIK3R2 of PI3-kinase as a significant negative prognosis factor for ovarian cancer. We conclude that targeting the IGF-IR and the PI3K pathways is a promising new strategy to treat cisplatin-resistant ovarian carcinomas.

Head and neck cancers are treated by a combination of surgery, radiotherapy and/or chemotherapy. The clinical success of cisplatin-based chemotherapy, mostly in combination with 5-FU or a taxane, is however limited by multifactorial intrinsic or acquired resistance. So far, known genes involved in cisplatin resistance do not sufficiently allow the prediction of cancer chemosensitivity. Thus, the purpose of this study was to search for further genes involved in cisplatin resistance by differential gene expression analysis of the parental tongue cancer cell line Cal27 and its 10-fold more resistant sub-cell line Cal27cis, which was obtained by treating Cal27 with increasing concentrations of cisplatin. As found by the suppression subtractive hybridization, expression of DKK1, an inhibitor of canonical WNT signaling, was decreased in Cal27cis. Microarray analysis, qPCR and ELISA confirmed the approximately 2-fold difference in expression. Cisplatin treatment and serum starvation increased by 2-fold the secretion of DKK1 in Cal27 and Cal27cis, thus rendering DKK1-levels significantly different in both cell lines under basal and stress conditions. Recombinant overexpression of DKK1 in Cal27 and Cal27cis resulted in clonal cell lines, which were both 2.2- to 3-fold more sensitive toward cisplatin in cell viability (MTT) and in proliferation (BrdU) assays. In conclusion, acquired (10-fold) resistance of Cal27 against cisplatin is associated with decreased DKK1 expression and could partially be reversed by DKK1 overexpression, thus suggesting DKK1 and the WNT signaling pathway as a marker and target for cisplatin chemosensitivity., ((c) 2008 Wiley-Liss, Inc.)

Current knowledge about molecular mechanisms underlying disease progression and drug resistance in multiple myeloma (MM) is still limited. Here, we analyzed the potential pathogenetic role of the Y-box binding protein YB-1 in MM. YB-1 is a member of the cold-shock domain protein superfamily and involved in various cellular functions such as proliferation. Immunohistochemical analyses revealed that neither normal bone marrow (BM) plasma cells (PCs), premalignant PCs of patients with monoclonal gammopathy of unknown significance (MGUS), nor MM cells with a mature morphology showed expression of YB-1 in situ. In contrast, YB-1 was strongly expressed in situ in normal PC precursor blasts as well as in a MM subset and in vitro in all of the evaluated MM cell lines. The YB-1-expressing MM cells were characterized by an immature morphology and a highly proliferative phenotype as defined by Ki 67 expression. We observed that siRNA-mediated knockdown of YB-1 decreased proliferation and induced apoptosis in MM cells even in the presence of BM stromal cells. Furthermore, we found that overexpression of YB-1 mediated resistance toward doxorubicin-induced apoptosis in MM cells. Thus, YB-1 contributes to disease progression, survival, and drug resistance in MM and might therefore provide an attractive therapeutic target.

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.

Isolated deletions of the long arm of chromosome 5, del(5q), are observed in 10% of myelodysplastic syndromes (MDS) and are associated with a more favorable prognosis, although the clinical course varies considerably. If one or more additional chromosomal aberrations are present, this correlates with a significantly shorter overall survival. To assess the frequency of hidden abnormalities in cases with an isolated cytogenetic del(5q), we have performed a genome wide high resolution 44 K 60mer oligonucleotide array comparative genomic hybridization (aCGH) study using DNA from bone marrow cells of 12 MDS and one AML patient. In one case a single additional hidden 5.6 Mb deletion of 13q14 and in another case multiple larger aberrations involving many chromosomes were found. Fluorescence in situ hybridization demonstrated that aberrations present in 35% of the bone marrow cells can be detected by aCGH. Furthermore with oligonucleotide aCGH the deletion end points in 5q were mapped precisely, revealing a cluster of proximal breakpoints in band q14.3 (n = 8) and a distal cluster between bands q33.2 and q34 (n = 11). This study shows the high resolution of oligonucleotide CGH arrays for precisely mapping genomic alterations and for refinement of deletion end points. In addition, the high sensitivity of this method enables the study of whole bone marrow cells from MDS patients, a disease with a low blast count., ((c) 2007 Wiley-Liss, Inc.)

Retinoblastoma protein (Rb) is a multifunctional tumor suppressor, frequently inactivated in certain types of human cancer. Nucleolin is an abundant multifunctional phosphoprotein of proliferating and cancerous cells, recently identified as cell cycle-regulated transcription activator, controlling expression of human papillomavirus type 18 (HPV18) oncogenes in cervical cancer. Here we find that nucleolin is associated with Rb in intact cells in the G1 phase of the cell cycle, and the complex formation is mediated by the growth-inhibitory domain of Rb. Association with Rb inhibits the DNA binding function of nucleolin and in consequence the interaction of nucleolin with the HPV18 enhancer, resulting in Rb-mediated repression of the HPV18 oncogenes. The intracellular distribution of nucleolin in epithelial cells is Rb-dependent, and an altered nucleolin localization in human cancerous tissues results from a loss of Rb. Our findings suggest that deregulated nucleolin activity due to a loss of Rb contributes to tumor development in malignant diseases, thus providing further insights into the molecular network for the Rb-mediated tumor suppression.

Conditionally replicating adenoviruses are a promising new modality for the treatment of cancer. However, early clinical trials demonstrate that the efficacy of current vectors is limited. Interestingly, DNA replication and production of viral particles do not always correlate with virus-mediated cell lysis and virus release depending on the vector utilized for infection. However, we have previously reported that nuclear accumulation of the human transcription factor YB-1 by regulating the adenoviral E2 late promoter facilitates viral DNA replication of E1-deleted adenovirus vectors which are widely used for cancer gene therapy. Here we report the promotion of virus-mediated cell killing as a new function of the human transcription factor YB-1. In contrast to the E1A-deleted vector dl312 the first-generation adenovirus vector AdYB-1, which overexpresses YB-1 under cytomegalovirus promoter control, led to necrosis-like cell death, virus production, and viral release after infection of A549 and U2OS tumor cell lines. Our data suggest that the integration of YB-1 in oncolytic adenoviruses is a promising strategy for developing oncolytic vectors with enhanced potency against different malignancies.

Vaults have been suggested to play a direct role in multidrug resistance (MDR) to anticancer drugs. The human major vault protein (MVP) also known as lung resistance-related protein (LRP) represents the predominant component of vaults that may be involved in the defense against xenobiotics. Here, we demonstrate that besides MDR-related cytostatics, also the non-MDR-related drug 5-fluorouracil (5-FU) was able to induce MVP mRNA and protein expression. Treatment with 5-FU amplified the binding activity and interaction of the transcription factor Y-box binding protein-1 (YB-1) with the Y-box of the human MVP gene promoter in a time-dependent manner. 5-FU also induced reporter expressions driven by a panel of newly generated MVP promoter deletion mutants. Interestingly, stably YB-1 overexpressing cell clones showed enhanced binding of YB-1 to the Y-box motif, associated with enhanced basal as well as 5-FU-inducible MVP promoter-driven reporter expressions. Moreover, transduction of YB-1 cDNA led to increased expression of endogenous MVP protein. Under physiological conditions, we observed a strong coexpression of MVP and YB-1 in human colon carcinoma specimen. In summary, our data demonstrate a direct involvement of YB-1 in controlling basal and 5-FU-induced MVP promoter activity. Therefore, YB-1 is directly linked to MVP-mediated drug resistance.

YB-1 protein levels are elevated in most human breast cancers, and high YB-1 levels have been correlated with drug resistance and poor clinical outcome. YB-1 is a stress-responsive, cell cycle-regulated transcription factor with additional functions in RNA metabolism and translation. In this study, we show in a novel transgenic mouse model that human hemagglutinin-tagged YB-1 provokes remarkably diverse breast carcinomas through the induction of genetic instability that emerges from mitotic failure and centrosome amplification. The increase of centrosome numbers proceeds during breast cancer development and explanted tumor cell cultures show the phenotype of ongoing numerical chromosomal instability. These data illustrate a mechanism that might contribute to human breast cancer development.

The serotonin transporter (5-HTT) gene contains a variable number tandem repeat (VNTR) domain within intron 2 that is often associated with a number of neurological conditions, including affective disorders. The implications of this polymorphism are not yet understood, however, we have previously demonstrated that the 5-HTT VNTR is a transcriptional regulatory domain, and the allelic variation supports differential reporter gene expression in vivo and in vitro. The aim of this study was to identify transcription factors responsible for the regulation of this VNTR. Using a yeast one-hybrid screen, we found the transcription factor Y box binding protein 1 (YB-1) interacts with the 5-HTT VNTR. Consistent with this, we demonstrate in a reporter gene assay that the polymorphic VNTR domains differentially respond to exogenous YB-1 and that YB-1 will bind to the VNTR in vitro in a sequence-specific manner. Interestingly, the transcription factor CCTC-binding factor (CTCF), previously shown to interact with YB-1, interferes with the ability of the VNTR to support YB-1-directed reporter gene expression. In addition, CTCF blocks the binding of YB-1 to its DNA recognition sequences in vitro, thus providing a possible mechanism of regulation of YB-1 activation of the VNTR by CTCF. Therefore, we have identified YB-1 and CTCF as transcription factors responsible, at least in part, for modulation of VNTR function as a transcriptional regulatory domain. Our data suggest a novel mechanism that explains, in part, the ability of the distinct VNTR copy numbers to support differential reporter gene expression based on YB-1 binding sites.

Resistance to chemotherapy is responsible for a failure of current treatment regimens in cancer patients. We have reported previously that the Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug resistant-tumor phenotype. YB-1 predicts drug resistance and patient outcome in breast cancer. Thus, YB-1 is a promising target for new therapeutic approaches to defeat multidrug resistance. In drug-resistant cancer cells and in adenovirus-infected cells YB-1 is found in the nucleus. Nuclear accumulation of YB-1 in adenovirus-infected cells is a function of the E1 region, and we have shown that YB-1 facilitates adenovirus replication. Here we report that E1A-deleted or mutant adenovirus vectors, such as Ad312 and Ad520, replicate efficiently in multidrug-resistant (MDR) cancer cells and induce an adenovirus cytopathic effect resulting in host cell lysis. Thus, replication-defective adenoviruses are a previously unrecognized vector system for a selective elimination of MDR cancer cells. Our work forms the basis for the development of novel oncolytic adenovirus vectors for the treatment of MDR malignant diseases in the clinical setting.

The human Y-box binding protein, YB-1, is a multifunctional protein that regulates gene expression. Nuclear expression of YB-1 has been associated with chemoresistance and poor prognosis of tumour patients. Representative samples from autopsied material of primary tumours from 77 patients with NSCLC were investigated by immunohistochemistry for subcellular distribution of YB-1 and p53, in order to evaluate the prognostic role of nuclear expression of YB-1. Cytoplasmic YB-1 expression was found in all tumour samples, whereas nuclear expression was only observed in 48%. There was no correlation with histological classification, clinical parameters or tumour size, stage and metastasis status. However, patients with positive nuclear YB-1 expression in tumours showed reduced survival times when compared with patients without nuclear expression. Including information about the histology and mutational status for p53 increased the prognostic value of nuclear YB-1. Patients with nuclear YB-1 expression and p53 mutations had the worst prognosis (median survival 3 months), while best outcome was found in patients with no nuclear YB-1 and wildtype p53 (median survival 15 months). This suggests that the combined analysis of both markers allows a better identification of subgroups with varying prognosis. Nuclear expression of Y-box binding protien seems to be an independent prognostic marker.

Expression of the Y-box protein YB-1 is increased in proliferating normal and cancer cells, but its role in cell proliferation and cell cycle progression is unclear. We have identified a cell cycle-dependent relocalization of YB-1 from the cytoplasm to the nucleus at the G1/S phase transition and demonstrate that both the charged zipper and the cold shock domain are involved in regulating this process. Using cell lines that constitutively overexpress YB-1, we show that nuclear accumulation of YB-1 is associated with increased cyclin A and cyclin B1 mRNA and protein expression. We provide evidence that deregulated YB-1 expression is linked to adhesion-independent cell proliferation through the induction of cyclin A. Thus, we have identified YB-1 as a cell cycle stage-specific transcription factor important for cell proliferation.

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18(+) cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16(+) SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18(+) precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18(+) carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis.

The adenovirus early proteins E1A and E1B-55kDa are key regulators of viral DNA replication, and it was thought that targeting of p53 by E1B-55kDa is essential for this process. Here we have identified a previously unrecognized function of E1B for adenovirus replication. We found that E1B-55kDa is involved in targeting the transcription factor YB-1 to the nuclei of adenovirus type 5-infected cells where it is associated with viral inclusion bodies believed to be sites of viral transcription and replication. We show that YB-1 facilitates E2 gene expression through the E2 late promoter thus controlling E2 gene activity at later stages of infection. The role of YB-1 for adenovirus replication was demonstrated with an E1-minus adenovirus vector containing a YB-1 transgene. In infected cells, AdYB-1 efficiently replicated and produced infectious progeny particles. Thus, adenovirus E1B-55kDa protein and the host cell factor YB-1 act jointly to facilitate adenovirus replication in the late phase of infection.

Sp1 binding sites have been identified in enhancer/promoter regions of several growth and cell cycle regulated genes, and it has been shown that Sp1 is increasingly phosphorylated in G1 phase of the cell cycle. Interactions of Sp1 with proteins involved in control of cell cycle and tumor formation have been reported. Here we show that expression of Sp1 protein predominates in the G1 phase of the cell cycle in epithelial cells. This is achieved by proteasome-dependent degradation. Inhibition of endogeneous Sp1 activity by a dominant-negative Sp1 mutant was associated with a cell cycle arrest in G1 phase, a strongly reduced expression of cyclin D1, the EGF-receptor and increased levels of p27Kip1. We have thus identified Sp1 as an important regulator of the cell cycle in G1 phase.

Intrinsic or acquired resistance to chemotherapy is responsible for failure of current treatment regimens in breast cancer patients. The Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug-resistant tumor phenotype. In human breast cancer, overexpression and nuclear localization of YB-1 is associated with upregulation of P-glycoprotein. In our pilot study, we analyzed the clinical relevance of YB-1 expression in breast cancer (n = 83) after a median follow-up of 61 months and compared it with tumor-biologic factors already used for clinical risk-group discrimination, i.e., HER2, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1). High YB-1 expression in tumor tissue and surrounding benign breast epithelial cells was significantly associated with poor patient outcome. In patients who received postoperative chemotherapy, the 5-year relapse rate was 66% in patients with high YB-1 expression. In contrast, in patients with low YB-1 expressions, no relapse has been observed so far. YB-1 expression thus indicates clinical drug resistance in breast cancer. Moreover, YB-1 correlates with breast cancer aggressiveness: in patients not treated with postoperative chemotherapy, those with low YB-1 expression are still free of disease, whereas the 5-year relapse rate in those with high YB-1 was 30%. There was no significant correlation between YB-1 expression and either HER2 expression or uPA and PAI-1 levels. Risk-group assessment achieved by YB-1 differed significantly from that by HER2 or uPA/PAI-1. In conclusion, YB-1 demonstrated prognostic and predictive significance in breast cancer by identifying high-risk patients in both the presence and absence of postoperative chemotherapy, independent of tumor-biologic factors currently available for clinical decision making., (Copyright 2001 Wiley-Liss, Inc.)

Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.

Hodgkin's disease is histopathologically characterized by the relative scarcity of neoplastic Hodgkin and Reed-Sternberg cells and for yet unknown reasons by an abundant reactive background of T lymphocytes and often eosinophils. Eotaxin is a CC-chemokine attracting eosinophils and T helper 2 (Th2) cells in allergic inflammation. We now report that eotaxin is strongly expressed in fibroblasts of Hodgkin's disease tissues, whereas Hodgkin/Reed-Sternberg cells do not express this chemokine. In tissue culture, Hodgkin's disease tumor cells induce eotaxin expression in cocultured dermal fibroblasts in a concentration leading to a specific chemotactic response of a Th2 cell clone. Production of tumor necrosis factor-alpha (TNF-alpha) by Hodgkin/Reed-Sternberg cells appears to be responsible for this induction, because blocking of TNF-alpha by neutralizing antibodies prevented fibroblast eotaxin expression. Our data suggest that eotaxin is involved in the pathobiology of Hodgkin's disease by contributing to eosinophil and T-lymphocyte recruitment.

The pathogenesis and etiology of Hodgkin's disease, a common human malignant lymphoma, is still unresolved. As a unique characteristic, we have identified constitutive activation of the transcription factor nuclear factor (NF)-kappaB p50-RelA in Hodgkin/Reed-Sternberg (H/RS) cells, which discriminates these neoplastic cells from most cell types studied to date. In contrast to other lymphoid and nonlymphoid cell lines tested, proliferation of H/RS cells depended on activated NF-kappaB. Furthermore, constitutive NF-kappaB p50-RelA prevented Hodgkin's lymphoma cells from undergoing apoptosis under stress conditions. Consistent with this dual function, Hodgkin's lymphoma cells depleted of constitutive nuclear NF-kappaB revealed strongly impaired tumor growth in severe combined immunodeficient mice. Our findings identify NF-kappaB as an important component for understanding the pathogenesis of Hodgkin's disease and for developing new therapeutic strategies against it.

Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.

Resistance to apoptosis plays an important role in malignancies that are refractory to chemotherapy treatment. Recently we have shown that the expression of bax-alpha, a death-promoting member of the bcl-2 family, is down-regulated in breast cancer and have provided evidence that low bax expression might contribute to the pathogenesis of breast cancer. In this study we were able to demonstrate the role of this gene in chemotherapy-induced apoptosis. We transfected bax-alpha into the breast-cancer cell lines R30C and MCF-7 under the control of an inducible tetracycline-dependent expression system. Induction of bax-alpha expression did not affect viability by itself but strongly increased chemosensitivity to epirubicin. We were able to demonstrate that this sensitization is due to apoptosis. These data might explain the recently published observation that reduced expression of bax is associated with poor response rates to chemotherapy in breast cancer.

We have studied the expression of members of the bcl-2 family in human breast cancer. The expression pattern of these genes in breast cancer tissue samples was compared with the expression pattern in normal breast epithelium. No marked difference with regard to bcl-2 and bcl-xL expression was observed between normal breast epithelium and cancer tissue. In contrast, bax-alpha, a splice variant of bax, which promotes apoptosis, is expressed in high amounts in normal breast epithelium, whereas only weak or no expression could be detected in 39 out of 40 cancer tissue samples examined so far. Of interest, downregulation of bax-alpha was found in different histological subtypes. Furthermore, we transfected bax-alpha into breast cancer cell lines under the control of a tetracycline-dependent expression system. We were able to demonstrate for the first time that induction of bax expression in breast cancer cell lines restores sensitivity towards both serum starvation and APO-I/Fas-triggered apoptosis and significantly reduces tumor growth in SCID mice. Therefore, we propose that dysregulation of apoptosis might contribute to the pathogenesis of breast cancer at least in part due to an imbalance between members of the bcl-2 gene family.

The nuclear factor p92, originally discovered by its interaction with the human papillomavirus type 18 enhancer, is a cellular protein whose activity is restricted to S phase in human primary fibroblasts. The human papillomavirus type 18 p92 binding sequence confers enhancer activity on a heterologous promoter, suggesting that p92 acts as a transcription factor. We have identified a class of nuclear inhibitory proteins, I-92s, which noncovalently associate with p92 but not with other transcription factors such as AP1, E2F, or NF-kappaB. Different I-92s occur in G1, G2, and G0, while no I-92 is detectable in S phase. Phase-specific inhibitors, therefore, are responsible for the cell cycle dependence of p92 activity and provide a novel mechanism linking transcription factor regulation with the cell cycle.

The transcription factor E2F is regulated during the cell cycle through interactions with the product of the retinoblastoma susceptibility gene and related proteins. It is thought that E2F-mediated gene regulation at the G1/S boundary and during S phase may be one of the rate-limiting steps in cell proliferation. It was reported that in vivo overexpression of E2F-1 in fibroblasts induces S phase entry and leads to apoptosis. This observation suggests that E2F plays a role in both cell cycle regulation and apoptosis. To further understand the role of E2F in cell cycle progression, cell death, and tumor development, we have blocked endogenous E2F activity in HBL-100 cells, derived from nonmalignant human breast epithelium, using dominant-negative mutants under the control of a tetracycline-dependent expression system. We have shown here that induction of dominant-negative mutants led to strong downregulation of transiently transfected E2F-dependent chloramphenicol acetyl transferase reporter constructs and of endogenous c-myc, which has been described as a target gene of the transcription factor E2F/DP. In addition, we have shown that blocking of E2F could efficiently protect from apoptosis induced by serum starvation within a period of 10 d, whereas control cells started to die after 24 h. Surprisingly, blocking of E2F did not alter the rate of proliferation or of DNA synthesis of these cells; this finding indicates that cell-cycle progression could be driven in an E2F-independent manner. In addition, we have been able to show that blocking of endogenous E2F in HBL-100 cells led to rapid induction of tumor growth in severe combined immunodeficiency mice. No tumor growth could be observed in mice that received mock-transfected clones or tetracycline to block expression of the E2F mutant constructs in vivo. Thus, it appears that E2F has a potential tumor-suppressive function under certain circumstances. Furthermore, we provide evidence that dysregulation of apoptosis may be an important step in tumorigenesis.

p92 is a novel sequence-specific octamer-binding factor interacting with the enhancer of human papillomavirus type 18. The nuclear inhibitor I-92 regulates the DNA binding activity of p92 during the cell cycle such that p92 DNA binding is restricted to S-phase. The sequence motif ++ 5'-AATTGCTTGCATAA, consisting of two partially overlapping octamer-related sequences, represents a recognition site for p92. It was the aim of this study to characterize the complexity of proteins interacting with the 5'-AATTGCTTGCATAA motif and to determine their regulation by I-92. UV cross-linking experiments showed that, besides p92, multiple novel proteins interact with the 5'-AATTGCTGCATAA motif. These novel proteins p84, p75, p73, p69, p61, p57, p49, and p46 specifically bind to this motif, although with different affinities. The inhibitor I-92 regulates, besides p92, the DNA-binding activities of p84, p75, p73, p69, and p57 but not of p61, p49, and p46. The association of I-92 with p92, p84, p75, p73, p69, and p57 was completely reversible after treatment with the detergent deoxycholate (DOC). Finally, we analyzed I-92 specificity and found that I-92 selectively inhibited DNA binding activities of partially purified octamer-binding proteins p84 and p92 whereas DNA binding of the POU factor Oct-1 was not regulated by I-92. Our results show that I-92 regulates multiple octamer-binding proteins and these findings provide an example how gene regulation could be linked to cell cycle regulation.