No abstract

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 G 1 /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.

Circadian clocks govern a wide range of cellular and physiological functions in various organisms. Recent evidence suggests distinct functions of local clocks in peripheral mammalian tissues such as immune responses and cell cycle control. However, studying circadian action in peripheral tissues has been limited so far to mouse models, leaving the implication for human systems widely elusive. In particular, circadian rhythms in human skin, which is naturally exposed to strong daytime-dependent changes in the environment, have not been investigated to date on a molecular level. Here, we present a comprehensive analysis of circadian gene expression in human epidermis. Whole-genome microarray analysis of suction-blister epidermis obtained throughout the day revealed a functional circadian clock in epidermal keratinocytes with hundreds of transcripts regulated in a daytime-dependent manner. Among those, we identified a circadian transcription factor, Krüp-pel-like factor 9 (Klf9), that is substantially up-regulated in a cortisol and differentiation-state-dependent manner. Gain-and loss-offunction experiments showed strong antiproliferative effects of Klf9. Putative Klf9 target genes include proliferation/differentiation markers that also show circadian expression in vivo, suggesting that Klf9 affects keratinocyte proliferation/differentiation by controlling the expression of target genes in a daytime-dependent manner.glucocorticoids | skin cancer B iological rhythms regulate cellular and physiological processes ranging from milliseconds to years. A well-studied timing system is the circadian (∼24-h) clockwork that allows organisms to anticipate diurnal variations in environmental conditions such as light, food availability, oxidative stress, pathogen exposure, or temperature. In mammals, the central circadian pacemaker resides in the suprachiasmatic nucleus (SCN), located in the anterior hypothalamus. Oscillations of SCN neurons are cell-autonomous, self-sustained, and synchronized to external time cues (Zeitgebers) such as light (1). The SCN, in turn, synchronizes peripheral clocks by systemic time cues such as neuronal input, hormonal signaling (e.g., cortisol), body temperature, and possibly many others (2). Interestingly, most cells in peripheral tissues also possess cell-autonomous clockworks with a similar molecular makeup to SCN neurons. These peripheral clocks are thought to generate or amplify daytime-dependent physiological and metabolic functions in a tissue-specific manner by circadian regulation of clock-controlled genes (3, 4).On a molecular level, circadian oscillation is generated by interlocked transcriptional-translational feedback loops. The transcription factor dimer CLOCK/BMAL1 drives expression of target genes such as Periods (Per1-3) and Cryptochromes (Cry1-2) by binding to E-box elements in their promoters. The negative feedback is formed by PER/CRY protein complexes that shuttle back into the nucleus, where they block CLOCK/BMAL1-mediated transactivation, thereby inhibiting their own transcr...

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. Multidrug resistance (MDR)1 of human malignant tumors represents a major cause of cancer chemotherapy failure. The development of the MDR phenotype is often associated with increased expression of certain ATP-binding cassette transporters (ABC transporters) such as P-glycoprotein and MRP1. The multidrug resistance gene (MDR1) encodes P-glycoprotein, which causes classical MDR. In contrast, the MRP1 gene encodes the multidrug resistance-related protein (MRP1), which is associated with an atypical non-P-glycoprotein-dependent MDR phenotype (1). Although both transporter proteins function as drug-efflux pumps, they share only 15% amino acid homology, transport a non-identical spectrum of anticancer substrates, and show a different distribution in human normal and cancer tissues (2, 3).Colorectal tumors are one of the most prevalent human malignancies and are highly drug resistant because of an MDR phenotype. Intrinsic expression of the ABC transporters Pglycoprotein and MRP1 in colon cancer has been reported (4 -6). To improve the response of colon cancer to chemotherapy, other treatment modalities such as radiation and hyperthermia when used in combination with chemotherapy may be able to increase the efficacy of cancer chemotherapy (7). However, environmental stresses such as drugs, radiation, and hyperthermia harbor the potential to induce or to enhance the MDR phenotype. Induction of MDR1 gene expression by exposure to drugs (8 -10) or radiation (11) were described earlier. In contrast, the influence of hyperthermia on the expression of MDR genes has not been clearly established. Two groups reported a heat-induced elevation of MDR1 gene expression, either observed following a single treatment or after repeated treatments with heat (12, 13). Induction of MRP1 expressi...

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 hemagglutinintagged 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. (Cancer Res 2005; 65(10): 4078-87)

ObjectiveTo assess whether reshaping of the immune balance by infusion of autologous natural regulatory T cells (nTregs) in patients after kidney transplantation is safe, feasible, and enables the tapering of lifelong high dose immunosuppression, with its limited efficacy, adverse effects, and high direct and indirect costs, along with addressing several key challenges of nTreg treatment, such as easy and robust manufacturing, danger of over immunosuppression, interaction with standard care drugs, and functional stability in an inflammatory environment in a useful proof-of-concept disease model.DesignInvestigator initiated, monocentre, nTreg dose escalation, phase I/IIa clinical trial (ONEnTreg13).SettingCharité-University Hospital, Berlin, Germany, within the ONE study consortium (funded by the European Union).ParticipantsRecipients of living donor kidney transplant (ONEnTreg13, n=11) and corresponding reference group trial (ONErgt11-CHA, n=9).InterventionsCD4+ CD25+ FoxP3+ nTreg products were given seven days after kidney transplantation as one intravenous dose of 0.5, 1.0, or 2.5-3.0×106 cells/kg body weight, with subsequent stepwise tapering of triple immunosuppression to low dose tacrolimus monotherapy until week 48.Main outcome measuresThe primary clinical and safety endpoints were assessed by a composite endpoint at week 60 with further three year follow-up. The assessment included incidence of biopsy confirmed acute rejection, assessment of nTreg infusion related adverse effects, and signs of over immunosuppression. Secondary endpoints addressed allograft functions. Accompanying research included a comprehensive exploratory biomarker portfolio.ResultsFor all patients, nTreg products with sufficient yield, purity, and functionality could be generated from 40-50 mL of peripheral blood taken two weeks before kidney transplantation. None of the three nTreg dose escalation groups had dose limiting toxicity. The nTreg and reference groups had 100% three year allograft survival and similar clinical and safety profiles. Stable monotherapy immunosuppression was achieved in eight of 11 (73%) patients receiving nTregs, while the reference group remained on standard dual or triple drug immunosuppression (P=0.002). Mechanistically, the activation of conventional T cells was reduced and nTregs shifted in vivo from a polyclonal to an oligoclonal T cell receptor repertoire.ConclusionsThe application of autologous nTregs was safe and feasible even in patients who had a kidney transplant and were immunosuppressed. These results warrant further evaluation of Treg efficacy and serve as the basis for the development of next generation nTreg approaches in transplantation and any immunopathologies.Trial registrationNCT02371434 (ONEnTreg13) and EudraCT:2011-004301-24 (ONErgt11).

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.The Y-box protein YB-1 is a member of the cold shock protein family, which exhibits pleiotropic functions. YB-1 specifically binds to a sequence motif termed Y-box. This motif is characterized by the presence of a core ATTGG sequence, which represents the inverted CCAAT-box. YB-1 controls the transcription of numerous genes that among others include MHC class II antigen, MDR1, MMP-2, and COL1A1 (1-4). DNA binding specificity is mediated through the evolutionarily conserved cold shock domain in conjunction with the adjacent C-terminal protein residues (5, 6). Interactions of YB-1 with numerous cellular and viral transcription factors including JC virus antigen (7), AP-2 (8), Pur␣ (9), CTCF (10), and p53 (11, 12) have been demonstrated. These interactions may in part explain cell-specific gene regulation, that is, stimulation and repression of transcription, even of the same gene (3). In addition, it has been proposed that YB-1 plays a role as an architectural protein by its propensity to sequence specifically unwind DNA duplexes and stabilize single-stranded templates, thereby altering sequence recognition motifs (1,4,8).In addition to their role in regulating gene transcription, cold shock proteins exhibit a wide spectrum of activities by virtue of sequence-specific and -nonspecific RNA binding. YB-1 has been identified as the major component of messenger ribonucleoprotein particles (mRNPs) in mammalian cells, which constitute templates for the translational machinery (13-15). At higher concentrations Y-box proteins Xenopus FRGY2 and human YB-1 act as repressors of translation in a process called mRNA masking (13, 16 -18), wh...

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.Adenoviruses have developed efficient strategies to force infected cells into the S phase of the cell cycle (1). This process involves the adenoviral E1A and E1B proteins, which are the first viral proteins to be expressed after infection, and both are essential for viral replication (2, 3). Replication of adenovirus DNA depends directly on interactions between the host cell replication factors NFI, NFII, and NFIII (4) and the three viral replication proteins encoded by the E2 region. The adenovirus E2 transcription unit consists of the E2A and E2B genes, which encode precursor terminal protein pTP, DNA polymerase, and DBP, a multifunctional DNA-binding protein (5). E2 gene expression is driven from two promoters. At early times of infection, E2 gene transcription is under control of the E2 early promoter. At intermediate stages of infection, E2 gene expression is controlled by the E2 late promoter (6). E2 early promoter activity is regulated by adenovirus E1A protein, which controls the activity of the E2F transcription factor by targeting the tumor suppressor protein pRB (7,8). In contrast, activity of the E2 late promoter is repressed by E1A (9). The E2 late promoter is characterized by the presence of a TATA box, two SP1 recognition sites, and three CCAAT boxes. Two of the inverted CCAAT boxes are located at positions Ϫ72 and Ϫ135 relative to the E2 late cap site in a 157-bp sequence of the of the E2 late promoter, which is sufficient for efficient E2 gene transcription (10, 11).Inverted CCAAT boxes have been identified as sites for Y box proteins, which are highly conserved through evolution from prokaryotes to eukaryotes, and they can function as transcriptional, translational, and developmental regulators (12)(13)(14). In eukaryotes, increased expression of Y box proteins in somatic cells is associated with drug resistance and a malignant phenotype (15), and it was discussed that Y box proteins are involved in activating certain genes that are expressed in the S phase of the cell cycle (16). Recently, it has...