Spiros Vlahopoulos

Requirement of a Novel Upstream Response Element in Respiratory Syncytial Virus-Induced IL-8 Gene Expression

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. In this study, we compare mechanisms for induction of the CXC chemokine IL-8, in human type II alveolar (A549) cells by RSV infection and by stimulation with the cytokine TNF. Promoter deletion and mutagenesis experiments indicate that although the region from −99 to −54 nt is sufficient for TNF-induced IL-8 transcription, this region alone is not sufficient for RSV-induced IL-8 transcription. Instead, RSV requires participation of a previously unrecognized element, spanning from −162 to −132 nt, that we term the RSV response element (RSVRE), and a previously characterized element at −132 to −99 nt, containing an AP-1 binding site. RSV infection of A549 cells induces increased RSVRE- and AP-1-binding activities and increased synthesis of IFN regulatory factor-1 protein, which is present in the RSVRE-binding complex. These data confirm that the IL-8 gene enhancers are controlled in a stimulus-specific fashion and participation of distinct promoter elements is required to activate gene transcription. These observations are important for rational design of inhibitors of RSV-induced lung inflammation.

The role of ATF‐2 in oncogenesis

Activating Transcription Factor‐2 is a sequence‐specific DNA‐binding protein that belongs to the bZIP family of proteins and plays diverse roles in the mammalian cells. In response to stress stimuli, it activates a variety of gene targets including cyclin A, cyclin D and c‐jun, which are involved in oncogenesis in various tissue types. ATF‐2 expression has been correlated with maintenance of a cancer cell phenotype. However, other studies demonstrate an antiproliferative or apoptotic role for ATF‐2. In this review, we summarize the signaling pathways that activate ATF‐2, as well as its downstream targets. We examine the role of ATF‐2 in carcinogenesis with respect to other bZIP proteins, using data from studies in human cancer cell lines, human tumours and mouse models, and we propose a potential model for its function in carcinogenesis, as well as a theoretical basis for its utility in anticancer drug design. BioEssays 30:314–327, 2008.

JNK: A Key Modulator of Intracellular Signaling

JNK is a family of stress activated protein kinase enzymes that is under intense study. JNK family members are involved in diverse phenomena, but the focus of research has been until now involvement of JNK in apoptosis. A great number of JNK substrates indeed play major roles in cell death. Conversely, accumulating data support a key role of JNK substrates in cell survival and proliferation. Continuous progress is being made, while several important questions remain unanswered. Does JNK cause cancer or prevent it? This paper attempts to evaluate the role of JNK in cell physiology and describe the effects of intracellular signaling pathways that are mediated by JNK family members.

Overexpression of Activating Transcription Factor-2 Is Required for Tumor Growth and Progression in Mouse Skin Tumors

Activating transcription factor (ATF)-2 is a member of the ATF/cyclic AMP-responsive element binding protein family of transcription factors. It has been shown, in vitro, to possess growth factor-independent proliferation and transformation capacity. The information concerning the involvement of ATF-2 in carcinogenesis is rather limited. In a previous report, we showed a progressive increase in the levels of various activator protein (AP)-1 components, including phosphorylated ATF-2, in a series of mouse skin cell lines that represented developmental stages of the mouse skin carcinogenesis system. In the present study, we examined in detail the role of ATF-2 in the development of mouse skin spindle cells A5 and CarB, which correspond to the late and most aggressive stage of the mouse skin carcinogenesis model. To address this issue, we overexpressed a dominant negative form of ATF-2 in the A5 and CarB cell lines and examined their behavior in vitro and in vivo at the molecular and cellular level. The stable transfectants expressed decreased levels of phosphorylated ATF-2 and c-Jun. Subsequently, we observed that dominant negative ATF-2 affected the composition and reduced the activity of AP-1. The above biochemical changes were followed, both in vitro and in vivo in BALB/c severe combined immunodeficient mice, by suppression of the aggressive characteristics of the A5 and CarB mouse skin spindle cells. We attributed this behavior to the significant down-regulation of cyclin D1, cyclin A, and ATF-3, known AP-1 targets implicated in cell cycle control and promotion. In conclusion, our findings underscore a key regulatory role of ATF-2 in tumor growth and progression of mouse skin tumors.

Estrogen receptor α and β in uterine fibroids: a basis for altered estrogen responsiveness

OBJECTIVE To investigate the relative expression and the DNA-binding status of estrogen receptors alpha and beta in fibroids and normal myometrial tissue to explore the molecular basis of altered estrogen responsiveness of leiomyomas. DESIGN Biopsy samples from uterine fibroids and adjacent normal myometrial tissue at the follicular phase of the menstrual cycle. SETTING Aretaieio University Hospital and the National Hellenic Research Foundation, Athens, Greece. PATIENT(S) Thirty-five patients who underwent hysterectomy or myomectomy because of myoma symptoms. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Deoxyribonucleic acid-binding status of estrogen receptors alpha and beta. RESULT(S) The level of messenger RNA expression of estrogen receptor alpha and beta and the level of estrogen receptor as a whole are increased on average to a similar extent in leiomyomas compared with normal myometrium. Occasionally, however, estrogen receptor alpha is disproportionately increased in leiomyomas, and this appears to increase the amount of estrogen receptor alpha that binds to the estrogen-responsive element of estrogen target genes as homodimer rather than as heterodimer with estrogen receptor beta. CONCLUSION(S) The estrogen receptor alpha-to-estrogen receptor beta expression ratio rather than the individual expression levels determines the fraction of DNA-binding homodimers of estrogen receptor alpha and possibly the growth potential of myomas.

Dynamic aberrant NF-κB spurs tumorigenesis: A new model encompassing the microenvironment

Recently it was discovered that a transient activation of transcription factor NF-κB can give cells properties essential for invasiveness and cancer initiating potential. In contrast, most oncogenes to date were characterized on the basis of mutations or by their constitutive overexpression. Study of NF-κB actually leads to a far more dynamic perspective on cancer: tumors caused by diverse oncogenes apparently evolve into cancer after loss of feedback regulation for NF-κB. This event alters the cellular phenotype and the expression of hormonal mediators, modifying signals between diverse cell types in a tissue. The result is a disruption of stem cell hierarchy in the tissue, and pervasive changes in the microenvironment and immune response to the malignant cells.

Oxidized guanine base lesions function in 8-oxoguanine DNA glycosylase-1-mediated epigenetic regulation of nuclear factor κB-driven gene expression

A large percentage of redox-responsive gene promoters contain evolutionarily conserved guanine-rich clusters; guanines are the bases most susceptible to oxidative modification(s). Consequently, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most abundant base lesions in promoters and is primarily repaired via the 8-oxoguanine DNA glycosylase-1 (OOG1)-initiated base excision repair pathway. In view of a prompt cellular response to oxidative challenge, we hypothesized that the 8-oxoG lesion and the cognate repair protein OGG1 are utilized in transcriptional gene activation. Here, we document TNFα-induced enrichment of both 8-oxoG and OGG1 in promoters of pro-inflammatory genes, which precedes interaction of NF-κB with its DNA-binding motif. OGG1 bound to 8-oxoG upstream from the NF-κB motif increased its DNA occupancy by promoting an on-rate of both homodimeric and heterodimeric forms of NF-κB. OGG1 depletion decreased both NF-κB binding and gene expression, whereas Nei-like glycosylase-1 and -2 had a marginal effect. These results are the first to document a novel paradigm wherein the DNA repair protein OGG1 bound to its substrate is coupled to DNA occupancy of NF-κB and functions in epigenetic regulation of gene expression.

Prednisolone exerts late mitogenic and biphasic effects on resistant acute lymphoblastic leukemia cells: Relation to early gene expression

Resistance or sensitivity to glucocorticoids is considered to be of crucial importance for disease prognosis in childhood acute lymphoblastic leukemia. Prednisolone exerted a delayed biphasic effect on the resistant CCRF-CEM leukemic cell line, necrotic at low doses and apoptotic at higher doses. At low doses, prednisolone exerted a pre-dominant mitogenic effect despite its induction on total cell death, while at higher doses, prednisolones mitogenic and cell death effects were counterbalanced. Early gene microarray analysis revealed notable differences in 40 genes. The mitogenic/biphasic effects of prednisolone are of clinical importance in the case of resistant leukemic cells. This approach might lead to the identification of gene candidates for future molecular drug targets in combination therapy with glucocorticoids, along with early markers for glucocorticoid resistance.

New use for old drugs? Prospective targets of chloroquines in cancer therapy.

During the last decade research is gradually repositioning the antimalarial drug chloroquine, and certain related quinoline derivatives, as anticancer agents. Chloroquine and hydroxychloroquine, in particular, have relatively well-characterized toxicity profiles due to several decades of use for treatment of malaria. Previously published review articles provide an excellent overview of the diversity of chloroquine effects on cancer cells, both in the cell culture as well as on human tumors grafted into mice; and an account of the increasing pace of incorporation of hydroxychloroquine in combination treatment schemes for clinical studies. In this review we present some features that are common between cancers that are sensitive to quinoline derivatives, in particular features that are amenable to pharmaceutical intervention.

Glucocorticoid and proteasome inhibitor impact on the leukemic lymphoblast: Multiple, diverse signals converging on a few key downstream regulators

Twenty years ago a proteasome inhibitor was suggested as therapy for glucocorticoid-resistant multiple myeloma, a disease that involves terminally differentiated B cells. Since then, research has proven that it has utility on a number of tumors resistant to chemotherapy. Hematologic malignancy, however, often involves lesser differentiated cells, which have a high potential to modulate their intrinsic machinery and thereby activate alternative rescue pathways. A corresponding multiplicity of therapies is not always practical. One approach to conditions with heterogeneous physiology is to identify key biochemical mediators, thereby reducing the number of treatment targets. Results from several ongoing studies indicate convergence of genomically diverse signal pathways to a limited number of key downstream regulators of apoptosis. Convergence of pathways can be exploited to address the problem of genetic heterogeneity in acute leukemia: this would mean treating multiple molecular aberrations with fewer drugs and enhanced therapeutic benefit.