Identification of Novel Cellular Transcription Factors that Regulate Early Promoters of Human Papillomavirus Types 18 and 16

Background. The long control region (LCR) of human papillomavirus (HPV) regulates early gene transcription by interaction with several viral and cellular transcription factors (TFs). Methods. To identify novel TFs that could influence early expression of HPV type 18 (HPV-18) and HPV type 16 (HPV-16), a high-throughput transfection array was used. Results. Among the 704 TFs tested, 28 activated and 36 inhibited the LCR of HPV-18 by more than 2-fold. For validation, C33 cells were cotransfected with increasing amounts of selected TF expression plasmids in addition to LCR-luciferase vectors of different molecular variants of HPV-18 and HPV-16. Among the TFs identified, only GATA3, FOXA1, and MYC have putative binding sites within the LCR sequence, as indicated using the TRANSFAC database. Furthermore, we demonstrated FOXA1 and MYC in vivo binding to the LCR of both HPV types using chromatin immunoprecipitation assay. Conclusions. We identified new TFs implicated in the regulation of the LCR of HPV-18 and HPV-16. Many of these factors are mutated in cancer or are putative cancer biomarkers and could potentially be involved in the regulation of HPV early gene expression.

Adipose tissue inflammation and cancer cachexia: Possible role of nuclear transcription factors

Cancer cachexia is a multifaceted syndrome whose aetiology is extremely complex and is directly related to poor patient prognosis and survival. Changes in lipid metabolism in cancer cachexia result in marked reduction of total fat mass, increased lipolysis, total oxidation of fatty acids, hyperlipidaemia, hypertriglyceridaemia, and hypercholesterolaemia. These changes are believed to be induced by inflammatory mediators, such as tumour necrosis factor-alpha (TNF-alpha) and other factors. Attention has recently been drawn to the current theory that cachexia is a chronic inflammatory state, mainly caused by the host's reaction to the tumour. Changes in expression of numerous inflammatory mediators, notably in white adipose tissue (WAT), may trigger several changes in WAT homeostasis. The inhibition of adipocyte differentiation by PPAR gamma is paralleled by the appearance of smaller adipocytes, which may partially account for the inhibitory effect of PPAR gamma on inflammatory gene expression. Furthermore, inflammatory modulation and/or inhibition seems to be dependent on the IKK/NF-kappa B pathway, suggesting that a possible interaction between NF-kappa B and PPAR gamma is required to modulate WAT inflammation induced by cancer cachexia. In this article, current literature on the possible mechanisms of NF-kappa B and PPAR gamma regulation of WAT cells during cancer cachexia are discussed. This review aims to assess the role of a possible interaction between NF-kappa B and PPAR gamma in the setting of cancer cachexia as well as its significant role as a potential modulator of chronic inflammation that could be explored therapeutically. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.

Glucocorticoid and Estrogen Receptors Are Reduced in Mitochondria of Lung Epithelial Cells in Asthma

Mitochondrial glucocorticoid (mtGR) and estrogen (mtER) receptors participate in the coordination of the cell's energy requirement and in the mitochondrial oxidative phosphorylation enzyme (OXPHOS) biosynthesis, affecting reactive oxygen species (ROS) generation and induction of apoptosis. Although activation of mtGR and mtER is known to trigger anti-inflammatory signals, little information exists on the presence of these receptors in lung tissue and their role in respiratory physiology and disease. Using a mouse model of allergic airway inflammation disease and applying confocal microscopy, subcellular fractionation, and Western blot analysis we showed mitochondrial localization of GR alpha and ER beta in lung tissue. Allergic airway inflammation caused reduction in mtGR alpha, mtER beta, and OXPHOS enzyme biosynthesis in lung cells mitochondria and particularly in bronchial epithelial cells mitochondria, which was accompanied by decrease in lung mitochondrial mass and induction of apoptosis. Confirmation and validation of the reduction of the mitochondrial receptors in lung epithelial cells in human asthma was achieved by analyzing autopsies from fatal asthma cases. The presence of the mitochondrial GR alpha and ER beta in lung tissue cells and especially their reduction in bronchial epithelial cells during allergic airway inflammation suggests a crucial role of these receptors in the regulation of mitochondrial function in asthma, implicating their involvement in the pathophysiology of the disease.

Frequent downregulation of the transcription factor Foxa2 in lung cancer through epigenetic silencing

Purpose: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). Methods: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. Results: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p < 0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173,55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122). Conclusions: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Signal transduction-related responses to phytohormones and environmental challenges in sugarcane

Abstract Background Sugarcane is an increasingly economically and environmentally important C4 grass, used for the production of sugar and bioethanol, a low-carbon emission fuel. Sugarcane originated from crosses of Saccharum species and is noted for its unique capacity to accumulate high amounts of sucrose in its stems. Environmental stresses limit enormously sugarcane productivity worldwide. To investigate transcriptome changes in response to environmental inputs that alter yield we used cDNA microarrays to profile expression of 1,545 genes in plants submitted to drought, phosphate starvation, herbivory and N2-fixing endophytic bacteria. We also investigated the response to phytohormones (abscisic acid and methyl jasmonate). The arrayed elements correspond mostly to genes involved in signal transduction, hormone biosynthesis, transcription factors, novel genes and genes corresponding to unknown proteins. Results Adopting an outliers searching method 179 genes with strikingly different expression levels were identified as differentially expressed in at least one of the treatments analysed. Self Organizing Maps were used to cluster the expression profiles of 695 genes that showed a highly correlated expression pattern among replicates. The expression data for 22 genes was evaluated for 36 experimental data points by quantitative RT-PCR indicating a validation rate of 80.5% using three biological experimental replicates. The SUCAST Database was created that provides public access to the data described in this work, linked to tissue expression profiling and the SUCAST gene category and sequence analysis. The SUCAST database also includes a categorization of the sugarcane kinome based on a phylogenetic grouping that included 182 undefined kinases. Conclusion An extensive study on the sugarcane transcriptome was performed. Sugarcane genes responsive to phytohormones and to challenges sugarcane commonly deals with in the field were identified. Additionally, the protein kinases were annotated based on a phylogenetic approach. The experimental design and statistical analysis applied proved robust to unravel genes associated with a diverse array of conditions attributing novel functions to previously unknown or undefined genes. The data consolidated in the SUCAST database resource can guide further studies and be useful for the development of improved sugarcane varieties.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

A possible new mechanism for the control of miRNA expression in neurons

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration,which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR,we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs “make sense” if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were oncurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression.

Influence of the dopaminergic system, CREB, and transcription factor-κB on cocaine neurotoxicity

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes.