Chemical studies on Escherichia coli DNA-dependent RNA polymerase

The interaction of E. coli DNA dependent RNA polymerase with nucleic acids was studied by chemical modification of both the enzyme and DNA. Chemical modification requires large quantities of a pure homogeneous preparation of a protein. Core enzyme was purified from up to 1 kg of E. coli, in yields of about 10 mg per 100 g cells, and to 95 % purity. Core-enzyme was used because it is the simplest active form, with most of the properties, of the complete enzyme. Work on the binding of synthetic peptides and proteins to nucleic acids suggests that tryptophan and tyrosine form stacking interactions with nucleic acid bases, and that lysine interacts ionically, N-bromosuccinimide was used to modify the tryptophan and tyrosine groups of RNA polymerase. A 30 fold molar excess of reagent rapidly and completely inactivated the enzyme. The modification resulted in oxidation of 1-2 tryptophans and 8 (+/-2) cysteines, loss of 9% of the protein fluorescence, and no loss of overall structure (as judged by the accessibility of thiols to reaction with 5, 5'-dithiobis(2-nitrobenzoic acid). Substrates did not protect against inactivation. The oxidation of 8 thiols, which accounted for up to 40 % of the inactivation, was limited to 4 thiols and 10 % inactivation by reversibly protecting the surface SH groups with 5,5'-dithiobis(2-nitrobenzoic acid). Inactivation of protected enzyme required a 30 fold molar excess of N-bromosuccinimide, and led to a loss of 11 % of the protein fluorescence, and no change (<2%) in the overall conformation (as judged by far ultraviolet circular dichroism). Amino acid analysis showed that between 3 and 6 tyrosines, and no methionines (+/-2) or histidines (+/-5) were oxidised. The error in determining tryptophan (+/-2) was too large to detect the oxidation previously observed by extinction changes. Stacking of aromatic amino acids with nucleic acid bases would contribute to the binding energy to DNA and nucleotides, and would help unwind the double helix, N-bromosuccinimide inactivation of thiol protected RNA polymerase resulted in no decrease in DNA binding activity (as judged by salt elution from DNA agarose), and no loss in ATP binding. No difference in inactivation was observed when assayed with native and denatured DNA, and inactivation was also independent of the initiation circumventing dinucleotide G-A, suggesting that the major cause of inactivation was not loss of unwinding or initiating activity. Further work is required to resolve the cause of inactivation. Methyl-[14C] acetimidate modification of 50 lysines resulted in complete inactivation of core enzyme. Both purine nucleoside triphosphates and denatured DNA protected against inactivation. ATP, GTP, CTP, and denatured DNA together resulted in maximal protection against inactivation, and protection of about 8 lysines against modification. Since amidination should not disrupt ionic interactions greatly, lysine may have another role in RNA polymerase. Protection against inactivation was used to obtain a dissociation constant of 80 +/- 20 ?M for denatured DNA from core enzyme. The kinetics of formaldehyde melting of DNA was studied as a probe of protein-DNA interactions. The kinetic analysis of Trifonov et al. (1968) did not adequately describe the unwinding of the small, double-helical, DNA from phage T7, despite taking precautions in the experimental techniques. Calf thymus DNA, and calf thymus DNA with nucleoside triphosphates and low concentrations of holoenzyme, gave interpretable kinetics. However the shortcomings of calf thymus DNA as a template, and the difficulties with explaining the kinetics with larger concentrations of enzyme, and with T7 DNA, detract from the method as a probe of RNA polymerase-DNA interactions

Regulation of MCP-1 chemokine transcription by p53

<p>Abstract</p> <p>Background</p> <p>Our previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. <it>In silico </it>analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process.</p> <p>Results</p> <p>The proposed p53 binding side could be confirmed <it>in vitro </it>by electrophoretic-mobility-shift assays and <it>in vivo </it>by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-α can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-α treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-α addition, directly confirming a crosstalk between p53 and MCP-1.</p> <p>Conclusion</p> <p>These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.</p

PLoS ONE

The molecular response to hypoxia is a critical cellular process implicated in cancer, and a target for drug development. The activity of the major player, HIF1α, is regulated at different levels by various factors, including the transcription factor ELK3. The molecular mechanisms of this intimate connection remain largely unknown. Whilst investigating global ELK3-chromatin interactions, we uncovered an unexpected connection that involves the microRNA hsa-miR-155-5p, a hypoxia-inducible oncomir that targets HIF1α. One of the ELK3 chromatin binding sites, detected by Chromatin Immuno-Precipitation Sequencing (ChIP-seq) of normal Human Umbilical Vein Endothelial Cells (HUVEC), is located at the transcription start site of the MIR155HG genes that expresses hsa-miR-155-5p. We confirmed that ELK3 binds to this promoter by ChIP and quantitative polymerase chain reaction (QPCR). We showed that ELK3 and hsa-miR-155-5p form a double-negative regulatory loop, in that ELK3 depletion induced hsa-miR-155-5p expression and hsa-miR-155-5p expression decreased ELK3 expression at the RNA level through a conserved target sequence in its 3'-UTR. We further showed that the activities of hsa-miR-155-5p and ELK3 are functionally linked. Pathway analysis indicates that both factors are implicated in related processes, including cancer and angiogenesis. Hsa-miR-155-5p expression and ELK3 depletion have similar effects on expression of known ELK3 target genes, and on in-vitro angiogenesis and wound closure. Bioinformatic analysis of cancer RNA-seq data shows that hsa-miR-155-5p and ELK3 expression are significantly anti-correlated, as would be expected from hsa-miR-155-5p targeting ELK3 RNA. Finally, hypoxia (0% oxygen) down-regulates ELK3 mRNA in a microRNA and hsa-miR-155-5p dependent manner. These results tie ELK3 into the hypoxia response pathway through an oncogenic microRNA and into a circuit implicated in the dynamics of the hypoxic response. This crosstalk could be important for the development of new treatments for a range of pathologies

Tubulin tyrosine ligase like 12 links to prostate cancer through tubulin posttranslational modification and chromosome ploidy

Prostate cancer is a common cause of death, and an important goal is to establish the pathways and functions of causative genes. We isolated RNAs that are differentially expressed in macrodissected prostate cancer samples. This study focused on 1 identified gene, TTLL12, which was predicted to modify tubulins, an established target for tumor therapy. TTLL12 is the most poorly characterized member of a recently discovered 14-member family of proteins that catalyze posttranslational modification of tubulins. We show that human TTLL12 is expressed in the proliferating layer of benign prostate. Expression increases during cancer progression to metastasis. It is highly expressed in many metastatic prostate cancer cell lines. It partially colocalizes with vimentin intermediate filaments and cellular structures containing tubulin, including midbodies, centrosomes, intercellular bridges and the mitotic spindle. Downregulation of TTLL12 affects several posttranslational modifications of tubulin (detyrosination and subsequent deglutamylation and polyglutamylation). Overexpression alters chromosomal ploidy. These results raise the possibility that TTLL12 could contribute to tumorigenesis through effects on the cytoskeleton, tubulin modification and chromosome number stability. This study contributes a step toward developing more selective agents targeting microtubules, an already successful target for tumor therapy.The European Union, The Ligue Nationale Française contre le Cancer, The Ligues De´partementales de Lutte contre le Cancer (Haut- and Bas-Rhin), The Association pour la Recherche sur le Cancer, The Centre National de la Recherche Scientifique, The Institut National de la Sante´ et de la Recherche Me´dicale, the Cance´ropoˆle Grand-Est (Axe IV and DKFZ-CGE projects) and INCaPeer reviewe

A novel ΔNp63-dependent immune mechanism improves prognosis of HPV-related head and neck cancer.

peer reviewed[en] BACKGROUND: Deconvoluting the heterogenous prognosis of Human Papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OSCC) is crucial for enhancing patient care, given its rapidly increasing incidence in western countries and the adverse side effects of OSCC treatments. METHODS: Transcriptomic data from HPV-positive OSCC samples were analyzed using unsupervised hierarchical clustering, and clinical relevance was evaluated using Kaplan-Meier analysis. HPV-positive OSCC cell line models were used in functional analyses and phenotypic assays to assess cell migration and invasion, response to cisplatin, and phagocytosis by macrophages in vitro. RESULTS: We found, by transcriptomic analysis of HPV-positive OSCC samples, a ΔNp63 dependent molecular signature that is associated with patient prognosis. ΔNp63 was found to act as a tumor suppressor in HPV-positive OSCC at multiple levels. It inhibits cell migration and invasion, and favors response to chemotherapy. RNA-Seq analysis uncovered an unexpected regulation of genes, such as DKK3, which are involved in immune response-signalling pathways. In agreement with these observations, we found that ΔNp63 expression levels correlate with an enhanced anti-tumor immune environment in OSCC, and ΔNp63 promotes cancer cell phagocytosis by macrophages through a DKK3/NF-κB-dependent pathway. CONCLUSION: Our findings are the first comprehensive identification of molecular mechanisms involved in the heterogeneous prognosis of HPV-positive OSCC, paving the way for much-needed biomarkers and targeted treatment

CTIP2 Expression in Human Head and Neck Squamous Cell Carcinoma Is Linked to Poorly Differentiated Tumor Status

We have demonstrated earlier that CTIP2 is highly expressed in mouse skin during embryogenesis and in adulthood. CTIP2 mutant mice die at birth with epidermal differentiation defects and a compromised epidermal permeability barrier suggesting its role in skin development and/or homeostasis. CTIP2 has also been suggested to function as tumor suppressor in cells, and several reports have described a link between chromosomal rearrangements of CTIP2 and human T cell acute lymphoblast leukemia (T-ALL). The aim of the present study was to look into the pattern of CTIP2 expression in Head and Neck Squamous Cell Carcinoma (HNSCC).In the present study, we analyzed CTIP2 expression in human HNSCC cell lines by western blotting, in paraffin embedded archival specimens by immunohistochemistry (IHC), and in cDNA samples of human HNSCC by qRT-PCR. Elevated levels of CTIP2 protein was detected in several HNSCC cell lines. CTIP2 staining was mainly detected in the basal layer of the head and neck normal epithelium. CTIP2 expression was found to be significantly elevated in HNSCC (p<0.01), and increase in CTIP2 expression was associated with poorly differentiated tumor status. Nuclear co-localization of CTIP2 protein and cancer stem cell (CSC) marker BMI1 was observed in most, if not all of the cells expressing BMI1 in moderately and poorly differentiated tumors.We report for the first time expression of transcriptional regulator CTIP2 in normal human head and neck epithelia. A statistically significant increase in the expression of CTIP2 was detected in the poorly differentiated samples of the human head and neck tumors. Actual CTIP2, rather than the long form of CTIP2 (CTIP2(L)) was found to be more relevant to the differentiation state of the tumors. Results demonstrated existence of distinct subsets of cancer cells, which express CTIP2 and underscores the use of CTIP2 and BMI1 co-labeling to distinguish tumor initiating cells or cancer stem cells (CSCs) from surrounding cancer cells

Etude d'un nouveau gène humain surexprimé dans les tumeurs de l'hypopharynx

Notre laboratoire a réalisé un criblage des gènes impliqués dans les tumeurs des voies aéro-digestives supérieures par les techniques de differential display (DD) et de micropuces à ADN. Dans cette étude, nous présentons la première caractérisation d un nouveau gène humain, LOC92912, identifié par DD comme surexprimé dans les tumeurs. LOC92912, un membre potentiel de la famille des enzymes E2 de conjugaison de l ubiquitine, code pour une protéine de 375 acides aminés contenant des domaines RWD, coiles-coil et E2. Des analyses bioinformatiques montrent qu il existe des protéines apparentées dans des espèces aussi diverses que le ver et l homme. La forte conservation de la séquence de LOC92012 entre les espèces, particulièrement au niveau du domaine putatif catalytique C-terminal, suggère une fonction importante de cette protéine. LOC92912 est surexprimé dans environ 85% des échantillons de tumeurs. Il est exprimé dans les masses tumorales et dans les épithéliums invasifs, avec une localisation subcellulaire cytoplasmique. Nous avons développé un anticorps polyclonal de lapin qui détecte spécifiquement LOC92912, endogène et transfecté, à la taille attendue (~43kDa). Pour comprendre quelles voies de signalisation sont affectées dans les tissus cancéreux surexprimant ce gène, nous avons établi des clones stables RPMI2650 surexprimant LOC92912. Dans ce système de surexpression, certaines fonctions normales de la cellule sont affectées. Nous avons observé une augmentation de la phase G0/G1 du cycle cellulaire, la formation de structures en foci en culture, une diminution de la clonogénicité et de la croissance cellulaire et une capacité de migration accrue en chambre de Boyden. Des données préliminaires montrent également des changements de la morphologie cellulaire et de l adhésion intercellulaire. Pour mieux comprendre les fonctions de LOC92912, nous avons identifié les partenaires potentiels d interaction par immunopurification de la protéine flaggée suivie par une analyse en spectrométrie de masse MALDI Peptide Mass Fingerprinting . L actine et des protéines liant l actine ont été identifiées comme partenaires potentiels d interaction, suggérant des fonctions de LOC92912 liées au cytosquelette. Ce nouveau gène humain pourrait représenter une nouvelle cible pour la thérapie anticancéreuse. Cette étude constitue une base solide qui devrait encourager les scientifiques et cliniciens à s intéresser à ce gène.Previous work, performed in our laboratory, screened for genes involved in head and neck squamous cell carcinoma (HNSCC) using differential display (DD) and DNA microarrays. In this study, we present the first analytical analysis on a novel human gene, LOC92912, identified by DD as a gene upregulated in HNSCC. LOC92912, which is a putative member of the E2 ubiquitin conjugating enzyme family, encodes a protein of 375 amino acids containing a RWD domain, a coiled-coil and an E2 domain. Bioinformatics analysis revealed that there are related proteins in organisms as diverse as humans and worms. The striking conservation of LOC92912 sequence homology among species, particularly in the predicted catalytic domain of the carboxy terminal half of the protein, suggests that it has an important catalytic function. LOC92912 is upregulated in about 85% of tumor samples. It is expressed in tumor masses and in invasive epithelium, and is located in the cytoplasm of cells. We raised a rabbit polyclonal antibody that specifically detects the transfected and endogenous LOC92912 polypeptide of the expected size (~43kDa). To understand which pathways are affected in cancer tissues that overexpress our gene, we established RPMI 2650 stable transfectant over-expressing LOC92912, aimed at the identification of the signatures that can be subsequently validated for their implication in cancer. In this over-expressing system some normal functions of the cells were altered. We observed: an increase in the G0/G1 phase of the cell cycle, formation of focus-like structures in the culture, a decrease in clonogenicity and cell growth and more migration in Boyden chamber. Preliminary data also showed changes in cell shape and cell-to-cell adhesion. To gain insights into LOC92912 functions, we identified potential interacting partners by immunoaffinity purification of the flag tagged protein followed by MALDI Peptide Mass Fingerprinting (PMF) mass spectrometry. Actin and 6 actin-binding proteins were unambiguously identified as potential interacting partners, suggesting that LOC92912 s functions may be linked with the cytoskeleton. This novel human gene may represent a new target for cancer therapeutics. This study provides a solid basis that should encourage scientists and clinicians to become interested in this gene.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

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STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF