Assessment of the Mutagenic Activity of Extracts of Brazilian Propolis in Topical Pharmaceutical Formulations on Mammalian Cells In Vitro and In Vivo

Propolis possesses various biological activities such as antibacterial, antifungal, anti-inflammatory, anesthetic and antioxidant properties. A topically applied product based on Brazilian green propolis was developed for the treatment of burns. For such substance to be used more safely in future clinical applications, the present study evaluated the mutagenic potential of topical formulations supplemented with green propolis extract (1.2, 2.4 and 3.6%) based on the analysis of chromosomal aberrations and of micronuclei. In the in vitro studies, 3-h pulse (G(1) phase of the cell cycle) and continuous (20 h) treatments were performed. In the in vivo assessment, the animals were injured on the back and then submitted to acute (24 h), subacute (7 days) and subchronic (30 days) treatments consisting of daily dermal applications of gels containing different concentrations of propolis. Similar frequencies of chromosomal aberrations were observed for cultures submitted to 3-h pulse and continuous treatment with gels containing different propolis concentrations and cultures not submitted to any treatment. However, in the continuous treatment cultures treated with the 3.6% propolis gel presented significantly lower mitotic indices than the negative control. No statistically significant differences in the frequencies of micronuclei were observed between animals treated with gels containing different concentrations of propolis and the negative control for the three treatment times. Under the present conditions, topical formulations containing different concentrations of green propolis used for the treatment of burns showed no mutagenic effect in either test system, but 3.6% propolis gel was found to be cytotoxic in the in vitro test.

Expression of the hepatitis C virus structural proteins in mammalian cells induces morphology similar to that in natural infection

Like many positive-strand RNA viruses, replication of the hepatitis C virus (HCV) is associated with cytoplasmic membrane rearrangements. However, it is unclear which HCV Proteins induce these ultrastructural features. This work examined the morphological changes induced by expression of the HCV structural proteins, core, E1 and E2, expressed from a Semliki Forest Virus (SFV) recombinant RNA replicon. Electron microscopy of cells expressing these proteins showed cytoplasmic vacuoles containing membranous and electron-dense material that were distinct from the type I cytoplasmic vacuoles induced during SFV replicon replication. Immunogold labelling showed that the core and E2 proteins localized to the external and internal membranes of these vacuoles. At times were also associated with some of the internal amorphous material. Dual immunogold labelling with antibodies raised against the core protein and against an endoplasmic reticulum (ER)-resident protein (protein disulphide isomerase) showed that the HCV-induced vacuoles were associated with ER-labelled membranes. This report has identified an association between the HCV core and E2 proteins with induced cytoplasmic vacuoles which are morphologically similar to those observed in HCV-infected liver tissue, suggesting that the HCV structural proteins may be responsible for the induction of these vacuoles during HCV replication in vivo.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Expression of the human carboxylesterase 2 enzyme (CES2) in mammalian cells

Dissertation to obtain a Master Degree in Biotechnology

Transcriptional regulation of RNA polymerase III in mammalian cells

L'ARN Polymérase III (Pol III) transcrit un ensemble de petits ARN non traduits impliqués dans des processus cellulaires tels que la biosynthèse des protéines, la maturation des ARNs ou le contrôle transcriptionnel. De ce fait, la Pol III joue un rôle important dans la régulation de la croissance et la prolifération cellulaire. L'initiation de la transcription par la Pol III nécessite l'interaction entre des facteurs de transcription et le complexe de la Pol III lui-même. Un sous- complexe de la Pol III, composé de 3 sous-unités, HsRPC3, HsRPC6 et HsRPC7 sert d'intermédiaire dans cette interaction. Dans cette étude, nous avons caractérisé une nouvelle sous-unité de la Pol III, HsRPC7-Like, homologue à HsRPC7. Nous avons montré que ces deux homologues se trouvent spécifiquement chez les vertébrés. Ils proviennent d'un ancêtre commun qui, après duplication il y a 600 millions d'années, a donné naissance à ces deux paralogues. Dans les cellules humaines, deux formes de Pol III coexistent : l'une contientt HsRPC7, l'autre HsRPC7-Like. Nous avons localisé, à l'échelle du génome entier, la présence de ces deux formes de Pol III dans des cellules humaines et dans le foie de souris. Les deux sous-unités ont démontré des caractéristiques identiques, suggérant qu'elles possèdent des fonctions similaires. Cependant, nous avons analysé les motifs d'expression des gènes codant pour RPC7 et RPC7-Like dans des lignées cellulaires dans des conditions variées telles que la concentration de sérum et la densité cellulaire, ainsi que les motifs d'expression dans le foie de souris et des cellules d'hépatocarcinome de souris. Nos résultats suggèrent que l'expression de ces deux sous-untiés varie en fonction de l'activité de prolifération de la cellule. - RNA polymerase III (Pol III) transcribes a set of genes coding for short untranslated RNAs involved in essential cellular processes as for example protein biosynthesis, RNA maturation, and transcriptional control. Thereby Pol III plays an important role in regulating cell growth and proliferation. Initiation of Pol III transcription requires interactions between transcription factors and the Pol III core complex. A Pol III sub-complex composed of three subunits, HsRPC3, HsRPC6, and HsRPC7 mediates this interaction. In this study, we have characterized a new Pol III subunit, HsRPC7-Like, an homologue of HsRPC7. We have shown that these two homologues are specific to vertebrates and originate from an ancestor gene that duplicated 600 mio years ago to give birth to two paralogues. In human cells, two forms of Pol III coexist, one containing HsRPC7 and the other HsRPC7-Like. We have localized, genome-wide, these two Pol III forms in human cells and mouse liver. Both subunits were found on all types of Pol III genes, suggesting that they share similar function. However, we analysed the expression patterns of the RPC7 and RPC7-Like coding genes under various conditions of serum concentration and cell density in different cell lines, as well as expression patterns in mouse liver and mouse hepatocarcinoma cells. Our results suggest that the expression of these two subunits varies with the proliferation rate of the cell.

High level expression of human neuropeptide Y receptors in mammalian cells infected with a recombinant vaccinia virus.

Neuropeptide Y (NPY) is a 36 amino acid peptide present in the central and peripheral nervous system. Numerous studies point to a role of NPY in cardiovascular regulation. NPY effects are mediated through stimulation of specific cell surface G protein-coupled receptors. To allow biochemical studies of the receptor and of its interaction with the ligand, we have developed a potent expression system for NPY receptors using a recombinant vaccinia virus. A human NPY receptor cDNA was fused to a strong vaccinia virus promoter and inserted into the viral genome by homologous recombination. Recombinant viruses were isolated and tested for their ability to induce NPY binding site expression following infection of mammalian cell lines. Using saturation and competition binding experiments we measured a Bmax of 5-10 x 10(6) NPY binding sites per cell. The Kd for the binding of NPY is about 20 nM. Labelling of infected cells with a fluorochrome-labelled NPY indicated that the recombinant protein integrates into the cell membrane.

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

The challenge of modeling nuclear receptor regulatory networks in mammalian cells.

Nuclear receptors are a major component of signal transduction in animals. They mediate the regulatory activities of many hormones, nutrients and metabolites on the homeostasis and physiology of cells and tissues. It is of high interest to model the corresponding regulatory networks. While molecular and cell biology studies of individual promoters have provided important mechanistic insight, a more complex picture is emerging from genome-wide studies. The regulatory circuitry of nuclear receptor regulated gene expression networks, and their response to cellular signaling, appear highly dynamic, and involve long as well as short range chromatin interactions. We review how progress in understanding the kinetics and regulation of cofactor recruitment, and the development of new genomic methods, provide opportunities but also a major challenge for modeling nuclear receptor mediated regulatory networks.

Epigenetic regulation of histone H3 serine 10 phosphorylation status by HCF-1 proteins in C. elegans and mammalian cells.

BACKGROUND: The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status. CONCLUSIONS/SIGNIFICANCE: These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation.

Rapid, simple and high yield production of recombinant proteins in mammalian cells using a versatile episomal system.

Many research projects in life sciences require purified biologically active recombinant protein. In addition, different formats of a given protein may be needed at different steps of experimental studies. Thus, the number of protein variants to be expressed and purified in short periods of time can expand very quickly. We have therefore developed a rapid and flexible expression system based on described episomal vector replication to generate semi-stable cell pools that secrete recombinant proteins. We cultured these pools in serum-containing medium to avoid time-consuming adaptation of cells to serum-free conditions, maintain cell viability and reuse the cultures for multiple rounds of protein production. As such, an efficient single step affinity process to purify recombinant proteins from serum-containing medium was optimized. Furthermore, a series of multi-cistronic vectors were designed to enable simultaneous expression of proteins and their biotinylation in vivo as well as fast selection of protein-expressing cell pools. Combining these improved procedures and innovative steps, exemplified with seven cytokines and cytokine receptors, we were able to produce biologically active recombinant endotoxin free protein at the milligram scale in 4-6weeks from molecular cloning to protein purification.

Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells.

Gene transfer in eukaryotic cells and organisms suffers from epigenetic effects that result in low or unstable transgene expression and high clonal variability. Use of epigenetic regulators such as matrix attachment regions (MARs) is a promising approach to alleviate such unwanted effects. Dissection of a known MAR allowed the identification of sequence motifs that mediate elevated transgene expression. Bioinformatics analysis implied that these motifs adopt a curved DNA structure that positions nucleosomes and binds specific transcription factors. From these observations, we computed putative MARs from the human genome. Cloning of several predicted MARs indicated that they are much more potent than the previously known element, boosting the expression of recombinant proteins from cultured cells as well as mediating high and sustained expression in mice. Thus we computationally identified potent epigenetic regulators, opening new strategies toward high and stable transgene expression for research, therapeutic production or gene-based therapies.

Epigenetic regulatory elements: Recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells.

Successful generation of high producing cell lines requires the generation of cell clones expressing the recombinant protein at high levels and the characterization of the clones' ability to maintain stable expression levels. The use of cis-acting epigenetic regulatory elements that improve this otherwise long and uncertain process has revolutionized recombinant protein production. Here we review and discuss new insights into the molecular mode of action of the matrix attachment regions (MARs) and ubiquitously-acting chromatin opening elements (UCOEs), i.e. cis-acting elements, and how these elements are being used to improve recombinant protein production. These elements can help maintain the chromatin environment of the transgene genomic integration locus in a transcriptionally favorable state, which increases the numbers of positive clones and the transgene expression levels. Moreover, the high producing clones tend to be more stable in long-term cultures even in the absence of selection pressure. Therefore, by increasing the probability of isolating a high producing clone, as well as by increasing transcription efficiency and stability, these elements can significantly reduce the time and cost required for producing large quantities of recombinant proteins.