Laurence A. Marchat

Protein Kinases and Transcription Factors Activation in Response to UV-Radiation of Skin: Implications for Carcinogenesis

Solar ultraviolet (UV) radiation is an important environmental factor that leads to immune suppression, inflammation, photoaging, and skin carcinogenesis. Here, we reviewed the specific signal transduction pathways and transcription factors involved in the cellular response to UV-irradiation. Increasing experimental data supporting a role for p38, MAPK, JNK, ERK1/2, and ATM kinases in the response network to UV exposure is discussed. We also reviewed the participation of NF-κB, AP-1, and NRF2 transcription factors in the control of gene expression after UV-irradiation. In addition, we discussed the promising chemotherapeutic intervention of transcription factors signaling by natural compounds. Finally, we focused on the review of data emerging from the use of DNA microarray technology to determine changes in global gene expression in keratinocytes and melanocytes in response to UV treatment. Efforts to obtain a comprehensive portrait of the transcriptional events regulating photodamage of intact human epidermis after UV exposure reveals the existence of novel factors participating in UV-induced cell death. Progress in understanding the multitude of mechanisms induced by UV-irradiation could lead to the potential use of protein kinases and novel proteins as specific targets for the prevention and control of skin cancer.

DNA methylation of leptin and adiponectin promoters in children is reduced by the combined presence of obesity and insulin resistance

Objective:Epigenetic alterations have been suggested to be associated with obesity and related metabolic disorders. Here we examined the correlation between obesity and insulin resistance with the methylation frequency of the leptin (LEP) and adiponectin (ADIPOQ) promoters in obese adolescents with the aim to identify epigenetic markers that might be used as tools to predict and follow up the physiological alterations associated with the development of the metabolic syndrome.Subjects:One hundred and six adolescents were recruited and classified according to body mass index and homeostasis model of assessment-insulin resistance index. The circulating concentrations of leptin, adiponectin and of several metabolic markers of obesity and insulin resistance were determined by standard methods. The methylation frequency of the LEP and ADIPOQ promoters was determined by methylation-specific PCR (MS-PCR) in DNA obtained from peripheral blood samples.Results:Obese adolescents without insulin resistance showed higher and lower circulating levels of, respectively, leptin and adiponectin along with increased plasmatic concentrations of insulin and triglycerides. They also exhibited the same methylation frequency than lean subjects of the CpG sites located at −51 and −31 nt relative to the transcription start site of the LEP gene. However, the methylation frequency of these nucleotides dropped markedly in obese adolescents with insulin resistance. We found the same inverse relationship between the combined presence of obesity and insulin resistance and the methylation frequency of the CpG site located at −283 nt relative to the start site of the ADIPOQ promoter.Conclusions:These observations sustain the hypothesis that epigenetic modifications might underpin the development of obesity and related metabolic disorders. They also validate the use of blood leukocytes and MS-PCR as a reliable and affordable methodology for the identification of epigenetic modifications that could be used as molecular markers to predict and follow up the physiological changes associated with obesity and insulin resistance.

Proteomic Profiling Reveals That Resveratrol Inhibits HSP27 Expression and Sensitizes Breast Cancer Cells to Doxorubicin Therapy

The use of chemopreventive natural compounds represents a promising strategy in the search for novel therapeutic agents in cancer. Resveratrol (3,4′,5-trans-trihydroxystilbilene) is a dietary polyphenol found in fruits, vegetables and medicinal plants that exhibits chemopreventive and antitumor effects. In this study, we searched for modulated proteins with preventive or therapeutic potential in MCF-7 breast cancer cells exposed to resveratrol. Using two-dimensional electrophoresis we found significant changes (FC >2.0; p≤0.05) in the expression of 16 proteins in resveratrol-treated MCF-7 cells. Six down-regulated proteins were identified by tandem mass spectrometry (ESI-MS/MS) as heat shock protein 27 (HSP27), translationally-controlled tumor protein, peroxiredoxin-6, stress-induced-phosphoprotein-1, pyridoxine-5′-phosphate oxidase-1 and hypoxanthine-guanine phosphoribosyl transferase; whereas one up-regulated protein was identified as triosephosphate isomerase. Particularly, HSP27 overexpression has been associated to apoptosis inhibition and resistance of human cancer cells to therapy. Consistently, we demonstrated that resveratrol induces apoptosis in MCF-7 cells. Apoptosis was associated with a significant increase in mitochondrial permeability transition, cytochrome c release in cytoplasm, and caspases -3 and -9 independent cell death. Then, we evaluated the chemosensitization effect of increasing concentrations of resveratrol in combination with doxorubicin anti-neoplastic agent in vitro. We found that resveratrol effectively sensitize MCF-7 cells to cytotoxic therapy. Next, we evaluated the relevance of HSP27 targeted inhibition in therapy effectiveness. Results evidenced that HSP27 inhibition using RNA interference enhances the cytotoxicity of doxorubicin. In conclusion, our data indicate that resveratrol may improve the therapeutic effects of doxorubicin in part by cell death induction. We propose that potential modulation of HSP27 levels using natural alternative agents, as resveratrol, may be an effective adjuvant in breast cancer therapy.

EhPgp5 mRNA Stability Is a Regulatory Event in the Entamoeba histolytica Multidrug Resistance Phenotype

The multidrug resistance (MDR) phenotype inEntamoeba histolytica is characterized by the overexpression of the EhPgp5 gene in trophozoites grown in high drug concentrations. Here we evaluated the role ofEhPgp5 mRNA stability on MDR using actinomycin D.EhPgp5 mRNA from trophozoites growing without emetine had a half-life of 2.1 h, which augmented to 3.1 h in cells cultured with 90 μm and to 7.8 h with 225 μm emetine. Polyadenylation sites were detected at 118-, 156-, and 189-nucleotide (nt) positions of the EhPgp5mRNA 3′-untranslated region. Interestingly, trophozoites grown with 225 μm emetine exhibited an extra polyadenylation site at 19 nt. The 3′-untranslated region sequence is AU-rich and has putative consensus sequences for RNA-binding proteins. We detected a RNA-protein complex in a region that contains a polypyrimidine tract (142–159 nt) and a cytoplasmic polyadenylation element (146–154 nt). A longer poly(A) tail in the EhPgp5 mRNA was seen in trophozoites grown with 225 μm emetine. Emetine stress may affect factors involved in mRNA turnover, including polyadenylation/deadenylation proteins, which could induce changes in the EhPgp5 mRNA half-life and poly(A) tail length. Novel evidence on mechanisms participating in E. histolytica MDR phenotype is provided.

Breast cancer proteomics reveals a positive correlation between glyoxalase 1 expression and high tumor grade

Breast cancer is the neoplasia with the highest incidence in women worldwide. Proteomics approaches have accelerated the discovery of diagnostic and prognostic biomarkers. Here, we compared the proteomic profiles of breast tumors versus non-tumoral tissues in order to identify modulated proteins, which could represent potential markers associated to clinical features. By two-dimensional electrophoresis, we detected 28 differentially expressed proteins. Among these, 21 proteins were up-regulated and 7 were down-regulated in tumors (p<0.05). Proteins were identified using LC/ESI-MS/MS tandem mass spectrometry. One protein was identified as glyoxalase 1 (GLO1), an enzyme involved in detoxification of methylglyoxal, a cytotoxic product of glycolysis. GLO1 overexpression was confirmed by western blot assays in paired normal and tumor breast tissues in clinical stages I-III, and by immunohistochemistry on tissue microarrays (TMA) comprising a cohort of 98 breast tumors and 20 healthy specimens. Results from TMA demonstrated that GLO1 is overexpressed in 79% of tumors. Interestingly, GLO1 up-regulation correlates with advanced tumor grade (p<0.05). These findings demonstrate the association of GLO1 overexpression with tumor grade and pointed out for additional studies to establish the importance of GLO1 in breast cancer.

Two CCAAT/enhancer binding protein sites are cis‐activator elements of the Entamoeba histolytica EhPgp1 (mdr‐like) gene expression

Here, we show the relevance of promoter regions (−74 to +24, −167 to −75 and −259 to −168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica, using mutated plasmids and transfection assays. We also demonstrate that both CCAAT/enhancer binding protein sites (−54 to −43 bp and −198 to −186 bp) are cis‐activating elements of gene expression in the drug‐resistant (clone C2) and ‐sensitive (clone A) trophozoites. Nuclear proteins from trophozoites of both clones and C/EBP sequences of the core promoter formed specific complexes, which were abolished by anti‐human C/EBPβ antibodies. UV cross‐linking and Western blot assays revealed 25 and 65 kDa bands in urea treated and untreated proteins respectively. The nuclear factors that bind to C/EBP sites were semi‐purified by affinity chromatography. They were immunodetected by anti‐human C/EBPβ antibodies and formed a specific complex with the C/EBP probe. The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments. Based on our results, we propose that the C/EBP site at −54 bp stabilizes the transcription pre‐initiation complex, whereas the other site at −198 bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription.

MetastamiRs: Non-Coding MicroRNAs Driving Cancer Invasion and Metastasis

MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that function as negative regulators of gene expression by either inhibiting translation or inducing deadenylation-dependent degradation of target transcripts. Notably, deregulation of miRNAs expression is associated with the initiation and progression of human cancers where they act as oncogenes or tumor suppressors contributing to tumorigenesis. Abnormal miRNA expression may provide potential diagnostic and prognostic tumor biomarkers and new therapeutic targets in cancer. Recently, several miRNAs have been shown to initiate invasion and metastasis by targeting multiple proteins that are major players in these cellular events, thus they have been denominated as metastamiRs. Here, we present a review of the current knowledge of miRNAs in cancer with a special focus on metastamiRs. In addition we discuss their potential use as novel specific markers for cancer progression.

Transcriptional profile of the homologous recombination machinery and characterization of the EhRAD51 recombinase in response to DNA damage in Entamoeba histolytica.

BackgroundIn eukaryotic and prokaryotic cells, homologous recombination is an accurate mechanism to generate genetic diversity, and it is also used to repair DNA double strand-breaks. RAD52 epistasis group genes involved in recombinational DNA repair, including mre11, rad50, nsb1/xrs2, rad51, rad51c/rad57, rad51b/rad55, rad51d, xrcc2, xrcc3, rad52, rad54, rad54b/rdh54 and rad59 genes, have been studied in human and yeast cells. Notably, the RAD51 recombinase catalyses strand transfer between a broken DNA and its undamaged homologous strand, to allow damaged region repair. In protozoan parasites, homologous recombination generating antigenic variation and genomic rearrangements is responsible for virulence variation and drug resistance. However, in Entamoeba histolytica the protozoan parasite responsible for human amoebiasis, DNA repair and homologous recombination mechanisms are still unknown.ResultsIn this paper, we initiated the study of the mechanism for DNA repair by homologous recombination in the primitive eukaryote E. histolytica using UV-C (150 J/m2) irradiated trophozoites. DNA double strand-breaks were evidenced in irradiated cells by TUNEL and comet assays and evaluation of the EhH2AX histone phosphorylation status. In E. histolytica genome, we identified genes homologous to yeast and human RAD52 epistasis group genes involved in DNA double strand-breaks repair by homologous recombination. Interestingly, the E. histolytica RAD52 epistasis group related genes were differentially expressed before and after UV-C treatment. Next, we focused on the characterization of the putative recombinase EhRAD51, which conserves the typical architecture of RECA/RAD51 proteins. Specific antibodies immunodetected EhRAD51 protein in both nuclear and cytoplasmic compartments. Moreover, after DNA damage, EhRAD51 was located as typical nuclear foci-like structures in E. histolytica trophozoites. Purified recombinant EhRAD51 exhibited DNA binding and pairing activities and exchanging reactions between homologous strands in vitro.ConclusionE. histolytica genome contains most of the RAD52 epistasis group related genes, which were differentially expressed when DNA double strand-breaks were induced by UV-C irradiation. In response to DNA damage, EhRAD51 protein is overexpressed and relocalized in nuclear foci-like structures. Functional assays confirmed that EhRAD51 is a bonafide recombinase. These data provided the first insights about the potential roles of the E. histolytica RAD52 epistasis group genes and EhRAD51 protein function in DNA damage response of this ancient eukaryotic parasite.

DNA repair mechanisms in eukaryotes: Special focus in Entamoeba histolytica and related protozoan parasites.

Eukaryotic cell viability highly relies on genome stability and DNA integrity maintenance. The cellular response to DNA damage mainly consists of six biological conserved pathways known as homologous recombination repair (HRR), non-homologous end-joining (NHEJ), base excision repair (BER), mismatch repair (MMR), nucleotide excision repair (NER), and methyltransferase repair that operate in a concerted way to minimize genetic information loss due to a DNA lesion. Particularly, protozoan parasites survival depends on DNA repair mechanisms that constantly supervise chromosomes to correct damaged nucleotides generated by cytotoxic agents, host immune pressure or cellular processes. Here we reviewed the current knowledge about DNA repair mechanisms in the most relevant human protozoan pathogens. Additionally, we described the recent advances to understand DNA repair mechanisms in Entamoeba histolytica with special emphasis in the use of genomic approaches based on bioinformatic analysis of parasite genome sequence and microarrays technology.

Putative DEAD and DExH-box RNA helicases families in Entamoeba histolytica

RNA helicases catalyze the unwinding of double-stranded RNA structures to perform numerous genetic processes. These enzymes are characterized by the presence of a conserved helicase domain with specific helicases motifs whose amino acid sequence allows the differentiation between DEAD and DExH-box RNA helicase families. Taking advantage of the availability of the complete genome sequence of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we have performed a genomic survey for DEAD and DExH-box RNA helicases encoding genes in this organism. By extensive in silico analysis, we identified 20 EhDEAD and 13 EhDExH-box RNA helicases, which contain almost all the conserved helicase motifs. Additionally, several EhDEAD and EhDExH proteins present specific N- and C-terminal domains that could be related to subcellular localization or function. Phylogenetic studies and sequences analysis suggested that this large EhDEAD/DExH-box RNA helicases family has been generated by gene or internal regions duplication, mutation events, introns formation and motif deletions. Interestingly, EhDexh1 and EhDeaxh10 genes seem to be formed by gene fusion of two ancestral bacterial genes, a mechanism that appears to be evolutionary conserved in the eukaryotic lineage of orthologous proteins. Finally, RT-PCR assays, microarrays and proteomics data analysis showed that several EhDead are differentially expressed in relation to distinct culture conditions. These computational and experimental data provide new information on the evolution of EhDEAD/EhDExH-box RNA helicases and their potential relevance for RNA metabolism in E. histolytica.