Luis A. López-Fernández

Molecular Characterization of Spontaneous Mesenchymal Stem Cell Transformation

Background We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC). Methodology/Principal Findings Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection. Conclusions/Significance In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell.

Cellular Gene Expression Survey of Vaccinia Virus Infection of Human HeLa Cells

ABSTRACT Vaccinia virus (VV) is a cytocidal virus that causes major changes in host cell machinery shortly after infecting cells. To define the consequences of virus infection on host gene expression, we used microarrays of approximately 15,000 human cDNAs to examine expression levels of mRNAs isolated at 2, 6, and 16 h postinfection from cultures of infected HeLa cells. The majority of profiling changes during VV infection corresponded to downregulation of genes at 16 h postinfection. Differentially expressed genes were clustered into seven groups to identify common regulatory pathways, with most of them (90%) belonging to clusters 6 and 7, which represent genes whose expression was repressed after infection. Cluster 1, however, contained 37 transcripts (2.81%) showing a robust pattern of induction that was maintained during the course of infection. Genes in cluster 1 included those for Wiskott-Aldrich syndrome protein (WASP) family member WASF1, thymosine, adenosine A2a receptor, glutamate decarboxylase 2, CD-80 antigen, KIAA0888 protein, selenophosphate synthetase, pericentrin, and attractin as well as several expressed sequence tags. We analyzed in more detail the fate of WASP protein in VV-infected cells, because a related family member, N-WASP, is involved in viral motility. WASP protein accumulated in the course of infection; its increase required viral DNA replication and de novo protein synthesis, and it localized in cytoplasmic structures distinct from uninfected cells. This study is the first quantitative analysis of host gene expression following VV infection of cultured human cells, demonstrating global changes in the expression profile, and identifies upregulated genes with potential roles in the virus replication cycle.

Microarray Analysis Reveals Characteristic Changes of Host Cell Gene Expression in Response to Attenuated Modified Vaccinia Virus Ankara Infection of Human HeLa Cells

ABSTRACT The potential use of the modified vaccinia virus Ankara (MVA) strain as a live recombinant vector to deliver antigens and elicit protective immune responses against infectious diseases demands a comprehensive understanding of the effect of MVA infection on human host gene expression. We used microarrays containing more than 15,000 human cDNAs to identify gene expression changes in human HeLa cell cultures at 2, 6, and 16 h postinfection. Clustering of the 410 differentially regulated genes identified 11 discrete gene clusters with altered expression patterns after MVA infection. Clusters 1 and 2 (accounting for 16.59% [68 of 410] of the genes) contained 68 transcripts showing a robust induction pattern that was maintained during the course of infection. Changes in cellular gene transcription detected by microarrays after MVA infection were confirmed for selected genes by Northern blot analysis and by real-time reverse transcription-PCR. Upregulated transcripts in clusters 1 and 2 included 20 genes implicated in immune responses, including interleukin 1A (IL-1A), IL-6, IL-7, IL-8, and IL-15 genes. MVA infection also stimulated the expression of NF-κB and components of the NF-κB signal transduction pathway, including p50 and TRAF-interacting protein. A marked increase in the expression of histone family members was also induced during MVA infection. Expression of the Wiskott-Aldrich syndrome family members WAS, WASF1, and the small GTP-binding protein RAC-1, which are involved in actin cytoskeleton reorganization, was enhanced after MVA infection. This study demonstrates that MVA infection triggered the induction of groups of genes, some of which may be involved in host resistance and immune modulation during virus infection.

Carbosilane Dendrimers to Transfect Human Astrocytes with Small Interfering RNA Targeting Human Immunodeficiency Virus

BackgroundHIV infection of the CNS is the principle cause of HIV-associated dementia in adults and encephalopathy in children. Gene therapy techniques such as small interfering RNA (siRNA) possess great potential in drug development, but first they must overcome the key obstacle of reaching the interior of the affected cells. A successful delivery vector for anti-HIV drugs that is capable of crossing the blood-brain barrier (BBB) could provide a way of addressing this issue. Non-viral vectors such as dendrimers offer a means for effectively delivering and transfecting siRNA to the target cells.ObjectiveTo evaluate the application of gene therapy for reducing HIV replication in human astrocytes.MethodsWe used the 2G-NN16 amino-terminated carbosilane dendrimer as a method for delivering siRNA to HIV-infected human astrocytes. We tested the cytotoxicity in human astrocytoma cells caused by 2G-NN16 and dendriplexes formed with siRNA (siRNA/2G-NN16) by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium-bromide (MTT) and lactate dehydrogenase assays. The ability to transfect human astrocytes with siRNA/2G-NN16 dendriplexes was tested by flow cytometry and immunofluorescence microscopy. To assess the potential capability of siRNA/2G-NN16 dendriplexes for crossing the BBB, we used an in vitro transcytosis assay with bovine brain microvascular endothelial cells. HIV-1 inhibition assays using 2G-NN16 and siRNA/2G-NN16 dendriplexes were determined by quantification of the viral load from culture supernatants of the astrocytes.ResultsA gradual time-controlled degradation of the 2G-NN16 dendrimer and liberation of its siRNA cargo between 12 and 24 hours was observed via gel electrophoresis. There was no cytotoxicity in HIV-infected or non-infected human astrocytoma cells when treated with up to 24 μg/mL of 2G-NN16 dendrimer or siRNA/2G-NN16 dendriplexes, and siRNA/2G-NN16 dendriplexes were seen to successfully transfect human astrocytes even after crossing an in vitro BBB model. More interestingly, transfected siRNA was observed to exert a biologic effect, as dendriplexes were shown to down-regulate the housekeeping gene GAPDH and to reduce replication of HIV-1 strains X4-HIV NL4-3 and R5-HIV BaL in human astrocytes.ConclusionsThe 2G-NN16 dendrimer successfully delivers and transfects siRNA to HIV-infected human astrocytes and achieves gene silencing without causing cytotoxicity.Both authors have contributed equally to this paper.

Gene Therapy in HIV‐Infected Cells to Decrease Viral Impact by Using an Alternative Delivery Method

The ability of dendrimer 2G‐[Si{O(CH2)2N(Me)2+(CH2)2NMe3+(I−)2}]8 (NN16) to transfect a wide range of cell types, as well as the possible biomedical application in direct or indirect inhibition of HIV replication, was investigated. Cells implicated in HIV infection such as primary peripheral blood mononuclear cells (PBMC) and immortalized suspension cells (lymphocytes), primary macrophages and dendritic cells, and immortalized adherent cells (astrocytes and trophoblasts) were analyzed. Dendrimer toxicity was evaluated by mitochondrial activity, cell membrane rupture, release of lactate dehydrogenase, erythrocyte hemolysis, and the effect on global gene expression profiles using whole‐genome human microarrays. Cellular uptake of genetic material was determined using flow cytometry and confocal microscopy. Transfection efficiency and gene knockdown was investigated using dendrimer‐delivered antisense oligonucleotides and small interfering RNA (siRNA). Very little cytotoxicity was detected in a variety of cells relevant to HIV infection and erythrocytes after NN16 dendrimer treatment. Imaging of cellular uptake showed high transfection efficiency of genetic material in all cells tested. Interestingly, NN16 further enhanced the reduction of HIV protein 24 antigen release by antisense oligonucleotides due to improved transfection efficiency. Finally, the dendrimer complexed with siRNA exhibited therapeutic potential by specifically inhibiting cyclooxygenase‐2 gene expression in HIV‐infected nervous system cells. NN16 dendrimers demonstrated the ability to transfect genetic material into a vast array of cells relevant to HIV pathology, combining high efficacy with low toxicity. These results suggest that NN16 dendrimers have the potential to be used as a versatile non‐viral vector for gene therapy against HIV infection.

Endocrine disruptors, gene deregulation and male germ cell tumors

Endocrine disruptors (EDs) belong to a large group of compounds, usually present as environmental pollutants, which can alter the homeostasis of living organisms by modifying hormonal balance and changing the normal patterns of gene regulation during development and cell differentiation. Hence, the development of male gonads and their functionality may be affected by exposure to specific EDs or their mixtures. The molecular mechanisms of action of these reprotoxicants leading to pathologies of the reproductive system such as testicular cancer, are complex and not well characterized. It is likely, however, that these compounds alter the interaction between the mechanisms of gene regulation and functional gene networks in windows of risk, mainly during embryonic development. Moreover, such changes could be transmitted through generations by epigenetic mechanisms. There are examples of the action of EDs on the expression of mRNAs, small non-coding RNAs and epigenetic marks in the developing testis associated with cellular and molecular alterations found in germ cell tumors. In the present review, we will discuss various aspects of genetic, transcriptomic and epigenetic changes related to testicular development, exposure to EDs and the occurrence of germ cell tumors.

Host Response to the Attenuated Poxvirus Vector NYVAC: Upregulation of Apoptotic Genes and NF-κB-Responsive Genes in Infected HeLa Cells

ABSTRACT NYVAC has been engineered as a safe, attenuated vaccinia virus (VV) vector for use in vaccination against a broad spectrum of pathogens and tumors. Due to the interest in NYVAC-based vectors as vaccines and current phase I/II clinical trials with this vector, there is a need to analyze the human host response to NYVAC infection. Using high-density cDNA microarrays, we found 368 differentially regulated genes after NYVAC infection of HeLa cells. Clustering of the regulated genes identified six discrete gene clusters with altered expression patterns. Clusters 1 to 3 represented 47.5% of the regulated genes, with three patterns of gene activation kinetics, whereas clusters 4 to 6 showed distinct repression kinetics. Quantitative real-time reverse transcription-PCR analysis of selected genes validated the array data. Upregulated transcripts correlated with genes implicated in immune responses, including those encoding interleukin-1 receptor 2 (IL-1R2), IL-6, ISG-15, CD-80, and TNFSF7. NYVAC upregulated several intermediates of apoptotic cascades, including caspase-9, correlating with its ability to induce apoptosis. NYVAC infection also stimulated the expression of NF-κB1 and NF-κB2 as well as that of NF-κB target genes. Expression of the VV host range K1L gene during NYVAC infection prevented NF-κB activation, but not the induction of apoptosis. This study is the first overall analysis of the transcriptional response of human cells to NYVAC infection and provides a framework for future functional studies to evaluate this vector and its derivatives as human vaccines.

The effects of different endocrine disruptors defining compound-specific alterations of gene expression profiles in the developing testis

Environmental contaminants considered endocrine disruptors have been shown to affect testis development and function but the mechanisms of action are not clear. We now have analyzed the effects on the transcriptome in testes of mice exposed to mono-(2-ethylhexyl)-phthalate (9.2; 46.3 or 92.7 mg/kg/d), zearalenone (1.3; 3.9 or 6.6 mg/kg/d), lindane (16.6; 32.2 or 64.4 mg/kg/d), bisphenol-A (0.16; 16 or 64 mg/kg/d) or 17β-estradiol (0.006; 0.012 or 0.048 mg/kg/d). The compounds were orally administered in the drinking water during distinct developmental periods: (A) mothers were exposed only during the two weeks before mating; (B) the exposure was continued during pregnancy until birth or (C) exposure was continued for a further four weeks after birth. Testes were studied at four weeks of age. Mono-(2-ethylhexyl)-phthalate and zearalenone, both produced specific alterations of gene signatures. Interestingly, this was irrespective of the concentration of the toxicant or the developmental period during which exposure occurred.

Fhx(Foxj2) expression is activated during spermatogenesis and very early in embryonic development

FHX (FOXJ2) is a recently characterized human fork head transcriptional activator that binds DNA with a dual sequence specificity. We have cloned the cDNA for the mouse orthologue Foxj2 and characterized its expression in the gonads and along the early pre-implantation development of the mouse. In the testis, Foxj2 is expressed from pachytene spermatocytes to round spermatids, but not in spermatogonia. In addition to the germ lineage, only Sertoli cells of the testis showed expression of Foxj2. In the ovary, only granulosa cells of the follicles express the factor. Neither mature spermatozoa nor oocytes showed expression of Foxj2. Foxj2 expression is early activated in zygotic development, being detected since as early as 8-cell stage embryos. Both cell layers of the blastocyst: the trophectoderm (TE) and the inner cell mass (ICM), express Foxj2.

The cytosolic aldehyde dehydrogenase gene (Aldh1) is developmentally expressed in Leydig cells

Cytosolic aldehyde dehydrogenase, ALDH1, participates in the oxidation of different aldehydes including that of all‐trans retinal to retinoic acid. The accumulation of mouse Aldh1 transcripts is characterized by having different patterns in different tissues. This paper reports the greatest expression of Aldh1 in testis and liver. It was demonstrated that in testis, Aldh1 is specifically expressed in Leydig cells and is under developmental regulation. In vitro studies of cultured Leydig TM3 cells confirmed these results though such gene expression was found not to be mediated by LH regulation. Previous investigations have associated androgen receptors, and hence the androgen insensitivity syndrome in man, with the presence of ALDH1 in genital skin fibroblasts. However, this relationship was not established in a functional cell type, as is reported here for Leydig cells. These results could suggest a model for a molecular pathway from androgen receptor to retinoic acid biogenesis in Leydig cells via the mediation of ALDH.