Lysosomal Activation Mediated by Endocytosis in J774 Cell Culture Treated with N-Trimethyl Chitosan Nanoparticles.

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 µg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Prevalence of sexually risky behaviors among Mexican medical students.

University students are at high risk of sexually transmitted infections due to the lack of adequate sexual education, as well as multiple associated factors, which lead to risky sexual practices. It is important to update data about sexual behaviors to identify the main factors associated with sexually risky behaviors. The present study aimed to evaluate the current prevalence of sexually risky practices in medical students. A cross-sectional study was conducted among medical students through an anonymous self-administered online questionnaire including demographic characteristics and sexual behaviors. We used descriptive statistics and multivariable regression to analyze the data collected. A total of 1520 undergraduate medical students aged between 18 and 28 years old were included in the study. Sixty percent of the students were sexually active with a higher proportion in men (70%), likewise, they had an earlier sexual debut (16.5 vs 16.9 years old), and a greater number of lifetime sexual partners than women (3.8 vs 2.2). The main sexual activity in both groups was vaginal sex with high use of condoms (75%), however, most of them (67%) reported having unprotected oral sex. Logistic regression analysis showed that condomless sex was associated with having oral sex, anal sex, and being female. The findings of this study showed that medical university students are involved in risky sexual behaviors, the major risk factor was unprotected oral sex. Based on these results, we recommended designing interventions to improve sexual education and preventive approaches from early stages such as in middle school students to mitigate sexually transmitted infections among medical university students.

Quantitative Proteomics for the Identification of Differentially Expressed Proteins in the Extracellular Vesicles of Cervical Cancer Cells.

The extracellular vesicles (EVs) in a tumoral microenvironment can exert different functions by transferring their content, which has been poorly described in cervical cancer. Here, we tried to clarify the proteomic content of these EVs, comparing those derived from cancerous HPV (+) keratinocytes (HeLa) versus those derived from normal HPV (-) keratinocytes (HaCaT). We performed a quantitative proteomic analysis, using LC-MS/MS, of the EVs from HeLa and HaCaT cell lines. The up- and downregulated proteins in the EVs from the HeLa cell line were established, along with the cellular component, molecular function, biological processes, and signaling pathways in which they participate. The biological processes with the highest number of upregulated proteins are cell adhesion, proteolysis, lipid metabolic process, and immune system processes. Interestingly, three of the top five signaling pathways with more up- and downregulated proteins are part of the immune response. Due to their content, we can infer that EVs can have a significant role in migration, invasion, metastasis, and the activation or suppression of immune system cells in cancer.

Human papillomavirus infection and seroprevalence among female university students in Mexico.

Human papillomavirus (HPV) is one of the most common causes of sexually transmitted diseases, and the main etiology of cervical cancer. This study was aimed to assess type-specific cervical HPV prevalence and their association with HPV-specific antibodies in a cohort of female university students. HPV genotyping was performed by amplifying and sequencing a fragment of the L1 protein. A BLAST search was performed to identify HPV types. HPV-specific IgG antibodies were measured by ELISA in serum samples. A total of 129 women participated, with an average age of 21.75 years. The prevalence of vaginal HPV infection was 74.42%. The most predominant high-risk HPV types were 18 (13.95%), 31 (10.85%), and 16 (9.3%). We found that early age at coitarche and a higher number of sexual partners were significantly associated with a high prevalence of HPV infection. In addition to sexual behavior, we observed that the presence of serum-specific IgG antibodies against HPV can impact the prevalence of the virus. Seropositivity to HPV-16 and HPV-18 was associated with a lower prevalence of HPV-16, but not for other HPV types. Of note, there was a lower proportion of HPV-specific seropositivity in women who had the presence of the same HPV type in a cervical specimen, suggesting an immunoregulatory mechanism associated with the viral infection. In conclusion, the prevalence of HPV in university women was higher than expected and it was associated with early age of sexual debut, an increasing number of sexual partners, and a low proportion of HPV seropositivity.

The Molecular Basis and Biologic Significance of the ß-Dystroglycan-Emerin Interaction.

ß-dystroglycan (ß-DG) assembles with lamins A/C and B1 and emerin at the nuclear envelope (NE) to maintain proper nuclear architecture and function. To provide insight into the nuclear function of ß-DG, we characterized the interaction between ß-DG and emerin at the molecular level. Emerin is a major NE protein that regulates multiple nuclear processes and whose deficiency results in Emery-Dreifuss muscular dystrophy (EDMD). Using truncated variants of ß-DG and emerin, via a series of in vitro and in vivo binding experiments and a tailored computational analysis, we determined that the ß-DG-emerin interaction is mediated at least in part by their respective transmembrane domains (TM). Using surface plasmon resonance assays we showed that emerin binds to ß-DG with high affinity (KD in the nanomolar range). Remarkably, the analysis of cells in which DG was knocked out demonstrated that loss of ß-DG resulted in a decreased emerin stability and impairment of emerin-mediated processes. ß-DG and emerin are reciprocally required for their optimal targeting within the NE, as shown by immunofluorescence, western blotting and immunoprecipitation assays using emerin variants with mutations in the TM domain and B-lymphocytes of a patient with EDMD. In summary, we demonstrated that ß-DG plays a role as an emerin interacting partner modulating its stability and function.

Eag1 Gene and Protein Expression in Human Retinoblastoma Tumors and its Regulation by pRb in HeLa Cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1. Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1. Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments.

Presence of HPV DNA in extracellular vesicles from HeLa cells and cervical samples.

INTRODUCTION: The main cause of cervical cancer is an infection of keratinocytes in the basal layer of the stratified epithelium of the cervix by human papillomavirus (HPV). Other than in cervical samples, HPV DNA has been found in serum and other fluids but its origin is unclear. Extracellular vesicles (EV) could be a conveyance of viral DNA given their emerging role in cellular communication. The content of EV derived from cervical cells has not been properly explored and it is not known whether or not they contain HPV DNA. METHODS: We evaluated the DNA content of exosomes purified from cultures of HeLa cells by Next Generation Sequencing (NGS) and confirmed its presence by PCR. The presence of HPV DNA was also evaluated by PCR and NGS in EV from HPV-positive cervical samples without apparent lesion or with LSIL. RESULTS: We detected the integrated form of viral-DNA in exosomes from HeLa cells by NGS and confirmed its presence by PCR. The search for HPV sequences in EV obtained from cervical exudate samples without apparent lesion or with LSIL, where we expected to find the viral genome as an episome, indicated that HPV DNA, including the E6 and E7 oncogenes, is present in these EV. CONCLUSION: HPV DNA, including the viral oncogenes E6/E7, is found in exosomes regardless of the integration status of the virus in the infected cell.

The combination astemizole-gefitinib as a potential therapy for human lung cancer.

Lung cancer is a major cause of cancer mortality. Thus, novel therapies are urgently needed. Repositioning of old drugs is gaining great interest in cancer treatment. Astemizole is an antihistamine proposed to be repositioned for cancer therapy. This drug targets several molecules involved in cancer including histamine receptors, ABC transporters and the potassium channels Eag1 and HERG. Astemizole inhibits the proliferation of different cancer cells including those from cervix, breast, leukemia and liver. Gefitinib is widely used to treat lung cancer; however, no response or drug resistance occurs in many cases. Here, we studied the combined effect of astemizole and gefitinib on the proliferation, survival, apoptosis and gene and protein expression of Eag1 channels in the human lung cancer cell lines A549 and NCI-H1975. Cell proliferation and survival were studied by the MTT method and the colony formation assay, respectively; apoptosis was investigated by flow cytometry. Gene expression was assessed by real-time polymerase chain reaction (RT-PCR), and protein expression was studied by Western blot analysis and immunocytochemistry. We obtained the inhibitory concentrations 20 and 50 (IC20 and IC50, respectively) values for each drug from the cell proliferation experiments. Drug combination at their IC20 had a superior effect by reducing cell proliferation and survival in up to 80% and 100%, respectively. The drugs alone did not affect apoptosis of H1975 cells, but the drug combination at their IC20 increased apoptosis roughly four times in comparison to the effect of the drugs alone. Eag1 mRNA levels and protein expression were decreased by the drug combination in A549 cells, and astemizole induced subcellular localization changes of the channel protein in these cells. Our in vitro studies strongly suggest that the combination astemizole-gefitinib may be a novel and promising therapy for lung cancer patients.

Mutations in TET2 and DNMT3A genes are associated with changes in global and gene-specific methylation in acute myeloid leukemia.

Acute myeloid leukemia is characterized by its high biological and clinical heterogeneity, which represents an important barrier for a precise disease classification and accurate therapy. While epigenetic aberrations play a pivotal role in acute myeloid leukemia pathophysiology, molecular signatures such as change in the DNA methylation patterns and genetic mutations in enzymes needed to the methylation process can also be helpful for classifying acute myeloid leukemia. Our study aims to unveil the relevance of DNMT3A and TET2 genes in global and specific methylation patterns in acute myeloid leukemia. Peripheral blood samples from 110 untreated patients with acute myeloid leukemia and 15 healthy control individuals were collected. Global 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from peripheral blood leukocytes were measured by using the MethylFlashTM Quantification kits. DNMT3A and TET2 expression levels were evaluated by real-time quantitative polymerase chain reaction. The R882A hotspot of DNMT3A and exons 6-10 of TET2 were amplified by polymerase chain reaction and sequenced using the Sanger method. Methylation patterns of 16 gene promoters were evaluated by pyrosequencing after treating DNA with sodium bisulfite, and their transcriptional products were measured by real-time quantitative polymerase chain reaction.Here, we demonstrate altered levels of 5-methylcytosine and 5-hydroxymethylcytosine and highly variable transcript levels of DNMT3A and TET2 in peripheral blood leukocytes from acute myeloid leukemia patients. We found a mutation prevalence of 2.7% for DNMT3A and 11.8% for TET2 in the Mexican population with this disease. The average overall survival of acute myeloid leukemia patients with DNMT3A mutations was only 4 months. In addition, we showed that mutations in DNMT3A and TET2 may cause irregular DNA methylation patterns and transcriptional expression levels in 16 genes known to be involved in acute myeloid leukemia pathogenesis. Our findings suggest that alterations in DNMT3A and TET2 may be associated with acute myeloid leukemia prognosis. Furthermore, alterations in these enzymes affect normal methylation patterns in acute myeloid leukemia- specific genes, which in turn, may influence patient survival.

Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing.

Our objective was to determine if whole genome amplification (WGA) provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3) or trophoblastic cells (Day 5) were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo's sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p < 0.001). Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL) was also used to determine sex. AMELY peak's height was higher and this peak's presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p < 0.001). Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89) and RPL17 for Trisomy 18 (AUC = 0.94). Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

HPV16-E2 protein modifies self-renewal and differentiation rate in progenitor cells of human immortalized keratinocytes.

BACKGROUND: Cervical cancer is the fourth cause of death worldwide by cancer in women and is a disease associated to persistent infection with human papillomavirus (HPV), particularly from two high-risk types HPV16 and 18. The virus initiates its replicative cycle infecting cells located in the basal layer of the epithelium, where a small population of epithelial stem cells is located performing important functions of renewal and maintenance of the tissue. Viral E2 gene is one of the first expressed after infection and plays relevant roles in the replicative cycle of the virus, modifying fundamental processes in the infected cells. Thus, the aim of the present study was to demonstrate the presence of hierarchic subpopulations in HaCaT cell line and evaluate the effect of HPV16-E2 expression, on their biological processes. METHODS: HaCaT-HPV16-E2 cells were generated by transduction of HaCaT cell line with a lentiviral vector. The α6-integrin-CD71 expression profile was established by immunostaining and flow cytometric analysis. After sorting, cell subpopulations were analyzed in biological assays for self-renewal, clonogenicity and expression of stemness factors (RT-qPCR). RESULTS: We identified in HaCaT cell line three different subpopulations that correspond to early differentiated cells (α6-integrindim), transitory amplifying cells (α6-integrinbri/CD71bri) and progenitor cells (α6-integrinbri/CD71dim). The last subpopulation showed stem cell characteristics, such as self-renewal ability, clonogenicity and expression of the well-known stem cell factors SOX2, OCT4 and NANOG, suggesting they are stem-like cells. Interestingly, the expression of HPV16-E2 in HaCaT cells changed its α6-integrin-CD71 immunophenotype modifying the relative abundance of the cell subpopulations, reducing significantly the percentage of α6-integrinbri/CD71dim cells. Moreover, the expression of the stem cell markers was also modified, increasing the expression of SOX2 and NANOG, but decreasing notably the expression of OCT4. CONCLUSIONS: Our data demonstrated the presence of a small subpopulation with epithelial "progenitor cells" characteristics in the HaCaT cell line, and that HPV16-E2 expression on these cells induces early differentiation.

Astemizole inhibits cell proliferation in human prostate tumorigenic cells expressing ether à-go-go-1 potassium channels.

Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether à-go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue®  assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells.  Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.

Characterization of cervical cancer stem cell-like cells: phenotyping, stemness, and human papilloma virus co-receptor expression.

Cancer stem cells (CSC) exhibit high tumorigenic capacity in several tumor models. We have now determined an extended phenotype for cervical cancer stem cells. Our results showed increased CK-17, p63+, AII+, CD49f+ expression in these cells, together with higher Aldehyde dehydrogenase (ALDHbright)activity in Cervical CSC (CCSC) enriched in cervospheres. An increase in stem cell markers, represented by OCT-4, Nanog, and ß-catenin proteins, was also observed, indicating that under our culture conditions, CCSC are enriched in cervospheres, as compared to monolayer cultures. In addition, we were able to show that an increased ALDHbright activity correlated with higher tumorigenic activity. Flow cytometry and immunflorescence assays demonstrated that CCSC in cervosphere cultures contain a sub-population of cells that contain Annexin II, a Human papillomavirus (HPV) co-receptor. Taken together, under our conditions there is an increase in the number of CCSC in cervosphere cultures which exhibit the following phenotype: CK-17, p63+, AII+, CD49f+ and high ALDH activity, which in turn correlates with higher tumorigenicity. The presence of Annexin II and CD49f in CCSC opens the possibility that normal cervical stem cells could be the initial target of infection by high risk HPV.

[Detection of microRNAs seed sequences within human papillomavirus genomes]. / Detección de secuencias de semillas de microRNAs celulares dentro del genoma de los virus del papiloma humano.

In this paper we are reporting for the first time the presence of seed sequences of human and viral microRNAs embedded within both high and low risk human papillomavirus (HPV) genomes. These seed sequences have high oncogenic potential. They were found using an in silico analysis based on the microRNA sequences added to Sanger's database. Among these sequences, it was observed a potential fingerprint harbouring several repeated sequences of microRNA 297 (miR-297) within the LCR region of HPV types 16, 18, 33, 45 and 52. Further analyses were performed for low risk HPV types 6 and 11 and we observed that the probable fingerprint was absent in HPV11, even when we detected other repeated sequences of miR-363. According to these findings, besides the fact that we detected the presence of microRNA sequences within HPV genomes, we suggest a common putative viral mechanism of gene expression regulation shared among human virus.

En el presente trabajo reportamos por primera vez la existencia de secuencias semilla de diferentes microRNAs (codificados en humano y de otros virus) en el genoma de los virus de papiloma humano (VPH). Estas secuencias tienen un alto poder oncogénico y se encontraron mediante un análisis in silico basado en las secuencias de microRNAs depositadas en la base de datos de Sanger. Entre ellas se detectó una posible huella que consiste en la presencia de varias repeticiones de la semilla del microRNA 297 (miR-297) en la región LCR y que fue detectada en los tipos virales 16, 18, 33, 45 y 52. Además, se realizó la búsqueda de semillas en los tipos virales de bajo poder oncogénico 6 y 11 y se observó que esta posible huella está ausente en el tipo 11, si bien se localizaron repeticiones de la semilla de otro microRNA, miR-363. Con base en este hallazgo, además de que se detectaron semillas de otros virus en las diferentes regiones de los seis tipos virales, se abre la posibilidad de la existencia de un mecanismo de regulación de la expresión de genes celulares a través de la transcripción de las diferentes regiones del genoma de los VPH de alto poder oncogénico que contienen las diferentes semillas de microRNAs.

Differential DNA methylation patterns in the CD86 gene controls its constitutive expression in keratinocytes.

The interaction of B7 family members with appropriate receptors is essential for an effective T cell response. CD80 and CD86 are the principal co-stimulatory molecules of this family and they are mainly expressed on professional antigen presenting cells (APCs), but also on several non-lymphoid cells. CD86 is constitutively expressed in keratinocytes from the spinous layer of normal cervical epithelium. However, the mechanisms that control the expression of this gene in epithelial cells remain unknown. We analyzed the DNA methylation status of the CD86 promoter and a CpG island located in the upstream intergenic region in keratinocyte-derived cell lines. In those cell lines where CD86 is expressed, a high degree of methylation in the CpG island was observed. However, a CpG dinucleotide within the cAMP response element (CRE) in the promoter region was consistently unmethylated and associated to the transcription factor CREB, as demonstrated by ChIP assays. The opposite methylation pattern was observed in cell lines where CD86 is not expressed, affecting also the binding of CREB. The analysis of the DNA methylation pattern of this gene in cells from the spinous and basal layers of normal cervical epithelium showed a similar profile to that observed in cell lines with and without expression of CD86 respectively. Our results indicate that the methylation pattern in the CD86 promoter and CpG island is closely related to the expression of this co-stimulatory molecule in keratinocytes.

In vitro effects of stromal cells expressing different levels of Jagged-1 and Delta-1 on the growth of primitive and intermediate CD34(+) cell subsets from human cord blood.

In trying to contribute to our knowledge on the role of Notch and its ligands within the human hematopoietic system, we have assessed the effects of the OP9 stroma cell line - naturally expressing Jagged-1 - transduced with either the Delta-1 gene (OP9-DL1 cells) or with vector alone (OP9-V), on the in vitro growth of two different hematopoietic cell populations. Primitive (CD34(+) CD38(-) Lin(-)) and intermediate (CD34(+) CD38(+) Lin(-)) CD34(+) cell subsets from human cord blood were cultured in the presence of 7 stimulatory cytokines under four different conditions: cytokines alone (control); cytokines and mesenchymal stromal cells; cytokines and OP9-V cells; cytokines and OP9-DL1 cells. Proliferation and expansion were determined after 7days of culture. Culture of CD34(+) CD38(-) Lin(-) cells in the presence of OP9-V or OP9-DL1 cells resulted in a significant increase in the production of new CD34(+) CD38(-) Lin(-) cells (expansion), which expressed increased levels of Notch-1; in contrast, production of total nucleated cells (proliferation) was reduced, as compared to control conditions. In cultures of CD34(+) CD38(+) Lin(-) cells established in the presence of OP9-V or OP9-DL1 cells, expansion was similar to that observed in control conditions, whereas proliferation was also reduced. Interestingly, in these latter cultures we observed production of CD34(+) CD38(-) Lin(-) cells. Our results indicate that, as compared to MSC, OP9 cells were more efficient at inducing self-renewal and/or de novo generation of primitive (CD34(+) CD38(-) Lin(-)) cells, and suggest that such effects were due, at least in part, to the presence of Jagged-1 and DL1.

HPV16 E2 could act as down-regulator in cellular genes implicated in apoptosis, proliferation and cell differentiation.

BACKGROUND: Human Papillomavirus (HPV) E2 plays several important roles in the viral cycle, including the transcriptional regulation of the oncogenes E6 and E7, the regulation of the viral genome replication by its association with E1 helicase and participates in the viral genome segregation during mitosis by its association with the cellular protein Brd4. It has been shown that E2 protein can regulate negative or positively the activity of several cellular promoters, although the precise mechanism of this regulation is uncertain. In this work we constructed a recombinant adenoviral vector to overexpress HPV16 E2 and evaluated the global pattern of biological processes regulated by E2 using microarrays expression analysis. RESULTS: The gene expression profile was strongly modified in cells expressing HPV16 E2, finding 1048 down-regulated genes, and 581 up-regulated. The main cellular pathway modified was WNT since we found 28 genes down-regulated and 15 up-regulated. Interestingly, this pathway is a convergence point for regulating the expression of genes involved in several cellular processes, including apoptosis, proliferation and cell differentiation; MYCN, JAG1 and MAPK13 genes were selected to validate by RT-qPCR the microarray data as these genes in an altered level of expression, modify very important cellular processes. Additionally, we found that a large number of genes from pathways such as PDGF, angiogenesis and cytokines and chemokines mediated inflammation, were also modified in their expression. CONCLUSIONS: Our results demonstrate that HPV16 E2 has regulatory effects on cellular gene expression in HPV negative cells, independent of the other HPV proteins, and the gene profile observed indicates that these effects could be mediated by interactions with cellular proteins. The cellular processes affected suggest that E2 expression leads to the cells in to a convenient environment for a replicative cycle of the virus.

Dp71f modulates GSK3-beta recruitment to the beta1-integrin adhesion complex.

Previously, it was shown that Dp71f binds to the beta1-integrin adhesion complex to modulate PC12 cell adhesion. The absence of Dp71f led to a failure in the beta1-integrin adhesion complex formation. One of the structural proteins which links the beta1-integrin cytoplasmic domain to the actin cytoskeleton is ILK. GSK3-beta is an ILK substrate and the carboxi-terminal region of dystrophin 427 is a substrate for hierarchical phosphorylation by GSK3-beta. Dp71f contains the carboxi-terminal domain present in dystrophin 427. By using co-immunoprecipitation assays, in the present work it is demonstrated that in the neuronal PC12 cell line an interaction between Dp71f and GSK3-beta occurs. This interaction was corroborated by in vitro pulldown assays. We show that GSK3-beta is recruited to the beta1-integrin complex and that a reduced expression of Dp71f induces a reduced GSK3-beta recruitment to the beta1-integrin complex. In addition, the present work establishes that adhesion of PC12 cells to laminin does not influence the phosphorylation status of Dp71f.

Benzoquinone activates the ERK/MAPK signaling pathway via ROS production in HL-60 cells.

Benzene (BZ) is a class I carcinogen and its oxidation to reactive intermediates is a prerequisite of hematoxicity and myelotoxicity. The generated metabolites include hydroquinone, which is further oxidized to the highly reactive 1,4-benzoquinone (BQ) in bone marrow. Therefore, we explored the mechanisms underlying BQ-induced HL-60 cell proliferation by studying the role of BQ-induced reactive oxygen species (ROS) in the activation of the ERK-MAPK signaling pathway. BQ treatment (0.01-30 microM) showed that doses below 10 microM did not significantly reduce viability. ROS production after 3 microM BQ treatment increased threefold; however, catalase addition reduced ROS generation to basal levels. FACS analysis showed that BQ induced a fivefold increase in the proportion of cells in S-phase. We also observed a high proportion of Bromodeoxyuridine (BrdU) stained cells, indicating a higher DNA synthesis rate. BQ also produced rapid and prolonged phosphorylation of ERK1/2 proteins. Simultaneous treatment with catalase or PD98059, a potent MEK protein inhibitor, reduced cell recruitment into the S-phase and also abolished the ERK1/2 protein phosphorylation induced by BQ, suggesting that MEK/ERK is an important pathway involved in BQ-induced ROS mediated proliferation. The prolonged activation of ERK1/2 contributes to explain the increased S-phase cell recruitment and to understand the leukemogenic processes associated with exposure to benzene metabolites. Thus, the possible mechanism by which BQ induce HL-60 cells to enter the cell cycle and proliferate is linked to ROS production and its growth promoting effects by specific activation of regulating genes known to be activated by redox mechanisms.