Infected HEK293 Cells Research Articles

Vinpocetine increases the microsporicidal effect of albendazole on Encephalitozoon intestinalis.

Microsporidia are obligate, intracellular, spore-forming eukaryotic fungi that infect humans and animals. In the treatment of disseminated microsporidiosis albendazole is the choice of drug. In recent years, antiparasitic activity of phosphodiesterase (PDE) enzyme inhibitors has been demonstrated against parasites and fungi, however, there is no information on microsporidia. Vinpocetine is currently used as a cerebral vasodilator drug and also as a dietary supplement to improve cognitive functions. Vinpocetine inhibits PDE1, so we aimed to investigate whether vinpocetine alone or in combination with albendazole has any effect on the spore load of Encephalitozoon intestinalis (E. intestinalis)-infected HEK293 cells. After determining the noncytotoxic concentrations of vinpocetine and albendazole on the host cell by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, HEK293 cells were infected with E. intestinalis spores. Then, two different concentrations of vinpocetine, albendazole, and a combination of both drugs were applied to the cells with an interval of 72 h for 15 days. Spore load of the cells was analyzed by real-time PCR. After the last treatment, spore Deoxyribonucleic Acid (DNA) load was significantly reduced only in the group treated with 14ng/ml albendazole. It was not different from control in groups treated with 7ng/ml albendazole and 4-20 µM vinpocetine. However, the combination of vinpocetine significantly increased the effect of albendazole at both concentrations. To our knowledge, this is the first study to investigate the microsporicidal activity of vinpocetine as well as its combinations with albendazole. However, further studies are needed to investigate the mechanism of action and also confirm in vivo conditions.

Exosome-specific loading Sox10 for the treatment of Cuprizone-induced demyelinating model

Demyelination is a pathological feature commonly observed in various central nervous system diseases. It is characterized by the aggregation of oligodendrocyte progenitor cells (OPCs) in the lesion area, which face difficulties in differentiating into mature oligodendrocytes (OLGs). The differentiation of OPCs requires the presence of Sox10, but its expression decreases under pathological conditions. Therefore, we propose a therapeutic strategy to regulate OPCs differentiation and achieve myelin repair by endogenously loading Sox10 into exosomes. To accomplish this, we generated a lentivirus-armed Sox10 that could anchor to the inner surface of the exosome membrane. We then infected HEK293 cells to obtain exosomes with high expression of Sox10 (exosomes-Sox10, ExoSs). In vitro, experiments confirmed that both Exos and ExoSs can be uptaken by OPCs, but only ExoSs exhibit a pro-differentiation effect on OPCs. In vivo, we administered PBS, Exos, and ExoSs to cuprizone-induced demyelinating mice. The results demonstrated that ExoSs can regulate the differentiation of PDGFRα+ OPCs into APC+ OLGs and reduce myelin damage in the corpus callosum region of the mouse brain compared to other groups. Further testing suggests that Sox10 may have a reparative effect on the myelin sheath by enhancing the expression of MBP, possibly facilitated by the exosome delivery of the protein into the lesion. This endogenously loaded technology holds promise as a strategy for protein-based drugs in the treatment of demyelinating diseases.

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ON THE BACK COVER: Septin‐caged Published online 7 September 2023 Shigella flexneri in infected HEK293 cells. Bacteria and actin are labelled in red, SEPT7 in green, and DNA in blue.Credit: Gizem Ozbaykal‐Guler and Serge Mostowy, London School of Hygiene & Tropical Medicineimage

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ON THE INNER FRONT COVER: Septin‐caged Shigella flexneri in infected HEK293 cells. Bacteria and actin are labelled in red, SEPT7 in green, and DNA in blue.Credit: Gizem Ozbaykal‐Guler and Serge Mostowy, London School of Hygiene & Tropical Medicineimage

Investigation of novel de novo KCNC2 variants causing severe developmental and early-onset epileptic encephalopathy

Purpose The voltage-gated potassium channel Kv3.2, encoded by KCNC2, facilitates fast-spiking GABAergic interneurons to fire action potentials at high frequencies. It is pivotal to maintaining excitation/inhibition balance in mammalian brains. This study identified two novel de novo KCNC2 variants, p.Pro470Ser (P470S) and p.Phe382Leu (F382L), in patients with early onset developmental and epileptic encephalopathy (DEE).Methods To examine the molecular basis of DEE, we studied the functional characteristics of variant channels using patch-clamp techniques and computational modeling.Results Whole-cell patch clamp recordings from infected HEK293 cells revealed that channel activation and deactivation kinetics strongly decreased in both Kv3.2 P470S and F382L variant channels. This decrease also occurred in Kv3.2 p.Val471Leu (V471L) channels, known to be associated with DEE. In addition, Kv3.2 F382L and V471L variants exhibited a significant increase in channel conductance and a ∼20 mV negative shift in the threshold for voltage-dependent activation. Simulations of model GABAergic interneurons revealed that all variants decreased neuronal firing frequency. Thus, the variants’ net loss-of-function effects disinhibited neural networks.Conclusion Our findings provide compelling evidence supporting the role of KCNC2 as a disease-causing gene in human neurodevelopmental delay and epilepsy.

The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells.

As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2′3′ cyclic phosphate instead of the common 5′ triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5′triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response.

Abstract 713: Inhibition of pseudo SARS-CoV-2 binding activity of a anti-cancer polypharmacological agent analogs

Abstract Severe acute respiratory syndrome coronavirus (SARS-CoV-2), a single-stranded RNA virus with largest genomes (∼ 29,800 bases) of all viruses, causes the infectious disease, COVID-19. SARS-CoV-2 is a zoonotic betacoronavirus transmitted through airborne and fecal-oral routes infecting nasal, lung, and intestinal tissues causing over 26 million confirmed coronavirus disease 2019 (COVID-19) cases and nearly 900,000 associated deaths worldwide as of September 2020. The surface of SARS-CoV-2 is covered with copies of spike glycoprotein (SGP) that plays a pivotal role in virus entry in host cells utilizing angiotensin-converting enzyme 2 (ACE2) as an attachment and entry receptor. We have successfully made a high titer replication-incompetent third-generation lentiviral vector pseudotyped with SARS-2-CoV SGP (pLV-S). Upon successful attachment and entry, the lentiviral vector causes enhanced green fluorescent protein (eGFP) to be expressed in the infected cell. Here, we show how the polypharmacological agent, 2-Deoxy-D-glucose (2-DG), a glucose mimic, and well characterized anti-cancer drug, and its analogs inhibit spike protein binding to the ACE2 receptors. This is as suggested from the 2DG and two analogs inhibition, with a low micromolar affinity, the pLV-S attachment and entry resulting in reduced cell fluorescence of infected HEK293 cells. In addition, we describe 2DG and analogs has the potential to deliver polypharmacological effects on the COVID-through inhibition of glycolysis (at energy status), modulation of inflammatory responses (cytokine storm) and alterations in glycosylation of viral proteins. A unique class of adjuvants and mitigants in the anti-SARS-CoV-2 activity is presented here. Citation Format: Rao V. Papineni, Amitava Adhikary, Ritesh Tandon, Dipanwita Mitra. Inhibition of pseudo SARS-CoV-2 binding activity of a anti-cancer polypharmacological agent analogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 713.

Characterization and Cytotoxic Evaluation of Bacteriocins Possessing Antibiofilm Activity Produced by Lactobacillus plantarum SJ33

Biofilm forming pathogens are among the major causes of hospital-acquired infections and are not much affected by antibiotic treatment. Consequently, novel agents and therapeutics are required urgently that possess antibacterial and antibiofilm activities. This study analyzed two bacteriocins from Lactobacillus plantarum subsp. argentoratensis SJ33 strain for their antibacterial and antibiofilm activity as well as cytotoxic properties. BacF1 and BacF2 showed broad spectrum activity against both Gram-positive (Listeria monocytogenes, Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa, Escherichia coli) bacteria. Significant bactericidal action was also observed on S. aureus cells by pore formation. Additionally, bacteriocins disrupted biofilms formed by S. aureus and P. aeruginosa which were shown by crystal violet staining assay and visualized by fluorescence as well as scanning electron microscopy. Quantitative Real-Time PCR study revealed changes in gene expression of biofilm formation in S. aureus (ica) and P. aeruginosa (pelA, psl, rhlA). Cytotoxicity of bacteriocins was further analyzed on normal mammalian cells and Caenorhabditis elegans. Notably, bacteriocins showed no major effect on HEK-293 cell line and enhanced the survival of S. aureus infected HEK-293 cells. Similarly, no cytotoxic effect was visible on C. elegans even after treatment with higher concentration than MIC at different time intervals.

Novel XTENylated AWRK6 analog with hypoglycemic activity, and anti-HSV-2 potential in combination with double shRNA

AimsTo design and evaluate a novel AWRK6 peptide-based long-acting GLP-1 receptor agonist (GLP-1RA) conjugated a recombinant polyethylene glycol mimetic (XTEN protein) with significant therapeutic potential on type 2 diabetes mellitus (T2DM) alone as well as Herpes simplex virus type 2 (HSV-2) infection in combination with double shRNA. Main methodsFirst, four AWRK6 analogs (termed XA-1 to XA-4) were designed and produced by solid phase synthesis strategy. Further surface plasmon resonance (SPR) measurement and in vitro cAMP accumulation assay were performed to detect the GLP-1R binding affinities and GLP-1R activation, respectively. The in vivo efficacy evaluation including pharmacokinetic test, oral glucose tolerance test (OGTT), hypoglycemic duration test and chronic pharmacodynamics study in rodent animals were all carefully performed. Key findingsFour XA peptides were synthesized with purity >99%. High binding affinity as well as activation potency of XA-4 for GLP-1R were demonstrated by SPR and cell-based luciferase reporter assay, respectively. Additionally, XA-4 exhibited the long-lasting antidiabetic effects in the multiple OGTTs, hypoglycemic duration test and chronic study in mice. Furthermore, combined treatment of XA-4 and double shRNA (D-shRNA) achieved potent antiviral effects in HSV-2 infected HEK293 cells. SignificanceXA-4 exhibited promising pharmaceutical potential to be a therapeutic drug for treating T2D, and also held potential to against the HSV-2 infection, which is really an accidental discovery. The strategy of recombinant XTENylation can also be applied to other peptides or small molecules for the development of long-acting therapeutic drugs.

A Bivalent Human Adenovirus Type 5 Vaccine Expressing the Rabies Virus Glycoprotein and Canine Distemper Virus Hemagglutinin Protein Confers Protective Immunity in Mice and Foxes.

The development of a safe and efficient multivalent vaccine has great prospects for application. Both rabies virus (RABV) and canine distemper virus (CDV) are highly infectious antigens, causing lethal diseases in domestic dogs and other carnivores worldwide. In this study, a replication-deficient human adenovirus 5 (Ad5)-vectored vaccine, rAd5-G-H, expressing RABV glycoprotein (G) and CDV hemagglutinin (H) protein was constructed. The RABV G and CDV H protein of rAd5-G-H were expressed and confirmed in infected HEK-293 cells by indirect immunofluorescence assay. The rAd5-G-H retained a homogeneous icosahedral morphology similar to rAd5-GFP under an electron microscope. A single dose of 108 GFU of rAd5-G-H administered to mice by intramuscular injection elicited rapid and robust neutralizing antibodies against RABV and CDV. Flow cytometry assays indicated that the dendritic cells and B cells in inguinal lymph nodes were significantly recruited in rAd5-G-H-immunized mice in comparison with the mock and rAd5-GFP groups. rAd5-G-H also activated the Th1- and Th2-mediated cell immune responses against RABV and CDV in mice, which contributed to 100% survival of a lethal-dose RABV challenge without any clinical signs. In foxes, a single dose of 109 GFU of rAd5-G-H could elicit high levels of neutralizing antibodies against both RABV and CDV in comparison with the mock and rAd5-GFP groups. All foxes in the rAd5-GFP and mock groups died, while the foxes inoculated with rAd5-G-H all survived and showed no clinical signs of disease after being challenged with a lethal wild-type CDV strain. These results suggested that rAd5-G-H has great potential as a bivalent vaccine against rabies and canine distemper in highly susceptible dogs and wildlife animals.

Molecular Approach for HIV-1 Replication Inhibition: Assessment of Different siRNAs Targeting Tat and Nef Genes to Effectively Suppress their Expression.

Inhibition of viral genes through siRNA seems to be promising for treatment of complicated viral infections like human immunodeficiency virus (HIV-1). HIV-1 Tat (Trans Activator of Transcription) and Nef (Negative regulatory Factor) proteins are very interesting targets for designing siRNAs. The effectiveness of suppressing Tat and Nef was investigated using three specific siTATs and three siNEFs. They were used to transfect the developed stable and infected Human Embryonic Kidney cells (HEK293) as an ex-vivo model. Both stable and virus infected HEK293 cells were transfected with each siTAT and siNEF. The inhibitory effect was evaluated using qRT-PCR, western blot analysis, and HIV P24 ELISA. siTAT-100, siTAT-162, and siNEF-136 and at a concentration of 100 nM/mL showed the most inhibitory effect on their target genes. Utilization of more developed molecular inhibition strategies such as RNAi or even a combination of different molecular approaches could be promising to overcome emerging HIV escape mutants.

Differential Reovirus-Specific and Herpesvirus-Specific Activator Protein 1 Activation of Secretogranin II Leads to Altered Virus Secretion.

Viruses utilize host cell machinery for propagation and manage to evade cellular host defense mechanisms in the process. Much remains unknown regarding how the host responds to viral infection. We recently performed global proteomic screens of mammalian reovirus TIL- and T3D-infected and herpesvirus (herpes simplex virus 1 [HSV-1])-infected HEK293 cells. The nonenveloped RNA reoviruses caused an upregulation, whereas the enveloped DNA HSV-1 caused a downregulation, of cellular secretogranin II (SCG2). SCG2, a member of the granin family that functions in hormonal peptide sorting into secretory vesicles, has not been linked to virus infections previously. We confirmed SCG2 upregulation and found SCG2 phosphorylation by 18 h postinfection (hpi) in reovirus-infected cells. We also found a decrease in the amount of reovirus secretion from SCG2 knockdown cells. Similar analyses of cells infected with HSV-1 showed an increase in the amount of secreted virus. Analysis of the stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) pathway indicated that each virus activates different pathways leading to activator protein 1 (AP-1) activation, which is the known SCG2 transcription activator. We conclude from these experiments that the negative correlation between SCG2 quantity and virus secretion for both viruses indicates a virus-specific role for SCG2 during infection. Mammalian reoviruses affect the gastrointestinal system or cause respiratory infections in humans. Recent work has shown that all mammalian reovirus strains (most specifically T3D) may be useful oncolytic agents. The ubiquitous herpes simplex viruses cause common sores in mucosal areas of their host and have coevolved with hosts over many years. Both of these virus species are prototypical representatives of their viral families, and investigation of these viruses can lead to further knowledge of how they and the other more pathogenic members of their respective families interact with the host. Here we show that secretogranin II (SCG2), a protein not previously studied in the context of virus infections, alters virus output in a virus-specific manner and that the quantity of SCG2 is inversely related to amounts of infectious-virus secretion. Herpesviruses may target this protein to facilitate enhanced virus release from the host.

Non-thermal microwave argon plasma affects interactions of Listeria monocytogenes with mammalian cells but it does not kill the intracellular pathogen

Effects of microwave argon non-thermal plasma (NTP) on the facultative intracellular pathogen Listeria monocytogenes were studied. NTP treatment for 2min caused a 3log10 decrease of wild type strain EGDe extracellular bacteria. Similar treatment of infected HEK293 cells caused a 1.5-fold decrease of intracellular EGDe bacteria. The intraphagosomal EGDeΔhly lacked the pore-forming toxin LLO was resistant to NTP. Cell pretreatment with NTP before infection increased EGDe invasion of human HEK293 and murine C26 cells 2.5 and 1.7-fold, respectively. HEK293 cell pretreatment did not affect invasion by EGDe ΔinlA that lacked the invasion factor InlA. Taken together, obtained results demonstrated that a direct NTP effect on intracellular L. monocytogenes was low, still NTP affected interactions between L. monocytogenes and the host cell.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.

Small RNA Analysis in Sindbis Virus Infected Human HEK293 Cells

IntroductionIn contrast to the defence mechanism of RNA interference (RNAi) in plants and invertebrates, its role in the innate response to virus infection of mammals is a matter of debate. Since RNAi has a well-established role in controlling infection of the alphavirus Sindbis virus (SINV) in insects, we have used this virus to investigate the role of RNAi in SINV infection of human cells.ResultsSINV AR339 and TR339-GFP were adapted to grow in HEK293 cells. Deep sequencing of small RNAs (sRNAs) early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral sRNAs (vsRNAs), with no size, sequence or location specific patterns characteristic of Dicer products nor did they possess any discernible pattern to ascribe to a specific RNAi biogenesis pathway. This was supported by multiple variants for each sequence, and lack of hot spots along the viral genome sequence. The abundance of the best defined vsRNAs was below the limit of Northern blot detection. The adaptation of the virus to HEK293 cells showed little sequence changes compared to the reference; however, a SNP in E1 gene with a preference from G to C was found.Deep sequencing results showed little variation of expression of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed no difference in expression by Northern blot analysis.ConclusionsWe show that, unlike SINV infection of invertebrates, generation of Dicer-dependent svRNAs and change in expression of cellular miRNAs were not detected as part of the Human response to SINV.

Recombinant adeno-associated virus type 8 mediated dual-luciferase gene expression in mouse

Recombinant adeno-associated virus type 8 (rAAV8) mediating transgene expression in mice was investigated using co-expressed report gene of secreted Gaussia princeps luciferase (Gluc) and non-secreted firefly luciferase(Fluc). rAAV8-Gluc/Fluc was prepared and infected HEK293 cells to test its performance in vitro. BALB/c mice were received rAAV8-Gluc/Fluc at different doses by intravenous injection (iv) or intramuscular injection (im). Then Glue activities in blood were measured,the whole-body images for Flue activities were performed and Flue activities of tissue lysate were also detected. rAAV8-Gluc/Fluc was successfully prepared and could infected HEK293 cells. The Glue was mainly detected in the culture media while the Flue was mainly detected within cells. The blood Glue activities of mice received rAAV8-Gluc/Fluc by iv or im peaked at 10-20 d post injection and persisted for at least 120d. The blood Gluc activities increased at the rAAV8-Gluc/Fluc dose-dependent manner. For mice received rAAV8 by iv, Fluc mainly expressed in liver and minor Fluc expression was also found in cardiac muscle and skeletal muscle. For mice received rAAV8-Gluc/Fluc by im, Fluc mainly expressed in local skeletal muscle and secondly in liver. rAAV8-Gluc/Fluc has been prepared successfully and its mediating transgene expression in mice has been investigated. This research will facilitate preclinical studies for rAAV8-mediated gene therapy.

Proteome analysis of vaccinia virus IHD-W-infected HEK 293 cells with 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS of on solid phase support N-terminally sulfonated peptides

BackgroundDespite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach.ResultsThe cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses.ConclusionsThe proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.

P27 Suppresses Arsenite-induced Hsp27/Hsp70 Expression through Inhibiting JNK2/c-Jun- and HSF-1-dependent Pathways

p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27.

Global profile of genes regulated by securin

Securin is a proto-oncogene that plays a pivotal role in cell division, sister chromatids separation and pancreatic beta cell proliferation. Over-expression of securin in normal cells results in cellular transformation and development on tumor in nude mice. On the other hand, depletion of securin from cancer cells results in suppression of tumor and reversal of cancer phenotype in vitro and in vivo. In many cases securin acts as a transcription factor and regulates the expression of important target genes. However, only a few target genes have been identified. Therefore, we used a quantitative microarray assay to identify the genes that are regulated by securin in a human embryonic kidney cell line (HEK293) that do not express securin or express at a very low level. We infected HEK293 cells with empty adenovirus, adenovirus expressing GFP or adenovirus expressing securin. Infection of cells with adenovirus expressing securin for 48 h resulted in alterations in the levels of expression of genes that ranged in magnitude from 1.1- to 42-fold, with a total of 313 genes exhibiting a p value < 0.05 compared to control adenovirus or adenovirus expressing GFP protein. Infection of HEK293 with adenovirus expressing securin was found to affect the expression of a diverse range of genes, including oncogenes and those that encode transcription factors, ion channel proteins, and cytoskeletal proteins as well as other proteins that are involved in signal transduction, the cell cycle, cell proliferation and apoptosis. The altered expression of some of these genes that were found by microarray analysis to be regulated by securin was confirmed by semiquantitative reverse transcriptase-polymerase chain reaction. The application of the microarray technique should prove to be a powerful tool for future analysis of the mechanisms by which securin regulates tumorigenesis.

Mycobacterium bovisBacillus Calmette-Guérin Induces CCL5 Secretion via the Toll-Like Receptor 2-NF-κB and -Jun N-Terminal Kinase Signaling Pathways

In response to Mycobacterium bovis bacillus Calmette-Guérin (BCG), CC chemokines are secreted from host cells to attract components of the innate and adaptive immune systems to the site of infection. Toll-like receptor 2 (TLR2) has been shown to recognize M. bovis BCG and to initiate signaling pathways that result in enhanced secretion of CC chemokines. Despite the essential requirement of TLR2 in M. bovis BCG infection, the mechanisms by which it induces secretion of CC chemokines are not well defined. In this study, we report that stimulation of HEK293 cells expressing human TLR2 with M. bovis BCG resulted in increased CCL2 and CCL5 secretion, as determined by an enzyme-linked immunosorbent assay. M. bovis BCG infection resulted in the activation of c-Jun N-terminal kinase (JNK), and the inhibition of JNK activity had a significant effect on M. bovis BCG-dependent CCL5 secretion in TLR2-expressing cells but no effect on M. bovis BCG-dependent CCL2 secretion from infected HEK293 cells expressing human TLR2. The M. bovis BCG-induced CCL5 release was attenuated by sulfasalazine (a well-described inhibitor of NF-kappaB activity), BAY 11-7082 (an IkappaB phosphorylation inhibitor), and ALLN (a well-described inhibitor of NF-kappaB activation that prevents degradation of IkappaB and eventually results in a lack of translocated NF-kappaB in the nucleus). In addition, stimulation of TLR2-expressing cells with M. bovis BCG resulted in translocation of NF-kappaB subunits from the cytoplasmic to the nuclear fraction, and stimulation of cells with M. bovis BCG activated IkappaB kinase alphabeta. These findings indicate that M. bovis BCG induces CCL5 production through mechanisms that include a TLR2-dependent component that requires JNK and NF-kappaB activities.