High-risk HPV E5-induced cell fusion: a critical initiating event in the early stage of HPV-associated cervical cancer

<p>Abstract</p> <p>Background</p> <p>Cervical cancer is strongly associated with high-risk human papillomavirus (HPV) and viral oncoproteins E5, E6 and E7 can transform cells by various mechanisms. It is proposed that oncogenic virus-induced cell fusion may contribute to oncogenesis if p53 or apoptosis is perturbed simultaneously. Recently, HPV-16 E5 was found to be necessary and sufficient for the formation of tetraploid cells, which are frequently found in precancerous cervical lesions and its formation is strongly associated with HPV state.</p> <p>Presentation of the hypothesis</p> <p>We propose that high-risk HPV E5-induced cell fusion is a critical initiating event in the early stage of HPV-associated cervical cancer.</p> <p>Testing the hypothesis</p> <p>Our hypothesis can be tested by comparing the likelihood for colony formation or tumorigenic ability in nude mice between normal HaCaT cells expressing all three oncogenic proteins and E5-induced bi-nucleated HaCaT cells expressing E6 and E7. Moreover, investigating premature chromosome condensation (PCC) in HPV-positive and negative precancerous cervical cells is another way to assess this hypothesis.</p> <p>Implication of the hypothesis</p> <p>This viewpoint would change our understanding of the mechanisms by which HPV induces cervical cancer. According to this hypothesis, blocking E5-induced cell fusion is a promising way to prevent the progression of cervical cancer. Additionally, establishment of a role of cell fusion in cervical carcinogenesis is of reference value for understanding the pathogenesis of other virus-associated cancers.</p

Establishment of Functioning Human Corneal Endothelial Cell Line with High Growth Potential

Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na+- and K+-dependent ATPase (Na+/K+-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na+/K+-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology

Alternative HER/PTEN/Akt Pathway Activation in HPV Positive and Negative Penile Carcinomas

Copyright: 2011 Stankiewicz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: The pathogenesis of penile squamous cell carcinoma (PSCC) is not well understood, though risk factors include human papillomavirus (HPV). Disruption of HER/PTEN/Akt pathway is present in many cancers; however there is little information on its function in PSCC. We investigated HER family receptors and phosphatase and tension homolog (PTEN) in HPV-positive and negative PSCC and its impact on Akt activation using immunohistochemistry and fluorescent in situ hybridisation (FISH). Methodology/Principal Findings: 148 PSCCs were microarrayed and immunostained for phosphorylated EGFR (pEGFR), HER2, HER3, HER4, phosphorylated Akt (pAkt), Akt1 and PTEN proteins. EGFR and PTEN gene status were also evaluated using FISH. HPV presence was assessed by PCR. pEGFR expression was detected significantly less frequently in HPV-positive than HPV-negative tumours (p = 0.0143). Conversely, HER3 expression was significantly more common in HPV-positive cases (p = 0.0128). HER4, pAkt, Akt and PTEN protein expression were not related to HPV. HER3 (p = 0.0054) and HER4 (p = 0.0002) receptors significantly correlated with cytoplasmic Akt1 immunostaining. All three proteins positively correlated with tumour grade (HER3, p = 0.0029; HER4, p = 0.0118; Akt1, p = 0.0001). pEGFR expression correlated with pAkt but not with tumour grade or stage. There was no EGFR gene amplification. HER2 was not detected. PTEN protein expression was reduced or absent in 62% of tumours but PTEN gene copy loss was present only in 4% of PSCCs. Conclusions/Significance: EGFR, HER3 and HER4 but not HER2 are associated with penile carcinogenesis. HPV-negative tumours tend to express significantly more pEGFR than HPV-positive cancers and this expression correlates with pAkt protein, indicating EGFR as an upstream regulator of Akt signalling in PSCC. Conversely, HER3 expression is significantly more common in HPV-positive cases and positively correlates with cytoplasmic Akt1 expression. HER4 and PTEN protein expression are not related to HPV infection. Our results suggest that PSCC patients could benefit from therapies developed to target HER receptors.Peer reviewedFinal Published versio

Signaling pathways responsible for the rapid antidepressant-like effects of a GluN2A-preferring NMDA receptor antagonist

In a previous study we found that the preferring GluN2A receptor antagonist, NVP-AAM077, elicited rapid antidepressant-like effects in the forced swim test that was related to the release of glutamate and serotonin in the medial prefrontal cortex. In the present work we sought to examine the duration of this behavioral effect as well as the molecular readouts involved. Our results showed that NVP-AAM077 reduced the immobility in the forced swim test 30?min and 24?h after its administration. However, this effect waned 7 days later. The rapid antidepressant-like response seems to be associated with increases in the GluA1 subunit of ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, mammalian target of rapamycin (mTOR) signaling, glia markers such as glial fibrillary acidic protein (GFAP) and excitatory amino acid transporter 1 (EAAT1), and a rapid mobilization of intracellular stores of brain-derived neurotrophic factor (BDNF) in the medial prefrontal cortex.Acknowledgements: M.G.-S. was recipient of a contract from the Sistema Nacional de Garantía Juvenil co-funded by the European Social Fund. We also thank Novartis for the generous gift of NVP-AAM077. This work was supported by the Instituto de Salud Carlos III, Subdirección General del Evaluación y Fomento de la Investigación (FIS Grants PI13/00038 and PI16/00217) that were co-funded by the European Regional Development Fund (‘A way to build Europe’). Funding from the Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Instituto de Salud Carlos III is also acknowledged

Novel Anti-Metastatic Action of Cidofovir Mediated by Inhibition of E6/E7, CXCR4 and Rho/ROCK Signaling in HPV+ Tumor Cells

Cervical cancer is frequently associated with HPV infection. The expression of E6 and E7 HPV oncoproteins is a key factor in its carcinogenicity and might also influence its virulence, including metastatic conversion. The cellular mechanisms involved in metastatic spread remain elusive, but pro-adhesive receptors and their ligands, such as SDF-1α and CXCR4 are implicated. In the present study, we assessed the possible relationship between SDF-1α/CXCR4 signaling, E6/E7 status and the metastatic process. We found that SDF-1α stimulated the invasion of E6/E7-positive cancer cell lines (HeLa and TC-1) in Matrigel though CXCR4 and subsequent Rho/ROCK activation. In pulmonary metastatic foci generated by TC-1 cells IV injection a high proportion of cells expressed membrane-associated CXCR4. In both cases models (in vitro and in vivo) cell adhesion and invasion was abrogated by CXCR4 immunological blockade supporting a contribution of SDF-1α/CXCR4 to the metastatic process. E6 and E7 silencing using stable knock-down and the approved anti-viral agent, Cidofovir decreased CXCR4 gene expression as well as both, constitutive and SDF-1α-induced cell invasion. In addition, Cidofovir inhibited lung metastasis (both adhesion and invasion) supporting contribution of E6 and E7 oncoproteins to the metastatic process. Finally, potential signals activated downstream SDF-1α/CXCR4 and involved in lung homing of E6/E7-expressing tumor cells were investigated. The contribution of the Rho/ROCK pathway was suggested by the inhibitory effect triggered by Cidofovir and further confirmed using Y-27632 (a small molecule ROCK inhibitor). These data suggest a novel and highly translatable therapeutic approach to cervix cancer, by inhibition of adhesion and invasion of circulating HPV-positive tumor cells, using Cidofovir and/or ROCK inhibition

Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR)(cy17) (killer line), which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+) fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+) signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+) fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+) fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and provides a valuable tool to visualize and quantify the anti-apoptotic potential of interesting target genes in vivo. The current work identifies a potential use for transgenic zebrafish as a high-throughput platform to validate potential apoptosis modulators in vivo

Small molecule activators of the Trk receptors for neuroprotection

The neurotophin signaling network is critical to the development and survival of many neuronal populations. Especially sensitive to imbalances in the neurotrophin system, cholinergic neurons in the basal forebrain are progressively lost in Alzheimer's disease. Therapeutic use of neurotrophins to prevent this loss is hampered, however, by a number of pharmacological challenges. These include a lack of transport across the blood-brain barrier, rapid degradation in the circulation, and difficulty in production. In this review we discuss the evidence supporting the neurotrophin system's role in preventing neurodegeneration and survey some of the pharmacological strategies being pursued to develop effective therapeutics targeting neurotrophin function

The dopamine β-hydroxylase -1021C/T polymorphism is associated with the risk of Alzheimer's disease in the Epistasis Project

Contains fulltext : 88930.pdf (publisher's version ) (Open Access)BACKGROUND: The loss of noradrenergic neurones of the locus coeruleus is a major feature of Alzheimer's disease (AD). Dopamine beta-hydroxylase (DBH) catalyses the conversion of dopamine to noradrenaline. Interactions have been reported between the low-activity -1021T allele (rs1611115) of DBH and polymorphisms of the pro-inflammatory cytokine genes, IL1A and IL6, contributing to the risk of AD. We therefore examined the associations with AD of the DBH -1021T allele and of the above interactions in the Epistasis Project, with 1757 cases of AD and 6294 elderly controls. METHODS: We genotyped eight single nucleotide polymorphisms (SNPs) in the three genes, DBH, IL1A and IL6. We used logistic regression models and synergy factor analysis to examine potential interactions and associations with AD. RESULTS: We found that the presence of the -1021T allele was associated with AD: odds ratio = 1.2 (95% confidence interval: 1.06-1.4, p = 0.005). This association was nearly restricted to men < 75 years old: odds ratio = 2.2 (1.4-3.3, 0.0004). We also found an interaction between the presence of DBH -1021T and the -889TT genotype (rs1800587) of IL1A: synergy factor = 1.9 (1.2-3.1, 0.005). All these results were consistent between North Europe and North Spain. CONCLUSIONS: Extensive, previous evidence (reviewed here) indicates an important role for noradrenaline in the control of inflammation in the brain. Thus, the -1021T allele with presumed low activity may be associated with misregulation of inflammation, which could contribute to the onset of AD. We suggest that such misregulation is the predominant mechanism of the association we report here

Cellular binding partners of the human papillomavirus E6 protein

The high-risk strains of human papillomavirus (HR-HPV) are known to be causative agents of cervical cancer and have recently also been implicated in cancers of the oropharynx. E6 is a potent oncogene of HR-HPVs, and its role in the progression to malignancy has been and continues to be explored. E6 is known to interact with and subsequently inactivate numerous cellular proteins pivotal in the mediation of apoptosis, transcription of tumor suppressor genes, maintenance of epithelial organization, and control of cell proliferation. Binding of E6 to these proteins cumulatively contributes to the oncogenic potential of HPV. This paper provides an overview of these cellular protein partners of HR-E6, the motifs known to mediate oncoprotein binding, and the agents that have the potential to interfere with E6 expression and activity and thus prevent the subsequent progression to oncogenesis