CRL4A-FBXW5-mediated degradation of DLC1 Rho GTPase-activating protein tumor suppressor promotes non-small cell lung cancer cell growth

The DLC1 tumor suppressor gene is commonly lost in cancer by genomic deletion or epigenetic silencing, leading to loss of gene transcription. DLC1 encodes a GTPase-activating protein for the RhoA small GTPase, and DLC1 loss of expression results in aberrant RhoA activation and signaling. Unexpectedly, we found that a subset of non-small cell lung cancer patient tumors and cell lines retained DLC1 mRNA but not protein expression. We determined that the CUL4A–DDB1–FBXW5 E3 ubiquitin ligase complex is responsible for loss of DLC1 protein expression. Suppression of FBXW5 function restored DLC1-dependent lung cancer cell growth suppression. Our observations identify a mechanism for posttranslational loss of DLC1 function in cancer and substrate for CRL4A-FBXW5–driven cancer growth

Lung adenocarcinoma originates from retrovirus infection of proliferating type 2 pneumocytes during pulmonary post-natal development or tissue repair

Jaagsiekte sheep retrovirus (JSRV) is a unique oncogenic virus with distinctive biological properties. JSRV is the only virus causing a naturally occurring lung cancer (ovine pulmonary adenocarcinoma, OPA) and possessing a major structural protein that functions as a dominant oncoprotein. Lung cancer is the major cause of death among cancer patients. OPA can be an extremely useful animal model in order to identify the cells originating lung adenocarcinoma and to study the early events of pulmonary carcinogenesis. In this study, we demonstrated that lung adenocarcinoma in sheep originates from infection and transformation of proliferating type 2 pneumocytes (termed here lung alveolar proliferating cells, LAPCs). We excluded that OPA originates from a bronchioalveolar stem cell, or from mature post-mitotic type 2 pneumocytes or from either proliferating or non-proliferating Clara cells. We show that young animals possess abundant LAPCs and are highly susceptible to JSRV infection and transformation. On the contrary, healthy adult sheep, which are normally resistant to experimental OPA induction, exhibit a relatively low number of LAPCs and are resistant to JSRV infection of the respiratory epithelium. Importantly, induction of lung injury increased dramatically the number of LAPCs in adult sheep and rendered these animals fully susceptible to JSRV infection and transformation. Furthermore, we show that JSRV preferentially infects actively dividing cell in vitro. Overall, our study provides unique insights into pulmonary biology and carcinogenesis and suggests that JSRV and its host have reached an evolutionary equilibrium in which productive infection (and transformation) can occur only in cells that are scarce for most of the lifespan of the sheep. Our data also indicate that, at least in this model, inflammation can predispose to retroviral infection and cancer

Development of an All-in-One Lentiviral Vector System Based on the Original TetR for the Easy Generation of Tet-ON Cell Lines

Lentiviral vectors (LVs) are considered one of the most promising vehicles to efficiently deliver genetic information for basic research and gene therapy approaches. Combining LVs with drug-inducible expression systems should allow tight control of transgene expression with minimal side effect on relevant target cells. A new doxycycline-regulated system based on the original TetR repressor was developed in 1998 as an alternative to the TetR-VP16 chimeras (tTA and rtTA) to avoid secondary effects due to the expression of transactivator domains. However, previously described TetR-based systems required cell cloning and/or antibiotic selection of tetracycline-responsive cells in order to achieve good regulation. In the present manuscript we have constructed a dual Tet-ON system based on two lentiviral vectors, one expressing the TetR through the spleen focus forming virus (SFFV) promoter (STetR) and a second expressing eGFP through the regulatable CMV-TetO promoter (CTetOE). Using these vectors we have demonstrated that the TetR repressor, contrary to the reverse transactivator (rtTA), can be expressed in excess to bind and modulate a high number of TetO operons. We have also showed that this dual vector system can generate regulatable bulk cell lines (expressing high levels of TetR) that are able to modulate transgene expression either by varying doxycycline concentration and/or by varying the amount of CTetOE vector genomes per cell. Based on these results we have developed a new all-in-one lentiviral vector (CEST) driving the expression of TetR through the SFFV promoter and the expression of eGFP through the doxycycline-responsive CMV-TetO operon. This vector efficiently produced Tet-ON regulatable immortalized (293T) and primary (human mesenchymal stem cells and human primary fibroblasts) cells. Bulk doxycycline-responsive cell lines express high levels of the transgene with low amount of doxycycline and are phenotypically indistinct from its parental cells

Rosiglitazone synergizes anticancer activity of cisplatin and reduces its nephrotoxicity in 7, 12-dimethyl benz{a}anthracene (DMBA) induced breast cancer rats

<p>Abstract</p> <p>Background</p> <p>Antineoplastic drug cisplatin remains the drug of choice for various solid tumours including breast cancer. But dose dependent nephrotoxicity is the major drawback in majority of platinum based chemotherapy regimens. Recent reports have shown that inflammatory pathways are the main offender for cisplatin induced nephrotoxicity. The present study was undertaken to assess the effect of rosiglitazone, a PPARγ agonist and an anti-inflammatory agent, on cisplatin induced nephrotoxicity, and its anticancer activity in DMBA induced breast cancer rats.</p> <p>Methods</p> <p>Mammary tumours were induced in female Sprague-Dawley rats by feeding orally with dimethylbenz [a]anthracene (DMBA) (60 mg/kg). Cisplatin induced nephropathy was assessed by measurements of blood urea nitrogen, albumin and creatinine levels. Posttranslational modifications of histone H3, mitogen-activated protein (MAP) kinase p38 expression and PPAR-γ expression were examined by western blotting.</p> <p>Results</p> <p>Our data shows involvement of TNF-α in preventing cisplatin induced nephrotoxicity by rosiglitazone. Rosiglitazone pre-treatment to cisplatin increases the expression of p38, PPAR-γ in mammary tumours and shows maximum tumour reduction. Furthermore, cisplatin induced changes in histone acetylation, phosphorylation and methylation of histone H3 in mammary tumours was ameliorated by pre-treatment of rosiglitazone. Suggesting, PPAR-γ directly or indirectly alters aberrant gene expression in mammary tumours by changing histone modifications.</p> <p>Conclusion</p> <p>To best of our knowledge this is the first report which shows that pre-treatment of rosiglitazone synergizes the anticancer activity of cisplatin and minimizes cisplatin induced nephrotoxicity in DMBA induced breast cancer.</p

CCAAT/Enhancer-Binding Protein γ Is a Critical Regulator of IL-1β-Induced IL-6 Production in Alveolar Epithelial Cells

CCAAT/enhancer binding protein γ (C/EBPγ) is a member of the C/EBP family of transcription factors, which lacks known activation domains. C/EBPγ was originally described as an inhibitor of C/EBP transactivation potential. However, previous study demonstrates that C/EBPγ augments the C/EBPβ stimulatory activity in lipopolysaccharide induction of IL-6 promoter in a B lymphoblast cell line. These data indicate a complexing functional role for C/EBPγ in regulating gene expression. Furthermore, the expression and function of C/EBPγ during inflammation are still largely unknown. In this study, we demonstrate that C/EBPγ activation was induced by IL-1β treatment in lung epithelial cells. Importantly, we demonstrate for the first time that C/EBPγ plays a critical role in regulating IL-1β-induced IL-6 expression in both mouse primary alveolar type II epithelial cells and a lung epithelial cell line, MLE12. We further provide the evidence that C/EBPγ inhibits IL-6 expression by inhibiting C/EBPβ but not NF-κB stimulatory activity in MLE12 cells. These findings suggest that C/EBPγ is a key transcription factor that regulates the IL-6 expression in alveolar epithelial cells, and may play an important regulatory role in lung inflammatory responses

Site of Allergic Airway Narrowing and the Influence of Exogenous Surfactant in the Brown Norway Rat

Background: The parameters RN (Newtonian resistance), G (tissue damping), and H (tissue elastance) of the constant phase model of respiratory mechanics provide information concerning the site of altered mechanical properties of the lung. The aims of this study were to compare the site of allergic airway narrowing implied from respiratory mechanics to a direct assessment by morphometry and to evaluate the effects of exogenous surfactant administration on the site and magnitude of airway narrowing. Methods: We induced airway narrowing by ovalbumin sensitization and challenge and we tested the effects of a natural surfactant lacking surfactant proteins A and D (InfasurfH) on airway responses. Sensitized, mechanically ventilated Brown Norway rats underwent an aerosol challenge with 5 % ovalbumin or vehicle. Other animals received nebulized surfactant prior to challenge. Three or 20 minutes after ovalbumin challenge, airway luminal areas were assessed on snap-frozen lungs by morphometry. Results: At 3 minutes, RN and G detected large airway narrowing whereas at 20 minutes G and H detected small airway narrowing. Surfactant inhibited RN at the peak of the early allergic response and ovalbumin-induced increase in bronchoalveolar lavage fluid cysteinyl leukotrienes and amphiregulin but not IgE-induced mast cell activation in vitro. Conclusion: Allergen challenge triggers the rapid onset of large airway narrowing, detected by RN and G, and subsequen

Purinergic signalling links mechanical breath profile and alveolar mechanics with the pro-inflammatory innate immune response causing ventilation-induced lung injury

Severe pulmonary infection or vigorous cyclic deformation of the alveolar epithelial type I (AT I) cells by mechanical ventilation leads to massive extracellular ATP release. High levels of extracellular ATP saturate the ATP hydrolysis enzymes CD39 and CD73 resulting in persistent high ATP levels despite the conversion to adenosine. Above a certain level, extracellular ATP molecules act as danger-associated molecular patterns (DAMPs) and activate the pro-inflammatory response of the innate immunity through purinergic receptors on the surface of the immune cells. This results in lung tissue inflammation, capillary leakage, interstitial and alveolar oedema and lung injury reducing the production of surfactant by the damaged AT II cells and deactivating the surfactant function by the concomitant extravasated serum proteins through capillary leakage followed by a substantial increase in alveolar surface tension and alveolar collapse. The resulting inhomogeneous ventilation of the lungs is an important mechanism in the development of ventilation-induced lung injury. The high levels of extracellular ATP and the upregulation of ecto-enzymes and soluble enzymes that hydrolyse ATP to adenosine (CD39 and CD73) increase the extracellular adenosine levels that inhibit the innate and adaptive immune responses rendering the host susceptible to infection by invading microorganisms. Moreover, high levels of extracellular adenosine increase the expression, the production and the activation of pro-fibrotic proteins (such as TGF-β, α-SMA, etc.) followed by the establishment of lung fibrosis

Management of high-risk corneal grafts

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