Differential p38-dependent signalling in response to cellular stress and mitogenic stimulation in fibroblasts

p38 MAP kinase is known to be activated by cellular stress finally leading to cell cycle arrest or apoptosis. Furthermore, a tumour suppressor role of p38 MAPK has been proposed. In contrast, a requirement of p38 for proliferation has also been described. To clarify this paradox, we investigated stress- and mitogen-induced p38 signalling in the same cell type using fibroblasts. We demonstrate that - in the same cell line - p38 is activated by mitogens or cellular stress, but p38-dependent signalling is different. Exposure to cellular stress, such as anisomycin, leads to a strong and persistent p38 activation independent of GTPases. As a result, MK2 and downstream the transcription factor CREB are phosphorylated. In contrast, mitogenic stimulation results in a weaker and transient p38 activation, which upstream involves small GTPases and is required for cyclin D1 induction. Consequently, the retinoblastoma protein is phosphorylated and allows G1/S transition. Our data suggest a dual role of p38 and indicate that the level and/or duration of p38 activation determines the cellular response, i.e either proliferation or cell cycle arrest

Expression Profiling of CYP1B1 in Oral Squamous Cell Carcinoma: Counterintuitive Downregulation in Tumors

Oral Squamous Cell Carcinoma (OSCC) has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy. CYP1B1, a member of cytochrome P450 family, has been implicated in chemical carcinogenesis. There exists a general accordance that this protein is overexpressed in a variety of cancers, making it an ideal candidate for a prodrug therapy. The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits. This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This report therefore clearly demonstrates the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers and also suggest the possibility of a prophylactic therapy for oral cancer

Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Toxicology and Applied Pharmacology 254 (2011): 1-7, doi:10.1016/j.taap.2011.04.015.This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-­‐induced oxidative stress. In the 1-­‐100 nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2 nM [3H]2, 3, 7, 8-­‐tetrachloro-­‐dibenzo-­‐p-­‐dioxin from rainbow trout AHR2α. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50 days of feeding 3 μg of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-­‐O-­‐deethylase (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50 days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3 days and remained elevated for up to 28 days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50 days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14 days of feeding both PAHs that was not significant after 50 days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-­‐ nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28 days. There was no clear association between AHR2α affinity and BaP and BeP-­‐induced oxidative stress.The Oregon Agricultural Experiment Station, Northwest Fisheries Science Center, and RO1ES006272 from the National Institute of Health supported this work

A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

<p>Abstract</p> <p>Background</p> <p>Allergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power.</p> <p>Results</p> <p>We have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization.</p> <p>Conclusions</p> <p>A gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.</p

Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology

More stories on Th17 cells

For more than two decades, immunologists have been using the so-called Th1/Th2 paradigm to explain most of the phenomena related to adaptive immunity. the Th1/Th2 paradigm implied the existence of two different, mutually regulated, CD4(+) T helper subsets: Th1 cells, driving cell-mediated immune responses involved in tissue damage and fighting infection against intracellular parasites; and Th2 cells that mediate IgE production and are particularly involved in eosinophilic inflammation, allergy and clearance of helminthic infections. A third member of the T helper set, IL-17-producing CD4(+) T cells, now called Th17 cells, was recently described as a distinct lineage that does not share developmental pathways with either Th1 or Th2 cells. the Th17 subset has been linked to autoimmune disorders, being able to produce IL-17, IL-17F and IL-21 among other inflammatory cytokines. Interestingly, it has been reported that there is not only a cross-regulation among Th1, Th2 and Th17 effector cells but there is also a dichotomy in the generation of Th17 and T regulatory cells. Therefore, Treg and Th17 effector cells arise in a mutually exclusive fashion, depending on whether they are activated in the presence of TGF-beta or TGF-beta plus inflammatory cytokines such as IL-6. This review will address the discovery of the Th17 cells, and recent progress on their development and regulation.Crohn's and Colitis Foundation of AmericaNIHLa Jolla Inst Allergy & Immunol, La Jolla, CA 92037 USAUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilNIH: RO1 AI050265-06Web of Scienc

Glutathione S-transferase T1 and M1 gene defects in ovarian carcinoma

Glutathione S-transferases (GSTs) M1 and T1 are known to be polymorphic in humans. Both polymorphisms are due to gene deletions, which are responsible for the existence of null genotypes. The gene defect of GSTT1 has been reported to be associated with an increased risk of myelodysplastic syndromes, astrocytoma and meningioma. A lack of GSTM1 was associated with tobacco smoke-induced lung and bladder cancer. In this study we examined whether the GSTT1 and/or GSTM1 homozygous null genotypes were associated with an increased risk of ovarian cancer using a multiplex polymerase chain reaction protocol. The GSTT1 null genotype was observed in 14% of the control subjects that had never suffered from neoplastic disease (n = 115) and in 16% of the patients affected with ovarian cancer (n = 103, OR 0.87, 95% CI 0.39-1.92, P = 0.73). A lack of GSTM1 was observed in 38% of the control subjects and in 46% of the patients (OR 0.77, 95% CI 0.44-1.32). This difference was not significant (P = 0.34). Similarly, no significant differences were obtained if GSTT1 and/or GSTM1 null genotypes were analyzed in subgroups of control subjects and ovarian cancer patients between the ages of 20-40, 41-70 and 71-90 years and in individuals with a positive family history of neoplastic disease. GSTT1 and/or GSTM1 null genotypes were not significantly associated with the histologic type and grade or FIGO (International Federation of Gynecology and Obstetrics) stages of the ovarian carcinomas. In conclusion, GSTT1 and/or GSTM1 null genotypes are not markers for an increased risk of ovarian cancer