ERG Functionally Overlaps with Other Ets Proteins in Promoting TH9 Cell Expression of Il9 during Allergic Lung Inflammation.

CD4+ TH cells develop into subsets that are specialized in the secretion of particular cytokines to mediate restricted types of inflammation and immune responses. Among the subsets that promote development of allergic inflammatory responses, IL-9-producing TH9 cells are regulated by a number of transcription factors. We have previously shown that the E26 transformation-specific (Ets) family members PU.1 and Ets translocation variant 5 (ETV5) function in parallel to regulate IL-9. In this study we identified a third member of the Ets family of transcription factors, Ets-related gene (ERG), that mediates IL-9 production in TH9 cells in the absence of PU.1 and ETV5. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during murine TH9 polarization. Knockdown or knockout of ERG during murine or human TH9 polarization in vitro led to a decrease in IL-9 production in TH9 cells. Deletion of ERG in vivo had modest effects on IL-9 production in vitro or in vivo. However, in the absence of PU.1 and ETV5, ERG was required for residual IL-9 production in vitro and for IL-9 production by lung-derived CD4 T cells in a mouse model of chronic allergic airway disease. Thus, ERG contributes to IL-9 regulation in TH9 cells.

Pretreatment Inflammation-Based Markers Predict Survival Outcomes in Patients with Early Stage Hepatocellular Carcinoma After Radiofrequency Ablation.

The prognostic significance of various systemic inflammation-based markers has been explored in different cancers after surgery. This study aimed to investigate whether these markers could predict outcomes in patients with early-stage hepatocellular carcinoma (HCC) undergoing radiofrequency ablation (RFA). One hundred eighteen patients with newly diagnosed HCC within the Milan criteria receiving RFA as initial therapy were retrospectively enrolled. Pretreatment inflammation-based markers including the neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR) and prognostic nutritional index (PNI), together with other clinicopathologic parameters were collected. Cumulative overall survival (OS) and recurrence-free survival (RFS) were estimated by the Kaplan-Meier method and by multivariate analysis using Cox proportional hazard model. The 1-, 3-, and 5-year OS rates of patients were 90%, 67%, and 52%, respectively. Kaplan-Meier curves showed that baseline high NLR ≥ 2.5 (p = 0.006), low PNI < 40 (p = 0.005), history of end-stage renal disease (ESRD) (p = 0.005), non-Child-Pugh class A (p = 0.001) and elevated alpha-fetoprotein (AFP) ≥ 200 ng/mL (p = 0.005) significantly associated with the poor OS, whereas high PLR ≥ 100 did not. By multivariate analysis, high NLR ≥ 2.5 (hazard ratio (HR) 1.94; 95% confidence interval (CI), 1.05-3.59; p = 0.034), low PNI < 40 (HR 0.38; 95% CI, 0.20-0.72; p = 0.003), ESRD history (HR 3.60; 95% CI, 1.48-8.76; p = 0.005) and elevated AFP ≥ 200 ng/mL (HR 4.61; 95% CI, 1.75-12.13; p = 0.002) were independent factors. An elevated AFP level of ≥200 ng/mL was the significant factor associated with intrahepatic new RFS by univariate and multivariate analyses. In conclusion, pretreatment NLR and PNI are simple and useful predictors for OS in patients with early-stage HCC after RFA.

Primary Prevention Using Cholesterol-Lowering Medications in Patients Meeting New Treatment Guidelines: A Retrospective Cohort Analysis.

BACKGROUND: The American College of Cardiology and American Heart Association (ACC/AHA) issued new cholesterol treatment guidelines in 2013. Two of the groups designated for primary prevention were analyzed: patients with a low-density lipoprotein cholesterol (LDL-C) level ≥ 190 mg per dL and diabetic patients aged 40-75 years. OBJECTIVE: To estimate the effects of primary prevention as specified in the 2013 guidelines on cardiovascular event risk and cost. METHODS: Primary prevention patients were identified using laboratory and diagnostic data for Humana members from 2007 to 2013. Potential study patients were classified into 3 risk groups: elevated LDL-C, diabetes, and elevated LDL-C and diabetes. Patients receiving cholesterol-lowering medications before their index date were excluded. Eligible patients were divided into 2 treatment groups: (1) primary prevention patients who initiated treatment before experiencing any cardiovascular disease (CVD)-related event, and (2) patients who either did not initiate treatment until after experiencing a CVD event or never initiated treatment. The associations between initiating cholesterol-lowering medications for primary prevention and the risk for acute myocardial infarction, stroke, coronary angioplasty, or coronary artery bypass graft surgery were estimated using Cox proportional hazards models. The effect of primary prevention on health care costs was estimated using generalized linear models. RESULTS: 91,066 patients met study selection criteria. Primary prevention rates were the lowest in diabetic patients (35%), who were newly designated for treatment in the 2013 guidelines. Primary prevention rates were higher for patients designated for treatment under earlier guidelines: 65% for patients with elevated LDL-C and 78% for the combined LDL-C and diabetes group. Primary prevention treatment was associated with significant reductions in cardiovascular event risk (up to 37%) and lower total all-cause costs (by $673) in the first post-index year. CONCLUSIONS: Initiating cholesterol-lowering medications for primary prevention, as specified in the ACC/AHA 2013 guidelines, for patients with high LDL-C and diabetes is associated with reduced CVD event risks and lower health care costs. DISCLOSURES: No outside funding supported this study. Han received fellowship support from the Pharmaceutical Research and Manufacturers Association Foundation (PhRMA) during the conduct of this study. Dougherty is employed by PhRMA. The authors have nothing to disclose.

Inferring relative numbers of human leucocyte genome replications.

Genome duplication inevitably results in replication errors. A priori, the more times a genome is copied, the greater the average number of replication errors. This principle could be used to 'count' mitotic divisions. Although somatic mutations are rare, cytosine methylation is also copied after DNA replication, but measurably increases with aging at certain CpG rich sequences in mitotic tissues, such as the colon. To further test whether such age-related methylation represents replication errors, these CpG rich 'clock' sequences were measured in leucocytes. Leucocytes within an individual have identical chronological ages (time since birth) but their mitotic ages (numbers of divisions since the zygote) may differ. Neutrophils, B-lymphocytes, and red cell progenitors are produced from relatively quiescent stem cells throughout life, but T-lymphocyte production largely ceases after puberty when the thymus disappears. However, T-lymphocyte genomes may continue to replicate throughout life in response to immunological stimulation. Consistent with this biology, clock methylation significantly increased with aging for T-lymphocyte genomes, but no significant increase was measured in other cell populations. Moreover, this methylation was greater in genomes isolated from their corresponding neoplastic populations. These studies tentatively support the hypothesis that methylation at certain CpG rich sequences in leucocytes could record their mitotic ages.

Persistent nicotine treatment potentiates amplification of the dihydrofolate reductase gene in rat lung epithelial cells as a consequence of Ras activation.

Although nicotine has been suggested to promote lung carcinogenesis, the mechanism of its action in this process remains unknown. The present investigation demonstrates that the treatment of rat lung epithelial cells with nicotine for various periods differentially mobilizes multiple intracellular pathways. Protein kinase C and phosphoinositide 3-OH-kinase are transiently activated after the treatment. Also, Ras and its downstream effector ERK1/2 are activated after long term exposure to nicotine. The activation of Ras by nicotine treatment is responsible for the subsequent perturbation of the methotrexate (MTX)-mediated G1 cell cycle restriction as well as an increase in production of reactive oxygen species. When p53 expression is suppressed by introducing E6, persistent exposure to nicotine enables dihydrofolate reductase gene amplification in the presence of methotrexate (MTX) and the formation of the MTX-resistant colonies. Altering the activity of phosphoinositide 3-OH-kinase has no effect on dihydrofolate reductase amplification. However, the suppression of protein kinase C dramatically affects the colony formation in soft agar. Thus, our data suggest that persistent exposure to nicotine perturbs the G1 checkpoint and causes DNA damage through the increase of the production of reactive oxygen species. However, a third element rendered by loss of p53 is required for the initiation of the process of gene amplification. Under p53-deficient conditions, the establishment of a full oncogenic transformation, in response to long term nicotine exposure, is achieved through the cooperation of multiple signaling pathways.

Long-term exposure to nicotine, via ras pathway, induces cyclin D1 to stimulate G1 cell cycle transition.

Nicotine, a major component in tobacco, has been implicated as a potential factor that promotes the development of lung cancer. However, the molecular mechanism of its action is still unclear. In this study, we have shown that, via nicotinic acetylcholine receptors, persistent exposure of mouse epithelial cells to nicotine elicits Ras signaling and subsequent Raf/MAP kinase activity, accompanied by a significant increase in cyclin D1 promoter activity and its protein expression. AP-1 is required for activation of the cyclin D1 promoter. The induction of cyclin D1 expression and its promoter activity by nicotine is abolished by the suppression of Raf/MAP kinase signaling. Furthermore, upon nicotine treatment, the cells do not arrest in the G(1) phase of the cell cycle following serum starvation. The perturbation of the G(1) cell cycle checkpoint is caused by the deregulation of retinoblastoma/E2F activity. Therefore, our data indicated that by targeting the Ras pathway, long-term exposure to nicotine disrupts cell cycle restriction machinery and thus potentiates tumor development.

A p53-independent G1 cell cycle checkpoint induced by the suppression of protein kinase C alpha and theta isoforms.

The protein kinase C (PKC) family consists of multiple isoforms that are involved in the regulation of diverse cellular responses. Suppression of PKC induces growth arrest in various types of cells. However, the underlying molecular mechanisms have not been thoroughly investigated. In this report, we demonstrated that the concurrent inhibition, rather than separate inhibition, of phorbol ester-dependent PKC alpha and theta isoforms is crucial for the induction of G1 cell cycle arrest and that this negative cell cycle regulation is via p53-independent mechanisms. PKC suppression-mediated growth arrest is associated with the induction of cell cycle inhibitor p21WAF1/CIP1 and the occurrence of hypophosphorylated Rb. The G1 checkpoint induced by the suppression of PKC occurs not only in murine Swiss3T3 but also in p53-deficient cells and human lung cancer cells containing mutated p53. Luciferase and nuclear run-off assays demonstrated that p21WAF1/CIP1 is, in part, transcriptionally regulated in response to the suppression of PKC alpha and theta. However, the stability of p21 mRNA is also augmented after the addition of PKC alpha and theta antisense oligonucleotides, indicating the involvement of post-transcriptional mechanisms in p21WAF1/CIP1 expression. These data suggest the existence of a cell cycle checkpoint pathway regulated by PKC alpha and theta isoforms. Furthermore, our findings support the notion that G1 checkpoint control can be restored in tumor cells containing abnormal p53, by targeting the PKC-regulated p21WAF1/CIP1 induction.