Bmi1 is required for tumorigenesis in a mouse model of intestinal cancer

The epigenetic regulator BMI1 is upregulated progressively in a wide variety of human tumors including colorectal cancer. In this study, we assessed the requirement for Bmi1 in intestinal tumorigenesis using an autochthonous mouse model in which Apc was conditionally ablated in the intestinal epithelium. Germline mutation of Bmi1 significantly reduced both the number and size of small intestinal adenomas arising in this model, and it acted in a dose-dependent manner. Moreover, in contrast to wild-type controls, Bmi1[superscript −/−] mice showed no increase in median tumor size, and a dramatic decrease in tumor number, between 3 and 4 months of age. Thus, Bmi1 is required for both progression and maintenance of small intestinal adenomas. Importantly, Bmi1 deficiency did not disrupt oncogenic events arising from Apc inactivation. Instead, the Arf tumor suppressor, a known target of Bmi1 epigenetic silencing, was upregulated in Bmi1 mutant tumors. This was accompanied by significant upregulation of p53, which was confirmed by sequencing to be wild-type, and also elevated apoptosis within the smallest Bmi1[superscript −/−] adenomas. By crossing Arf into this cancer model, we showed that Arf is required for the induction of both p53 and apoptosis, and it is a key determinant of the ability of Bmi1 deficiency to suppress intestinal tumorigenesis. Finally, a conditional Bmi1 mutant strain was generated and used to determine the consequences of deleting Bmi1 specifically within the intestinal epithelium. Strikingly, intestinal-specific Bmi1 deletion suppressed small intestinal adenomas in a manner that was indistinguishable from germline Bmi1 deletion. Thus, we conclude that Bmi1 deficiency impairs the progression and maintenance of small intestinal tumors in a cell autonomous and highly Arf-dependent manner.Virginia and D.K. Ludwig Fund for Cancer ResearchNational Science Foundation (U.S.)National Cancer Institute (U.S.

Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1

Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are creditedThe impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe 3H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. 3H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 was measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on 3H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in 3H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.Peer reviewe

Immune Responses to RHAMM in Patients with Acute Myeloid Leukemia after Chemotherapy and Allogeneic Stem Cell Transplantation

Leukemic blasts overexpress immunogenic antigens, so-called leukemia-associated antigens like the receptor for hyaluronan acid-mediated motility (RHAMM). Persistent RHAMM expression and decreasing CD8+ T-cell responses to RHAMM in the framework of allogeneic stem cell transplantation or chemotherapy alone might indicate the immune escape of leukemia cells. In the present study, we analyzed the expression of RHAMM in 48 patients suffering from acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Furthermore, we correlated transcripts with the clinical course of the disease before and after treatment. Real-time quantitative reverse transcriptase polymerase chain reaction was performed from RNA of peripheral blood mononuclear cells. T cell responses against RHAMM were assessed by tetramer staining (flow cytometry) and enzyme-linked immunospot (ELISPOT) assays. Results were correlated with the clinical outcome of patients. The results of the present study showed that almost 60% of the patients were RHAMM positive; specific T-cells recognizing RHAMM could be detected, but they were nonfunctional in terms of interferon gamma or granzyme B release as demonstrated by ELISPOT assays. Immunotherapies like peptide vaccination or adoptive transfer of RHAMM-specific T cells might improve the immune response and the outcome of AML/MDS patients

High Rates of Nicotine Use Relapse and Ulcer Development Following Roux-en-Y Gastric Bypass

PURPOSE: Given that smoking is known to contribute to gastrojejunal anastomotic (GJA) ulcers, cessation is recommended prior to laparoscopic Roux-en-Y gastric bypass (LRYGB). However, smoking relapse rates and the exact ulcer risk remain unknown. This study aimed to define smoking relapse, risk of GJA ulceration, and complications after LRYGB. MATERIALS AND METHODS: We performed a retrospective cohort study of patients who underwent primary LRYGB during 2011-2015. Initially, three patient categories were identified: lifetime non-smokers, patients who were smoking during the initial visit at the bariatric clinic or within the prior year (recent smokers), and patients who had ceased smoking more than a year prior to their initial clinic visit (former smokers). Smoking relapse, GJA ulcer occurrences, reinterventions, and reoperations were recorded and compared. RESULTS: A total of 766 patients were included in the analysis. After surgery, 53 (64.6%) recent smokers had resumed smoking. Out of these relapsed smokers, 51% developed GJA ulcers compared with 14.8% in non-relapsed recent smokers, 16.1% in former smokers, and 6% in lifetime nonsmokers (p < 0.001). Furthermore, relapsed smokers required more frequently endoscopic reinterventions (60.4%) compared with non-relapsed smokers (20.8%, p < 0.001), former smokers (20.7%, p < 0.001), and lifetime non-smokers (15.4%, p < 0.001). Additionally, relapsed smokers required a reoperation (18.9%) more often than non-relapsed recent smokers (5.7%, p < 0.001) and lifetime non-smokers (1.3%, p < 0.001). CONCLUSION: Smokers relapse frequently after LRYGB, and the majority experience GJA complications. They should be counseled about this risk preoperatively and directed towards less ulcerogenic procedures when possible. Alternatively, longer periods of preoperative smoking abstinence might be needed

How are Bariatric Patients Coping During the COVID-19 Pandemic? Analysis of Factors Known to Cause Weight Regain Among Postoperative Bariatric Patients

Background The global coronavirus disease 2019 (COVID-19) pandemic is wreaking havoc on society. Bariatric patients are more prone to severe infection due to their high body mass index (BMI) and are more vulnerable to the effects of isolation, such as depression or disruption of their health habits. Objectives To quantify the impact of self-quarantine on bariatric patients and self-quarantine’s relationship with weight gain. Setting Academic hospital, United States. Methods A 30-item survey examining several known contributors to weight regain was distributed among the postoperative bariatric patients of our clinic. Changes in eating habits, exercise, depression, social support, loneliness, and anxiety were studied, among others. Results A total of 208 patients completed the survey (29.3% response rate). A large percentage of patients reported increases in their depression (44.2%), loneliness (36.2%), nervousness (54.7%), snacking (62.6%), loss of control when eating (48.2%), and binge eating (19.5%) and decreases in their social support (23.2%), healthy food eating (45.5%), and activity (55.2%). Difficulty in accessing vitamins was reported by 13%. Patients more than 18 months out of surgery regained more than 2 kg during an average of 47 days. Risk factors for weight regain were found to be loss of control when eating, increases in snacking and binge eating, reduced consumption of healthy food, and reduced physical activity. Conclusion Bariatric patients are negatively affected by the COVID-19 pandemic and subsequent social isolation on many levels. This patient population is vulnerable to crisis situations; thus, additional intervention is needed to address behaviors that lead to weight regain

Rollover cyclometalation vs nitrogen coordination in Tetrapyridyl Anticancer Gold(III) complexes: effect on protein interaction and toxicity

In this work, a pair of gold(III) complexes derived from the analogous tetrapyridyl ligands H(2)biqbpy1 and H(2)biqbpy2 was prepared: the rollover, bis-cyclometalated [Au(biqbpy1)Cl ([1]Cl) and its isomer [Au(biqbpy2)Cl ([2]Cl). In [1](+), two pyridyl rings coordinate to the metal via a Au-C bond ((CNNC)-N-boolean AND-N-boolean AND-C-boolean AND coordination) and the two noncoordinated amine bridges of the ligand remain protonated, while in [2](+) all four pyridyl rings of the ligand coordinate to the metal via a Au-N bond ((NNNN)-N-boolean AND-N-boolean AND-N-boolean AND coordination), but both amine bridges are deprotonated. As a result, both complexes are monocationic, which allowed comparison of the sole effect of cyclometalation on the chemistry, protein interaction, and anticancer properties of the gold(III) compounds. Due to their identical monocationic charge and similar molecular shape, both complexes [1]Cl and [2]Cl displaced reference radioligand [H-3]dofetilide equally well from cell membranes expressing the K(v)11.1 (hERG) potassium channel, and more so than the tetrapyridyl ligands H(2)biqbpy1 and H(2)biqbpy2. By contrast, cyclometalation rendered [1]Cl coordinatively stable in the presence of biological thiols, while [2]Cl was reduced by a millimolar concentration of glutathione into metastable Au(I) species releasing the free ligand H(2)biqbpy2 and TrxR-inhibiting Au+ ions. The redox stability of [1]Cl dramatically decreased its thioredoxin reductase (TrxR) inhibition properties, compared to [2]Cl. On the other hand, unlike [2]Cl, [1]Cl aggregated into nanoparticles in FCS-containing medium, which resulted in much more efficient gold cellular uptake. [1]Cl had much more selective anticancer properties than [2]Cl and cisplatin, as it was almost 10 times more cytotoxic to human cancer cells (A549, A431, A375, and MCF7) than to noncancerous cells (MRC5). Mechanistic studies highlight the strikingly different mode of action of the two compounds: while for [1]Cl high gold cellular uptake, nuclear DNA damage, and interaction with hERG may contribute to cell killing, for [2]Cl extracellular reduction released TrxR-inhibiting Au+ ions that were taken up in minute amounts in the cytosol, and a toxic tetrapyridyl ligand also capable of binding to hERG. These results demonstrate that bis-cyclometalation is an appealing method to improve the redox stability of Au(III) compounds and to develop gold-based cytotoxic compounds that do not rely on TrxR inhibition to kill cancer cells.Medicinal ChemistryMetals in Catalysis, Biomimetics & Inorganic Material

P2Y₁₂-dependent activation of hematopoietic stem and progenitor cells promotes emergency hematopoiesis after myocardial infarction

Emergency hematopoiesis is the driving force of the inflammatory response to myocardial infarction (MI). Increased proliferation of hematopoietic stem and progenitor cells (LSK) after MI enhances cell production in the bone marrow (BM) and replenishes leukocyte supply for local cell recruitment to the infarct. Decoding the regulation of the inflammatory cascade after MI may provide new avenues to improve post-MI remodeling. In this study, we describe the influence of adenosine diphosphate (ADP)-dependent P2Y12-mediated signaling on emergency hematopoiesis and cardiac remodeling after MI. Permanent coronary ligation was performed to induce MI in a murine model. BM activation, inflammatory cell composition and cardiac function were assessed using global and platelet-specific gene knockout and pharmacological inhibition models for P2Y12. Complementary in vitro studies allowed for investigation of ADP-dependent effects on LSK cells. We found that ADP acts as a danger signal for the hematopoietic BM and fosters emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. We were able to detect P2Y12 in LSK, implicating a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 knockout and P2Y12 inhibitor treatment with prasugrel reduced emergency hematopoiesis and the excessive inflammatory response to MI, translating to lower numbers of downstream progeny and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition preserved cardiac function and reduced chronic adverse cardiac remodeling after MI. P2Y12-dependent signaling is involved in emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel, non-canonical value for P2Y12 antagonists beyond inhibition of platelet-mediated atherothrombosis

Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels

Aims Macrophages (MΦ), known for immunological roles, such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrioventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterize passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM. Methods and results We combined classic electrophysiological approaches with 3D florescence imaging, RNA-sequencing, pharmacological interventions, and computer simulations. We used Cx3creYFP/+1 mice wherein cardiac MΦ are fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2±0.1 GΩ (all data mean±SEM), capacitance 18.3±0.1 pF, resting membrane potential −39.6±0.3 mV, and several voltage-activated, outward or inwardly rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, and DIDS), flow cytometry, immuno-staining, and RNA-sequencing, we identified Kv1.3, Kv1.5, and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarize resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1. Conclusion Our results provide a first electrophysiological characterization of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ–CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources

Downregulation of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular carcinoma and their prognostic significance

<p>Abstract</p> <p>Background</p> <p>Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates and detoxification of xenobiotics and chemotherapeutics. The transporters became pharmaceutically interesting, because OCTs are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. No data exist about the relevance of OCTs in hepatocellular carcinoma (HCC).</p> <p>Methods</p> <p>OCT1 (<it>SLC22A1</it>) and OCT3 (<it>SLC22A3</it>) mRNA expression was measured in primary human HCC and corresponding non neoplastic tumor surrounding tissue (TST) by real time PCR (n = 53). Protein expression was determined by western blot analysis and immunofluorescence. Data were correlated with the clinicopathological parameters of HCCs.</p> <p>Results</p> <p>Real time PCR showed a downregulation of <it>SLC22A1 </it>and <it>SLC22A3 </it>in HCC compared to TST (p ≤ 0.001). A low <it>SLC22A1 </it>expression was associated with a worse patient survival (p < 0.05). Downregulation was significantly associated with advanced HCC stages, indicated by a higher number of T3 tumors (p = 0.025) with a larger tumor diameter (p = 0.035), a worse differentiation (p = 0.001) and higher AFP-levels (p = 0.019). In accordance, <it>SLC22A1 </it>was less frequently downregulated in tumors with lower stages who underwent transarterial chemoembolization (p < 0.001) and liver transplantation (p = 0.001). Tumors with a low <it>SLC22A1 </it>expression (< median) showed a higher <it>SLC22A3 </it>expression compared to HCC with high <it>SLC22A1 </it>expression (p < 0.001). However, there was no significant difference in tumor characteristics according to the level of the <it>SLC22A3 </it>expression.</p> <p>In the western blot analysis we found a different protein expression pattern in tumor samples with a more diffuse staining in the immunofluorescence suggesting that especially OCT1 is not functional in advanced HCC.</p> <p>Conclusion</p> <p>The downregulation of OCT1 is associated with tumor progression and a worse patient survival.</p