Widespread distribution of bisphenol-A (BPA) complicates epidemiological studies of possible carcinogenic effects on the breast because there are few unexposed controls. To address this challenge, we ...previously developed non-cancerous human high-risk donor breast epithelial cell (HRBEC) cultures, wherein BPA exposure could be controlled experimentally. BPA consistently induced activation of the mammalian target of rapamycin (mTOR) pathway--accompanied by dose-dependent evasion of apoptosis and increased proliferation--in HRBECs from multiple donors. Here, we demonstrate key molecular changes underlying BPA-induced cellular reprogramming. In 3/3 BPA-exposed HRBEC cell lines, and in T47D breast cancer cells, proapoptotic negative regulators of the cell cycle (p53, p21(WAF1) and BAX) were markedly reduced, with concomitant increases in proliferation-initiating gene products (proliferating cell nuclear antigen, cyclins, CDKs and phosphorylated pRb). However, simultaneous exposure to BPA and the polyphenol, curcumin, partially or fully reduced the spectrum of effects associated with BPA alone, including mTOR pathway proteins (AKT1, RPS6, pRPS6 and 4EBP1). BPA exposure induced an increase in the ERα (Estrogen Receptor): ERβ ratio--an effect also reversed by curcumin (analysis of variance, P < 0.02 for all test proteins). At the functional level, concurrent curcumin exposure reduced BPA-induced apoptosis evasion and rapid growth kinetics in all cell lines to varying degrees. Moreover, BPA extended the proliferation potential of 6/6 primary finite-life HRBEC cultures--another effect reduced by curcumin. Even after removal of BPA, 1/6 samples maintained continuous growth--a hallmark of cancer. We show that BPA exposure induces aberrant expression of multiple checkpoints that regulate cell survival, proliferation and apoptosis and that such changes can be effectively ameliorated.
Objectives
To assess the accuracy and interobserver agreement of tele‐ultrasonography for the diagnosis of gastrointestinal obstruction in small animals by radiologists with different levels of ...experience.
Materials and Methods
A retrospective cross‐sectional study including dogs and cats admitted with gastrointestinal signs, between 2017 and 2019, that had abdominal ultrasonographic (US) examination performed and images saved for review. Patients were classified into two categories based on final diagnosis: animals with or without complete or partial gastrointestinal obstruction. Observers with four experience levels interpreted the archived ultrasound examinations, simulating a tele‐ultrasonography consultation. Analyses of accuracy, sensitivity, specificity, positive and negative predictive values were obtained for each observer for detection of gastrointestinal obstruction. Agreement between observers for the gastrointestinal obstruction diagnosis was assessed using Fleiss's Kappa statistics.
Results
Ninety patients with gastrointestinal signs were included. Of these, 23 of 90 had partial or complete gastrointestinal obstruction. Interpretation of the images by observers via tele‐ultrasonography showed intervals of accuracy, sensitivity, specificity, positive and negative predictive values, respectively, of 78.9% to 87.8%, 73.9% to 100%, 77.6% to 89.6%, 55.9% to 70.8% and 90.9% to 100% for diagnosis of gastrointestinal obstruction. Agreement for the gastrointestinal obstruction diagnosis across all reviewers was moderate (Kappa 0.6).
Clinical Significance
Tele‐ultrasonography had good accuracy for detection of gastrointestinal obstruction, however had a rather low positive predictive value and only moderate interobserver agreement. Therefore, this technique should be used with caution in this clinical context, given the potential surgical decision at hand.
But L’éducation thérapeutique des diabétiques de type 2 peut être pratiquée de plusieurs façons avec des acteurs différents. Notre but a été d’évaluer l’impact d’une d’éducation de groupe dispensées ...au sein d’un réseau ville-hôpital de prise en charge du diabète de type 2 sur l’acquisition des connaissances sur la maladie. Patients et Méthodes L’éducation se compose de 3 séances collectives de 5 à 10 participants, d’un atelier cuisine et d’une sortie supermarché. Elles sont proposées aux patients adhérents du réseau en sus de consultations individuelles avec une diététicienne ou une infirmière d’éducation. Un auto-questionnaire de 23 items et 3 parties (généralités, suivi et complications, diététique), a été adressé à l’ensemble des patients du réseau. Le niveau de connaissance a été défini comme le pourcentage de bonnes réponses obtenues. Résultats Le taux de retour s’élevait à 38,4 % des 440 questionnaires envoyés. Le niveau de connaissance de 86 patients ayant bénéficié d’une ou plusieurs séances d’éducation collective (SE +) a été comparés à celui de 76 patients n’ayant pas souhaité y assister (SE-). Les deux populations étaient comparables sur l’âge (65/62,4 ans), le sexe masculin (62/52 %), l’ancienneté de la maladie (15/ 12 ans) et le traitement par insuline (33 %/30 %). Les patients SE + ont obtenu de meilleurs résultats que le groupe SE- pour le score global et les 3 parties du test (généralités, suivi et complications, et diététique) avec respectivement 68 % vs 55 %, 76 % vs 71 %, 63 % vs 53 %, et 67 % vs 63 % (p < 0,001). Nous avons ajusté sur deux facteurs qui étaient liés au critère de jugement, l’âge et le niveau d’étude. Les patients plus jeunes ont obtenu de meilleurs résultats et les patients avec un niveau d’étude supérieur étaient plus performants. Après ajustement les patients SE + obtenaient encore de meilleurs résultats. Conclusion Notre travail a permis de mettre en valeur l’intérêt d’associer des séances collectives et individuelles dans l’éducation thérapeutique proposée par le réseau. Il a également montré l’intérêt de composer des groupes homogènes en terme d’âge et de niveau d’étude pour diffuser des informations mieux ciblées avec un meilleur impact.
In late mitosis and early G1, replication origins are licensed for subsequent use by loading complexes of the minichromosome maintenance proteins 2-7 (Mcm2-7). The number of Mcm2-7 complexes loaded ...onto DNA greatly exceeds the number of replication origins used during S phase, but the function of the excess Mcm2-7 is unknown. Using Xenopus laevis egg extracts, we show that these excess Mcm2-7 complexes license additional dormant origins that do not fire during unperturbed S phases because of suppression by a caffeine-sensitive checkpoint pathway. Use of these additional origins can allow complete genome replication in the presence of replication inhibitors. These results suggest that metazoan replication origins are actually comprised of several candidate origins, most of which normally remain dormant unless cells experience replicative stress. Consistent with this model, using Caenorhabditis elegans, we show that partial RNAi-based knockdown of MCMs that has no observable effect under normal conditions causes lethality upon treatment with low, otherwise nontoxic, levels of the replication inhibitor hydroxyurea.
Activation of the p53 tumour suppressor protein can lead to cell-cycle arrest or apoptosis. p53 function is controlled by the mdm2 oncogene product, which targets p53 for proteasomal degradation. In ...this report we demonstrate that Mdm2 induces translation of the p53 mRNA from two alternative initiation sites, giving full-length p53 and another protein with a relative molecular mass (Mr) of approximately 47K; we designate this protein as p53/47. This translation induction requires Mdm2 to interact directly with the nascent p53 polypeptide. The alternatively translated p53/47 does not contain the Mdm2-binding site and it lacks the most amino-terminal transcriptional-activation domain of p53. Increased expression of p53/47 stabilizes p53 in the presence of Mdm2, and alters the expression levels of p53-induced gene products. These results show how the interaction of Mdm2 with p53 leads to a change in the ratio of full-length p53 to p53/47 by inducing translation of both p53 proteins and the subsequent selective degradation of full-length p53. Thus, Mdm2 controls the expression levels of p53 through a dual mechanism that involves induction of synthesis and targeting for degradation.
Colorectal cancer (CRC) is the most common gastrointestinal malignancy. Most of the clinical data on CRC prevention have come from the use of aspirin. Besides inhibition of cyclooxygenases, aspirin ...has a diversity of molecular effects that counteract colon carcinogenesis. Aspirin restrains cell proliferation by inducing a G1 arrest in colorectal cells. To determine which cell cycle checkpoint pathways are involved in this response, colorectal cell lines wild-type or defective for p53 and p21Waf1/Cip1 were treated with aspirin or the anti-proliferative drug sulindac sulfide, then assayed for proliferative activity, for cell cycle progression and apoptosis, for the activation and phosphorylation of checkpoint components and for the transcriptional up-regulation of p21Waf1/Cip1 and Bax. Aspirin and sulindac sulfide induced a G1 arrest within 48 h. While all cell lines responded in a comparable way to sulindac sulfide, the aspirin-induced G1 arrest was dependent on p21Waf1/Cip1—as cells lacking the cyclin-dependent kinase inhibitor failed to show this arrest—and on ataxia-telangiectasia-mutated kinase (ATM)—as the inhibitor caffeine abrogated the checkpoint. Moreover, aspirin induced cell death mainly in cells expressing p53. Aspirin induced the phosphorylation of p53 at residue Ser15 within 8 h in a caffeine-dependent manner, and also caused the activation of checkpoint kinase 2 and the cleavage of caspase 7. Our results suggest that aspirin induces a G1 arrest and apoptosis by activating p53 and p21Waf1/Cip1 in an ATM-dependent way. By activating these checkpoint pathways, aspirin may restrain uncontrolled proliferation of colorectal cells, enhance their response to stresses such as DNA damage and promote entry of abnormal cells into apoptosis.
Objective: Chronic inflammation in ulcerative colitis is associated with increased risk for colorectal cancer. Its molecular pathway of cancer development is poorly understood. We investigated the ...role of neutrophil-derived cellular stress in an in vitro model of neutrophils as effectors and colon epithelial cells as targets, and tested for changes in cell cycle distribution and the appearance of replication errors. Design: Colon epithelial cells with different mismatch repair phenotypes were co-cultured with activated neutrophils. Target cells were analysed for cell cycle distribution and replication errors by flow cytometry. Changes in nuclear and DNA-bound levels of mismatch repair- and checkpoint-related proteins were analysed by western blotting. Results: Activated neutrophils cause an accumulation of target cells in G2/M, consistent with the installation of a DNA-damage checkpoint. Cells that do not express hMSH2, p53 or p21waf1/cip1 failed to undergo the G2/M arrest. Phosphorylation of p53 at site Ser15 and Chk1 at Ser317, as well as accumulation of p21waf1/cip1, was observed within 8–24 h. Superoxide dismutase and catalase were unable to overcome this G2/M arrest, possibly indicating that neutrophil products other than superoxide or H2O2 are involved in this cellular response. Finally, exposure to activated neutrophils increased the number of replication errors. Conclusions: By using an in vitro co-culture model that mimics intestinal inflammation in ulcerative colitis, we provide molecular evidence for an hMSH2-dependent G2/M checkpoint arrest and for the presence of replication errors.
In embryonic stem cells, promoters of key lineage-specific differentiation genes are found in a bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them ...poised for transcription upon loss of H3K27me3. Whether cancer-initiating cells (C-ICs) have similar epigenetic mechanisms that prevent lineage commitment is unknown. Here we show that colorectal C-ICs (CC-ICs) are maintained in a stem-like state through a bivalent epigenetic mechanism. Disruption of the bivalent state through inhibition of the H3K27 methyltransferase EZH2, resulted in decreased self-renewal of patient-derived C-ICs. Epigenomic analyses revealed that the promoter of Indian Hedgehog (IHH), a canonical driver of normal colonocyte differentiation, exists in a bivalent chromatin state. Inhibition of EZH2 resulted in de-repression of IHH, decreased self-renewal, and increased sensitivity to chemotherapy in vivo. Our results reveal an epigenetic block to differentiation in CC-ICs and demonstrate the potential for epigenetic differentiation therapy of a solid tumour through EZH2 inhibition.
Estrogenic overstimulation is carcinogenic to the human breast. Personal care products (PCPs) commonly contain xenoestrogens (XE), such as parabens and phthalates. Here, we identified the adverse ...effects of persistent exposure to such PCPs directly within human estrogen responsive breast tissue of subjects enrolled in a regimen of reduced XE use (REDUXE). Pre- and post-intervention fine needle aspirates (FNAs) of the breast were collected from healthy volunteers who discontinued the use of paraben and phthalate containing PCPs over a 28 d period. Based on high-dimensional gene expression data of matched FNA pairs of study subjects, we demonstrate a striking reversal of cancer-associated phenotypes, including the PI3K-AKT/mTOR pathway, autophagy, and apoptotic signaling networks within breast cells of REDUXE compliant subjects. These, and other altered phenotypes were detected together with a significant reduction in urinary parabens and phthalate metabolites. Moreover, in vitro treatment of paired FNAs with 17β-estradiol (E2), displayed a ‘normalizing’ impact of REDUXE on gene expression within known E2-modulated pathways, and on functional endpoints, including estrogen receptor alpha: beta ratio, and S-phase fraction of the cell cycle. In a paradigm shifting approach facilitated by community-based participatory research, REDUXE reveals unfavorable consequences from exposure to XEs from daily-use PCPs. Our findings illustrate the potential for REDUXE to suppress pro-carcinogenic phenotypes at the cellular level towards the goal of breast cancer prevention.
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•The REDUXE intervention reveals the in vivo molecular impact of xenoestrogens within healthy human tissue.•Substitution with paraben and phthalate-free product use reverses pro carcinogenic gene expression profiles.•Estrogen-mediated functional signaling is normalized within post-REDUXE breast cells of participants.
Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology ...target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.
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