We further demonstrate that the two compounds bind to ⌬N3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for ⌬N3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the ⌬N3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.
DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organspecific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.
The roles of DNA methyltransferase-2 (DNMT2) enzymes are controversial; whether DNMT2 functions primarily as a nuclear DNA methyltransferase or as a cytoplasmic tRNA methyltransferase, and whether DNMT2 activity impacts development, as dnmt2 mutant mice or Drosophila lack phenotypes. Here we show that morpholino knockdown of Dnmt2 protein in zebrafish embryos confers differentiation defects in particular organs, including the retina, liver, and brain. Importantly, proper organ differentiation required Dnmt2 activity in the cytoplasm, not in the nucleus. Furthermore, zebrafish Dnmt2 methylates an RNA species of ∼80 bases, consistent with tRNA methylation. Thus, Dnmt2 promotes zebrafish development, likely through cytoplasmic RNA methylation.Supplemental material is available at http://www.genesdev.org.
To detect targeted antileukemia agents we have designed a novel, high-content in vivo screen using genetically engineered, T-cell reporting zebrafish. We exploited the developmental similarities between normal and malignant T lymphoblasts to screen a small molecule library for activity against immature T cells with a simple visual readout in zebrafish larvae. After screening 26 400 molecules, we identified Lenaldekar (LDK), a compound that eliminates immature T cells in developing zebrafish without affecting the cell cycle in other cell types. LDK is well tolerated in vertebrates and induces longterm remission in adult zebrafish with cMYCinduced T-cell acute lymphoblastic leukemia (T-ALL). LDK causes dephosphorylation of members of the PI3 kinase/AKT/mTOR pathway and delays sensitive cells in late mitosis. Among human cancers, LDK selectively affects survival of hematopoietic malignancy lines and primary leukemias, including therapy-refractory B-ALL and chronic myelogenous leukemia samples, and inhibits growth of human T-ALL xenografts. This work demonstrates the utility of our method using zebrafish for antineoplastic candidate drug identification and suggests a new approach for targeted leukemia therapy. Although our efforts focused on leukemia therapy, this screening approach has broad implications as it can be translated to other cancer types involving malignant degeneration of developmentally arrested cells. (Blood. 2012;119(24):5621-5631) IntroductionThe yearly incidence in the US for all leukemia types, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myelogenous leukemia (CML), was estimated at more than 40 000 men and women in 2010, with a yearly death rate of 50%. 1 More than 2000 cases of ALL are diagnosed in US children every year, making it the most common childhood cancer. 2 T-cell ALL (T-ALL) represents approximately 15% and 25% of pediatric and adult ALL cases, respectively. 3 Although leukemia treatment has become increasingly efficient over the past 50 years, mortality from ALL is still 20% for children and more than 40% for adults, and T-ALL has been more difficult to treat than B-cell ALL (B-ALL). 4 Currently, research efforts are devoted to molecularbased risk stratification of patients and the development of targeted therapies to limit side effects [5][6][7] and to increase treatment efficacy.Development of targeted cancer therapies typically requires knowledge of the molecular target. 8 In the absence thereof, an alternative approach may use a robust readout designed to screen large numbers of compounds for specific effects 9 against the malignant cell type in question. More than 50% of patients with T-ALL have deregulated NOTCH1, 10 and in a recent study 47% had mutations in the PI3 kinase/AKT/mTOR (P/A/mT) pathway. 11 NOTCH1 signaling requires proteolytic cleavage by ␥-secretase and other proteases 12 to release the intracytoplasmic domain, providing severalpotential targets for therapeutic intervention. Targeted treatment approaches for T-ALL using...
Neutrophils respond to infection by degranulation, release of reactive oxygen intermediates, and secretion of chemokines and cytokines; however, activation of neutrophil transcriptional machinery has been little appreciated. Recent findings suggest that gene expression may represent an additional neutrophil function after exposure to lipopolysaccharide (LPS). We performed microarray gene expression analysis of 4,608 mostly nonredundant genes on LPS-stimulated human neutrophils. Analysis of three donors indicated some variability but also a high degree of reproducibility in gene expression. Twenty-eight verifiable, distinct genes were induced by 4 h of LPS treatment, and 13 genes were repressed. Genes other than cytokines and chemokines are regulated; interestingly, genes involved in cell growth regulation and survival, transcriptional regulation, and interferon response are among those induced, whereas genes involved in cytoskeletal regulation are predominantly repressed. In addition, we identified monocyte chemoattractant protein-1 as a novel LPS-regulated chemokine in neutrophils. Included in these lists are five clones with no defined function. These data suggest molecular mechanisms by which neutrophils respond to infection and indicate that the transcriptional potential of neutrophils is greater than previously thought.
Mutations in the human adenomatous polyposis coli (APC) gene are thought to initiate colorectal tumorigenesis. The tumor suppressor function of APC is attributed primarily to its ability to regulate the WNT pathway by targeting the destruction of -catenin. We report here a novel role for APC in regulating degradation of the transcriptional co-repressor C-terminalbinding protein-1 (CtBP1) through a proteasome-dependent process. Further, CtBP1 suppresses the expression of intestinal retinol dehydrogenases, which are required for retinoic acid production and intestinal differentiation. In support of a role for CtBP1 in initiation of colorectal cancer, adenomas taken from individuals with familial adenomatous polyposis contain high levels of CtBP1 protein in comparison with matched, uninvolved tissue. The relationship between APC and CtBP1 is conserved between humans and zebrafish and provides a mechanistic model explaining APC control of intestinal retinoic acid biosynthesis.Germline mutations in the adenomatous polyposis coli (APC) 2,3 tumor suppressor invariably result in familial adenomatous polyposis coli (FAP), a syndrome characterized by early onset colorectal cancer (1). The mechanism by which APC mutations cause colon tumorigenesis is attributed primarily to its role in negatively regulating canonical WNT signaling (2, 3). In this role, APC functions by targeting the transcriptional coactivator -catenin for intracellular degradation through a proteasome-dependent pathway, thereby limiting its ability to associate with T cell factor/lymphoid enhancer factor nuclear transcription factors. Current evidence indicates that following APC mutation, -catenin accumulates and translocates into the nucleus, where it partners with T cell factor/lymphoid enhancer factors to drive a program of cellular proliferation. As evidence for a genetic relationship between APC and WNT signaling, some studies cite the existence of rare, -cateninactivating mutations in colon adenocarcinomas (4, 5). Importantly, however, these mutations do not appear to fully recapitulate the clinical phenotypes associated with APC mutation (6). This discrepancy raises the possibility of additional, -cateninindependent functions for APC.A number of reports suggest that the functions of APC are not limited to its well established role in regulating canonical WNT signaling. For example, APC is reported to bind to microtubules, to regulate asymmetric cell division in Drosophila male germline stem cells, and to promote proper T-cell differentiation in mice (7-11). Further, we recently demonstrated that sporadic human colorectal carcinomas lack retinol dehydrogenases and that introduction of APC into human colon carcinoma cells lines induced the expression of the retinol dehydrogenase DHRS9 in a -catenin-independent manner (12). In addition, apc mcr zebrafish lack expression of intestinal enzymes, such as rdh1l, that are required for retinoic acid production. Injection of apc mcr zebrafish embryos with mRNA encoding rdh1l or treatment with exogenous...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.