RESUMO
Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.
Assuntos
Aminoglicosídeos/farmacologia , Códon sem Sentido/genética , Ribossomos/metabolismo , Aminoglicosídeos/metabolismo , Aminoglicosídeos/fisiologia , Linhagem Celular , Quimiocinas CXC/efeitos dos fármacos , Quimiocinas CXC/metabolismo , Códon sem Sentido/metabolismo , Códon de Terminação , Gentamicinas/farmacologia , Humanos , Mutação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores da Síntese de Proteínas , Ribossomos/efeitos dos fármacosRESUMO
Nonsense mutations underlie about 10% of rare genetic disease cases. They introduce a premature termination codon (PTC) and prevent the formation of full-length protein. Pharmaceutical gentamicin, a mixture of several related aminoglycosides, is a frequently used antibiotic in humans that can induce PTC readthrough and suppress nonsense mutations at high concentrations. However, testing of gentamicin in clinical trials has shown that safe doses of this drug produce weak and variable readthrough activity that is insufficient for use as therapy. In this study we show that the major components of pharmaceutical gentamicin lack PTC readthrough activity but the minor component gentamicin B1 (B1) is a potent readthrough inducer. Molecular dynamics simulations reveal the importance of ring I of B1 in establishing a ribosome configuration that permits pairing of a near-cognate complex at a PTC. B1 induced readthrough at all three nonsense codons in cultured cancer cells with TP53 (tumor protein p53) mutations, in cells from patients with nonsense mutations in the TPP1 (tripeptidyl peptidase 1), DMD (dystrophin), SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like 1), and COL7A1 (collagen type VII alpha 1 chain) genes, and in an in vivo tumor xenograft model. The B1 content of pharmaceutical gentamicin is highly variable and major gentamicins suppress the PTC readthrough activity of B1. Purified B1 provides a consistent and effective source of PTC readthrough activity to study the potential of nonsense suppression for treatment of rare genetic disorders.
Assuntos
Antibacterianos/farmacologia , Códon sem Sentido/genética , Gentamicinas/farmacologia , Mutação/efeitos dos fármacos , Aminopeptidases/genética , Antibacterianos/química , Linhagem Celular Tumoral , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Distrofina/genética , Gentamicinas/química , Humanos , Serina Proteases/genética , Tripeptidil-Peptidase 1 , Proteína Supressora de Tumor p53/genéticaRESUMO
Nonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglycosides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations.
Assuntos
Aminoglicosídeos/farmacologia , Códon sem Sentido/genética , Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Alelos , Aminoglicosídeos/química , Doenças Genéticas Inatas/genética , Células HCT116 , Homozigoto , Humanos , Paromomicina/farmacologia , Ftalimidas/química , Ftalimidas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Fatores de Tempo , Tripeptidil-Peptidase 1 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Individual tumor cells utilize one of two modes of motility to invade the extracellular matrix, mesenchymal or amoeboid. We have determined that the diterpenoid genkwanine M (GENK) enhances the mesenchymal mode of cell motility that is intrinsic to HT-1080 osteosarcoma cells, stimulates a mesenchymal mode of motility in stationary MDA-MB-453 breast carcinoma cells, and induces a shift to a mesenchymal mode of cell motility in LS174T colorectal adenocarcinoma cells that normally utilize the alternate amoeboid mode of motility. The ability of GENK to stimulate or induce mesenchymal motility was preceded by a rapid cell spreading, elongation and polarization that did not require new gene expression. However, these initial morphologic changes were integrin dependent and they were associated with a reorganization of focal contacts and focal adhesions as well as an activation of the focal adhesion kinase. Therefore, GENK induces a mesenchymal mode of cell motility in a wide variety of tumor cell types that may be mediated, at least in part, by an activation of integrin-associated signaling.
Assuntos
Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Flavonas/farmacologia , Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Polaridade Celular , Flavonas/química , Adesões Focais , Humanos , Cadeias alfa de Integrinas/química , Cadeias beta de Integrinas/química , Invasividade Neoplásica/patologia , Osteossarcoma/química , Osteossarcoma/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Transcrição Gênica , Wikstroemia/químicaRESUMO
T cells show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to provide a renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains an important challenge. Here, we report an efficient serum- and feeder-free system for differentiating human PSCs into hematopoietic progenitors and T cells. This fully defined approach allowed us to study the impact of individual proteins on blood emergence and differentiation. Providing DLL4 and VCAM1 during the endothelial-to-hematopoietic transition enhanced downstream progenitor T cell output by ~80-fold. These two proteins synergized to activate notch signaling in nascent hematopoietic stem and progenitor cells, and VCAM1 additionally promoted an inflammatory transcriptional program. We also established optimized medium formulations that enabled efficient and chemically defined maturation of functional CD8αß+, CD4-, CD3+, TCRαß+ T cells with a diverse TCR repertoire.
RESUMO
Two synthetic approaches to the microtubule-stabilizing ceratamine alkaloids are described. The first approach involved attempts to graft an aminoimidazole moiety onto an azepine ring to form partially hydrogenated versions of the unprecedented aromatic imidazo[4,5-d]azepine core of the ceratamines. This route ultimately failed because it was not possible to aromatize the partially hydrogenated ceratamine intermediates. A second approach started with tribromoimidazole that was sequentially metalated and functionalized to efficiently generate a key imidazole intermediate containing vinyl bromide and amide functionalities. An intramolecular Buchwald vinyl amidation reaction converted this key intermediate into a bicyclic imidazo[4,5-d]azepine that was at the same oxidation state as the aromatic core of the ceratamines. The 2-amino functionality present on the imidazole ring of the ceratamines was installed using a Buchwald/Hartwig amination reaction on a 2-chloroimidazole precursor. Deprotection and aromatization resulted in the first synthesis of desbromoceratamine A (55) and desmethyldesbromoceratamine A (60). An unanticipated addition of atmospheric oxygen was encountered during deprotection of the imidazole ring in the last step of the synthesis leading to C-11 oxygenated ceratamine analogues as byproducts. Evaluation of the synthetic ceratamines in a TG3 cell-based assay for mitotic arrest revealed that the C-14 and C-16 bromine substituents in ceratamine A (1) play a major role in the antimitotic potency of the natural product. The synthetic route to ceratamine analogues has provided sufficient quantities of desbromoceratamine A (55) for testing in mouse models of cancer.
Assuntos
Alcaloides/síntese química , Azepinas/síntese química , Imidazóis/síntese química , Animais , Bromobenzenos/síntese química , Poríferos/químicaRESUMO
Research on the retention of women in academia has focused on challenges, including a "chilly climate," devaluation, and incivility. The unique consequences of workplace ostracism - being ignored and excluded by others in an organizational setting - require focus on this experience as another interpersonal challenge for women in academia. The purpose of this study is to examine differences in the faculty experiences and outcomes of workplace ostracism, and to determine if these experiences are affected significantly by the gender composition of an employee's specific department. Participants were recruited at two time points to complete campus climate surveys that were distributed to faculty at a large, public, research university. We examined the number of reported ostracism experiences (Study 1) and perceived information sharing (Study 2) among male and female university faculty. The findings indicated that female faculty members perceived more workplace ostracism than male faculty members. Analyses of department gender ratios suggested that the proportion of women in the department did not reduce the amount of workplace ostracism experienced by women. No gender differences were found in perceived information sharing. However, we found that Faculty of Color, both men and women, reported more frequent information exclusion than White faculty. These results have important implications for theoretical and practical understandings of workplace demography and suggest that it is necessary to look at subtle, ambiguous forms of discrimination in order to increase retention of faculty from underrepresented groups in academia.
RESUMO
BACKGROUND: Acidification of the cytoplasm and the extracellular environment is associated with many physiological and pathological conditions, such as intense exercise, hypoxia and tumourigenesis. Acidification affects important cellular functions including protein synthesis, growth, and proliferation. Many of these vital functions are controlled by mTORC1, a master regulator protein kinase that is activated by various growth-stimulating signals and inactivated by starvation conditions. Whether mTORC1 can also respond to changes in extracellular or cytoplasmic pH and play a role in limiting anabolic processes in acidic conditions is not known. METHODOLOGY/FINDINGS: We examined the effects of acidifying the extracellular medium from pH 7.4 to 6.4 on human breast carcinoma MCF-7 cells and immortalized mouse embryo fibroblasts. Decreasing the extracellular pH caused intracellular acidification and rapid, graded and reversible inhibition of mTORC1, assessed by measuring the phosphorylation of the mTORC1 substrate S6K. Fibroblasts deleted of the tuberous sclerosis complex TSC2 gene, a major negative regulator of mTORC1, were unable to inhibit mTORC1 in acidic extracellular conditions, showing that the TSC1-TSC2 complex is required for this response. Examination of the major upstream pathways converging on the TSC1-TSC2 complex showed that Akt signaling was unaffected by pH but that the Raf/MEK/ERK pathway was inhibited. Inhibition of MEK with drugs caused only modest mTORC1 inhibition, implying that other unidentified pathways also play major roles. CONCLUSIONS: This study reveals a novel role for the TSC1/TSC2 complex and mTORC1 in sensing variations in ambient pH. As a common feature of low tissue perfusion, low glucose availability and high energy expenditure, acidic pH may serve as a signal for mTORC1 to downregulate energy-consuming anabolic processes such as protein synthesis as an adaptive response to metabolically stressful conditions.
Assuntos
Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos , Fosforilação/genética , Fosforilação/fisiologia , Proteínas/genética , Transdução de Sinais/genética , Serina-Treonina Quinases TOR , Proteína 1 do Complexo Esclerose Tuberosa , Proteína 2 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Desbromoceratamine A (3) exhibits significantly less potent activity than the natural product ceratamine A (1) in a cell-based assay for antimitotic activity. Synthesis of the ceratamine A analogue 4 has shown that replacing the bromine atoms in the natural product with methyl groups generates an analogue that is more active than natural ceratamine A (1). Further enhancement of the antimitotic activity of the ceratamine pharmacophore has been achieved in the synthetic analogue 33, which has both bromine atoms replaced with methyl groups and an additional methyl substituent on the amino nitrogen at C-2. An efficient synthetic route has been developed to 33 that should enable the first in vivo evaluation of the new ceratamine microtubule-stabilizing pharmacophore and has provided several additional analogues for structure-activity relationship evaluation.
Assuntos
Alcaloides/farmacologia , Azepinas/farmacologia , Imidazóis/farmacologia , Moduladores de Tubulina/farmacologia , Alcaloides/química , Azepinas/síntese química , Azepinas/química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/síntese química , Imidazóis/química , Mitose/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/químicaRESUMO
Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to lead to protracted cell death.
Assuntos
Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Flavonas/farmacologia , Mitose/efeitos dos fármacos , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Cisteína/análogos & derivados , Cisteína/farmacologia , Humanos , Indóis/farmacologia , Microtúbulos/efeitos dos fármacos , Paclitaxel/farmacologia , Sulfonamidas/farmacologia , Triflupromazina/farmacologia , Vimblastina/farmacologiaRESUMO
The intracellular pathways by which inflammatory mediators transmit their angiogenic signals is not well studied. The effects of a potent inflammatory mediator, bacterial lipopolysaccharide (LPS), are transmitted through Toll-like receptors (TLRs). A major, although not exclusive, LPS/TLR intracellular signaling pathway is routed through TNF (tumor necrosis factor) receptor associated factor 6 (TRAF6). In this report we demonstrate that LPS directly stimulates endothelial sprouting in vitro. By blocking TRAF6 activity using retroviral expression of a dominant-negative TRAF6 in endothelial cells, we show that TRAF6 is absolutely required for the LPS-initiated angiogenic response in vitro and in vivo. Inhibition of either c-Jun N-terminal kinase (JNK) activity or nuclear factor kappaB (NF-kappaB) activity, downstream of TRAF6, is sufficient to inhibit LPS-induced endothelial sprouting. In contrast, only inhibition of NF-kappaB, but not JNK, activity blocks basic fibroblast growth factor (bFGF)-induced angiogenesis. Our findings thus demonstrate a direct endothelial-stimulatory role of LPS in initiating angiogenesis through activation of TRAF6-dependent signaling pathways.