scholarly journals Effect of spermine synthase on the sensitivity of cells to anti-tumour agents

2003 ◽  
Vol 373 (3) ◽  
pp. 885-892 ◽  
Author(s):  
Yoshihiko IKEGUCHI ◽  
Caroline A. MACKINTOSH ◽  
Diane E. McCLOSKEY ◽  
Anthony E. PEGG
Keyword(s):  
Cell Growth Rate ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Polyamine Analogues ◽  
Spermine Synthase ◽  
Site Of Action ◽  
Cross Links ◽  
Sites Of Action ◽  
Paired Cell

The role of spermine in the sensitivity of cells to various established and experimental anti-tumour agents was examined, using paired cell lines that possess or lack spermine synthase. All spermine-synthase-deficient cells had no detectable spermine, and elevated spermidine, content. Spermine content did not alter the cell growth rate. There was little or no difference in sensitivity of immortalized mouse embryonic fibroblasts to doxorubicin, etoposide, cisplatin, methylglyoxal bis(guanylhydrazone) or H2O2 and only a slight increase in sensitivity to vinblastine and nocodazole. However, the absence of spermine clearly increased the sensitivity to 1,3-bis(2-chloroethyl)-N-nitrosourea, suggesting that depletion of spermine may be a useful way to increase the anti-neoplastic effects of anti-tumour agents that form chloroethyl-mediated interstrand DNA cross-links. The effects of spermine on the response to polyamine analogues (which have been proposed to be useful anti-neoplastic agents) were complex, and depended on the compound examined and on the cells tested. Sensitivity to CHENSpm {N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane} was substantially greater in immortalized fibroblasts that lack spermine. In contrast, BE-3-4-3 [N1,N12-bis(ethyl)spermine] and BE-3-3-3 [N1,N11-bis(ethyl)norspermine] were more active against cells that contained spermine. The presence of spermine correlated with a greater induction of spermidine/spermine-N1-acetyltransferase by BE-3-3-3, which is consistent with suggestions that this induction is important for the response to this drug. These findings support the concepts that different polyamine analogues have different sites of action and that CHENSpm has a different site of action from BE-3-3-3.

scholarly journals Critical Role of PDE4D in β2-Adrenoceptor-dependent cAMP Signaling in Mouse Embryonic Fibroblasts

2008 ◽  
Vol 283 (33) ◽  
pp. 22430-22442 ◽  
Author(s):  
Matthew D. Bruss ◽  
Wito Richter ◽  
Kathleen Horner ◽  
S.-L. Catherine Jin ◽  
Marco Conti
Keyword(s):  
Critical Role ◽  
Camp Signaling ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts

scholarly journals Quantitative Proteome Analysis of Atg5-Deficient Mouse Embryonic Fibroblasts Reveals the Range of the Autophagy-Modulated Basal Cellular Proteome

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Kiran Bala Sharma ◽  
Manish Sharma ◽  
Suruchi Aggarwal ◽  
Amit Kumar Yadav ◽  
Shinjini Bhatnagar ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Inflammatory Response ◽  
Transforming Growth Factor ◽  
Proteome Analysis ◽  
Interferon Response ◽  
Type I ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
The Impact

ABSTRACT Basal autophagy is crucial for maintenance of cellular homeostasis. ATG5 is an essential protein for autophagosome formation, and its depletion has been extensively used as a tool to disrupt autophagy. Here, we characterize the impact of Atg5 deficiency on the cellular proteome of mouse embryonic fibroblasts (MEFs). Using a tandem mass tagging (TMT)-based quantitative proteomics analysis, we observe that 14% of identified proteins show dysregulated levels in atg5−/− MEFs. These proteins were distributed across diverse biological processes, such as cell adhesion, development, differentiation, transport, metabolism, and immune responses. Several of the upregulated proteins were receptors involved in transforming growth factor β (TGF-β) signaling, JAK-STAT signaling, junction adhesion, and interferon/cytokine-receptor interactions and were validated as autophagy substrates. Nearly equal numbers of proteins, including several lysosomal proteins and enzymes, were downregulated, suggesting a complex role of autophagy/ATG5 in regulating their levels. The atg5−/− MEFs had lower levels of key immune sensors and effectors, including Toll-like receptor 2 (TLR2), interferon regulatory factor 3 (IRF3), IRF7, MLKL, and STAT1/3/5/6, which were restored by reexpression of ATG5. While these cells could efficiently mount a type I interferon response to the double-stranded RNA (dsRNA) mimic poly(I·C), they were compromised in their inflammatory response to the bacterial pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and Pam3CSK4. Transcriptional activation and secretion of interleukin-6 (IL-6) in these cells could be recovered by ATG5 expression, supporting the role of autophagy in the TLR2-induced inflammatory response. This study provides a key resource for understanding the effect of autophagy/ATG5 deficiency on the fibroblast proteome. IMPORTANCE Autophagy performs housekeeping functions for cells and maintains a functional mode by degrading damaged proteins and organelles and providing energy under starvation conditions. The process is tightly regulated by the evolutionarily conserved Atg genes, of which Atg5 is one such crucial mediator. Here, we have done a comprehensive quantitative proteome analysis of mouse embryonic fibroblasts that lack a functional autophagy pathway (Atg5 knockout). We observe that 14% of the identified cellular proteome is remodeled, and several proteins distributed across diverse cellular processes with functions in signaling, cell adhesion, development, and immunity show either higher or lower levels under autophagy-deficient conditions. These cells have lower levels of crucial immune proteins that are required to mount a protective inflammatory response. This study will serve as a valuable resource to determine the role of autophagy in modulating specific protein levels in cells.

scholarly journals TIMELESS mutation alters phase responsiveness and causes advanced sleep phase

2019 ◽  
pp. 201819110 ◽  
Author(s):  
Philip Kurien ◽  
Pei-Ken Hsu ◽  
Jacy Leon ◽  
David Wu ◽  
Thomas McMahon ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Molecular Clock ◽  
Mutant Mice ◽  
Circadian Period ◽  
Negative Regulators ◽  
Mouse Embryonic Fibroblasts ◽  
Sleep Phase ◽  
Embryonic Fibroblasts ◽  
Light Entrainment

Many components of the circadian molecular clock are conserved from flies to mammals; however, the role of mammalian Timeless remains ambiguous. Here, we report a mutation in the human TIMELESS (hTIM) gene that causes familial advanced sleep phase (FASP). Tim CRISPR mutant mice exhibit FASP with altered photic entrainment but normal circadian period. We demonstrate that the mutation prevents TIM accumulation in the nucleus and has altered affinity for CRY2, leading to destabilization of PER/CRY complex and a shortened period in nonmature mouse embryonic fibroblasts (MEFs). We conclude that TIM, when excluded from the nucleus, can destabilize the negative regulators of the circadian clock, alter light entrainment, and cause FASP.

scholarly journals Essential Role of Cyclin-G–associated Kinase (Auxilin-2) in Developing and Mature Mice

2008 ◽  
Vol 19 (7) ◽  
pp. 2766-2776 ◽  
Author(s):  
Dong-won Lee ◽  
Xiaohong Zhao ◽  
Yang-In Yim ◽  
Evan Eisenberg ◽  
Lois E. Greene
Keyword(s):  
Essential Gene ◽  
Inducible Promoter ◽  
Cre Recombinase ◽  
Conditional Knockout ◽  
Mouse Embryonic Fibroblasts ◽  
Coated Pits ◽  
Embryonic Fibroblasts ◽  
Adult Mice ◽  
Cyclin G

Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.

scholarly journals <i>Gsta</i>4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity

2013 ◽  
Vol 02 (01) ◽  
pp. 1-11 ◽  
Author(s):  
Kevin E. McElhanon ◽  
Chhanda Bose ◽  
Rajendra Sharma ◽  
Liping Wu ◽  
Yogesh C. Awasthi ◽  
...  
Keyword(s):  
Null Mouse ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Enhanced Sensitivity

scholarly journals Translocation of CrkL to Focal Adhesions Mediates Integrin-Induced Migration Downstream of Src Family Kinases

2003 ◽  
Vol 23 (8) ◽  
pp. 2883-2892 ◽  
Author(s):  
Leiming Li ◽  
Deborah L. Guris ◽  
Masaya Okura ◽  
Akira Imamoto
Keyword(s):  
G Proteins ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Adapter Protein ◽  
Related Protein ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Src Signaling ◽  
Downstream Mediator

ABSTRACT The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130 Cas (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.

scholarly journals HIF-1 regulates hypoxia- and insulin-induced expression of apelin in adipocytes

2007 ◽  
Vol 293 (6) ◽  
pp. E1590-E1596 ◽  
Author(s):  
Alexander J. Glassford ◽  
Patrick Yue ◽  
Ahmad Y. Sheikh ◽  
Hyung J. Chun ◽  
Shirin Zarafshar ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Cobalt Chloride ◽  
Hypoxia Inducible Factor ◽  
Metabolic Responses ◽  
Quantitative Real Time Pcr ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Targeted Deletion ◽  
Hypoxia Inducible Factor 1

Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O2 environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1α gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CoCl2) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl2-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1α-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia- and insulin-induced apelin expression.

scholarly journals Cdk2 and Cdk4 Activities Are Dispensable for Tumorigenesis Caused by the Loss of p53

2009 ◽  
Vol 29 (10) ◽  
pp. 2582-2593 ◽  
Author(s):  
V. C. Padmakumar ◽  
Eiman Aleem ◽  
Cyril Berthet ◽  
Mary Beth Hilton ◽  
Philipp Kaldis
Keyword(s):  
Cyclin D ◽  
P53 Mutations ◽  
Spontaneous Tumors ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Oncogenic Potential ◽  
Cyclin Dependent Kinases ◽  
Cell Cycle Inhibitors

ABSTRACT The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G1 deregulation or a combination of these effects. In order to determine the role of proteins involved in G1 control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G1/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2 − / − p53 − / − mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53 − / − mice. In contrast, the genotypes Cdk4 − / − p53 − / − were mostly lethal, with few exceptions, and Cdk2 − / − Cdk4 − / − p53 − / − mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53 − / −, Cdk2 − / − p53 − / −, Cdk4 − / − p53 − / −, and Cdk2 − / − Cdk4 − / − p53 − / − MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.

The role of COX-2 in mediating the effect of PTEN on BMP9 induced osteogenic differentiation in mouse embryonic fibroblasts

Biomaterials ◽  
2014 ◽  
Vol 35 (36) ◽  
pp. 9649-9659 ◽  
Author(s):  
Jun Huang ◽  
Shuang-Xue Yuan ◽  
Dong-Xu Wang ◽  
Qiu-Xiang Wu ◽  
Xing Wang ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Cox 2
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