A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells

Nichols, Ralph C.; Botson, John; Wang, Xiao Wei; Hamilton, Byron B.; Collins, Jane; Uribe, Victoria; Brooks, S. E. H.; Zan, Moe; Rigby, William F.C.

doi:10.1007/s12033-010-9360-8

Cited by 7 publications

(5 citation statements)

References 31 publications

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“…4h, i and 6e, f). This is consistent with previous studies on AREs in cancers [14,30,59,[97][98][99][100]. These data suggest that phospho-TTP level or TTP activity is an important regulator of inflammatory response-mediated chemoresistance, which can be harnessed as a marker and target against AML resistance.…”

Section: Discussionsupporting

confidence: 92%

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells

et al. 2020

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Background: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. Results:We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo.Conclusions: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.

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Section: Discussionsupporting

confidence: 92%

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells

et al. 2020

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“…Like VEGF, the tumor necrosis factor and glucose transporter-1 genes are regulated by a 3′UTR-dependent mechanism controlled by AURE [5,23,24]. To determine if AUF1-RGG peptides affect other AURE-regulated genes, AUF1-RGG stable cells were transfected with TNF full-length 3′UTR (TNF-FL) or GLUT1 full-length 3′UTR (GLUT1-FL) luciferase reporter plasmids.…”

Section: Resultsmentioning

confidence: 99%

“…A central mediator of the innate immune response is the macrophage cell [1]. Macrophages are activated by bacterial cell wall components and by endogenous cytokines, including vascular endothelial growth factor (VEGF) [2,3] and TNF [4,5]. Both VEGF and the macrophage play a critical role in inflammatory processes including sepsis [6], synovial joint inflammation [7], atherosclerosis [8], tumorigenesis [9], corneal neovascularization [10], and nephritis [11].…”

Section: Introductionmentioning

confidence: 99%

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1)

2013

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The macrophage is essential to the innate immune response, but also contributes to human disease by aggravating inflammation. Under severe inflammation, macrophages and other immune cells over-produce immune mediators, including vascular endothelial growth factor (VEGF). The VEGF protein stimulates macrophage activation and induces macrophage migration. A natural inhibitor of VEGF, the soluble VEGF receptor (sFlt-1) is also produced by macrophages and sFlt-1 has been used clinically to block VEGF. In macrophages, we have shown that the mRNA regulatory protein AUF1/hnRNP D represses VEGF gene expression by inhibiting translation of AURE-regulated VEGF mRNA. Peptides (AUF1-RGG peptides) that are modeled on the arginine- glycine-glycine (RGG) motif in AUF1 also block VEGF expression. This report shows that the AUF1-RGG peptides reduce two other AURE-regulated genes, TNF and GLUT1. Three alternative splice variants of sFlt-1 contain AURE in their 3′UTR, and in an apparent paradox, AUF1-RGG peptides stimulate expression of these three sFlt-1 variants. The AUF1-RGG peptides likely act by distinct mechanisms with complimentary effects to repress VEGF gene expression and over-express the endogenous VEGF blocking agent, sFlt-1. The AUF1-RGG peptides are novel reagents that reduce VEGF and other inflammatory mediators, and may be useful tools to suppress severe inflammation.

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“…Regulation by mRNA-binding proteins on 3′ UTR elements is analyzed by introducing the 3′ UTR from regulated mRNA (VEGF, GLUT1, tumor necrosis factor-α [TNF]) into the 3′ UTR of reporter genes (firefly luciferase or green fluorescent protein; Griffin et al , 2004 ; Du et al , 2006 ; Nichols et al , 2010 ). In this study, the 3′ UTR of VEGF was placed in the 3′ UTR of a luciferase reporter, as described previously ( Du et al , 2006 ).…”

Section: Discussionmentioning

confidence: 99%

AUF1/hnRNP D represses expression of VEGF in macrophages

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A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells

Cited by 7 publications

References 31 publications

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells

A post-transcriptional program of chemoresistance by AU-rich elements and TTP in quiescent leukemic cells

AUF1-RGG peptides up-regulate the VEGF antagonist, soluble VEGF receptor-1 (sFlt-1)

AUF1/hnRNP D represses expression of VEGF in macrophages

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