Stable inhibition of human thymidylate synthase expression following retroviral introduction of an siRNA gene

Yang, Zhaogang; Cloud, Ashley S.; Hughes, D; Johnson, Lee F.

doi:10.1038/sj.cgt.7700880

Cited by 9 publications

(10 citation statements)

References 26 publications

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“…Of note, addition of 10 μM thymidine to the cell culture medium completely reversed the growth inhibitory effects after 10 nM ssiRNA/siRNA transfection (96% ± 5 vs LF2000-treated cells). This protective effect has been previously reported by us and others using the same siRNA sequence [4,18]. …”

Section: Resultssupporting

confidence: 86%

Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors

Schmitz

Chu

2011

Silence

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BackgroundSmall interfering RNAs (siRNAs) are double-stranded RNAs that effectively inhibit expression of its complimentary target mRNA. Standard siRNAs contain two nucleotide overhangs on their 3' end. While these overhangs are usually comprised of deoxythymidines (dT), it has been shown that any nucleotide can be used on the 3' end without affecting RNAi silencing.ResultsIt was recently shown that extension of the 3' end to five or eight dT molecules allows siRNAs to be effectively complexed with linear polyethylenimine (PEI), leading to enhanced cellular uptake and intracellular release. Here, we provide further evidence that only extended or 'sticky' siRNAs complexed with PEI result in significant target knockdown. However, when investigating the potential effects of these extended siRNAs on growth of human colon cancer RKO cells, we observed a dose-dependent reversal of cytotoxicity of a thymidylate synthase-targeted siRNA. In contrast, siRNAs with uridine overhangs maintained their growth inhibitory effects. We further demonstrated that dT-containing siRNAs prevented the cytotoxic effects of thymidylate synthase (TS) inhibitor compounds, such as ZD1694 and 5'-fluoro-deoxyuridine, while having no deleterious effect on cisplatin toxicity. We show that this rescue effect results from the rapid degradation of the siRNA.ConclusionsGiven that TS is an important enzyme for cell growth and proliferation and that its expression is controlled by multiple pathways, the rescue of its growth inhibitory effects may have unintended consequences. As siRNAs are being developed as therapeutic molecules, it will be important to avoid such off-target effects due to dT release. Hence, siRNAs should contain only uridine residues in their 3'-end overhangs.

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Section: Resultssupporting

confidence: 86%

Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors

Schmitz

Chu

2011

Silence

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“…TS, which converts dUMP to dTMP, is the only de novo source of intracellular thymidylate for DNA synthesis [30] . Inhibition of the enzyme results in chromosome breaks and cell death [31] .…”

Section: Discussionmentioning

confidence: 99%

Pretreatment with insulin enhances anticancer functions of 5-fluorou-racil in human esophageal and colonic cancer cells

Zou

Xie

2007

Acta Pharmacologica Sinica

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Aim: To investigate the effects of insulin on enhancing 5-fluorouracil (5-FU) anticancer functions and its mechanisms in the human esophageal cancer cell line (Eca 109) and human colonic cancer cell line (Ls-174-t). Methods: The effect of insulin/5-FU combination treatment on the growth of Eca 109 and Ls-174-t cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After insulin treatment or insulin/5-FU treatment, cell cycle distribution of both cell lines was analyzed by flow cytometry. Western blot assay was used to assess the expression of caspase-3 and thymidylate synthase (TS). Apoptosis was detected by flow cytometry, DNA fragmentation assay, and terminal transferase dUTP nick end labeling assay (TUNEL). Moreover, the changes of 5-FU uptake after insulin pretreatment were detected by HPLC assay and Western blot analysis. Results: We found that insulin enhanced the inhibitory effect of 5-FU on cell proliferation when Eca 109 cells and Ls-174-t cells were pretreated with insulin for the appropriate time. Insulin increased the cell number of the S phase and the uptake of 5-FU. Insulin/5-FU treatment enhanced apoptosis of tumor cells and upregulated the expression of cleaved caspase-3 compared with 5-FU treatment. Moreover, insulin/5-FU treatment induced the changes of free TS and the TS ternary complex level compared with 5-FU treatment in Eca 109 and Ls-174-t cells. Conclusion: These data suggest that insulin enhances anticancer functions of 5-FU when it is treated before 5-FU for the appropriate time in human esophageal and colonic cancer cell lines.

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“…Thus, TS-targeted drugs must be delivered at high doses and sustained over long periods of time to reach the sought-after goals of reducing thymidylate pools and promoting cell death. Also, the high stability of TS potentially constrains attempts to down-regulate enzyme synthesis via siRNA or other antisense strategies (49,50), as such down-regulation will occur slowly due to the enzyme long half-life. In all, improving the effectiveness of TS-targeted therapies will likely require destabilization of the protein.…”

Section: Discussionmentioning

confidence: 99%

The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway

Peña

Melo²,

Xing

et al. 2009

Journal of Biological Chemistry

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The ubiquitin-independent proteasomal degradation pathway is increasingly being recognized as important in regulation of protein turnover in eukaryotic cells. One substrate of this pathway is the pyrimidine biosynthetic enzyme thymidylate synthase (TS; EC 2.1.1.45), which catalyzes the reductive methylation of dUMP to form dTMP and is essential for DNA replication during cell growth and proliferation. Previous work from our laboratory showed that degradation of TS is ubiquitin-independent and mediated by an intrinsically disordered 27-residue region at the N-terminal end of the molecule. In the current study we show that this region, in cooperation with an ␣-helix formed by the next 15 residues, functions as a degron, i.e. it is capable of destabilizing a heterologous protein to which it is fused. Comparative analysis of the primary sequence of TS from a number of mammalian species revealed that the N-terminal domain is hypervariable among species yet is conserved with regard to its disordered nature, its high Pro content, and the occurrence of Pro at the penultimate site. Characterization of mutant proteins showed that Pro-2 protects the N terminus against N ␣ -acetylation, a post-translational process that inhibits TS degradation. However, although a free amino group at the N terminus is necessary, it is not sufficient for degradation of the polypeptide. The implications of these findings to the proteasome-targeting function of the N-terminal domain, particularly with regard to its intrinsic flexibility, are discussed.Regulated protein degradation within the cell is carried out primarily by the 26 S proteasome, a large 2-MDa complex consisting of several dozen proteins that function in recognizing and degrading its target substrates (1, 2). Typically, covalent attachment of polyubiquitin chains serves as the primary signal for target recognition by the proteasome (1, 2). However, in recent years several proteasomal substrates have been shown to be degraded without a requirement for ubiquitin modification (for a recent review, see Ref.3). Such substrates include ornithine decarboxylase (ODC) 3 (4 -6), c-Fos (7, 8), p21 Cip1 (9, 10), hepatitis virus F protein (11), and c-Jun (12), among others. Although the number of substrates identified as degraded by a ubiquitin-independent mechanism remains small, recent biochemical analyses indicate that the process may be more widespread than previously thought and contributes significantly to the regulation of protein turnover (13).Among the known substrates of the ubiquitin-independent degradation pathway, ODC has been the most studied. The degradation signal for ODC is composed of a disordered, flexible domain formed by a 37-residue region at the C terminus (4 -6). The region mediates docking of the ODC polypeptide to the proteasome and initiates its entry into the proteasomal chamber, where proteolysis proceeds in a C-to N-terminal direction (4 -6). This process is stimulated by an accessory protein termed antizyme, which binds ODC and increases the availability of i...

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Stable inhibition of human thymidylate synthase expression following retroviral introduction of an siRNA gene

Cited by 9 publications

References 26 publications

Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors

Effect of small interfering RNA 3'-end overhangs on chemosensitivity to thymidylate synthase inhibitors

Pretreatment with insulin enhances anticancer functions of 5-fluorou-racil in human esophageal and colonic cancer cells

The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway

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