The 1p-Encoded Protein Stathmin and Resistance of Malignant Gliomas to Nitrosoureas

Ngo, Teri T.B.; Peng, Tien; Lü, Xing; Akeju, Oluwaseun; Pastorino, Sandra; Zhang, Wei; Kotliarov, Yuri; Zenklusen, Jean C.; Fine, Howard A.; Maric, Dragan; Wen, Patrick Y.; Girolami, Umberto De; Black, Peter M.; Wu, Wells W.; Shen, Rong; Jeffries, Neal; Kang, Dong Won; Park, John K.

doi:10.1093/jnci/djk135

Cited by 50 publications

(41 citation statements)

References 40 publications

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“…[15][16][17] STMN1 knockdown also produced an additive to synergistic interaction with chemotherapeutic agents such as the taxanes. [18][19][20] The bi-sh-STMN1 incorporates two stem-loop structures in an expression construct promoting both cleavagedependent and cleavage-independent RNA-induced silencing complex (RISC) assemblies thereby generating RISC-associated mature effector small RNAs with multiple independent gene silencing activities. On the basis of the observation that miRNAs are associated with both non-nucleolytic (Ago1, 3,4) and nucleolytic Ago2-containing RISC and siRNAs with the nucleolytic Ago2, the novel bifunctional strategy specifically promotes the loading of miRNA-like effector molecules onto the cleavage-independent RISC as well as the accumulation of siRNA effector molecules by the cleavagedependent (Ago2 containing) RISC.…”

Section: Introductionmentioning

confidence: 99%

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

et al. 2010

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RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

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Section: Introductionmentioning

confidence: 99%

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

et al. 2010

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“…The contention was further supported by the demonstration that stathmin knockdown increased the sensitivity of glioma cells in vitro and in vivo [11]. Whether a single modification at the most sensitive lysine or modification at multiple lysine residues in the proteins is required to confer chemosensitivity of 1p −/+ gliomas remains to be elucidated.…”

Section: Discussionmentioning

confidence: 97%

“…We have previously conducted proteomic profiling on two types of malignant glioma cell lines, one with normal chromosome 1p (1p +/+ ) and resistant to nitrosourea chemotherapy and the other missing a region of chromosome 1p (1p +\− ) and sensitive to nitrosourea chemotherapy. We showed that stathmin was significantly down-regulated in the 1p +\− cells [11]. Since stathmin gene is localized at chromosome 1p36.11, we suspected that a decrease in stathmin protein might account for sensitivity of 1p +/− gliomas to chemotherapy with nitrosourea derivatives such as CCNU (lomustine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-urea) or BCNU (1,3-bis (2-chloroethyl)-1-nitrosourea).…”

Section: Introductionmentioning

confidence: 83%

“…His-tagged human stathmin protein was prepared as previously described [11]. Briefly, the nucleotide sequence 5′-CACCACCATCACCATCATTAA-3′, which encodes for six histidines, was inserted at the 3′ end to replace the stop codon of a full-length human stathmin cDNA (Origene Technologies; Rockville, MD).…”

Section: Expression and Purification Of His-tagged Human Stathminmentioning

confidence: 99%

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Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides

Wang

Liang

et al. 2008

Biochemical and Biophysical Research Communications

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Chemical modification of proteins is often carried out to generate protein-small molecule conjugates for various applications. The high resolution and mass accuracy of a Fourier transform mass spectrometer is particularly useful for assessing the extent or sites of covalent modifications. As protein-small molecule reactions often produce products with variable numbers of the compound incorporated at different sites, a direct mass analysis of the reaction products at times yields mass spectra hard to interpret. Chromatographic separation at protein level could reduce the complexity of a sample, thus allowing more accurate mass spectrometric analysis. In this report, we demonstrate the utility of reversed-phase protein chromatography and FT-ICR mass spectrometry in analyzing CCNU (lomustine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitroso-urea, MW: 233.7 Da) modification of stathmin. With this combined approach, we determined the stoichiometry as well as sites of CCNU incorporation into the protein, demonstrating differential reactivity of several lysyl residues to CCNU alkylation.

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“…Further, overexpression of SIRT2 resulted in perturbation of microtubule network and reduced colony formation suggesting SIRT2 may act as tumor suppressor in glioma. Ngo et al, used a 2D-DIGE based proteomic approach to identify differences in protein expression between two glioma cells which differ in their chromosome 1p status (Ngo et al, 2007). The rational for this study is that 1p+/-anaplastic oligodendroglioma patients respond better to chemotherapeutic agents like procarbazine, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and vincristine.…”

Section: Glioma Tumor Tissue and Cell Line Based Studiesmentioning

confidence: 99%

Glioma Proteomics: Methods and Current Perspective

Somasundaram¹,

Nijaguna²,

Kumar³

2011

Brain Tumors - Current and Emerging Therapeutic Strategies

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The 1p-Encoded Protein Stathmin and Resistance of Malignant Gliomas to Nitrosoureas

Abstract: Loss of heterozygosity for the stathmin gene may be associated with improved outcomes of patients with 1p+/- anaplastic oligodendroglioma tumors.

Cited by 50 publications

References 40 publications

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

Covalent modification of stathmin by CCNU determined by FTMS analysis of modified proteins and tryptic peptides

Glioma Proteomics: Methods and Current Perspective

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