GeneBlocs Are Powerful Tools to Study and Delineate Signal Transduction Processes That Regulate Cell Growth and Transformation

Sternberger, Maria; Schmiedeknecht, Anett; Kretschmer, Anny; Gebhardt, Frank; Leenders, Frauke; Czauderna, Frank; Carlowitz, Ira von; Engle, Mike; Giese, Klaus; Beigelman, Leonid; Klippel, Anke

doi:10.1089/108729002760220734

Cited by 34 publications

(31 citation statements)

References 40 publications

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“…This antisense RNA:DNA hybrid oligonucleotides recruit RNaseH to the Smad2 or Smad3 target mRNA, causing its degradation. Several lines of evidence have indicated already that this RNaseH-mediated antisense strategy is a valid and alternative approach for siRNA-mediated depletion of gene products (15,25). Moreover, we have validated some of our results obtained with GBs also with the siRNA-based approach and the same results were obtained using either tool, except for the effect of Smad2 depletion on MMP-9 gene induction by TGFβ.…”

Section: Discussionsupporting

confidence: 83%

“…In our experiments with Nme cells, the most discernible responses to TGFβ began to occur 48 to 72 hours after treatment, but the changes in cell morphology could already be detected 24 hours after stimulation (data not shown). To carry out transient knockdown of Smads in Nme cells, modified single-stranded AS-ONs (called GBs) were used, which induce RNaseH-mediated degradation of target Smad transcripts (15). As a negative control in each experiment, cells were transfected with control oligonucleotides, which do not induce Smad mRNA degradation [mismatch (MM) control].…”

Section: Specific Knockdown Of Smad2 and Smad3 Using Gbsmentioning

confidence: 99%

“…We mainly used a transient loss-of-function approach using GeneBlocs (GB), which are antisense oligonucleotides (AS-ON) of the third generation (15). These hybrid DNA:RNA and 21 nucleotide-long AS-ONs hybridize to their target mRNA and induce its degradation by RNaseH.…”

Section: Introductionmentioning

confidence: 99%

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Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

Dzwonek

Preobrazhenska

Cazzola

et al. 2009

Molecular Cancer Research

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Smad2 and Smad3 are intracellular mediators of transforming growth factor β (TGFβ) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFβ as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFβ-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFβ-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFβ-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein. (Mol Cancer Res 2009;7(8):1342-53)

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Section: Discussionsupporting

confidence: 83%

Section: Specific Knockdown Of Smad2 and Smad3 Using Gbsmentioning

confidence: 99%

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Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

Dzwonek

Preobrazhenska

Cazzola

et al. 2009

Molecular Cancer Research

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“…Under the conditions used the transfection efficiency was optimized to be at least 95% (Sternberger et al, 2002). Three different GB molecules (A, B, C) were selected for each target and further tested for efficacy in a dose-response experiment.…”

Section: Gb Treatment Ofmentioning

confidence: 99%

“…The need for an unbiased approach in such a comparative study is reflected by the observation that Smad2-and Smad3-deficient mouse embryo fibroblasts exhibit disturbances in basal expression levels of target genes as well as in their basal proliferation rates compared to wild-type cells (Piek et al, 2001). In this report, we have therefore used improved antisense molecules, the so-called GeneBlocs (GBs) (Sternberger et al, 2002), to inhibit selectively expression of Smad2, Smad3 and Smad4 in parallel in two differentiationcompetent human cell lines, HaCaT keratinocytes and MCF-10A breast epithelial cells. Both are immortalized, nontransformed human cell lines that respond to TGF-b (Boukamp et al, 1988;Soule et al, 1990;Game et al, 1992;Basolo et al, 1994) and retain the capacity for further differentiation (Blaschke et al, 1994;Schoop et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

Differential regulation of TGF-β signaling through Smad2, Smad3 and Smad4

Kretschmer¹,

Moepert²,

Dames³

et al. 2003

Oncogene

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100

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Smad transcription factors mediate the growth inhibitory effect of transforming growth factor-b (TGF-b) in many cell types. Mutational inactivation of Smads has been correlated with loss of responsiveness to TGF-b-mediated signal transduction. In this study, we compare the contribution of individual Smads to TGF-b-induced growth inhibition and endogenous gene expression in isogenic cellular backgrounds. Smad2, Smad3 and Smad4 expression were selectively inhibited in differentiationcompetent cells by using improved antisense molecules. We found that TGF-b mediates its inhibitory effect on HaCaT keratinocyte cell growth predominantly through Smad3. Inhibition of Smad3 expression was sufficient to interfere with TGF-b-induced cell cycle arrest and to induce or suppress endogenous cell cycle regulators. Inhibition of Smad4 expression exhibited a partial effect, whereas inhibition of Smad2 expression had no effect. By gene expression profiling, we identified TGF-b-dependent genes that are differentially regulated by Smad2 and Smad3 under regular growth conditions on a genome-wide scale. We show that Smad2, Smad3 and Smad4 contribute to the regulation of TGF-b responses to varying extents, and demonstrate, in addition, that these Smads exhibit distinct roles in different cell types.

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PTEN: A crucial mediator of mitochondria-dependent apoptosis

et al. 2006

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The highly frequent mutation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in various cancers has attracted much attention to study its role in tumorigenesis. As an important tumor suppressor, the pro-apoptotic function of PTEN has been linked to its capacity antagonizing the PI3K/Akt signaling pathway. However, less data are available concerning its role in neurodegeneration in which apoptotic processes are also involved. In the present study, we attempted to study the role and the underlying mechanism of PTEN in neuronal apoptosis. Using primary rat hippocampal cultures, staurosporine (STS, 100 nM) induced a time-dependent apoptosis, accompanied by a marked production of reactive oxygen species (ROS), release of cytochrome c and activation of caspase 9 and 3. However, the expression of PTEN, and the levels of phospho-PTEN and phospho-Akt were not changed at all time points tested (0.5-24 h) after STS stimulation, suggesting that the protein level as well as the phosphorylation status of PTEN were not related to the procession of apoptosis. Interestingly, immunostaining revealed a punctate intracellular distribution of PTEN from 2 to 8 h after adding STS. Double labeling and Western blotting of mitochondrial fraction demonstrated a mitochondrial location and accumulation of PTEN, respectively, after challenging with STS. Furthermore, we provide evidence for the first time that PTEN was associated with Bax in the absence and the presence of STS. Of note, the STS-induced marked increase in the cellular ROS level, release of cytochrome c and activation of caspase 3 were inhibited in cultured hippocampal cells when PTEN was knocked down by a specific antisense. Moreover, knockdown of PTEN significantly protected hippocampal cells from apoptotic damage. These findings demonstrated that PTEN is a crucial mediator of mitochondria-dependent apoptosis, and thus could become a molecular target for interfering with neurodegenerative diseases.

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GeneBlocs Are Powerful Tools to Study and Delineate Signal Transduction Processes That Regulate Cell Growth and Transformation

Cited by 34 publications

References 40 publications

Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

Differential regulation of TGF-β signaling through Smad2, Smad3 and Smad4

PTEN: A crucial mediator of mitochondria-dependent apoptosis

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