siRNA-mediated Knockdown (siRNA-mediat + knockdown)

Distribution by Scientific Domains

Selected Abstracts

Anti-apoptotic effect of the basic helix-loop-helix (bHLH) transcription factor DEC2 in human breast cancer cells

GENES TO CELLS, Issue 4 2010
Yang Liu
DEC1 (BHLHB2/Stra13/Sharp2) and DEC2 (BHLHB3/Sharp1) are basic helix-loop-helix (bHLH) transcription factors that are involved in circadian rhythms, differentiation and the responses to hypoxia. We examined whether DEC1 and DEC2 are involved in apoptosis regulation, in human breast cancer MCF-7 cells. We found that siRNA-mediated knockdown of DEC2 resulted in marked enhancement of apoptosis compared with that in control cells transfected with nonspecific siRNA. However, knockdown of DEC1 by siRNA did not affect cell survival. Knockdown of DEC2 affected the expression of mRNA or proteins related to apoptosis, such as Fas, c-Myc, caspase-8, poly (ADP-ribose) polymerase (PARP) and Bax. We also showed that tumor necrosis factor-, (TNF-,) up-regulates the expression of DEC1 and DEC2. DEC2 over-expression caused by the transfection of an expression vector reduced the amounts of cleaved PARP and caspase-8 induced by TNF-, treatment, whereas DEC1 over-expression increased it. Finally, we revealed that treatment with double knockdown against both DEC1 and DEC2 decreased the amounts of cleaved PARP and caspase-8 induced by DEC2 siRNA with or without TNF-,. These data indicate that DEC2 has an anti-apoptotic effect, whereas DEC1 has a pro-apoptotic effect, which are involved in the balance of survival of human breast cancer MCF-7 cells. [source]

SEI family of nuclear factors regulates p53-dependent transcriptional activation

GENES TO CELLS, Issue 8 2005
Rie Watanabe-Fukunaga
SEI family proteins, p34SEI-1 and SEI-2(TRIP-Br2), are nuclear factors that are implicated in cell cycle regulation through interaction with CDK4/CyclinD and E2F-1/DP-1 complexes. Here we report that the SEI family proteins regulate transcriptional activity of p53 tumor suppressor protein. Expression of SEI-1, SEI-2 or SEI-3 strongly stimulates p53-dependent gene activation in HeLa and U2OS cells but not in p53-deficient Saos2 or p53-knockdown HeLa cells. SEI proteins possess an intrinsic transactivation activity, interact with the coactivator CREB-binding protein, and cooperate synergistically with the ING family of chromatin-associated proteins to stimulate the transactivation function of p53. Doxycycline-induced expression of SEI proteins results in activation of the p21 gene and inhibition of cell growth, but the growth arrest was not suppressed by the siRNA-mediated knockdown of the endogenous p53 protein. These results indicate that the SEI family of nuclear proteins regulates p53 transcriptional activity and a p53-independent signaling pathway leading to growth inhibition. [source]

Mechanical stretching induces osteoprotegerin in differentiating C2C12 precursor cells through noncanonical Wnt Pathways,

Hsiao-Chi Yu
Abstract Mechanical loading is known to be important for maintaining the formation and resorption rates of bone. To study the mechanisms by which mechanical loading regulates osteogenesis, we investigated the role of the Wnt pathway in C2C12 cells committed to osteogenic differentiation in response to cyclic mechanical stretching. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL to inhibit osteoclastogenesis and resorption of bone. Our results demonstrate that stretching leads to a sustained increase in OPG expression in C2C12 cells. The expression of osteogenic marker genes, such as osteocalcin and alkaline phosphatase, was transiently decreased by stretching at 24 hours and returned to control levels at 48 hours. The addition of inhibitors of the canonical Wnt/,-catenin pathways, such as the secreted FZD-related peptide sRFP2, as well as siRNA-mediated knockdown, did not inhibit the effect of stretching on OPG expression. In contrast, treatment with inhibitors of noncanonical Wnt signaling, including KN93, and siRNA for Nemo-like kinase (NLK) blocked most of the mechanical inductive effect on OPG. Furthermore, stretching-induced OPG production in the culture medium was able to inhibit the osteoclast formation of bone marrow macrophages. These results suggest that mechanical stretching may play an important role in bone remodeling through the upregulation of OPG and that the mechanical signaling leading to OPG induction involves the noncanonical Wnt pathway. 2010 American Society for Bone and Mineral Research [source]

Functional analysis of CBP/p300 in embryonic orofacial mesenchymal cells

D.R. Warner
Abstract CREB binding protein (CBP) and the close structural homolog, p300, are nuclear coactivators of multiple signaling pathways that play important roles in embryonic development and cellular homeostasis. TGF, regulates the proliferation rate of many cell types and has been demonstrated to inhibit the growth rate of mouse embryonic maxillary mesenchymal (MEMM) cells. The role of CBP and p300 in TGF,-mediated control of proliferation of MEMM cells was thus investigated using an in vitro gene knockdown approach. TGF, reporter assays demonstrated that p300 mRNA knockdown via targeted siRNAs led to a reduction in the response to TGF,, whereas knockdown of CBP by the same approach had an insignificant effect. In MEMM cell proliferation assays, siRNA-mediated knockdown of CBP and/or p300 had little impact upon TGF,-mediated growth inhibition; however, the basal rate of proliferation was increased. Inhibition of p300 activity via overexpression of a dominant-negative mutant (p300,C/H3) led to significant inhibition of TGF,-mediated activation of p3TP-lux. As with the siRNA knockdown approach, p300,C/H3 also increased the basal rate of cell proliferation of MEMM cells. CBP/p300 siRNA knockdown had a significant but incomplete inhibition of TGF,-induction of matrix metalloproteinase-9 (gelatinase B) expression. These data demonstrate that p300 is involved in Smad-mediated transcription of p3TP-lux, however, its role (and that of CBP) in biological processes such as the control of cell proliferation and extracellular matrix metabolism is more complex and may be mediated via mechanisms beyond coactivator recruitment. J. Cell. Biochem. 99: 1374,1379, 2006. 2006 Wiley-Liss, Inc. [source]

siRNA-mediated Knockdown of the Heme Synthesis and Degradation Pathways: Modulation of Treatment Effect of 5-Aminolevulinic Acid-based Photodynamic Therapy in Urothelial Cancer Cell Lines

Makito Miyake
Photodynamic therapy mediated by 5-aminolevulinic acid (ALA-PDT) has been developed as a therapeutic modality for refractory superficial bladder cancers. Here, in experiments using urothelial cancer cell lines, we investigated the effects of siRNA modulating heme-synthetic and degradation pathways for ALA-PDT. Targeted knockdown of ferrochelatase (FECH) suppressed heme synthesis and significantly increased intracellular protoporphyrin IX (PpIX) accumulation, leading to enhanced phototoxicity in four of five cell lines. Heme oxygenase-1 (HO-1) is recognized as important for cytoprotection against oxidative stress such as PDT. Targeted knockdown of HO-1 leads to decreased intracellular PpIX accumulation, resulting in a failure to enhance ALA-PDT effect in four cell lines. Knockdown of HO-1 caused marked growth inhibition in UM-UC-2 overexpressing HO-1, whereas no inhibitory effect was observed in UM-UC-3 lacking HO-1 expression. Moreover, HO-1 protein levels and (GT)n repeat polymorphism of the HO-1 gene promoter region were examined with the implication that the constitutive expressions of HO-1 protein were associated with a shorter (GT)n repeat. Our results suggested that (1) FECH siRNA improved the phototoxicity of ALA-PDT, (2) overexpression of HO-1 was associated with shorter (GT)n repeat of the promoter region, and (3) siRNA-mediated knockdown of HO-1 could suppress the growth of bladder cancer cells overexpressing HO-1. [source]

Off-Target Effects Related to the Phosphorothioate Modification of Nucleic Acids

CHEMMEDCHEM, Issue 8 2010
Johannes Winkler Dr.
Abstract Phosphorothioate antisense oligonucleotides have been widely used in clinical studies for rational sequence-specific gene silencing. However, several sequence-unspecific off-target effects have been recently described for this compound class. In contrast to siRNA-mediated knockdown of the same gene, the bcl-2 -targeted oblimersen (Genasense, G3139) downregulates a number of proteins involved in apoptotic resistance and several glycolytic enzymes in 607B human melanoma cells. Regardless of their target, phosphorothioate-modified antisense and siRNA compounds, but not oligonucleotides with a phosphodiester backbone, resulted in a similar impact on the proteome. Unspecifically downregulated proteins include cancer markers involved in apoptotic resistance and endoplasmatic reticulum (ER) stress such as the 78,kDa glucose regulated protein (GRP,78), protein disulfide isomerase,A3 (PDIA3, GRP,58), calumenin, and galectin-1, as well as the glycolytic enzymes triose phosphate isomerase, glyceraldehyde phosphodehydrogenase, and phosphoglycerate mutase. The depletion of the glycolytic enzymes is reflected by a decrease in L -lactate production, indicating a partial reversal of the Warburg effect. Compared with other phosphorothioate oligonucleotides, oblimersen generally led to a more pronounced effect both in terms of the number of influenced proteins and the extent of downregulation, suggesting a synergistic effect of Bcl-2 downregulation. [source]