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PubMed2005

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference.

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.

Animals↗
PubMed2005

Enhanced sensitivity to inhibition of SHP2, STAT5, and Gab2 expression in chronic myeloid leukemia (CML).

Although targeting the BCR-ABL tyrosine kinase activity by imatinib mesylate has rapidly become first-line therapy in chronic myeloid leukemia (CML), drug resistance suggests that combination therapy directed to a complementing target may significantly improve treatment results. To identify such potential targets, we used lentivirus-mediated RNA interference (RNAi) as a tool for functional genomics in cell lines as well as primary normal and CML CD34+ cells. In a conditional cell culture model, we demonstrate that RNAi-mediated reduction of SHP2, STAT5, and Gab2 protein expression inhibits BCR-ABL-dependent but not cytokine-dependent proliferation in a dose-dependent manner. Similarly, colony formation of purified primary CML but not of normal CD34+ colony-forming cells is specifically reduced by inhibition of SHP2, STAT5, and Gab2 expression, respectively. In addition, coexpression of both anti-BCR-ABL and anti-SHP2 shRNAs from a single lentiviral vector induces stronger inhibition of colony formation as compared to either shRNA alone. The data indicate that BCR-ABL expression may affect the function of normal signaling molecules. Targeting these molecules may harbor significant therapeutic potential for the treatment of patients with CML.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro.

The tyrosine kinase receptor fetal liver kinase 1 (Flk-1) plays a crucial role in vasculogenesis and angiogenesis, but its target genes remain elusive. Comparing Flk-1(+/+) with Flk-1(-/-) embryonic stem (ES) cells, we identified transcripts regulated by the vascular endothelial growth factor A (VEGF-A)/Flk-1 pathway at an early stage of their differentiation to endothelial and mural precursors. Further analysis of a number of these genes (Nm23-M1, Nm23-M2, Slug, Set, pp32, Cbp, Ship-1, Btk, and Pim-1) showed that their products were transiently up-regulated in vivo in endothelial cells (ECs) during angiogenesis of the ovary, and their mRNA was rapidly induced in vitro by VEGF-A in human umbilical cord vein endothelial cells (HUVECs). Functional analysis by RNA interference (RNAi) in ES cells induced to differentiate demonstrated that Pim-1 is required for their differentiation into ECs and smooth muscle cells (SMCs). In HUVECs, RNAi showed that Pim-1 is required in ECs for VEGF-A-dependent proliferation and migration. The identification of Flk-1 target genes should help in elucidating the molecular pathways that govern the vasculogenesis and angiogenesis processes.

Animals↗
PubMed2004

Knockdown of Chk1, Wee1 and Myt1 by RNA interference abrogates G2 checkpoint and induces apoptosis.

Mammalian cells undergo cell cycle arrest in response to DNA damage due to the existence of multiple checkpoint response mechanisms. One such checkpoint pathway operating at the G(1) phase is frequently lost in cancer cells due to mutation of the p53 tumor suppressor gene. However, cancer cells often arrest at the G(2) phase upon DNA damage, due to activation of another checkpoint pathway that prevents the activation Cdc2 kinase. The kinases, Chk1, Wee1, and Myt1 are key regulators of this G(2) checkpoint, which act directly or indirectly to inhibit Cdc2 activity. Here we show that RNA interference (RNAi)-mediated downregulation of Wee1 kinase abrogated an Adriamycin trade mark -induced G(2) checkpoint in human cervical carcinoma Hela cells that are defective in G(1) checkpoint response. Wee1 downregulation sensitized HeLa cells to Adriamycin trade mark -induced apoptosis. Downregulation of Chk1 kinase in Hela cells also caused a significant amount of cell death in dependent of DNA damage. In contrast, Myt1 downregulation also abrogated Adriamycin trade mark -induced G(2) arrest but did not cause substantial apoptosis. Reduction in Wee1, Chk1, or Myt1 levels did not sensitize normal human mammary epithelial cells (HMEC) cells to Adriamycin trade mark -induced apoptosis unlike the situation in Hela cells. Our study reveals distinct roles for Chk1, Wee1, and Myt1 in G(2) checkpoint regulation. The data reported here support the attractiveness of Wee1 and Chk1 is as molecular targets for abrogating the G(2) DNA damage checkpoint arrest, a situation that may selectively sensitize p53-deficient tumor cells to radiation or chemotherapy treatment.

Apoptosis↗
PubMed2004

DEQOR: a web-based tool for the design and quality control of siRNAs.

RNA interference (RNAi) is a powerful tool for inhibiting the expression of a gene by mediating the degradation of the corresponding mRNA. The basis of this gene-specific inhibition is small, double-stranded RNAs (dsRNAs), also referred to as small interfering RNAs (siRNAs), that correspond in sequence to a part of the exon sequence of a silenced gene. The selection of siRNAs for a target gene is a crucial step in siRNA-mediated gene silencing. According to present knowledge, siRNAs must fulfill certain properties including sequence length, GC-content and nucleotide composition. Furthermore, the cross-silencing capability of dsRNAs for other genes must be evaluated. When designing siRNAs for chemical synthesis, most of these criteria are achievable by simple sequence analysis of target mRNAs, and the specificity can be evaluated by a single BLAST search against the transcriptome of the studied organism. A different method for raising siRNAs has, however, emerged which uses enzymatic digestion to hydrolyze long pieces of dsRNA into shorter molecules. These endoribonuclease-prepared siRNAs (esiRNAs or 'diced' RNAs) are less variable in their silencing capabilities and circumvent the laborious process of sequence selection for RNAi due to a broader range of products. Though powerful, this method might be more susceptible to cross-silencing genes other than the target itself. We have developed a web-based tool that facilitates the design and quality control of siRNAs for RNAi. The program, DEQOR, uses a scoring system based on state-of-the-art parameters for siRNA design to evaluate the inhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i) regions in a gene that show high silencing capacity based on the base pair composition and (ii) siRNAs with high silencing potential for chemical synthesis. In addition, each siRNA arising from the input query is evaluated for possible cross-silencing activities by performing BLAST searches against the transcriptome or genome of a selected organism. DEQOR can therefore predict the probability that an mRNA fragment will cross-react with other genes in the cell and helps researchers to design experiments to test the specificity of esiRNAs or chemically designed siRNAs. DEQOR is freely available at http://cluster-1.mpi-cbg.de/Deqor/deqor.html.

Internet↗
PubMed2006

The kinase Mirk/Dyrk1B mediates cell survival in pancreatic ductal adenocarcinoma.

Ductal adenocarcinoma of the pancreas is almost uniformly lethal as this cancer is invariably detected at an advanced stage and is resistant to treatment. The serine/threonine kinase Mirk/Dyrk1B has been shown to be antiapoptotic in rhabdomyosarcomas. We have now investigated whether Mirk might mediate survival in another cancer in which Mirk is widely expressed, pancreatic ductal adenocarcinoma. Mirk was an active kinase in each pancreatic cancer cell line where it was detected. Mirk knockdown by RNA interference (RNAi) reduced the clonogenicity of Panc1 pancreatic cancer cells 4-fold and decreased tumor cell number, showing that Mirk mediates survival in these cells. Mirk knockdown by synthetic duplex RNAis in Panc1, AsPc1, and SU86.86 pancreatic cancer cells induced apoptosis and enhanced the apoptosis induced by gemcitibine. Mirk knockdown did not increase the abundance or activation of Akt. However, four of five pancreatic carcinoma cell lines exhibited either elevated Mirk activity or elevated Akt activity, suggesting that pancreatic cancer cells primarily rely on Mirk or Akt for survival signaling. Mirk protein was detected by immunohistochemistry in 25 of 28 cases (89%) of pancreatic ductal adenocarcinoma, with elevated expression in 11 cases (39%). Increased expression of Mirk was seen in pancreatic carcinomas compared with primary cultures of normal ductal epithelium by serial analysis of gene expression and by immunohistochemistry. Thus, Mirk is a survival factor for pancreatic ductal adenocarcinoma. Because knockout of Mirk does not cause embryonic lethality, Mirk is not essential for normal cell growth and may represent a novel therapeutic target.

Aged↗
PubMed2006

Targeting PIM kinases impairs survival of hematopoietic cells transformed by kinase inhibitor-sensitive and kinase inhibitor-resistant forms of Fms-like tyrosine kinase 3 and BCR/ABL.

Previous studies have shown that activation of the signal transducer and activator of transcription 5 (STAT5) plays an essential role in leukemogenesis mediated through constitutive activated protein tyrosine kinases (PTK). Because PIM-1 is a STAT5 target gene, we analyzed the role of the family of PIM serine/threonine kinases (PIM-1 to PIM-3) in PTK-mediated transformation of hematopoietic cells. Ba/F3 cells transformed to growth factor independence by various oncogenic PTKs (TEL/JAK2, TEL/TRKC, TEL/ABL, BCR/ABL, FLT3-ITD, and H4/PDGFbetaR) show abundant expression of PIM-1 and PIM-2. Suppression of PIM-1 activity had a negligible effect on transformation. In contrast, expression of kinase-dead PIM-2 mutant (PIM-2KD) led to a rapid decline of survival in Ba/F3 cells transformed by FLT3-ITD but not by other oncogenic PTKs tested. Coexpression of PIM-1KD and PIM-2KD abrogated growth factor-independent growth of Ba/F3 transformed by several PTKs, including BCR/ABL. Targeted down-regulation of PIM-2 by RNA interference (RNAi) selectively abrogated survival of Ba/F3 cells transformed by various Fms-like tyrosine kinase 3 (FLT3)-activating mutants [internal tandem duplication (ITD) and kinase domain] and attenuated growth of human cell lines containing FLT3 mutations. Interestingly, cells transformed by FLT3 and BCR/ABL mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM-2, or PIM-1 and PIM-2 by RNAi. Our observations indicate that combined inactivation of PIM-1 and PIM-2 interferes with oncogenic PTKs and suggest that PIMs are alternative therapeutic targets in PTK-mediated leukemia. Targeting the PIM kinase family could provide a new avenue to overcome resistance against small-molecule tyrosine kinase inhibitors.

Animals↗
PubMed2005

Reversal of the phenotype by K-rasval12 silencing mediated by adenovirus-delivered siRNA in human pancreatic cancer cell line Panc-1.

AIM: To investigate the in vitro antitumor effect of adenovirus-mediated small interfering RNAs (siRNAs) on pancreatic cancer and the associated mechanism. METHODS: A 63-nucleotide (nt) oligonucleotide encoding K-ras(val12) and specific siRNA were introduced into pSilencer 3.1-H1, then the H1-RNA promoter and siRNA coding insert were subcloned into pAdTrack to get plasmid pAdTrackH1-K-ras(val12). After homologous recombination in bacteria and transfections of such plasmids into a mammalian packaging cell line 293, siRNA expressing adenovirus AdH1-K-ras(val12) was obtained. Stable suppression of K-ras(val12) was detected by Northern blot and Western blot. Apoptosis in Panc-1 cells was detected by flow cytometry. RESULTS: We obtained adenovirus AdH1-K-ras(val12) carrying the pSilencer 3.1-H1 cassette, which could mediate gene silencing. Through siRNA targeted K-ras(val12), the oncogenic phenotype of cancer cells was reversed. Flow cytometry showed that apoptotic index of Panc-1 cells was significantly higher in the AdH1-K-ras(val12)-treatment group (18.70% at 72 h post-infection, 49.55% at 96 h post-infection) compared to the control groups (3.47%, 3.98% at 72 and 96 h post-infection of AdH1-empty, respectively; 4.21%, 3.78% at 72 and 96 h post-infection of AdH1-p53, respectively) (P<0.05). CONCLUSION: These results demonstrate that adenoviral vectors can be used to mediate RNA interference (RNAi) to induce persistent loss of functional phenotypes. In gene therapy, the selective down-regulation of only the mutant version of a gene allows for highly specific effects on tumor cells, while leaving the normal cells untouched. In addition, the apoptosis of pancreatic cancer cell line Panc-1 can be induced after AdH1-K-ras(val12) infection. This kind of adenovirus based on RNAi might be a promising vector for cancer therapy.

Adenoviridae↗
PubMed2002

Regeneration in planarians and other worms: New findings, new tools, and new perspectives.

Molecular biology, recombinant DNA techniques, and new methods of cell lineage have reignited the interest of planarians and other worms (mainly annelids and nemerteans) as invertebrate model systems of regeneration. Here, the mean results produced in the last five years are reviewed, an update of the genes and molecules involved in planarian regeneration is provided, and a new morphallactic-epimorphic model of pattern formation is suggested. Moreover, and most importantly, we highlight the new strides brought upon by genomic/proteomic analyses, RNA interference (RNAi) to inactivate gene function, and Bromodeoxyuridine (BrdU) cell labelling. The raising hope to obtain transformed neoblasts and transgenic planarians is also stressed. Altogether, such approaches will eventually lead to solve the long-standing open questions on regeneration which still baffles us. Finally, we warn against overlooking the evident links between regeneration processes and those controlling the daily wear and tear of tissues and cells. Both processes act, at least in planarians, upon a unique stem-cell endowed with an unrivaled developmental potential in the animal kingdom-the neoblast. This cell could be considered the forebear and a model system for stem-cell analysis.

Animals↗
PubMed2006

Tuba stimulates intracellular N-WASP-dependent actin assembly.

Tuba is a multidomain scaffolding protein that links cytoskeletal dynamics and membrane trafficking pathways. The N-terminus of Tuba binds dynamin1, and the C-terminus contains domains that can interact with signaling pathways and cytoskeletal regulatory elements. We investigated Tuba localization, distribution and function in B16 melanoma cells. Tuba overexpression stimulated dorsal ruffles that occurred independently of dynamin function. Tuba expression induced actin-driven motility of small puncta that required the C-terminal SH3, GEF and BAR domains. Additionally, Tuba was recruited to lipid vesicles generated by overexpression of phosphatidylinositol-4-phosphate 5-kinase type Ialpha (PIP5Kalpha), localizing prominently to the head of the comets and at lower levels along the actin tail. We propose that Tuba facilitates dorsal ruffling of melanoma cells through direct interaction with actin-regulatory proteins and the recruitment of signaling molecules to lipid microdomains for the coordinated assembly of a cytoskeletal network. Knockdown of Tuba by RNA interference (RNAi) attenuated PIP5Kalpha-generated comet formation and the invasive behavior of B16 cells, implying that Tuba function is required for certain aspects of these processes. These results suggest first that Tuba-stimulated dorsal ruffling might represent a novel mechanism for the coordination of N-WASP-dependent cytoskeletal rearrangements and second that Tuba function is implicated in motility processes.

Actin-Related Protein 2-3 Complex↗
PubMed2002

Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA.

Double-stranded RNA (dsRNA) induces gene-specific silencing in organisms from fungi to animals, a phenomenon known as RNA interference (RNAi). RNAi represents an evolutionarily conserved system to protect against aberrant expression of genes and a powerful tool for gene manipulation. Despite reports that RNAi can be induced in vertebrates, severe sequence-non-specific effects of long dsRNA have been documented in various systems. It has recently been shown in cultured mammalian cells that small interfering RNAs (siRNAs) of 21-23 nt can mediate RNAi but bypass the non-specific response induced by longer dsRNAs. However, the effectiveness of siRNAs has not been demonstrated in living vertebrates. In addition, the mechanism of siRNA suppression of gene expression in vertebrate cells remains to be elucidated. Here we show that synthetic 21 nt siRNAs can specifically inhibit the expression of exogenously introduced as well as endogenous genes in the embryos of Xenopus laevis. siRNAs significantly reduced the steady-state amount of both the mRNA and protein of the cognate gene target. Moreover, co-injection of siRNA with the target RNA transcript specifically suppressed the activity of the latter. Taken together, our findings establish siRNA-mediated post-transcriptional suppression of gene expression in Xenopus embryos.

Animals↗
PubMed2006

hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells.

Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited telomerase activity but had no effect on the expressions of telomerase core subunits. RNA interference (RNAi) and in vitro phosphorylation studies revealed that PKC isoforms alpha, beta, delta, epsilon, zeta specifically involved in telomerase regulation, and the phosphorylation target was on hTERT. Treatment with the hsp-90 inhibitor novobiocin dissociated hsp90 and hTERT as revealed by immunoprecipitation and immunoblot analysis and reduced telomerase activity. Treatment with the PKC activator SC-10 restored the association of hsp90 and hTERT and reactivate telomerase, suggesting that hTERT phosphorylation by PKC is essential for telomerase holoenzyme integrity and function. Analysis on clinical normal and tumour tissues reveal that the expressions of PKC alpha, beta, delta, epsilon, zeta were higher in the tumour tissues, correlated with telomerase activity. Disruption of PKC phosphorylation by BIS significantly increased chemosensitivity to cisplatin. In conclusion, PKC isoenzymes alpha, beta, delta, epsilon, zeta regulate telomerase activity in head and neck cancer cells by phosphorylating hTERT. This phosphorylation is essential for telomerase holoenzyme assembly, leading to telomerase activation and oncogenesis. Manipulation of telomerase activity by PKC inhibitors is worth exploring as an adjuvant therapeutic approach.

Cell Transformation, Neoplastic↗
PubMed2006

Coactivator cross-talk specifies transcriptional output.

Cells often fine-tune gene expression at the level of transcription to generate the appropriate response to a given environmental or developmental stimulus. Both positive and negative influences on gene expression must be balanced to produce the correct level of mRNA synthesis. To this end, the cell uses several classes of regulatory coactivator complexes including two central players, TFIID and Mediator (MED), in potentiating activated transcription. Both of these complexes integrate activator signals and convey them to the basal apparatus. Interestingly, many promoters require both regulatory complexes, although at first glance they may seem to be redundant. Here we have used RNA interference (RNAi) in Drosophila cells to selectively deplete subunits of the MED and TFIID complexes to dissect the contribution of each of these complexes in modulating activated transcription. We exploited the robust response of the metallothionein genes to heavy metal as a model for transcriptional activation by analyzing direct factor recruitment in both heterogeneous cell populations and at the single-cell level. Intriguingly, we find that MED and TFIID interact functionally to modulate transcriptional response to metal. The metal response element-binding transcription factor-1 (MTF-1) recruits TFIID, which then binds promoter DNA, setting up a "checkpoint complex" for the initiation of transcription that is subsequently activated upon recruitment of the MED complex. The appropriate expression level of the endogenous metallothionein genes is achieved only when the activities of these two coactivators are balanced. Surprisingly, we find that the same activator (MTF-1) requires different coactivator subunits depending on the context of the core promoter. Finally, we find that the stability of multi-subunit coactivator complexes can be compromised by loss of a single subunit, underscoring the potential for combinatorial control of transcription activation.

Animals↗
PubMed2002

Small interfering RNA-mediated gene silencing in T lymphocytes.

Introduction of small interfering RNAs (siRNAs) into a cell can cause a specific interference of gene expression known as RNA interference (RNAi). However, RNAi activity in lymphocytes and in normal primary mammalian cells has not been thoroughly demonstrated. In this report, we show that siRNAs complementary to CD4 and CD8alpha specifically reduce surface expression of these coreceptors and their respective mRNA in a thymoma cell line model. We show that RNAi activity is only caused by a subset of siRNAs complementary to the mRNA target and that ineffective siRNAs can compete with effective siRNAs. Using primary differentiated T lymphocytes, we provide the first evidence of siRNA-mediated RNAi gene silencing in normal nontransformed somatic mammalian lymphocytes.

Animals↗
PubMed2006

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference.

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.

Animals↗
PubMed2005

Splice variant-specific silencing of angiotensin II type 1a receptor messenger RNA by RNA interference in vascular smooth muscle cells.

In the rat, two distinct angiotensin II type 1a (rAT(1a)) receptor mRNAs are synthesized from a single rAT(1a) receptor gene by alternative splicing. These two transcripts are comprised of exons 1, 2, and 3 (E1,2,3) or exons 1 and 3 (E1,3). Since exon 3 contains the entire coding region, both transcripts encode identical rAT(1a) receptors. Real-time PCR revealed that in rat aortic smooth muscle cells (RASMC), E1,2,3 mRNA accounted for 69.5+/-0.9% of total rAT(1a) receptor mRNA. The aim of this study was to use RNA interference (RNAi) to selectively silence the rAT(1a) receptor splice variants. Forty-eight hour treatment of RASMC with E1,3-targeting siRNA (10nM; S1(E1,3)) resulted in a 91.2+/-0.5% (n=3, P<0.001) reduction in E1,3 mRNA and a 19.0+/-3.0% (n=4, P<0.05) reduction in AT(1) receptor specific binding compared with cells treated with a non-silencing control siRNA; under these conditions, no effect was observed on levels of E1,2,3 mRNA. Conversely, treatment with E1,2,3-targeting siRNA (S2(E2)) had no effect on E1,3 mRNA while reducing E1,2,3 mRNA by 73.9+/-4.2% (n=3, P<0.001), and AT(1) receptor binding by 39.4+/-5.4% (n=4, P<0.001) compared with control. These data show that the majority of functional AT(1) receptor expression in RASMC derives from the E1,2,3 splice variant. These data also demonstrate that rAT(1a) receptor mRNA can be silenced in a splice-variant specific manner using siRNA in RASMC, thus providing an excellent model system for investigating the role of alternative splicing in the regulation of rAT(1a) receptor expression.

Alternative Splicing↗
PubMed2006

Therapeutic potential of siRNA-mediated transcriptional gene silencing.

RNA interference (RNAi) and specifically the use of small interfering RNAs (siRNAs) represents a potentially new paradigm in gene knockout technology. Clearly siRNAs can be used to knockdown the expression of a targeted transcript in what has been termed posttranscriptional gene silencing (PTGS). While there are a plethora of reports applying siRNA-mediated PTGS the limitation of the duration of the effect remains. Recently, in human cells, siRNAs have been shown, similar to plants and Schizosaccharomyces pombe, to mediate transcriptional gene silencing (TGS). The observation that siRNAs can function in a TGS manner in human cells suggests that, similar to plants, human genes may also be able to be silenced more permanently via epigenetic modifications. The ramifications of siRNA-mediated TGS in humans suggest that longer term suppression of gene function can be obtained via siRNA-directed chromatin modifications. Undoubtedly the potential to employ siRNA technology is broader than once envisioned in human cells and suggests that siRNA-mediated TGS is not simply limited to PTGS. The potential to utilize siRNAs to direct epigenetic changes in local chromatin structure offers a new therapeutic avenue that could prove remarkably robust and of immeasurable therapeutic value in the directed control of target gene expression.

Animals↗
PubMed2006

Lamin A/C and emerin are critical for skeletal muscle satellite cell differentiation.

Mutations within LMNA, encoding A-type nuclear lamins, are associated with multiple tissue-specific diseases, including Emery-Dreifuss (EDMD2/3) and Limb-Girdle muscular dystrophy (LGMD1B). X-linked EDMD results from mutations in emerin, a lamin A-associated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) down-regulated for either A-type lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking A-type lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and M-cadherin; up-regulated Myf5; but no changes in Pax3, Pax7, MEF2C, MEF2D, c-met, and beta-catenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna(-/-) myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna(-/-) myoblasts resulted in increased differentiation potential. These studies indicate roles for A-type lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/C-deficient and emerin-deficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD.

Animals↗