Showing papers on "Small hairpin RNA published in 2007"

Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma

Abstract: Autophagy is a lysosome-dependent degradative pathway frequently activated in tumor cells treated with chemotherapy or radiation. Whether autophagy observed in treated cancer cells represents a mechanism that allows tumor cells to survive therapy or a mechanism for initiating a nonapoptotic form of programmed cell death remains controversial. To address this issue, the role of autophagy in a Myc-induced model of lymphoma generated from cells derived from p53ERTAM/p53ERTAM mice (with ER denoting estrogen receptor) was examined. Such tumors are resistant to apoptosis due to a lack of nuclear p53. Systemic administration of tamoxifen led to p53 activation and tumor regression followed by tumor recurrence. Activation of p53 was associated with the rapid appearance of apoptotic cells and the induction of autophagy in surviving cells. Inhibition of autophagy with either chloroquine or ATG5 short hairpin RNA (shRNA) enhanced the ability of either p53 activation or alkylating drug therapy to induce tumor cell death. These studies provide evidence that autophagy serves as a survival pathway in tumor cells treated with apoptosis activators and a rationale for the use of autophagy inhibitors such as chloroquine in combination with therapies designed to induce apoptosis in human cancers.

Loss of function RNA interference screens for molecular targets in cancer

Abstract: SY27-02 We have designed a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We are using this “Achilles heel” screen to identify those cellular proteins and pathways that are most vulnerable to therapeutic attack. A doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) was used to construct a library targeting 2,500 human genes, with each gene targeting by 3-6 shRNAs. Each vector was engineered to contain a unique 60-base pair “bar code”, allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar code sequences. In a typical screen, retroviral pools of 500-1000 vectors from this library are used to a infect cancer cell line. After selection for stable integration, doxycycline is added to half of the culture to induce shRNA expression, and the remaining half serves as an uninduced control. These two cultures are then grown in parallel for 3 weeks, during which time cells expressing toxic shRNAs will be depleted from the induced culture. DNA bar code microarrays are used to compare the abundance of each shRNA vector in the induced versus uninduced populations, thereby revealing which shRNAs inhibited the proliferation or survival of the cancer cells. In our genetic screens, we have use a “synthetic lethal” strategy in which we search for shRNAs that are only toxic to cancer types with particular underlying genetic lesions. To this end, we conducted parallel screens in two distinct molecular subgroups of diffuse large B cell lymphoma (DLBCL) termed activated B cell-like (ABC) DLBCL and germinal center B cell-like (GCB) DLBCL. These two subgroups are profoundly different in their pathogenesis in that they arise from different stages of normal B lymphocyte development and utilize distinct oncogenic mechanims. Notably, we had previously discovered that the ABC DLBCL subgroup depends upon the NF-kB pathway for survival, but the GCB DLBCL subgroup does not. However, we did not know which upstream signaling pathways in ABC DLBCLs were responsible for their constitutive activaty of the IkB kinase, the key regulator of the NF-kB pathway. Remarkably, shRNAs targeting the NF-kB pathway were depleted in screens of ABC DLBCL but not GCB DLBCL cells, in keeping with the essential role of this pathway in the survival of ABC DLBCL. Unexpectedly, this screen uncovered an upstream signaling pathway involving CARD11, MALT1 and BCL10 that is responsible for the constitutive NF-kB activity in ABC DLBCL. Knock down of any of these genes with shRNAs descreased IkB kinase activity, NF-kB target gene expression, and cell viability. The CARD11 pathway therefore represents an attractive new therapeutic target for the ABC DLBCL subgroup. Importantly, therapeutic targeting of this pathway is likely to have discrete and manageable toxicity since mice that are deficient in these genes are viable and only have defects in subsets of B and T lymphocytes. More recently, we have used the Achilles heel methodology to discover genes essential for cellular proliferation and survival in other forms of lymphoid malignancy. We believe that a new taxonomy of cancer may emerge in which cancers are defined by their critical dependence on particular signaling pathways for survival and proliferation. Another important use of this technology will be to evaluate which of the many somatically mutated genes in human cancer make the most important ongoing contributions to the biology of cancer cells. Finally, these genetic screens are revealing that cancers may “inherit” from their normal cellular counterparts a dependance on particular regulatory pathways for their proliferation and survival. These pathways may not necessary be affected by genetic alterations but the cancer cells may nevertheless be “addicted” to their continous activity. Therefore, this methodology has the potential to uncover a new class of therapeutic targets distinct from known oncogenes.

The adipokine lipocalin 2 is regulated by obesity and promotes insulin resistance

Abstract: OBJECTIVE—We identified lipocalin 2 (Lcn2) as a gene induced by dexamethasone and tumor necrosis factor-α in cultured adipocytes. The purpose of this study was to determine how expression of Lcn2 is regulated in fat cells and to ascertain whether Lcn2 could be involved in metabolic dysregulation associated with obesity. RESEARCH DESIGN AND METHODS—We examined Lcn2 expression in murine tissues and in 3T3-L1 adipocytes in the presence and absence of various stimuli. We used quantitative Western blotting to observe Lcn2 serum levels in lean and obese mouse models. To assess effects on insulin action, we used retroviral delivery of short hairpin RNA to reduce Lcn2 levels in 3T3-L1 adipocytes. RESULTS—Lcn2 is highly expressed by fat cells in vivo and in vitro. Expression of Lcn2 is elevated by agents that promote insulin resistance and is reduced by thiazolidinediones. The expression of Lcn2 is induced during 3T3-L1 adipogenesis in a CCAAT/enhancer-binding protein–dependent manner. Lcn2 serum levels are elevated in multiple rodent models of obesity, and forced reduction of Lcn2 in 3T3-L1 adipocytes improves insulin action. Exogenous Lcn2 promotes insulin resistance in cultured hepatocytes. CONCLUSIONS—Lcn2 is an adipokine with potential importance in insulin resistance associated with obesity.

Therapeutic silencing of mutant huntingtin with siRNA attenuates striatal and cortical neuropathology and behavioral deficits

Abstract: Huntington's disease (HD) is an autosomal dominant disease caused by a CAG repeat expansion in the Htt gene (1). Mutant Htt causes neuronal death, dementia, and movement dysfunction; there is no effective treatment. In an inducible transgenic mouse model of HD, turning off transgene expression reversed neuropathology and motor deficits (2). Lowering mutant Htt gene expression in brain may treat HD. In mice, viral vector delivery of short hairpin RNAs (shRNAs) against mutant Htt gene exon 1 or genes that cause other neurodegenerative disorders reduced neuropathology and motor deficits (3–10). Brain delivery of adeno-associated virus (AAV)-shRNA against mutant Htt improved signs of disease in HD transgenic models (7, 11). In the inaugural study on RNAi targeting Htt in vivo, shRNA against Htt in AAV2, delivered to the N171–82Q transgenic model of HD, improved ambulation at 4 months and rotarod performance at 10 and 18 weeks after injection (7). Five and one-half months after shRNA administration, quantitative RT-PCR revealed a 50% reduction in striatal Htt mRNA. Statistical changes in quantification of Htt protein reduction and inclusions were not reported. AAV5 delivery of shRNA against Htt in the R6/1 murine model of HD showed a 25% decrease in Htt protein and an 80% reduction in Htt mRNA 10 weeks after shRNA injection (10). The shRNA delayed onset of clasping by 2 weeks (20–22 weeks), and treated mice had fewer clasps. No difference in rotarod performance was detected. Inclusion size and number decreased in the striatum, but not in the cortex, compared with the corresponding contralateral brain regions. The authors provided an important caveat that one of the shRNAs had off-target effects; the cause of the off-target effects was not established. shRNA in AAV2 or AAV5 was used to target EGFP to knock down EGFP-Htt in another transgenic model of HD (11). shRNA reversed pathology after the onset of pathologic changes; however, behaviors were not studied. Administration of large amounts of siRNA against Htt in a Lipofectamine 2000 suspension into the lateral ventricle of newborn R6/2 transgenic mice (exon 1 of Htt) reduced whole-brain levels of mutant Htt in two mice and Htt mRNA up to 7 days posttreatment, delayed the onset of clasping, rotarod, and open-field phenotypes, and improved survival (12). Statistical quantification of neuropathology was not reported. Thus, prior studies examining RNAi against Htt provided the groundwork for therapeutic gene silencing in HD. Most of the studies used viral delivery of shRNA, and the study using siRNA required liposome delivery to newborns, with the potential liposome neuronal toxicity. Caveats attend the use of shRNAs, which can be toxic when integrated into the host genome (13, 14), in part because shRNA production is unregulated. Long siRNAs (>29 nt) and shRNAs are prone to activate off-target gene expression (15). For patient safety, shRNA will need to be able to be switched off, currently a hurdle in viral delivery systems. An alternative strategy for HD therapy is the use of small-interfering RNAs (siRNAs), ≈21-nt RNA duplexes. siRNA has been administered into cerebroventricles, vasculature, intrathecal space, and parenchyma (16–20). siRNAs were found effective and safe when introduced into mice and non-human primates (19, 21, 22). Several limitations impede progress in using siRNAs as a treatment for HD: entry and effectiveness in adult neurons without the use of potentially toxic transfection reagents; a clear demonstration that gene silencing reduces protein expression; and an improvement in behavioral deficits and neuropathology, especially neuron survival. Because bioactive molecules conjugated to cholesterol have improved cellular uptake in vitro (23), LDL receptors have been detected in brain (24), and cholesterol conjugation enhances siRNA uptake in cells outside of the central nervous system (16), we speculated that cholesterol-conjugated (cc) siRNA might enter neurons in vivo. An in vivo, rapid-onset model of HD would be optimal to test gene silencing in brain. The current rapid mouse model of HD shows mutant Htt-induced pathology after 2 months. Transgenic mice expressing exon 1 of mutant Htt [R6/2 (25)] develop nuclear inclusions throughout the brain at 2 months of age and exhibit a rapidly progressing, severe phenotype. Other transgenic or knock-in mice expressing mutant Htt exhibit late-onset, mild phenotypes, often after 6 months of age (26, 27), and lack prominent neuronal loss. Neither model is ideal to test transient effects of a single injection of siRNA against Htt introduced directly to the striatum. We therefore developed an acute, in vivo, HD mouse model tailored to addressing the efficacy of siRNA. Here, we used AAV to deliver a 1,395-nt cDNA fragment of human mutant Htt into mouse striatum to evaluate the effectiveness of an siRNA targeting human Htt.

The ubiquitin-specific protease USP28 is required for MYC stability

Abstract: The MYC proto-oncogene encodes a transcription factor that has been implicated in the genesis of many human tumours Here, we used a bar-code short hairpin RNA (shRNA) screen to identify multiple genes that are required for MYC function One of these genes encodes USP28, an ubiquitin-specific protease USP28 is required for MYC stability in human tumour cells USP28 binds to MYC through an interaction with FBW7alpha, an F-box protein that is part of an SCF-type ubiquitin ligase Therefore, it stabilizes MYC in the nucleus, but not in the nucleolus, where MYC is degraded by FBW7gamma High expression levels of USP28 are found in colon and breast carcinomas, and stabilization of MYC by USP28 is essential for tumour-cell proliferation

Artificial MicroRNA-Mediated Virus Resistance in Plants

Abstract: RNA silencing in plants is a natural defense system against foreign genetic elements including viruses. This natural antiviral mechanism has been adopted to develop virus-resistant plants through expression of virus-derived double-stranded RNAs or hairpin RNAs, which in turn are processed into small interfering RNAs (siRNAs) by the host's RNA silencing machinery. While these virus-specific siRNAs were shown to be a hallmark of the acquired virus resistance, the functionality of another set of the RNA silencing-related small RNAs, microRNAs (miRNAs), in engineering plant virus resistance has not been extensively explored. Here we show that expression of an artificial miRNA, targeting sequences encoding the silencing suppressor 2b of Cucumber mosaic virus (CMV), can efficiently inhibit 2b gene expression and protein suppressor function in transient expression assays and confer on transgenic tobacco plants effective resistance to CMV infection. Moreover, the resistance level conferred by the transgenic miRNA is well correlated to the miRNA expression level. Comparison of the anti-CMV effect of the artificial miRNA to that of a short hairpin RNA-derived small RNA targeting the same site revealed that the miRNA approach is superior to the approach using short hairpin RNA both in transient assays and in transgenic plants. Together, our data demonstrate that expression of virus-specific artificial miRNAs is an effective and predictable new approach to engineering resistance to CMV and, possibly, to other plant viruses as well.

RNAi for insect-proof plants

Abstract: RNA interference induced in insects after ingestion of plant-expressed hairpin RNA offers promise for managing devastating crop pests.

Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC

Abstract: Despite the great potential of RNAi, ectopic expression of shRNA or siRNAs holds the inherent risk of competition for critical RNAi components, thus altering the regulatory functions of some cellular microRNAs. In addition, specific siRNA sequences can potentially hinder incorporation of other siRNAs when used in a combinatorial approach. We show that both synthetic siRNAs and expressed shRNAs compete against each other and with the endogenous microRNAs for transport and for incorporation into the RNA induced silencing complex (RISC). The same siRNA sequences do not display competition when expressed from a microRNA backbone. We also show that TAR RNA binding protein (TRBP) is one of the sensors for selection and incorporation of the guide sequence of interfering RNAs. These findings reveal that combinatorial siRNA approaches can be problematic and have important implications for the methodology of expression and use of therapeutic interfering RNAs.

HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR

Abstract: RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA. In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing. HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency.

Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting.

Abstract: RNA interference (RNAi) is an evolutionarily conserved process by which double-stranded small interfering RNA (siRNA) induces sequence-specific, post-transcriptional gene silencing. Unlike other mRNA targeting strategies, RNAi takes advantage of the physiological gene silencing machinery. The potential use of siRNA as therapeutic agents has attracted great attention as a novel approach for treating severe and chronic diseases. RNAi can be achieved by either delivery of chemically synthesized siRNAs or endogenous expression of small hairpin RNA, siRNA, and microRNA (miRNA). However, the relatively high dose of siRNA required for gene silencing limits its therapeutic applications. This review discusses several strategies to improve therapeutic efficacy as well as to abrogate off-target effects and immunostimulation caused by siRNAs. There is an in-depth discussion on various issues related to the (1) mechanisms of RNAi, (2) methods of siRNA production, (3) barriers to RNAi-based therapies, (4) biodistribution, (5) design of siRNA molecules, (6) chemical modification and bioconjugation, (7) complex formation with lipids and polymers, (8) encapsulation into lipid particles, and (9) target specificity for enhanced therapeutic effectiveness.

DDX3 DEAD-Box RNA Helicase Is Required for Hepatitis C Virus RNA Replication

Abstract: DDX3, a DEAD-box RNA helicase, binds to the hepatitis C virus (HCV) core protein. However, the role(s) of DDX3 in HCV replication is still not understood. Here we demonstrate that the accumulation of both genome-length HCV RNA (HCV-O, genotype 1b) and its replicon RNA were significantly suppressed in HuH-7-derived cells expressing short hairpin RNA targeted to DDX3 by lentivirus vector transduction. As well, RNA replication of JFH1 (genotype 2a) and release of the core into the culture supernatants were suppressed in DDX3 knockdown cells after inoculation of the cell culture-generated HCVcc. Thus, DDX3 is required for HCV RNA replication.

Suppression of Hepatocyte Growth Factor Production Impairs the Ability of Adipose‐Derived Stem Cells to Promote Ischemic Tissue Revascularization

Abstract: The use of adipose-derived stem/stromal cells (ASCs) for promoting repair of tissues is a promising potential therapy, but the mechanisms of their action are not fully understood. We and others previously demonstrated accelerated reperfusion and tissue salvage by ASCs in peripheral ischemia models and have shown that ASCs secrete physiologically relevant levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor. The specific contribution of HGF to ASC potency was determined by silencing HGF expression. RNA interference was used to downregulate HGF expression. A dual-cassette lentiviral construct expressing green fluorescent protein (GFP) and either a small hairpin RNA specifically targeted to HGF mRNA (shHGF) or an inactive control sequence (shCtrl) were used to stably transduce ASCs (ASC-shHGF and ASC-shCtrl, respectively). Transduced ASC-shHGF secreted >80% less HGF, which led to a reduced ability to promote survival, proliferation, and migration of mature and progenitor endothelial cells in vitro. ASC-shHGF were also significantly impaired, compared with ASC-shCtrl, in their ability to promote reperfusion in a mouse hindlimb ischemia model. The diminished ability of ASCs with silenced HGF to promote reperfusion of ischemic tissues was reflected by reduced densities of capillaries in reperfused tissues. In addition, fewer GFP+ cells were detected at 3 weeks in ischemic limbs of mice treated with ASC-shHGF compared with those treated with ASC-shCtrl. These results indicate that production of HGF is important for the potency of ASCs. This finding directly supports the emerging concept that local factor secretion by donor cells is a key element of cell-based therapies. Disclosure of potential conflicts of interest is found at the end of this article.

LambdaN-GFP: an RNA reporter system for live-cell imaging.

Abstract: We describe a GFP-based RNA reporter system (lambdaN-GFP) to visualize RNA molecules in live mammalian cells. It consists of GFP fused to an arginine-rich peptide derived from the phage lambda N protein, lambdaN22, which binds a unique minimal RNA motif and can be used to tag any RNA molecule. LambdaN-GFP uses a small and easy to engineer RNA tag, reducing the likelihood of perturbing the function of the tagged RNA molecule.

c-Myc and Sp1 contribute to proviral latency by recruiting histone deacetylase 1 to the human immunodeficiency virus type 1 promoter.

Abstract: Histone deacetylase (HDAC) inhibitors such as valproic acid (VPA) induce the expression of quiescent proviral human immunodeficiency virus type 1 (HIV-1) and may deplete proviral infection in vivo. To uncover novel molecular mechanisms that maintain HIV latency, we sought cellular mRNAs whose expression was diminished in resting CD4(+) T cells of HIV-1-infected patients exposed to VPA. c-Myc was prominent among genes markedly downregulated upon exposure to VPA. c-Myc expression repressed HIV-1 expression in chronically infected cell lines. Chromatin immunoprecipitation (ChIP) assays revealed that c-Myc and HDAC1 are coordinately resident at the HIV-1 long terminal repeat (LTR) promoter and absent from the promoter after VPA treatment in concert with histone acetylation, RNA polymerase II recruitment, and LTR expression. Sequential ChIP assays demonstrated that c-Myc, Sp1, and HDAC1 coexist in the same DNA-protein complex at the HIV promoter. Short hairpin RNA inhibition of c-Myc reduces both c-Myc and HDAC1 occupancy, blocks c-Myc repression of Tat activation, and increases LTR expression. These results expand the understanding of mechanisms that recruit HDAC and maintain the latency of HIV-1, suggesting novel therapeutic approaches against latent proviral HIV infection.

Cutting Edge: Monarch-1 Suppresses Non-Canonical NF-κB Activation and p52-Dependent Chemokine Expression in Monocytes

Abstract: CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-κB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-κB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.

Regulation of Notch1 gene expression by p53 in epithelial cells.

Abstract: The E6 protein of cervical cancer-associated human papillomaviruses (HPVs) is known to suppress keratinocyte differentiation through unidentified mechanisms. Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis. Here, we report that the Notch1 gene is a novel target of p53 and can be down-regulated by E6 through p53 degradation in normal human epithelial cells. Thus, inactivation of p53 by E6 or short-hairpin RNA (shRNA) resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. The expression of E6, p53 shRNA, or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and its induction upon DNA damage. Furthermore, the induction of Notch1 and differentiation makers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type but not in p53-deficient mouse skin. Together, our findings not only demonstrate a novel link between p53 and Notch1 in keratinocyte differentiation upon genotoxic stress but also suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors.

RNA interference and inhibition of MEK-ERK signaling prevent abnormal skeletal phenotypes in a mouse model of craniosynostosis.

Abstract: Premature fusion of one or more of the cranial sutures (craniosynostosis) in humans causes over 100 skeletal diseases, which occur in 1 of approximately 2,500 live births. Among them is Apert syndrome, one of the most severe forms of craniosynostosis, primarily caused by missense mutations leading to amino acid changes S252W or P253R in fibroblast growth factor receptor 2 (FGFR2). Here we show that a small hairpin RNA targeting the dominant mutant form of Fgfr2 (Fgfr2(S252W)) completely prevents Apert-like syndrome in mice. Restoration of normal FGFR2 signaling is manifested by an alteration of the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2), implicating the gene encoding ERK and the genes downstream of it in disease expressivity. Furthermore, treatment of the mutant mice with U0126, an inhibitor of mitogen-activated protein (MAP) kinase kinase 1 and 2 (MEK1/2) that blocks phosphorylation and activation of ERK1/2, significantly inhibits craniosynostosis. These results illustrate a pathogenic role for ERK activation in craniosynostosis resulting from FGFR2 with the S252W substitution and introduce a new concept of small-molecule inhibitor-mediated prevention and therapy for diseases caused by gain-of-function mutations in the human genome.

Critical role for Ebf1 and Ebf2 in the adipogenic transcriptional cascade.

Abstract: The Ebf (O/E) family of helix-loop-helix transcription factors plays a significant role in B lymphocyte and neuronal development. The three primary members of this family, Ebf1, 2, and 3, are all expressed in adipocytes, and Ebf1 promotes adipogenesis when overexpressed in NIH 3T3 fibroblasts. Here we report that these three proteins have adipogenic potential in multiple cellular models and that peroxisome proliferator-activated receptor gamma (PPAR gamma) is required for this effect, at least in part due to direct activation of the PPAR gamma 1 promoter by Ebf1. Ebf1 also directly binds to and activates the C/EBP alpha promoter, which exerts positive feedback on C/EBP delta expression. Despite this, C/EBP alpha is dispensable for the adipogenic action of Ebf proteins. Ebf1 itself is induced by C/EBP beta and delta, which bind and activate its promoter. Reduction of Ebf1 and Ebf2 proteins by specific short hairpin RNA blocks differentiation of 3T3-L1 cells, suggesting a critical role for these factors and the absence of functional redundancy between members of this family. Altogether, these data place Ebf1 within the known transcriptional cascade of adipogenesis and suggest critical roles for Ebf1 and Ebf2. (Less)

Tissue-specific and reversible RNA interference in transgenic mice

Abstract: Genetically engineered mice provide powerful tools for understanding mammalian gene function. These models traditionally rely on gene overexpression from transgenes or targeted, irreversible gene mutation. By adapting the tetracycline (tet)-responsive system previously used for gene overexpression, we have developed a simple transgenic system to reversibly control endogenous gene expression using RNA interference (RNAi) in mice. Transgenic mice harboring a tet-responsive RNA polymerase II promoter driving a microRNA-based short hairpin RNA targeting the tumor suppressor Trp53 reversibly express short hairpin RNA when crossed with existing mouse strains expressing general or tissue-specific 'tet-on' or 'tet-off' transactivators. Reversible Trp53 knockdown can be achieved in several tissues, and restoring Trp53 expression in lymphomas whose development is promoted by Trp53 knockdown leads to tumor regression. By leaving the target gene unaltered, this approach permits tissue-specific, reversible regulation of endogenous gene expression in vivo, with potential broad application in basic biology and drug target validation.

MUC4 mucin potentiates pancreatic tumor cell proliferation, survival, and invasive properties and interferes with its interaction to extracellular matrix proteins

Abstract: MUC4, a transmembrane mucin, is aberrantly expressed in pancreatic adenocarcinomas while remaining undetectable in the normal pancreas. Recent studies have shown that the expression of MUC4 is associated with the progression of pancreatic cancer and is inversely correlated with the prognosis of pancreatic cancer patients. In the present study, we have examined the phenotypic and molecular consequences of MUC4 silencing with an aim of establishing the mechanistic basis for its observed role in the pathogenesis of pancreatic cancer. The silencing of MUC4 expression was achieved by stable expression of a MUC4-specific short hairpin RNA in CD18/HPAF, a highly metastatic pancreatic adenocarcinoma cell line. A significant decrease in MUC4 expression was detected in MUC4-knockdown (CD18/HPAF-siMUC4) cells compared with the parental and scrambled short interfering RNA-transfected (CD18/HPAF-Scr) control cells by immunoblot analysis and immunofluorescence confocal microscopy. Consistent with our previous observation, inhibition of MUC4 expression restrained the pancreatic tumor cell growth and metastasis as shown in an orthotopic mouse model. Our in vitro studies revealed that MUC4-associated increase in tumor cell growth resulted from both the enhanced proliferation and reduced cell death. Furthermore, MUC4 expression was also associated with significantly increased invasiveness (P < or = 0.05) and changes in actin organization. The presence of MUC4 on the cell surface was shown to interfere with the tumor cell-extracellular matrix interactions, in part, by inhibiting the integrin-mediated cell adhesion. An altered expression of growth- and metastasis-associated genes (LI-cadherin, CEACAM6, RAC1, AnnexinA1, thrombomodulin, epiregulin, S100A4, TP53, TP53BP, caspase-2, caspase-3, caspase-7, plakoglobin, and neuregulin-2) was also observed as a consequence of the silencing of MUC4. In conclusion, our study provides experimental evidence that supports the functional significance of MUC4 in pancreatic cancer progression and indicates a novel role for MUC4 in cancer cell signaling.

Effect of target secondary structure on RNAi efficiency

Abstract: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful tool for gene knockdown studies. However, the levels of knockdown vary greatly. Here, we examine the effect of target disruption energy, a novel measure of target accessibility, along with other parameters that may affect RNAi efficiency. Based on target secondary structures predicted by the Sfold program, the target disruption energy represents the free energy cost for local alteration of the target structure to allow target binding by the siRNA guide strand. In analyses of 100 siRNAs and 101 shRNAs targeted to 103 endogenous human genes, we find that the disruption energy is an important determinant of RNAi activity and the asymmetry of siRNA duplex asymmetry is important for facilitating the assembly of the RNA-induced silencing complex (RISC). We estimate that target accessibility and duplex asymmetry can improve the target knockdown level significantly by nearly 40% and 26%, respectively. In the RNAi pathway, RISC assembly precedes target binding by the siRNA guide strand. Thus, our findings suggest that duplex asymmetry has significant upstream effect on RISC assembly and target accessibility has strong downstream effect on target recognition. The results of the analyses suggest criteria for improving the design of siRNAs and shRNAs.

Histone deacetylase 2 modulates p53 transcriptional activities through regulation of p53-DNA binding activity.

Abstract: Histone deacetylase (HDAC) inhibitors are emerging as promising cancer therapeutics. HDAC inhibitors have been found to induce cellular activities that are strikingly similar to p53-mediated responses to genotoxic stress. For example, HDAC inhibitors induce cell cycle arrest, apoptosis, and cellular senescence. Because at least 11 HDACs are affected by the current HDAC inhibitors, the HDAC critical for tumor cell survival and proliferation remains unknown. Thus, we sought to characterize the distinct roles of HDACs in the p53 pathway. Through the use of stable MCF7 cell lines which inducibly express short hairpin RNA targeting HDAC2, we found that HDAC2 plays important roles in the p53 pathway. Specifically, we found that knockdown of HDAC2 inhibited cellular proliferation in a dose-dependent manner which was also partly p53-dependent. Furthermore, knockdown of HDAC2 induced cellular senescence. Importantly, we found that knockdown of HDAC2 enhanced p53-dependent trans-repression and trans-activation of a subset of target genes. We found that the enhancement was due to increased p53-DNA binding activity but not alterations in p53 stability or posttranslational modification(s). Thus, for the first time, our data suggest that HDAC inhibitors function through the p53 pathway, at least in part, by activating p53-DNA binding activity.

Integrin α11 regulates IGF2 expression in fibroblasts to enhance tumorigenicity of human non-small-cell lung cancer cells

Abstract: Integrin α11 (ITGA11/α11) is localized to stromal fibroblasts and commonly overexpressed in non-small-cell lung carcinoma (NSCLC). We hypothesized that stromal α11 could be important for the tumorigenicity of NSCLC cells. SV40 immortalized mouse embryonic fibroblasts established from wild-type (WT) and Itga11-deficient [knockout (KO)] mice were tested for their tumorigenicity in immune-deficient mice when implanted alone or coimplanted with the A549 human lung adenocarcinoma cells. A549 coimplanted with the fibroblasts showed a markedly enhanced tumor growth rate compared with A549, WT, or KO, which alone formed only small tumors. Importantly, the growth was significantly greater for A549+WT compared with A549+KO tumors. Reexpression of human α11 cDNA in KO cells rescued a tumor growth rate to that comparable with the A549+WT tumors. These findings were validated in two other NSCLC cell lines, NCI-H460 and NCI-H520. Gene expression profiling indicated that IGF2 mRNA expression level was >200 times lower in A549+KO compared with A549+WT tumors. Stable short-hairpin RNA (shRNA) down-regulation of IGF2 in WT (WTshIGF2) fibroblasts resulted in a decreased growth rate of A549+WTshIGF2, compared with A549+WT tumors. The results indicate that α11 is an important stromal factor in NSCLC and propose a paradigm for carcinoma–stromal interaction indirectly through interaction between the matrix collagen and stromal fibroblasts to stimulate cancer cell growth.

Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis

Abstract: Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2−/− mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2−/− mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

Gene silencing by small regulatory RNAs in mammalian cells.

Abstract: Since some years, activation of RNA interference (RNAi) has been developed into a widely used and powerful biological tool to functionally annotate genomes. Several variants of small regulatory RNAs can trigger this evolutionary highly conserved cellular pathway leading to specific hybridisation to and subsequent degradation or translational repression of target mRNAs. These regulatory RNAs include synthetic double-stranded small interfering RNAs (siRNAs), pol III transcribed small hairpin RNAs (shRNAs), or endogenous or artificial micro RNAs (miRNAs) which are expressed from pol II promoters as primary pri-miRNA transcripts subsequently processed into mature miRNAs in a regulated multi-step process. Depending on the mode of activation RNA-processing and efficacy as well as kinetics of RNAi may differ from transient effects to long-lasting gene silencing. However, one of the major challenges for in vitro and in vivo application of RNAi still remains the efficient delivery of suitable RNAi-triggers to target cells. This review highlights the mechanism of RNAi and its activation, the use of plasmid-based and retro-/ lentiviral vector-based systems to mediate long-term RNAi, kinetic aspects of RNAi and their impact on selection of cell populations with modified gene expression.