Biomedical subjects

Flossie Wong-Staal

Publications and source records attributed to Flossie Wong-Staal.

At least 19 recordsLinked to original sources

PubMed2007

Ribozyme technology for cancer gene target identification and validation.

Ribozymes are naturally occurring RNAs with catalytic activities including cis- or trans- cleavage of RNA at predefined sequence sites. This activity has been exploited for specific gene inactivation in cells during the last two decades, and ribozymes have been important functional genomics tools, especially in the pre-RNAi era. It has also been broadly applied in drug target identification and validation in pharmaceutical R&D. This chapter covers many application principles and case studies of ribozyme technology in the areas of cancer research. We also described RNAi applications in some of the same studies for comparison. Although RNAi may be more effective than ribozymes in many respects, they are nonetheless built on many of the same principles.

Animals↗

PubMed2006

A new inducible RNAi xenograft model for assessing the staged tumor response to mTOR silencing.

Human xenograft tumor models are widely used for efficacy evaluation of potential cancer targets. siRNA is usually stably introduced into tumor cells prior to transplantation. However, silencing of the cancer therapeutic target usually results in reduced cell growth/survival in vitro and/or failure to establish tumors in vivo, thus hindering tumor response-based efficacy evaluation. The present study explored a new tumor response model based on regulated RNAi, which is more relevant from a clinical standpoint. As a proof of principle, an inducible lentiviral RNAi vector was used to silence the known cancer therapeutic target mTOR upon induction with Doxycycline (DOX). The responses to DOX-induced mTOR silencing were tested both in vitro and in vivo for prostate cancer PC3 models. Significant reduction in cancer cell survival was observed due to cell cycle arrest and apoptosis when mTOR silencing was induced in vitro. mTOR silencing also caused tumor regression for the early-staged PC3 tumors (100% tumor regressed and 45% became tumor-free). The advanced-staged tumors also demonstrated significant responses (100% regressed). Therefore, our results demonstrate the powerful utility of this new inducible xenograft tumor model for efficacy evaluation of cancer targets, and it provides a direct in vivo efficacy validation of mTOR as a cancer therapeutic target.

Animals↗

PubMed2006

A novel RNAi library based on partially randomized consensus sequences of nuclear receptors: identifying the receptors involved in amyloid beta degradation.

Combinatorial gene inactivation using an RNAi library is a powerful approach to discovering novel functional genes. However, generation of a comprehensive RNAi library remains technically challenging. In this report, we describe a simple and novel approach to designing gene-family-specific RNAi libraries by targeting conserved motifs using degenerate oligonucleotides. We created an siRNA library in the pHUMU vector using partially randomized sequences targeting the consensus region in the ZnF_C4 signature motif of the nuclear hormone receptors and thus against the entire receptor superfamily. For proof of principle, we adapted a reporter assay to screen this library for receptors that might be involved in reducing amyloid beta peptide accumulation. We modified a previously described luciferase reporter assay to measure the amyloid beta precursor cleavages occurring only between beta- and gamma-secretase cleavage sites, thus excluding the major gamma-secretase activities that could generate neurotoxic Abeta peptides. Our screen using this assay identified siRNA vectors that specifically increase the Abeta40/42 cleavage and pointed to a potential receptor target, ROR-gamma. SiRNAs targeting other regions of ROR-gamma not only confirmed the observed reporter activity but also reduced the level of the toxic Abeta peptides. The results demonstrated a general principle for the creation and application of this RNAi library approach for functional gene discovery within a predefined protein family. The discovered negative effect of ROR-gamma on the degradation of the toxic Abeta peptides may also provide a potential drug target or targetable pathway for intervention of Alzheimer disease.

Alzheimer Disease↗

PubMed2006

PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway.

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.

Amino Acid Sequence↗

PubMed2006

MDDD, a 4,9-diazapyrenium derivative, is selectively toxic to glioma cells by inducing growth arrest at G0/G1 independently of p53.

4-Methyl-2,7-diamino-5,10-diphenyl-4,9-diaz-apyrenium chloride (MDDD), a stable and water soluble nucleic acid-intercalating agent, was shown to be toxic to cancer cells with IC50 around 10 microM. IC(50) We tested MDDD for its potential antitumor activities and found it inhibited cancer cell growth with IC(50) in the micromolar range for the majority of cancer cells tested, with the exception of glioma cells, for which the IC(50) is in the submicromolar range. This unique selectivity of MDDD to glioma cells can potentially be exploited for anti-glioma therapeutics. Although the underlying mechanisms for the apparent glioma specificity remain to be elucidated, our analysis indicates that MDDD significantly reduces cell clonogenicity and blockes cell proliferation at the G1 phase. MDDD treatment also triggers induction of p53 and p21 at the protein levels, suggesting the activation of DNA damage response. However, MDDD mediated growth inhibition does not require the p53 pathway since p53+/- isogenic cell pairs display the same sensitivity. These properties of MDDD favor its candidacy for evaluation as a new anti-tumor agent, particularly for glioma.

Animals↗

PubMed2006

Therapy with ribozyme to the proliferating cell nuclear antigen-ribozyme and 5-fluorouracil of experimental choroidal neovascularization in rats.

PURPOSE: The aim of this study was to evaluate the efficacy of hammerhead ribozyme to the proliferating cell nuclear antigen (PCNA-Rz) and 5-fluorouracil (5-FU) in experimental choroidal neovascularization (CNV) model in rats. METHODS: Laser was used to induce CNV in each eye of 44 rats. For angiography studies, injections of either a mixture of PCNA-Rz 10 microg/microL and 5-FU 1.5 microg/microL, versus the same dose of either drug alone versus a control injection of Hanks' Balanced Salt Solution (HBSS) were performed. We also studied this regimen to evaluate scar size and volume. RESULTS: There was significantly less angiographic leakage for the treated eyes compared to the controls by 3.53 grading points (P = 0.0005); CNV leakage was reduced in the combination group compared to 5-FU alone by 1.75 grading units (P = 0.04) and compared to PCNARz by 2.22 grading units (P = 0.07). The scar size and volume were smaller (diameter 354.6 +/- 174.2 microm vs 477.3 +/- 157.0 microm), (thickness 52.7 +/- 43.0 microm versus 79.6 +/- 46.2 microm) with a reduction in scar volume of 44.8%. CONCLUSIONS: Subretinal injection of PCNA-Rz and 5-FU mixture is more effective as treatment of laser-induced CNV, than either drug alone. The majority of the antiangiogenic effect is a result of 5-FU activity with a contribution by the PCNA ribozyme.

Animals↗

PubMed2006

RNA interference and potential applications.

RNA interference (RNAi) is the process of using specific sequences of double-stranded RNA (dsRNA) to knock down the expression level of sequence-homologous genes. Such ability of small interfering RNA (siRNA) in mammalian cells will undoubtedly revolutionize the study of functional genomics, the discovery of drug targets and even the treatment of human diseases. In this review we briefly describe the history of RNAi discovery, the RNAi mechanism and the general guideline for siRNA design as well as various methods for siRNA production and delivery. We also introduce the potential applications of siRNA, inducible siRNA and siRNA library in speeding up basic biomedical research and in acting as potential therapeutic agents for treatment of numerous human diseases.

Animals↗

PubMed2005

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli.

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.

Animals↗

PubMed2005

A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.

Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.

Base Sequence↗

PubMed2004

Nuclear hormone receptor NR4A2 is involved in cell transformation and apoptosis.

HeLaHF cells are transformation revertants of cervical cancer HeLa cells and have lost anchorage-independent growth potential and tumorigenicity. Activation of tumor suppressor(s) was implicated previously in this transformation reversion. In this study, expression profiling analysis was carried out to identify potential oncogenes that are down-regulated in HeLaHF cells. We found that all three members of the NR4A1/Nur77/NGFIB orphan nuclear hormone receptor subfamily (NR4A1, NR4A2, and NR4A3) were down-regulated in the HeLaHF revertant. Small interfering RNA-mediated down-regulation of NR4A2 in HeLa cells, either transiently or stably, resulted in reduced anchorage-independent growth that was largely attributable to increased anoikis. Furthermore, down-regulation of NR4A2 as well as NR4A1 promoted intrinsic apoptosis. These phenotypes were also observed in several other experimental cancer cells, suggesting the observed apoptosis suppression is a more general property of NR4A2 and NR4A1. These phenotypes also suggest that the Nur77/NGFIB subfamily of orphan receptors exhibit certain oncogenic functionalities with regards to cell proliferation and apoptosis and could therefore be evaluated as potential cancer therapeutic targets.

Apoptosis↗

PubMed2004

Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus.

Short interfering RNAs (siRNAs) directed against different regions of genes display marked variation in their potency in mediating mRNA degradation. Various factors have been proposed to affect the efficacy of siRNA. We explored some of the factors by evaluating in cultured human cells 28 randomly selected siRNAs targeting the GPR39 and MGC29643 transcripts derived from the same genetic locus but transcribed in opposite directions. Twenty of the 24 siRNAs targeting the overlapping regions of the transcripts simultaneously reduced the levels of both transcripts. Single nucleotide changes in either of the siRNA strands significantly reduced the gene-silencing efficiency of the siRNA on targeted sense transcript without affecting the antisense transcript. Overall, we observed a greater gene-silencing efficiency on the MGC29643 transcript than on the GPR39 transcript in HeLa cells. Since MGC29643 transcript is more abundant than the GPR39 transcript [0.24 versus 0.008% relative to 100% for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)], the results suggest that the abundance of the mRNA affects the efficiency of silencing. Two additional observations supported this hypothesis. First, GAPDH whose intracellular level is the highest of the three was the most efficiently silenced. Second, a reversal of gene-silencing efficiency was observed in U-138 MG cells in which the relative abundance of the GPR39 and MGC29643 transcripts is also reversed. Our study suggests that low-abundant transcripts are less susceptible to siRNA-mediated degradation than medium- and high-abundant transcripts.

Cell Line, Tumor↗

PubMed2004

The HIV-1 HLA-A2-SLYNTVATL is a help-independent CTL epitope.

The CTL response to the HLA-A*0201-restricted, HIV-1 p17 Gag(77-85) epitope (SLYNTVATL; SL9) has been extensively studied in patients. Although this reactivity is exceptionally prominent in chronically infected patients and inversely correlated to viral load, SL9-specific CTLs (SL9-CTLs) are rarely detected in acute infection. To explore the cellular basis for this unusual manifestation, SL9-CTLs primed ex vivo from naive circulating CD8(+) T cells of healthy, seronegative donors were generated and characterized. SL9 appeared to differ from other well-studied A*0201-restricted epitopes in several significant respects. In contrast to published reports for influenza and melanoma peptides and the HIV gag IV9 epitope studied here in parallel, SL9-CTLs were primed by immature but not mature autologous dendritic cells. Highly activated SL9-CTLs produce sufficient autocrine mediators to sustain clonal expansion and CTL differentiation for months without CD4(+) T cells or exogenous IL-2. Moreover, SL9-CTLs were sensitive to paracrine IL-2-induced apoptosis. IL-2 independence and sensitivity to paracrine IL-2 were also characteristic of SL9-CTLs immunized by dendritic cells transduced by a nonreplicating lentiviral vector encoding full-length Gag. In vitro-primed SL9-CTLs resembled those derived from patients in degeneracy of recognition and functional avidities for both SL9 and its natural mutations. Together, these data show that SL9 is a highly immunogenic, help-independent HIV epitope. The scarcity of SL9-CTLs in acute infection may result from cytokine-induced apoptosis with the intense activation of the innate immunity. In contrast, SL9-CTLs that constitutively produce autocrine help would predominate during CD4-diminished chronic infection.

Antigen Presentation↗

PubMed2004

Arginine methylation of RNA helicase a determines its subcellular localization.

RNA helicase A (RHA) undergoes nuclear translocation via a classical import mechanism utilizing karyopherin beta. The nuclear transport domain (NTD) of RHA is known to be necessary and sufficient for its bi-directional nuclear trafficking. We report here that arginine methylation is a novel requirement for NTD-mediated nuclear import. Nuclear translocation of glutathione S-transferase (GST)-NTD fusion proteins is abrogated by arginine-methylation inhibitors. However, in vitro arginine-methylation of GST-NTD prior to injection allows the fusion protein to localize to the nucleus in the presence of methylation inhibitors. Removal of the arginine-rich C-terminal region negates the effects of the methylation inhibitors on NTD import, suggesting that methylation of the NTD C terminus the relieves the cytoplasmic retention of RHA. The NTD physically interacts with PRMT1, the major protein arginine methyltransferase. These findings provide evidence for a novel arginine methylation-dependent regulatory pathway controlling the nuclear import of RHA.

Arginine↗

PubMed2004

Transduction of cell lines and primary cells by FIV-packaged HIV vectors.

Human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and feline immunodeficiency virus (FIV) are capable of packaging viral RNA derived from heterologous as well as homologous lentiviruses, a phenomenon referred to as "cross packaging." To remove the possibility of seroconversion to HIV proteins, and to avoid potential problems arising due to targeting of vector or packaging construct by antiviral genes, we investigated the feasibility of using an FIV-based packaging system to deliver human immunodeficiency virus type 2 (HIV-2)-based vectors bearing anti-HIV-1 RNA expression cassettes to target cells. In the absence of FIV rev, FIV was packaged by HIV-2 at only 3% the efficiency of FIV packaging by FIV, but this was increased to 39% of homologous controls by supplying FIV rev in trans. HIV-2 vectors were packaged by FIV at levels equal to or exceeding the homologous HIV-2 packaging system in the absence of HIV-1 tat and rev, and levels increased approximately four- to fivefold with the addition of tat and rev in trans. HIV-2 vectors bearing a polyribozyme cassette targeting multiple regions of HIV RNA were efficiently packaged by FIV and transferred to target cells. Upon challenge with cell-free HIV-1 (m.o.i. = 0.1) a significant reduction in replication was observed. These findings demonstrate that packaging HIV vectors with FIV is a viable alternative, which avoids use of HIV structural proteins.

Cell Line↗

PubMed2004

The development and applications of nonradioactive plate-formatted DNA-binding assay for Ku70/80, a multifunctional DNA-binding protein complex.

Ku is a heterodimer composed of p70 and p80, and is the regulatory subunit of DNA-dependent protein kinase. As a multifunctional DNA-binding protein complex, Ku plays important roles in DNA damage repair through non-homologous end joining and in V(D)J recombination. In addition, Ku has also been implicated in various biological functions including growth control, cell proliferation, cell cycle, chromosome maintenance, transcriptional regulation, apoptosis, and viral infection. In particular, using our Inverse Genomics (Immusol, Inc., San Diego, CA) platform technology, we recently identified Ku80 as an essential co-factor for human immunodeficiency virus replication. Although Ku has been studied extensively in the past years, its in-depth study as well as development as a drug target has been limited by conventional DNA-binding activity assay. Here we describe the development and applications of a nonradioactive DNA binding assay in the 96-well format. We show that this plate-formatted assay is more sensitive and allows for direct quantification when compared with an electrophoretic mobility shift assay. The establishment of this assay will not only facilitate structure and function studies on Ku, but also help the development of Ku protein or its DNA repair enzyme complex as a drug target.

Animals↗

PubMed2004

RNA helicase A interacts with nuclear factor kappaB p65 and functions as a transcriptional coactivator.

RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export. RHA has been reported to bind directly to the transcriptional coactivator, CREB-binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex. Using yeast two-hybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor kappaB (NF-kappaB). The interaction between p65 and RHA was confirmed by glutathione-S transferase pull-down assay in vitro, and by co-immunoprecipitation assay in vivo. In transient transfection assays, RHA enhanced NF-kappaB dependent reporter gene expression induced by p65, tumor necrosis factor-alpha, or NF-kappaB inducing kinase. The mutant form of RHA lacking ATP-binding activity inhibited NF-kappaB dependent reporter gene expression induced by these activators. Moreover, depletion of RHA using short interfering RNA reduced the NF-kappaB dependent transactivation. These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF-kappaB.

Autoantigens↗

PubMed2004

Identification of cellular cofactors for human immunodeficiency virus replication via a ribozyme-based genomics approach.

Ribozymes are small, catalytic RNA molecules that can be engineered to down-regulate gene expression by cleaving specific mRNA. Here we report the selection of hairpin ribozymes that inhibit human immunodeficiency virus (HIV) replication from a combinatorial ribozyme library. We identified a total of 17 effective ribozymes, each capable of inhibiting HIV infection of human CD4(+) cells. These ribozymes target diverse steps of the viral replication cycle, ranging from entry to transcription. One ribozyme suppressed HIV integration and transcription by inhibiting the expression of the Ku80 subunit of the DNA-activated protein kinase. Another ribozyme specifically inhibited long terminal repeat transactivation, while two additional ones blocked a step that can be bypassed by vesicular stomatitis virus G-protein pseudotyping. The function of Ku80 in HIV replication and its mechanism of action were further confirmed using short interfering RNA. Identification of the gene targets of these and other selected ribozymes may reveal novel therapeutic targets for combating HIV infection.

Apoptosis↗

PubMed2004

One-week 96-well soft agar growth assay for cancer target validation.

Soft agar growth, used to measure cell anchorage-independent proliferation potential, is one of the most important and most commonly used assays to detect cell transformation. However, the traditional soft agar assay is time-consuming, labor-intensive, and plagued with inconsistencies due to individual subjectivity. It does not, therefore, meet the increasing demands of today's oncology drug target screening or validation processes. This report describes an alternative 96-well soft agar growth assay that can function as a replacement for the traditional method and overcomes the aforementioned limitations. It offers the following advantages: a shortened assay duration (1 week instead of 4 weeks) that makes transient transfection or treatment possible; plate reader quantification of soft agar growth (measuring cloning efficiency and colony size); and a significant reduction in required labor. Higher throughput also makes it possible to process large numbers of samples and treatments simultaneously and in a much more efficient manner, while saving precious workspace and overall cost.

Agar↗