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Mouldy Sioud

Publications and source records attributed to Mouldy Sioud.

At least 37 records · Page 2Linked to original sources
PubMed2005

Identification of a novel centrosome/microtubule-associated coiled-coil protein involved in cell-cycle progression and spindle organization.

Here we describe the identification of a novel vertebrate-specific centrosome/spindle pole-associated protein (CSPP) involved in cell-cycle regulation. The protein is predicted to have a tripartite domain structure, where the N- and C-terminal domains are linked through a coiled-coil mid-domain. Experimental analysis of the identified domains revealed that spindle association is dependent on the N-terminal and the coiled-coil mid domain. The expression of CSPP at the mRNA level was detected in all tested cell lines and in testis tissue. Ectopic expression of CSPP in HEK293T cells blocked cell-cycle progression in early G1 phase and in mitosis in a dose-dependent manner. Interestingly, mitosis-arrested cells contained aberrant spindles and showed impairment of chromosome congression. Inhibition of CSPP gene expression by small interfering RNAs induced cell-cycle arrest/delay in S phase. This phenotype was characterized by elevated levels of cyclin A, decreased levels of cyclin E and hyperphosphorylation of the S-phase checkpoint kinase Chk1. The activation of Chk1 may indicate a replication stress response due to an inappropriate G1/S-phase transition. Taken together, we demonstrate that CSPP is associated with centrosomes and microtubules and may play a role in the regulation of G(1)/S-phase progression and spindle assembly.

Amino Acid Sequence↗
PubMed2005

On the delivery of small interfering RNAs into mammalian cells.

RNA interference is becoming the technique of choice for analysing gene function and drug target validation. In this process, sequence-specific gene inhibition is initiated by small RNA duplexes, known as small interfering RNAs (siRNAs). The possibility that exogenously delivered siRNAs or endogenously expressed hairpin siRNAs can cause the destruction of specific target mRNA in vitro and in animal models has been demonstrated. However, the key challenges for the development of siRNAs as human therapeutics is largely dependent on the development of suitable delivery agents and improved siRNA specificity. This review highlights recent advances in siRNA delivery, as well as challenging problems related to immune stimulation.

Animals↗
PubMed2005

Ribozymes, DNAzymes and small interfering RNAs as therapeutics.

Selective gene silencing by nucleic acid enzymes has provided researchers with a new strategy to block gene expression and drug target validation. Ribozymes, DNAzymes and small interfering RNAs (siRNAs) are being explored as genetic inhibitors of gene expression as well as potential therapeutics against viral infections, inflammatory disorders, haematological diseases and cancers. We review the mode of action of these molecules, with special emphasis on their construction and the possibility to enhance their serum half-lives via specific chemical modifications. Their potential use in cell cultures and in animal models for disease is also highlighted.

Animals↗
PubMed2005

Systematic search for natural antisense transcripts in eukaryotes (review).

The availability of several complete genome sequences and large numbers of expressed sequences, has led to the development of bioinformatic strategies for large-scale predictions of natural antisense transcripts. In the past two years, this has given at least 1,600 human pairs and 2,500 murine pairs of natural antisense transcripts. However, due to limitations in bioinformatic search tools, experimental validation of the predicted antisense transcripts is crucial. This has been performed only for a small fraction of the large number of predicted natural antisense transcripts. Additionally, bioinformatic approaches will not allow systematic identification of natural antisense transcripts in specific cell or tissue types. More recently, an experimental approach for identification of sense-antisense transcript pairs has been developed. The principle of the strategy is based upon the selection of double-stranded cDNAs as a result of hybridization between first-strand cDNAs that should be generated in the presence of actinomycin D. This experimental strategy allows systematic analysis of sense and antisense partners in any cell or tissue type.

Animals↗
PubMed2004

Colony-stimulating factor-1 blockade by antisense oligonucleotides and small interfering RNAs suppresses growth of human mammary tumor xenografts in mice.

Colony-stimulating factor (CSF)-1 is the primary regulator of tissue macrophage production. CSF-1 expression is correlated with poor prognosis in breast cancer and is believed to enhance mammary tumor progression and metastasis through the recruitment and regulation of tumor-associated macrophages. Macrophages produce matrix metalloproteases (MMPs) and vascular endothelial growth factor, which are crucial for tumor invasion and angiogenesis. Given the important role of CSF-1, we hypothesized that blockade of CSF-1 or the CSF-1 receptor (the product of the c-fms proto-oncogene) would suppress macrophage infiltration and mammary tumor growth. Human MCF-7 mammary carcinoma cell xenografts in mice were treated with either mouse CSF-1 antisense oligonucleotide for 2 weeks or five intratumoral injections of either CSF-1 small interfering RNAs or c-fms small interfering RNAs. These treatments suppressed mammary tumor growth by 50%, 45%, and 40%, respectively, and selectively down-regulated target protein expression in tumor lysates. Host macrophage infiltration; host MMP-12, MMP-2, and vascular endothelial growth factor A expression; and endothelial cell proliferation within tumors of treated mice were decreased compared with tumors in control mice. In addition, mouse survival significantly increased after CSF-1 blockade. These studies demonstrate that CSF-1 and CSF-1 receptor are potential therapeutic targets for the treatment of mammary cancer.

Animals↗
PubMed2004

Analysis of the humoral immune response to immunoselected phage-displayed peptides by a microarray-based method.

We describe a novel approach for high-throughput analysis of the immune response in cancer patients using phage-based microarray technology. The recombinant phages used for fabricating phage arrays were initially selected via the use of random peptide phage libraries and breast cancer patient serum antibodies. The peptides displayed by the phages retained their ability to be recognized by serum antibodies after immobilization. The recombinant phage microarrays were screened against either breast cancer or healthy donor serum antibodies. A model-based statistical method is proposed to estimate significant differences in serum antibody reactivity between patients and normals. A significant tumor effect was found with most of the selected phage-displayed peptides, suggesting that recombinant phage microarrays can serve as a tool in monitoring humoral responses towards phage-displayed peptides.

Amino Acid Sequence↗
PubMed2004

Therapeutic siRNAs.

The ability of small-interfering RNAs (siRNAs) to silence gene expression in somatic mammalian cells has provided researchers with a novel tool to block the expression of disease-causing genes, provided that their mRNA sequences are known. siRNAs can be delivered to cells either exogenously as synthetic agents or endogenously as gene-encoding siRNAs. Recent studies demonstrate the general application of siRNAs to silence gene expression in a range of cell types and in whole mammals. Beyond their value for dissecting gene functions and target validation, siRNAs also hold great potential as gene-specific therapeutic agents.

Animals↗
PubMed2004

Inhibition of gene expression by nucleic acid enzymes in rodent models of human disease.

Nucleic acid enzymes have emerged as a versatile technique for sequence-specific gene silencing in a wide range of cells. However, the question remains as to whether, for example, DNA enzymes and ribozymes are functional in animals. In this chapter, we describe two different rodent models of human diseases--namely, leukemia and chronic heart failure. We specifically reduced Raf-1 expression in leukemic mice using an anti-Raf-1 DNA enzyme. A continuous supply of this catalytic molecule led to a substantial reduction in leukemic-cell burden and survival. Rats with postinfarction heart failure were treated with a DNA enzyme targeting TNFa, and this led to a substantial improvement of cardiac function concomitant with a restoration of the hemodynamic status of the animals. The described protocols should facilitate the in vivo evaluation of other oligonucleotide-based therapy such as small interfering RNAs (siRNAs).

Animals↗
PubMed2004

Potential design rules and enzymatic synthesis of siRNAs.

Small interfering RNAs (siRNAs) have emerged as a powerful technique for sequence-specific gene silencing in a wide variety of organisms. However, the base composition of the siRNA sequence is not the only determinant of efficacy. Intrinsic factors related to mRNA structures are likely to be crucial determinants for siRNA activity. Indeed, placing the recognition site of an active siRNA into a structured mRNA region has abrogated the siRNA activity. Therefore, a successful gene-targeting project may require the design of many distinct siRNAs at a high cost. Here, potential design rules, cost-effective strategies for producing siRNAs by T7 RNA polymerase, and expression cassettes for in vivo testing are described.

Base Sequence↗
PubMed2004

Gene-array analysis of glioma cells after treatment with an anti-PKCalpha siRNA: a general protocol.

Post-transcriptional gene silencing is a powerful tool to reveal gene function. In this chapter, we have used gene-array technology to analyze changes in gene expression after specific targeting of the protein kinase Calpha isoform with small interfering RNAs (siRNAs). Expression profiles were investigated on a human gene array of 588 key genes involved in DNA synthesis, apoptosis, cell-cell communication, intracellular signal-transduction pathways, cell-surface molecules and transcription factors. The data indicate that siRNAs are an ideal tool for target validation, and show that RNA-dependent RNA polymerase is not required for RNAi in mammalian cells.

Cell Line, Tumor↗
PubMed2004

Systemic delivery of synthetic siRNAs.

The ability to regulate endogenous gene expression by cleaving mRNA is important in basic and applied biological research. This is now driven predominantly by small interfering RNAs (siRNAs) as they induce sequence-specific gene silencing. However, the major obstacle to the use of siRNAs as therapeutics is the difficulty involved in effective in vivo delivery. This chapter describes the liposomal delivery of siRNAs into adult mice.

Animals↗
PubMed2004

Selection of peptides for specific delivery of oligonucleotides into cancer cells.

In this chapter, phage-display peptide technology has been used to select peptides that internalize into breast-cancer cells. The used biopanning procedure provides information on the best peptide to be used. When one of the selected peptides was conjugated to an antisense oligonucleotide against the ErbB2 receptor, specific delivery to breast-cancer cells was demonstrated. The established biopanning procedure should help in the rational selection of cancer-targeting peptides for specific delivery of DNA and RNA oligonucleotides into cancer cells.

Amino Acid Sequence↗
PubMed2004

Profiling microRNA expression using sensitive cDNA probes and filter arrays.

MicroRNAs (miRNAs) are small noncoding RNAs (approximately 22 nucleotides) that have recently emerged as important regulators of gene expression in both plants and animals. With few exceptions, however, the target genes and the expression levels of most miRNAs are unknown. Here we show that direct random-primed cDNA synthesis on either chemically synthesized small RNAs (21-22 nucleotides) or gel-purified mature miRNAs from human cells can produce specific and sensitive full-length cDNA probes. Using oligonucleotide filter arrays, we demonstrate that the internally labeled cDNA probes are sensitive for detecting differential miRNA expression between untreated and O-tetradecanoylphorbol-13-acetate (TPA)-treated HL60 cells. The present study should facilitate a high-throughput analysis of miRNA expression between samples.

DNA Probes↗
PubMed2004

Identification of genes differentially expressed in breast cancer cell line SKBR3: potential identification of new prognostic biomarkers.

The identification of differentially expressed genes in tumour cells should have important implications in understanding carcinogenesis and developing new therapeutic and prognostic biomarkers. We have combined PCR-based cDNA subtraction and Northern blotting to identify truly differentially expressed genes in breast cancer cell line SKBR3 as compared to normal human mammary epithelial cells (HMEC). Hybridizing probe molecules were rescued from the Hybond N+ membranes and then PCR reamplified. The PCR reamplification is possible due to the fact that all probe molecules contain the same pair of adapter sequences on both ends. After cloning and sequencing three known genes, ribosomal protein L19 (RPL19), ADP/ATP carrier protein and ErbB-2 with high-elevated mRNA levels in SKBR3 were identified. In addition, two overexpressed genes with unknown functions, CXYorf1-related protein and hypothetical protein PRO2605, were found. High-titer andibodies against the recombinant RPL19 were detected in 5 patients out of 50 patients investigated. Thus, the present novel strategy based on the combination of PCR-based cDNA subtraction and Northern blotting should facilitate the identification of truly differentially expressed biomarkers, which may offer the potential to determine the proper drug for an individual patient at a given stage of disease or treatment.

Biomarkers, Tumor↗