Activation of systemic acquired silencing by localised introduction of DNA.
BACKGROUND: In plants, post-transcriptional gene silencing results in RNA degradation after transcription. Among tobacco transformants carrying a nitrate reductase (Nia) construct under the control of the cauliflower mosaic virus 35S promoter (35S-Nia2), one class of transformants spontaneously triggers Nia post-transcriptional gene silencing (class II) whereas another class does not (class I). Non-silenced plants of both classes become silenced when grafted onto silenced stocks, indicating the existence of a systemic silencing signal. Graft-transmitted silencing is maintained in class II but not in class I plants when removed from silenced stocks, indicating similar requirements for spontaneous triggering and maintenance. RESULTS: Introduction of 35S-Nia2 DNA by the gene transfer method called biolistics led to localised acquired silencing (LAS) in bombarded leaves of wild-type, class I and class II plants, and to systemic acquired silencing (SAS) in class II plants. SAS occurred even if the targeted leaf was removed 2 days after bombardment, indicating that the systemic signal is produced, transmitted and amplified rapidly. SAS was activated by sense, antisense and promoterless Nia2 DNA constructs, indicating that transcription is not required although it does stimulate SAS. CONCLUSIONS: SAS was activated by biolistic introduction of promoterless constructs, indicating that the DNA itself is a potent activator of post-transcriptional gene silencing. The systemic silencing signal invaded the whole plant by cell-to-cell and long-distance propagation, and reamplification of the signal. (+info)
Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo.
Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Angiogenic factors are potentially optimal targets for therapeutic strategies because they are essential for tumor growth and progression. In this study, we sought a strategy for efficiently delivering an antisense cDNA molecule of the vascular endothelial growth factor (VEGF) to glioma cells. The recombinant adenoviral vector Ad5CMV-alphaVEGF carried the coding sequence of wild-type VEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignant glioma cells with the Ad5CMV-alphaVEGF resulted in reduction of the level of the endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein. Treatment of s.c. human glioma tumors established in nude mice with intralesional injection of Ad5CMV-alphaVEGF inhibited tumor growth. Taken together, these findings indicate that the efficient down-regulation of the VEGF produced by tumoral cells using antisense strategies has an antitumor effect in vivo. This is the first time that an adenoviral vector is used to transfer antisense VEGF sequence into glioma cells in an animal model, and our results suggest that this system may have clinical and therapeutic utility. (+info)
Antisense expression of the CK2 alpha-subunit gene in Arabidopsis. Effects on light-regulated gene expression and plant growth.
The protein kinase CK2 (formerly casein kinase II) is thought to be involved in light-regulated gene expression in plants because of its ability to phosphorylate transcription factors that bind to the promoter regions of light-regulated genes in vitro. To address this possibility in vivo and to learn more about the potential physiological roles of CK2 in plants, we transformed Arabidopsis with an antisense construct of the CK2 alpha-subunit gene and investigated both morphological and molecular phenotypes. Antisense transformants had a smaller adult leaf size and showed increased expression of chs in darkness and of cab and rbcS after red-light treatment. The latter molecular phenotype implied that CK2 might serve as one of several negative and quantitative effectors in light-regulated gene expression. The possible mechanism of CK2 action and its involvement in the phytochrome signal transduction pathway are discussed. (+info)
A novel imprinted gene, encoding a RING zinc-finger protein, and overlapping antisense transcript in the Prader-Willi syndrome critical region.
We describe a complex imprinted locus in chromosome 15q11-q13 that encodes two genes, ZNF127 and ZNF127AS. The ZNF127 gene encodes a protein with a RING (C3HC4) zinc-finger and multiple C3H zinc-finger motifs, the former being closely related to a protein from variola major virus, the smallpox etiological agent. These motifs allow prediction of ZNF127 function as a ribonucleoprotein. The intronless ZNF127 gene is expressed ubiquitously, but the entire coding sequence and 5' CpG island overlaps a second gene, ZNF127AS, that is transcribed from the antisense strand with a different transcript size and pattern of expression. Allele-specific analysis shows that ZNF127 is expressed only from the paternal allele. Consistent with this expression pattern, in the brain the ZNF127 5' CpG island is completely unmethylated on the paternal allele but methylated on the maternal allele. Analyses of adult testis, sperm and fetal oocytes demonstrates a gametic methylation imprint with unmethylated paternal germ cells. Recent findings indicate that ZNF127 is part of the coordinately regulated imprinted domain affected in Prader-Willi syndrome patients with imprinting mutations. Therefore, ZNF127 and ZNF127AS are novel imprinted genes that may be associated with some of the clinical features of the polygenic Prader-Willi syndrome. (+info)
Imprinting of a RING zinc-finger encoding gene in the mouse chromosome region homologous to the Prader-Willi syndrome genetic region.
A novel locus in the human Prader-Willi syndrome (PWS) region encodes the imprinted ZNF127 and antisense ZNF127AS genes. Here, we show that the mouse ZNF127 ortholog, Zfp127, encodes a homologous putative zinc-finger polypeptide, with a RING (C3HC4) and three C3H zinc-finger domains that suggest function as a ribonucleoprotein. By the use of RT-PCR across an in-frame hexamer tandem repeat and RNA from a Mus musculus x M.spretus F1interspecific cross, we show that Zfp127 is expressed only from the paternal allele in brain, heart and kidney. Similarly, Zfp127 is expressed in differentiated cells derived from androgenetic embryonic stem cells and normal embryos but not those from parthogenetic embryonic stem cells. We hypothesize that the gametic imprint may be set, at least in part, by the transcriptional activity of Zfp127 in pre- and post-meiotic male germ cells. Therefore, Zfp127 is a novel imprinted gene that may play a role in the imprinted phenotype of mouse models of PWS. (+info)
Antisense and nuclear medicine.
Despite many uncertainties concerning mechanism, synthetic single-strand antisense deoxyribonucleic acids (DNAs) are now in clinical trials for the chemotherapy of viral infections such as human immunodeficiency virus (HIV) and human papilloma virus; several cancers, including follicular lymphoma and acute myelogenous leukemia; inflammatory processes such as Crohn's disease and rheumatoid arthritis and in allergic disorders. There are approximately 10 trials, and early results are generally encouraging. Therefore, the expectation is that antisense DNAs will be important to future chemotherapy. The question considered here is whether antisense DNAs will also be important to future nuclear medicine imaging. While efforts toward developing antisense imaging are comparatively nonexistent thus far, investigations into the mechanisms of cellular transport and localization and the development of a second generation of antisense DNAs have occurred largely within the antisense chemotherapy industry. Fortunately, many of the properties of DNA for antisense imaging, such as high in vivo stability and adequate cell membrane transport, are the same as those for antisense chemotherapy. Unfortunately, interests diverge in the case of several other key properties. For example, rapid localization and clearance kinetics of the radiolabel and prolonged retention in the target are requirements unique to nuclear medicine. No doubt the development of antisense imaging will continue to benefit from improvements in the antisense chemotherapy industry. However, a considerable effort will be required to optimize this approach for imaging (and radiotherapy). The potential of specifically targeting virtually any disease or normal tissue should make this effort worthwhile. (+info)
Elevation of alpha2(I) collagen, a suppressor of Ras transformation, is required for stable phenotypic reversion by farnesyltransferase inhibitors.
Farnesyltransferase inhibitors (FTIs) are a novel class of anticancer drugs that can reverse Ras transformation. One of the intriguing aspects of FTI biology is that continuous drug exposure is not necessary to maintain phenotypic reversion. For example, after a single exposure to FTIs, Ha-Ras-transformed fibroblasts revert to a flat and anchorage-dependent phenotype that persists for many days after processed Ras has returned to pretreatment levels. In this study, we show that persistence of the reverted state is mediated by elevated expression of the collagen isoform alpha2(I), a suppressor of Ras transformation the transcription of which is repressed by activated Ras and derepressed by FTI treatment. To our knowledge, this is the first report identifying an FTI-regulated gene which is linked to phenotypic reversion. The finding that extracellular matrix alterations can influence the kinetics of reversion supports our assertion that Rho-regulated cell adhesion parameters are a crucial determinant of the cellular response to FTIs. (+info)
Expression of human alpha(1,3)fucosyltransferase antisense sequences inhibits selectin-mediated adhesion and liver metastasis of colon carcinoma cells.
The initial steps of leukocyte and tumor cell adhesion involve selectin receptor/ligand interactions. The selectin ligand components sialyl Lewis x and sialyl Lewis a are oncodevelopmental antigens involved in progression of adenocarcinoma. Interrupting biosynthesis of these surface glycans by inhibition of alpha(1,3)fucosyltransferase (FUT) gene expression is an attractive goal for functional and therapeutic studies. We report here the inhibition of E-selectin-mediated adenocarcinoma cell adhesion by stable transfection of antisense sequences directed at the human Lewis alpha(1,3/1,4)fucosyltransferase gene, FUT3. The metastatic parental cell line, HT-29LMM, expressed high levels of sialyl Lewis x, sialyl Lewis a, alpha(1,3/1,4)fucosyltransferase activity, and FUT3 transcript, but antisense transfectant cell lines did not. When injected into the spleens of nude mice, the stable antisense clones were unable to colonize the liver. These results provide target validation for inhibition of carcinoma metastasis with antisense FUT sequences and confirm the primacy of alpha(1,3)fucosyltransferases in the synthesis of selectin ligands. (+info)