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At least 55 records · Page 3Linked to original sources
PubMed2005

Oncogene-targeted antisense oligonucleotides for the treatment of Ewing sarcoma.

The genetic hallmark of the Ewing sarcoma family of tumours (ESFT) is the presence of the t(11;22)(q24;q12) translocation, present in up to 85% of cases of ESFT, which creates the EWS/FLI1 fusion gene and results in the expression of a chimeric protein regulating many other genes. The inhibition of this protein by antisense strategies has shown its predominant role in the transformed phenotype of Ewing cells. In addition, the junction point at the mRNA level offers a target for short therapeutic nucleic acids that is present only in the cancer cells and not in the normal tissues of a patient. Several teams have, therefore, investigated the activity of antisense oligonucleotides and siRNAs targeted against the junction point in mRNA; thus, inhibiting EWS/FLI1 synthesis. Generally speaking, the molecules induce a cell growth inhibition in culture. Apoptosis has also been reported. One laboratory has reported the in vivo tumour inhibitory effect of phosphorothioate antisense oligonucleotide directed against the EWS part of EWS/FlI1 when injected intratumourally. Independently, a tumour inhibitory effect of oligonucleotides targeting the junction point has been demonstrated provided they are delivered by polymeric nanoparticles through the intratumoural route. Alongside this target, other genes participating to the maintenance of the transformed phenotype of Ewing cells have been downregulated by antisense strategies.

Down-Regulation↗
PubMed2001

Prevention of human prostate tumor metastasis in athymic mice by antisense targeting of human angiogenin.

PURPOSE: Angiogenin is a potent positive mediator of neovascularization, a process required for both primary tumor growth and metastasis. In the present study, the effect of a fully phosphorothioated antisense oligodeoxynucleotide, designated JF2S, targeting the AUG translation initiation codon region of human angiogenin, on human prostate tumor development and metastasis in athymic mice was examined. EXPERIMENTAL DESIGN: JF2S was evaluated for its capacity to affect in vitro synthesis of angiogenin and subsequent tumorigenicity of transiently transfected prostate tumor cells in mice. In vivo treatment experiments were then conducted in which JF2S was used to prevent formation of tumors in an ectopic model and metastasis in an orthotopic model. RESULTS: Transient transfection of tumor cells with JF2S inhibited both angiogenin gene expression in vitro and tumorigenicity of these transfected cells in athymic mice. In therapy experiments, local treatment with JF2S completely protected mice from developing prostate tumors after s.c. injection of PC-3 human prostate tumor cells (P < 0.0001, survivor analysis). Most importantly, systemic prophylactic administration of JF2S prevented, in 47% of mice, formation of regional iliac lymph node micrometastases arising from primary tumors growing in the more natural orthotopic prostate setting (P = 0.0003, Fisher's exact test). Furthermore, total protection from regional metastasis occurred in those mice in which JF2S treatment successfully diminished human angiogenin expression in vivo. Tumor-associated angiogenesis was also impaired by JF2S treatment. When therapy was delayed until all of the mice harbored primary tumors in the prostate, the incidence of regional metastasis was still significantly decreased (P < 0.005, survivor analysis). CONCLUSIONS: These findings demonstrate that human prostate cancer establishment and spread in athymic mice is extremely susceptible to targeted disruption of tumor-derived human angiogenin gene expression. Therefore, angiogenin is a valid target against which to devise preventative strategies for prostate cancer metastasis.

Animals↗
PubMed2003

HER-2-targeted antisense oligonucleotide results in sensitization of head and neck cancer cells to chemotherapeutic agents.

Existing HER-2 targeted therapies for human head and neck cancers, usually administered in combination with chemotherapeutic drugs or irradiation, include monoclonal antibodies to HER-2, receptor tyrosine kinase inhibitors and HER-2 specific immunotoxins. Instead of targeting the existing protein, interference with HER-2 mRNA translation by antisense oligonucleotides may be a more efficient method to downregulate levels of HER-2 protein for combination therapy. To test this hypothesis we have used a phosphorothioate pentadecamer, complementary to the HER-2 mRNA initiation codon region (AS HER-2 ODN), to increase sensitivity to four chemotherapeutic agents in human head and neck cancer cell lines, all of which express low levels of the HER-2 protein. To improve delivery into tumor cells, the AS HER-2 ODN was complexed with our previously established folate-liposome delivery system. Cell survival assays and Western blot analysis data demonstrated that folate-liposome mediated AS HER-2 oligonucleotide treatment inhibited cell growth and HER-2 expression, and induced apoptosis in SCC-25CP cells. Moreover, there was a synergistic effect on the percent of apoptotic cells. Additionally, the combination of folate-liposome-AS HER-2 ODN and CDDP had a synergistic effect on the induction of apoptosis. Using confocal microscopy, FITC labeled ODN (FITC-ODN) in complex with folate-liganded, rhodamine (Rh) labeled, cationic liposomes was observed to enter SCC-25CP head and neck tumor cells within 3 to 6 h. Intracellularly, the FITC-ODN separated from the Rh-folate-liposomes, and FITC-ODN accumulated in the nucleus while Rh-liposomes remained in punctate cytoplasmic structures. Thus, folate-liposome-mediated delivery of AS HER-2 ODN has potential as a new means of increasing the responsiveness of head and neck cancer to conventional chemotherapy.

Antineoplastic Agents↗
PubMed2000

An unusual structure formed by antisense-target RNA binding involves an extended kissing complex with a four-way junction and a side-by-side helical alignment.

The antisense RNA CopA binds to the leader region of the repA mRNA (target: CopT). Previous studies on CopA-CopT pairing in vitro showed that the dominant product of antisense RNA-mRNA binding is not a full RNA duplex. We have studied here the structure of CopA-CopT complex, combining chemical and enzymatic probing and computer graphic modeling. CopI, a truncated derivative of CopA unable to bind CopT stably, was also analyzed. We show here that after initial loop-loop interaction (kissing), helix propagation resulted in an extended kissing complex that involves the formation of two intermolecular helices. By introducing mutations (base-pair inversions) into the upper stem regions of CopA and CopT, the boundaries of the two newly formed intermolecular helices were delimited. The resulting extended kissing complex represents a new type of four-way junction structure that adopts an asymmetrical X-shaped conformation formed by two helical domains, each one generated by coaxial stacking of two helices. This structure motif induces a side-by-side alignment of two long intramolecular helices that, in turn, facilitates the formation of an additional intermolecular helix that greatly stabilizes the inhibitory CopA-CopT RNA complex. This stabilizer helix cannot form in CopI-CopT complexes due to absence of the sequences involved. The functional significance of the three-dimensional models of the extended kissing complex (CopI-CopT) and the stable complex (CopA-CopT) are discussed.

Bacterial Proteins↗
PubMed2004

Evaluation of developmental phenotypes produced by morpholino antisense targeting of a sea urchin Runx gene.

BACKGROUND: Runx transcription factors are important regulators of metazoan development. The sea urchin Runx gene SpRunt was previously identified as a trans-activator of the CyIIIa actin gene, a differentiation marker of larval aboral ectoderm. Here we extend the functional analysis of SpRunt, using morpholino antisense oligonucleotides (morpholinos) to interfere with SpRunt expression in the embryo. RESULTS: The developmental effects of four different SpRunt-specific morpholinos were evaluated. The two morpholinos most effective at knocking down SpRunt produce an identical mitotic catastrophe phenotype at late cleavage stage that is an artifact of coincidental mis-targeting to histone mRNA, providing a cautionary example of the insufficiency of two different morpholinos as a control for specificity. The other two morpholinos produce gastrula stage proliferation and differentiation defects that are rescued by exogenous SpRunt mRNA. The expression of 22 genes involved in cell proliferation and differentiation was analyzed in the latter embryos by quantitative polymerase chain reaction. Knockdown of SpRunt was found to perturb the expression of differentiation markers in all of the major tissue territories as well as the expression of cell cycle control genes, including cyclin B and cyclin D. CONCLUSIONS: SpRunt is essential for embryonic development, and is required globally to coordinate cell proliferation and differentiation.

Animals↗
PubMed1992

Specific inhibition of hepatitis B viral gene expression in vitro by targeted antisense oligonucleotides.

A 21-mer oligodeoxynucleotide complementary to the polyadenylation signal for human hepatitis B virus (HBV) was complexed to a soluble DNA-carrier system that is targetable to hepatocytes via asialoglycoprotein receptors present on those cells. A cell line, HepG2 (2.2.15) that possesses asialoglycoprotein receptors and is permanently transfected with hepatitis B virus (ayw subtype) was exposed to complexed antisense DNA or controls. In the presence of complexed antisense DNA, the concentration of hepatitis B surface antigen in medium was 80% lower than controls after 24 h. Furthermore, during the next 6 days, there was no significant increase in surface antigen concentration in the presence of complexed antisense DNA. The inhibition could be effectively blocked by competition with an excess of free asialoglycoprotein. Total protein synthesis remained unchanged by exposure to complexed antisense sequences under identical conditions. In addition, HBV DNA in the medium and cell layers after 24-h exposure to complexed antisense sequences was 80% lower than in controls. The data indicate that antisense oligonucleotides complexed by a soluble DNA-carrier system can be targeted to cells via asialoglycoprotein receptors resulting in specific inhibition of hepatitis B viral gene expression and replication.

Asialoglycoprotein Receptor↗
PubMed2000

Oligonucleotide sequence-specific inhibition of gene expression, tumor growth inhibition, and modulation of cAMP signaling by an RNA-DNA hybrid antisense targeted to protein kinase A RIalpha subunit.

The primary mediator of cAMP action in mammalian cells is cAMP-dependent protein kinase (PKA). There are two types of PKA, type I (PKA-I) and type II (PKA-II), which share a common catalytic subunit but contain distinct regulatory subunits, RI and RII, respectively. Evidence suggests that increased expression of RIalpha/PKA-I correlates with neoplastic cell growth. Here, we show that sequence-specific oligonucleotide inhibition of RIalpha expression results in inhibition of growth and modulation of cAMP signaling in cancer cells. The antisense promoted growth inhibition in a time-dependent, concentration-dependent, and sequence-dependent manner in human cancer cells in monolayer culture, and it inhibited colony formation in soft agar and tumor growth in nude mice. Among the cancer cells are LS-174T, HCT-15, and Colo-205 colon carcinoma cells; A-549 lung carcinoma cells; LNCaP prostate adenocarcinoma cells; Molt-4 leukemia cells; and Jurkat T lymphoma cells. Northern blot and immunoprecipitation analyses revealed that the growth inhibitory effect of the antisense correlated with a decrease in RIalpha expression at both the mRNA and protein levels. Pulse-chase experiments revealed that the antisense-directed inhibition of RIalpha expression resulted in compensatory changes in expression of the isoforms of R and C subunits and cAMP signaling in a cell type-specific manner. These results demonstrate that cAMP is ubiquitous in the regulation of cell growth and that the antisense oligonucleotide, which inhibits the synthesis of the RIalpha subunit of PKA, can be targeted to a single gene for treatment of cancer in a variety of cell types.

3' Untranslated Regions↗
PubMed2001

Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated antisense targeting.

Hepatocyte nuclear factor 4 (HNF4) is a member of the nuclear receptor super-family that has shown activating effects on particular cytochrome P450 (CYP) promoters from several species. However, its role in the regulation of human CYPs in the liver is still poorly understood, as no comprehensive studies in human-relevant models have been performed. In the present study, we have investigated whether HNF4 plays a general role in the expression of 7 major CYP genes in primary cultured human hepatocytes. To this end, we developed an adenoviral vector for efficient expression of HNF4 antisense RNA. Transduction of human hepatocytes with the recombinant adenovirus resulted in a time-dependent increase in the antisense transcript, followed by a concomitant decrease in apolipoprotein C III mRNA (a target gene of HNF4). Specificity was confirmed by showing that increasing levels of HNF4 antisense RNA resulted in the reduction of HNF4 protein, whereas retinoic X receptoralpha-(RXRalpha), the closest homologous member of the nuclear receptor super-family, was unaffected. Analysis of CYP gene expression in human hepatocytes transfected with HNF4 antisense RNA revealed singular behaviors: (1) CYP3A4, CYP3A5, and CYP2A6 showed an important, dose-dependent down-regulation on blockage of HNF4 translation; (2) a moderate inhibition of CYP2B6, CYP2C9, and CYP2D6 expression was observed (40%-45% reduction); (3) the levels of CYP2E1 were not affected even in the absence of this transcription factor. In conclusion, using an original strategy (efficient antisense RNA expression vector), our study shows that HNF4 is a general regulator supporting the expression of major drug-metabolizing CYPs in human hepatocytes.

Adenoviridae↗
PubMed1995

[Inhibition of hepatitis B viral gene expression and replication in vitro by targeted antisense oligonucleotides].

We chose the 2.2. 15 cells as an in vitro cell culture assay system, and identified the surface antigen subtype of hepatitis B virus (HBV) DNA in the cells, by using the amplification of polymerase chain reaction and directed sequencing of amplified products. According to the result of the sequencing, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASON) directed against the HBV U5-unique region was synthesized and then linked with two liver-targeting ligands. The galactosylated human serum albumin coupled poly-L-lysine and the galactosylated poly-L-lysine. The effect of different modifications of ASONs on the expression of HBV gene was compared by using the 2.2. 15 cells. In the same experimental conditions, the inhibitary effects of surface antigen (HBsAg) and antigen (HBeAg) by PS-ASON were 70% and 58% respectively, and those of HBsAg and HBeAg by ligand-PS-ASONs were 90%-96% and 78%-82%. In the same time, the amount of HBV NDA in culture supernatant and cells was depressed significantly. Thus, the ligands targeted ASON to the hepatocytes were more effective inhibitors of HBV gene expression. ASONs were effective and specific inhibitors of HBV replication and expression, caused the dose-dependent inhibition of viral proteins and had no effect on cellular alpha-fetoprotein synthesis and no cytotoxicity.

Base Sequence↗
PubMed1996

Tissue-targeted antisense c-fos retroviral vector inhibits established breast cancer xenografts in nude mice.

The c-fos proto-oncogene has been implicated as a regulator of estrogen-mediated cell proliferation. We have tested the tissue specificity and antitumor efficacy of a mouse mammary tumor virus-regulated antisense c-fos retroviral vector. Systemically administered vector could be detected in several tissues but was only expressed in breast epithelium, thus supporting targeting to mouse mammary tumor virus-regulated tissues. Ex vivo transduction of 30-70% of MCF-7 human breast cancer cells produced expression of antifos RNA, decreased expression of the c-fos target mRNA, induction of differentiation, and inhibition of s.c. tumor growth and invasiveness. In vivo transduction of established i.p. MCF-7 tumors with a single injection of XM6:antifos inhibited tumor growth in athymic mice with a corresponding inhibition of c-fos, transforming growth factor beta1 and transforming growth factor alpha expression. Four daily injections with the antifos RNA induced a much larger MCF-7 i.p. tumor inhibition, with a marked prolongation of survival in the absence of any host tissue toxicity. These results indicate that inhibition of key nuclear genes such as c-fos may lead to disruption of paracrine factors and an antitumor effect, providing a strategy for cancer gene therapy.

Animals↗
PubMed1998

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Animals↗
PubMed2000

HER2/neu antisense targeting of human breast carcinoma.

Overexpression of the HER2/neu oncogene is observed in approximately 30% of human breast carcinoma specimens. HER2/neu overexpression is a negative prognostic factor in breast cancer patients. Cancer cells that overexpress HER2/neu may also be less sensitive to chemotherapy. In order to further define mechanisms by which HER2/neu overexpression drives neoplastic cell growth and chemoresistance, antisense oligonucleotides (ODNs) have been utilized to selectively down-regulate HER2/neu expression in human breast cancer cells. Such antisense ODNs suppress HER2/neu mRNA and protein levels in a dose-dependent, sequence-specific manner. Down-regulation of HER2/neu expression in HER2/neu overexpressing breast cancer cells inhibits cell cycle progression in G0/G1 and results in apoptotic cell death. In tissue culture studies, combined treatment of HER2/ neu overexpressing breast cancer cells with HER2/neu antisense ODNs and conventional chemotherapeutic agents results in synergistic inhibition of cancer cell growth and activation of apoptotic cell death mechanisms. These studies have been extended to demonstrate synergistic antitumor effects following systemic treatment with antisense ODNs plus doxorubicin in nude mice bearing human breast carcinoma xenografts. Collectively these findings demonstrate that HER2/neu overexpression stimulates anti-apoptotic cell survival mechanisms and suggest that HER2/neu antisense ODNs may be of use in cancer therapeutics.

Animals↗
PubMed2001

Antisense targeting of c-fos transcripts inhibits serum- and TGF-beta 1-stimulated PAI-1 gene expression and directed motility in renal epithelial cells.

Plasminogen activator inhibitor type-1 (PAI-1), the major regulator of pericellular plasmin generation, and the c-FOS transcription factor are expressed by migrating cells in response to monolayer wounding. Induced c-fos and PAI-1 transcripts were evident within 30 min and 2 h, respectively, of scrape injury to confluent, growth-arrested, cultures of NRK epithelial cells. Since c-FOS/AP-1 DNA-binding activity modulates both basal and inducible modes of PAI-1 gene control, and AP-1 motif binding factors were present in quiescent as well as stimulated NRK cells, a model of directionally regulated cell movement (migration into scrape-denuded "wounds") was used to assess the consequences of c-fos transcript targeting on PAI-1 expression and cell motility. This in vitro model of epithelial injury closely approximated in vivo wound repair with regard to the spatial and temporal emergence of cohorts of cells involved in migration, proliferation, and PAI-1 expression. Stable cell lines (NRKsof) were generated by transfection of parental NRK cells with a c-fos antisense expression vector. Serum-inducible c-fos transcripts and PAI-1 protein levels were significantly attenuated in NRKsof transfectants relative to parental controls or cells transfected with a neo(R) vector without the sof insert. NRKsof cells had a markedly impaired ability to repair scrape-generated monolayer wounds under basal, serum-stimulated, or TGF-beta 1-supplemented culture conditions. Since injury closure and PAI-1 induction were attenuated in c-fos antisense cells, it was important to clarify the role of specific AP-1 sites in serum-mediated PAI-1 transcription. PAI-1 "promoter"-driven CAT reporter expression was assessed within the real time of serum-stimulated PAI-1 induction. A segment of the PAI-1 promoter corresponding to nucleotides -533 to -764 upstream of the transcription start site functioned as a prominent serum-responsive region (SSR). The 9-fold increase in CAT mRNA levels attained with the -533 to -764 bp PAI-1 SRR ligated to a minimal PAI-1 promoter (i.e., 162 bp of 5' flanking sequence containing the basal transcription complex) closely approximated the serum-induced transcriptional activity of a fully responsive (1,230 bp) PAI-1 promoter construct as well as the endogenous PAI-1 gene. AP-1-like, CTF/NF-1-like, and AP-2 sites were identified in the SRR. The SRR AP-1 motif was homologous to the sequence TGACACA that mapped between nucleotides -740 and -703 in the human PAI-1 gene, a region essential for growth factor-inducible PAI-1 transcription. While the functionality of this AP-1 site in wound-regulated PAI-1 synthesis remains to be determined, antisense c-fos transcripts effectively attenuated PAI-1 induction and basal as well as growth factor-stimulated cell locomotion, indicating that expression of both the PAI-1 and c-fos genes is necessary for wound-initiated NRK cell migration.

Animals↗