Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Immunization with defined tumor antigens is currently limited to a small number of cancers where candidates for tumor rejection antigens have been identified. In this study we investigated whether pulsing dendritic cells (DC) with tumor-derived RNA is an effective way to induce CTL and tumor immunity. DC pulsed with in vitro synthesized chicken ovalbumin (OVA) RNA were more effective than OVA peptide-pulsed DC in stimulating primary, OVA-specific CTL responses in vitro. DC pulsed with unfractionated RNA (total or polyA+) from OVA-expressing tumor cells were as effective as DC pulsed with OVA peptide at stimulating CTL responses. Induction of OVA-specific CTL was abrogated when polyA+ RNA from OVA-expressing cells was treated with an OVA-specific antisense oligodeoxynucleotide and RNase H, showing that sensitization of DC was indeed mediated by OVA RNA. Mice vaccinated with DC pulsed with RNA from OVA-expressing tumor cells were protected against a challenge with OVA-expressing tumor cells. In the poorly immunogenic, highly metastatic, B16/F10.9 tumor model a dramatic reduction in lung metastases was observed in mice vaccinated with DC pulsed with tumor-derived RNA (total or polyA+, but not polyA- RNA). The finding that RNA transcribed in vitro from cDNA cloned in a bacterial plasmid was highly effective in sensitizing DC shows that amplification of the antigenic content from a small number of tumor cells is feasible, thus expanding the potential use of RNA-pulsed DC-based vaccines for patients bearing very small, possibly microscopic, tumors.
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PMID:Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. 876 Aug

Although many compounds have been found that bind to DNA in various ways and exhibit various biological activities, few compounds that specifically bind to RNA or RNA:DNA hybrids are known, even though such compounds are expected to have important biological properties. For example, one characteristic function of the retroviruses, which is generally not found in eukaryotic cells, is the production of an RNA:DNA hybrid in the viral replication phase. If an agent is designed to bind only to an RNA:DNA hybrid, and not to DNA or to RNA, such an agent might be able to inhibit specifically the RNase H activity of retroviral reverse transcriptase, and therefore suppress viral replication. Actinomycin D is known to bind to double-stranded DNA, but not to RNA, because steric hindrance between the 2-amino group of the phenoxazone ring and the 2'-hydroxyl group of RNA prevents intercalation of the compound. However, if the > C-H moiety at the 8-position of the phenoxazone ring is replaced by a > C-F, a possible hydrogen-bond acceptor, this analogue (8-fluoro-actinomycin D, F8AMD) might be able to bind intercalatively to an RNA:DNA hybrid by forming an additional hydrogen bond between F8 and the 2'-hydroxyl group of the guanosine ribose. To test this hypothesis, the crystal structure of d(GAAGCTTC)2-F8AMD has been determined at 3.0 A resolution. Based on this crystal structure, a model in which F8AMD binds into the hybrid r(GAAGCUUC):d(GAAGCTTC) has been built using molecular mechanics and dynamic methods. These structural studies indicate that F8AMD binds intercalatively to a B-form double-stranded DNA whereas the drug intercalates into an RNA:DNA hybrid taking an A-form conformation. In the RNA:DNA hybrid complex, the F8 atom is located so as to be able to interact to an O2' hydroxyl group with either an O-H...F hydrogen bond or H+...F- electrostatic interaction. This interaction might stabilize the F8AMD molecule in the RNA:DNA hybrid. A binding study indicates that both actinomycin D (AMD) and F8AMD bind intercalatively not only to double-stranded DNAs, but also to RNA:DNA hybrids. Although the overall binding capacity of F8AMD (k = 4.5 x 10(5) M-1) is reduced slightly in comparison with AMD itself (k = 1.8 x 10(6) M-1), F8AMD tends to bind relatively more favorably than AMD to the RNA:DNA hybrids. The drugs' effects on RNA synthesis in HeLa cells indicates that the binding capacities of AMD and F8AMD correlates strongly to their RNA synthesis inhibitory activities. F8AMD required a concentration of 78 nM to inhibit RNA polymerase activity in HeLa cells by 50%, whereas AMD reached the same inhibitory level at 30 nM. Surprisingly, F8AMD exhibits unique selectivity against leukemia cells as does another C8-derivatized AMD analogue, N8AMD. F8AMD inhibits 50% of leukemia cell growth at less than 1.0 nM whereas 10- to 130-fold-higher drug concentrations are required to inhibit the growth of other tumor cell lines by 50%. The GI50 value of F8AMD for leukemia cells is the lowest among the GI50 values for all other AMD derivatives tested. By contrast, AMD is quite potent and kills most cells at less than 50 nM concentration, but it does not show any selectivity for certain cell lines. This indicates that AMD should have very limited use as an antitumor agent. It is difficult to rationalize why F8AMD and N8AMD show such strong selectivity against leukemia cells. However, this study and our previous study (J. Am. Chem. Soc. 1994, 116, 7971) indicated that F8AMD and N8AMD tended to bind more favorably to RNA:DNA hybrids. Thus, the unique antileukemia selectivity shown by F8AMD and N8AMD might be used by the agents binding to RNA:DNA hybrids rather than to double-stranded DNA.
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PMID:Selectivity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia activity. 922 13

Human papillomavirus (HPV) type 16 E6 and E7 inactivate the tumor suppressors p53 and pRB, respectively. Both viral oncoproteins play important roles in maintaining the transformed phenotype of cells. In this study, we examine the effects of antisense oligodeoxynucleotides with polarity and anomeric center reversal (alpha/beta-ODNs). ODNs of the general structure 5'alphaN3'3'NNN5'5'alphaN3'3'NNNN5'5'alphaN3+ ++'3'N5' were synthesized using phosphoramidite DNA chemistry. These alpha/beta-ODNs were complementary in sequence to regions flanking the start codons of HPV type 16 E6 and E7 genes. The anti-HPV type 16 alpha/beta-ODNs were able to form stable duplexes with their complementary RNA, which then serve as substrates for RNase H hydrolysis. Anti-HPV type 16 alpha/beta-ODNs also specifically inhibited the growth of two cervical carcinoma cell lines, CaSki and SiHa, both of which harbor HPV type 16 DNA. A decrease in E7 protein expression was also observed. Injection of nude mice with SiHa cells induces tumors. Treatment of these tumor-bearing mice with anti-HPV type 16 alpha/beta-ODNs led to substantially smaller tumors. These results show that alpha/beta-ODNs can exert antisense activities both in vitro and in vivo on the E6 and E7 genes of HPV type 16.
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PMID:Biologic activity of oligonucleotides with polarity and anomeric center reversal. 959 47

Human papillomavirus type 16 (HPV-16) is the HPV type most frequently associated with cervical carcinomas. Based on our previous research with anti-HPV ribozymes, we developed a 16-nucleotide antisense oligodeoxynucleotide (AntiE6) able to direct RNase H activity on full-length HPV-16 E6/E7 mRNA. Although the precise mechanism is not completely understood, addition of 50 microM AntiE6 oligodeoxynucleotide in sterile water caused a significant decrease in the growth rate of CaSki and QGU cervical tumor cell lines. In contrast, addition of a mismatched mutant oligodeoxynucleotide (M7) did not affect cell growth after 72 hours. Treatment with AntiE6 resulted in down-regulation of E6/E7 mRNA and an increase in p53 levels in QGU cells. AntiE6 was also able to (>70%) inhibit significantly growth of transplanted cervical tumors in nude mice after 2 weeks treatment using constant delivery by osmotic pumps. These results indicate that the AntiE6 antisense oligodeoxynucleotides can act as a therapeutic agent against cervical carcinomas.
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PMID:Growth inhibition of cervical tumor cells by antisense oligodeoxynucleotides directed to the human papillomavirus type 16 E6 gene. 1055 51

This study was aimed at creating a more effective tumor cell vaccine by suppressing Ii protein in the presence of MHC class II molecules within a cancer cell. Absence of the Ii protein, which normally blocks the antigenic-peptide-binding site of MHC class II molecules at synthesis in the endoplasmic reticulum, presumably increases the range of cancer-related epitopes presented to CD4+ helper T cells. Effective suppression of Ii protein was achieved with an antisense, phosphorothioate oligonucleotide, which was selected on the basis of (1) the RNase H activation assay, (2) an assay for Ii protein suppression, and (3) a test for potency with respect to the extent of base sequence ("sequence walking"). The SaI murine sarcoma, which is MHC-class-I+ and MHC-class-II-, Ii-protein-, upon transfection with genes for either interferon gamma or the MHC class II transactivator, came to express MHC class II molecules and Ii protein. In each line of transfected tumor cells, the antisense oligonucleotide profoundly suppressed Ii protein in 35%-55% cells, without affecting expression of MHC class II molecules. Inoculation of mice with such Ii-protein-suppressed tumor vaccine cells, after either formaldehyde fixation or X-irradiation, led to much greater protection against challenge with the parental SaI sarcoma than did inoculation with untreated cells. This approach to cancer cell vaccination can be applied in a wide range of human tumors.
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PMID:Cancer immunotherapy by antisense suppression of Ii protein in MHC-class-II-positive tumor cells. 1060 87

Chemotherapy resistance is a significant obstacle in lung cancer therapy, and has been found to frequently correlate with amplification and overexpression of the c-myc oncogene. Earlier studies have shown that c-Myc inhibition alone is not always effective in cancer models. The purpose of this study was to test different dosing regimen, which included commonly used chemotherapeutic drugs in combination with c-Myc inhibition in a Lewis lung syngeneic drug-resistant murine tumor model. Inhibition of c-myc was specifically achieved by using phosphorodiamidate Morpholino oligomer (PMOs), a novel, non-toxic antisense DNA chemistry for inhibition of gene expression by an RNase H-independent mechanism. When administration of cisplatin overlapped with c-myc PMO (AVI-4126) treatment there was no additional effect on tumor growth inhibition compared to cisplatin alone. In contrast, using a dosing regimen in which cisplatin or taxol treatment preceded AVI-4126, a dramatic decrease in tumor growth rate was observed with tumor areas less then 0.5 cm2 in 60% of the animals at the end of the study. This effect was specific to c-Myc inhibition as other antisense PMOs against p21 or Rad51 showed no such effect in combination with chemotherapy. Immunoblot and HPLC-based analysis of tumor lysates at the end of the study confirmed c-Myc inhibition and detection of intact AVI-4126, respectively. In conclusion, AVI-4126 potentiates the efficacy of chemotherapeutic drugs in a manner that is schedule dependent.
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PMID:Resistance to chemotherapeutic drugs overcome by c-Myc inhibition in a Lewis lung carcinoma murine model. 1254 57

Allele-specific inhibition (ASI) is a new strategy to treat cancer through a vulnerability created by the loss of large segments of chromosomal material by loss of heterozygosity (LOH). Using antisense approaches, it is possible to target single nucleotide polymorphisms (SNP) in the remaining allele of an essential gene in the tumor, thus killing the tumor while the heterozygous patient survives at the expense of the other nontargeted allele lost by the tumor. In this study, the feasibility of using locked nucleic acid (LNA)-modified DNAzymes (LNAzymes) of the 10-23 motif as allele-specific drugs was investigated. We demonstrate that incorporation of LNA into 10-23 motif DNAzymes increases their efficacy in mRNA degradation and that, in a cell-free system, the 10-23 motif LNAzyme can adequately discriminate and recognize an SNP in the large subunit of RNA polymerase II (POLR2A), an essential gene frequently involved in LOH in cancer cells. However, the LNAzymes, optimized under in vitro conditions, are not always efficient in cleaving their RNA target in cell culture, and the efficiency of RNA cleavage in cell culture is cell type dependent. The cleavage rate of the LNAzyme is also much slower than RNase H-recruiting DNA phosphorothioate antisense oligonucleotides. Moreover, compared with DNA phosphorothioates, the ability of the LNAzymes to differentially knock down two POLR2A alleles in cultured cancer cells is limited.
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PMID:Evaluation of LNA-modified DNAzymes targeting a single nucleotide polymorphism in the large subunit of RNA polymerase II. 1639 19

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 '-deoxyinosine (dI) and 2 '-O-methylribonucleotide (2 '-OMe) residues: 5'-d(TITIT)-2'OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.
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PMID:Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2. 1690 21

Procathepsin D (pCD), a zymogen of lysosomal aspartic peptidase cathepsin D, overexpression is correlated with highly invasive malignancies, including breast cancer. Recently, different studies have shown the role of secreted pCD as mitogen acting both in an autocrine and a paracrine manner. The aim of the present study is to examine the anti-tumor effects elicited by a decrease in the protein level of pCD by ribozyme and to explore the therapeutic potential of this specific targeting. Using the mFold program, we designed seven anti-pCD ribozymes and checked the accessibility to target pCD mRNA by RNase H cleavage experiment in a cell-free system. The sequences of the 4 most effective ribozymes were cloned and stably transfected in a highly metastatic human breast cancer cell line, MDA-MB-231, to knock down the expression of pCD. Downregulation of pCD due to ribozyme expression was observed by Western blotting and real-time RT-PCR. Stably transfected cells with anti-pCD ribozymes exhibited a significant lowering of in vitro invasion (p<0.001) and reduction in lung colonization potential in nude mice when compared to control ribozyme transfected cells. We also found that downregulation of pCD by ribozyme promotes apoptosis of MDA-MB-231 cells on serum deprivation. These results suggest that we have generated a biologically functional ribozyme against pCD with possible therapeutic implications in breast cancer cells.
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PMID:Ribozyme-targeting procathepsin D and its effect on invasion and growth of breast cancer cells: an implication in breast cancer therapy. 1739 25

Oblimersen (Genasense is a Bcl-2 antisense compound that selectively targets Bcl-2 RNA for degradation by RNase H and thereby decreases Bcl-2 protein production. Bcl-2 protein plays a major role in preventing apoptosis and has been linked to chemotherapy resistance in melanoma. Preclinical studies with oblimersen in melanoma cell lines and xenograft models of melanoma have demonstrated downregulation of Bcl-2 protein, induction of apoptosis and enhanced tumor response when combined with chemotherapy. Results of a Phase I/II study have shown that reducing Bcl-2 with oblimersen coincident with the administration of dacarbazine may amplify apoptosis and improve therapeutic outcome. A subsequent Phase III trial showed that the addition of oblimersen to dacarbazine significantly improved multiple clinical outcomes relative to dacarbazine alone based on an intent-to-treat analysis of progression-free survival and response rate (overall, complete and durable), as well as overall survival in patients with normal lactate dehydrogenase. This article reviews the biochemistry, pharmacodynamics and pharmacokinetics, safety and efficacy data related to oblimersen in melanoma.
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PMID:Oblimersen in the treatment of metastatic melanoma. 1754 20


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