ISSN:1050-5261    

DOI:10.1089/ard.1992.2.1

年代:1992

数据来源: MAL

摘要:

In this study a ribozyme (catalytic RNA) was designed to site specifically cleave the mRNA of the activated H-rasgene expressed in human bladder carcinoma EJ cells. The optimal conditions for catalytic cleavage by the ribozyme were demonstratedin vitro. A synthetic DNA encoding the ribozyme was cloned into a mammalian expression vector (pHβAPr-1) and transfected into EJ cells. The expressed ribozyme significantly altered the morphology and suppressed the growth of EJ cellsin vitro. These cell lines were examined for their malignant potential in athymic (nude) mice by an orthotopic (transurethral) implantation model, which recapitulates the invasive potential of various bladder carcinomas. EJ tumors expressing the H-rasribozyme were characterized by a marked reduction in tumor take and invasion compared to those formed by control EJ cells. These differences resulted in almost a twofold increase in survival of mice implanted with ribozyme-containing EJ cells. These results further elucidate the role ofrasgenes in tumorigenicity and invasion, as well as introduce ribozymes as a new class of anticancer agents

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.3

年代:1992

数据来源: MAL

摘要:

Although antisense oligonucleotides have been widely used to inhibit gene expression, their mechanism of entry into cells and their site of action are still in some doubt. In this report, we describe a novel technique for kinetically analyzing oligonucleotide association with living cells as well as intracellular compartmentalization. The technique utilizes a photoactivatable, radiolabeled crosslinker, the Denny-Jaffe reagent. Oligonucleotides containing pendant amine groups were conjugated to this reagent, added to HL60 cells in culture, and photocrosslinked to associated proteins, which were analyzed electrophoretically. We find that several proteins are labeled, predominantly a 75 kD one that appears to be membrane-associated. Our results suggest that the majority of intracellular oligonucleotide is associated in vesicles with the same protein to which it bound on the cell surface, but only a small percentage of non-protein-bound cytosolic oligonucleotide can be detected. Additionally, oligonucleotides are readily accumulated by nuclei, and by treating whole nuclei, a unique set of nuclear binding proteins is detected.

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.17

年代:1992

数据来源: MAL

摘要:

An antisense oligonucleotide phosphorothioate, previously shown to inhibit HIV-1 viral expression in chronically infected H9 cells, was fluorescently labeled to study oligonucleotide fluxes and localization within living cells. Observations based on flow cytometry and fluorescence microscopy show the following: within around 0.5-2 h, an apparent steady-state distribution of the oligonucleotide is achieved in which the intracellular oligonucleotide concentration is less than that present in the external medium; following oligonucleotide uptake and resuspension of the cells in oligonucleotide-free medium, an oligonucleotide efflux, with a time constant similar to that for uptake, is observed (although a significant fraction of the phosphorothioate remains within the cell); cellular uptake as a function of the external oligonucleotide concentration is nonlinear, being more efficient at lower concentrations (<2 μM); and a predominant oligonucleotide localization within the cell nucleus and perinuclear organelles is observed

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.27

年代:1992

数据来源: MAL

摘要:

We have found that various mycoplasma species quickly and efficiently incorporate radiophosphorus into their RNA from labeled oligonucleotides added to the medium. The label can be in any of several positions in an oligodeoxynucleotide, and incorporation also occurs efficiently from labeled RNA. Mycoplasmas also incorporate the radiolabel when they infect a mammalian cell culture; the host cells do not. This incorporation presumably involves uptake of the oligodeoxynucleotide followed by digestion to mononucleotides, conversion to ribonucleotides, and incorporation in new RNA. We believe that the processing of oligodeoxynucleotides by mycoplasma could be a source of artifacts in antisense work in cell culture and could have implications for the development of antisense therapeutics. We also suggest ways to exploit the incorporation phenomenon in mycoplasma testing.

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.41

年代:1992

数据来源: MAL

摘要:

A natural DNA oligomer (15-mer) was synthesized with a sequence complementary to the translation initiation codon region of the human TGF-α mRNA and mixed with Lipofectin to form unilamellar complexes. It was found that tumor cell growth was inhibited when HCT116 cells were treated with Lipofectin-DNA oligomer complexes or with Lipofectin alone. Uptake of32P-labeled 15-mers into colon tumor cells was compared in the presence and absence of Lipofectin. The amount of labeled oligomer found in cells that received optimal ratios of Lipofectin to DNA was 4- to 10-fold higher than the amount found in cells that received32P-labeled DNA alone. Although Lipofectin—antisense DNA oligomer treatment of HCT116 cells caused a dose-dependent inhibition of cell growth, there was a subsequent rise in target mRNA product. Because the mechanism of growth inhibition could not involve an inhibition of TGF-α expression, it was concluded that Lipofectin probably exerts a nonspecific, detergent-like effect upon the cell membrane, producing an enhancement of TGF-α processing and rel

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.51

年代:1992

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.61

年代:1992

数据来源: MAL

ISSN:1050-5261    

DOI:10.1089/ard.1992.2.63

年代:1992

数据来源: MAL