The Detection of Oligodeoxynucleotide Molecules Following Uptake into Mammalian Cells

Hawley, Patricia; Gıbson, Ian

doi:10.1089/ard.1992.2.119

Cited by 15 publications

(5 citation statements)

References 21 publications

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“…It has been suggested that the stability of oligonucleotides in tissue culture cells is less than three days [27, 28]. Therefore, since we evaluated colony formation of human progenitors after 14 days, degradation of the antisense oligonucleotides could account for partial inhibition of colony formation of human BM cells.…”

Section: Resultsmentioning

confidence: 99%

Raf‐1 Protein is Required for Growth Factor‐Induced Proliferation of Primitive Hematopoietic Progenitors Stimulated with Synergistic Combinations of Cytokines

et al. 1997

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Raf-1 is a serine/threonine kinase that has been identified as a component of growth factor-activated signal transduction pathways, and is required for growth factorinduced proliferation of leukemic cell lines and colony formation of hematopoietic progenitors stimulated with single colony-stimulating factors, which promote the growth of committed hematopoietic progenitor cells. However, it is known that the most primitive progenitors in the bone marrow require stimulation with multiple cytokines to promote cell growth. We have determined that c-raf antisense oligonucleotides inhibit the growth of murine lineage-negative progenitors stimulated with two-, three-and four-factor combinations of growth factors, including GM-CSF + interleukin (

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Section: Resultsmentioning

confidence: 99%

Raf‐1 Protein is Required for Growth Factor‐Induced Proliferation of Primitive Hematopoietic Progenitors Stimulated with Synergistic Combinations of Cytokines

et al. 1997

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“…Endogenous uptake of ODNs is a time and energy dependent process (13,18,19,25,26,27). Thus to achieve a particular antisense effect long periods of in vitro exposure to relatively high concentrations of ODNs may be required.…”

Section: Discussionmentioning

confidence: 99%

Electroporation enhances c-myc antisense oligodeoxynucleotide efficacy

Bergan¹,

Connell²,

Fahmy³

et al. 1993

Nucl Acids Res

107

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Obtaining high transfection efficiencies and achieving appropriate intracellular concentrations and localization are two of the most important barriers to the implementation of gene targeted therapy. The efficiency of endogenous uptake of oligodeoxynucleotides (ODNs) varies from cell type to cell type and may be a limiting factor of antisense efficacy. The use of electroporation to obtain high intracellular concentrations of a synthetic ODN in essentially 100% of viable cells is described. It is also shown that the transfected ODNs initially localize to the nucleus and remain there for at least 48 hours. The cellular trafficking of electroporated ODNs is shown to be an energy dependent process. Targeting of the c-myc proto-oncogene of U937 cells by electroporation of phosphorothioate-modified ODNs results in rapid and specific suppression of this gene at ODN concentrations much lower than would otherwise be required. This technique appears to be applicable to a variety of cell types and may represent a powerful new investigate tool as well as a promising approach to the ex vivo treatment of hematologic disorders.

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“…The 20-tof sequence (5' ACA CCC AAT TCT GAA AAT GG 3') was synthesized using standard phosphoramidite chemistry in PO and PS forms using reagents from Applied Biosystems and Cruachem. In addition, a phosphorothioate end-capped oligonucleotide was synthesized and purified at Norwich as previously described (Hawley and Gibson, 1992). This 3' phosphorothioate end-capped phosphodiester oligomer is a 16-mer identical to the first 16 bases of the normal human N-ras translated sequence where * shows the position of the sulfur-substituted phosphate groups, 5' ATGACT-GAGTACAA*A*C 3'.…”

Section: Methodsmentioning

confidence: 99%

The Influence of Polarized Epithelial (Caco-2) Cell Differentiation on the Cellular Binding of Phosphodiester and Phosphorothioate Oligonucleotides

Akhtar¹,

Beck²,

Hawley³

et al. 1996

Antisense and Nucleic Acid Drug Development

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Cell aging and the degree of cellular differentiation are thought to be important variables governing uptake of oligonucleotides but remain poorly understood. The Caco-2 colon carcinoma cell line has the ability to spontaneously differentiate into enterocytes in vitro and serves as a useful model to further investigate the effect of differentiation on oligonucleotide binding and uptake. In this study, we report that the extent of oligonucleotide association and the expression of cell surface binding proteins are governed by the age and thus the degree of differentiation of Caco-2 epithelial cells in culture. Cellular association (normalized for cell number) of an all phosphodiester (PO), all phosphorothioate (PS), and a phosphodiester oligonucleotide containing two terminal phosphorothioate internucleotide linkages at the 3' end (EC-PO) gradually increased from day 3 to around day 17 of the culture, followed by a plateau, or slight decrease, up to day 21 of the cell aging study. Overall, a threefold to fourfold increase in binding was observed from day 3 to day 17. Oligonucleotide binding was temperature and pH dependent, but the magnitude of the effect was influenced by cell aging and the degree of differentiation. PS oligonucleotides exhibited greater binding (up to threefold) at the basolateral surface compared with the apical surface within the pH range 5-7. These findings could be directly correlated with the expression levels of cell surface oligonucleotide binding proteins during the aging study. A Caco-2 cell surface protein binding complex of around 46 kDa was identified as the major site of binding for both PO and PS oligonucleotides, although the latter also bound to several other proteins, especially at low pH.

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The Detection of Oligodeoxynucleotide Molecules Following Uptake into Mammalian Cells

Cited by 15 publications

References 21 publications

Raf‐1 Protein is Required for Growth Factor‐Induced Proliferation of Primitive Hematopoietic Progenitors Stimulated with Synergistic Combinations of Cytokines

Raf‐1 Protein is Required for Growth Factor‐Induced Proliferation of Primitive Hematopoietic Progenitors Stimulated with Synergistic Combinations of Cytokines

Electroporation enhances c-myc antisense oligodeoxynucleotide efficacy

The Influence of Polarized Epithelial (Caco-2) Cell Differentiation on the Cellular Binding of Phosphodiester and Phosphorothioate Oligonucleotides

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