Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

Beltinger, Christian; Saragovi, H. Uri; Smith, Richard M.; LeSauteur, Lynne; Shah, Neelima; DeDionisio, Larry; Christensen, Laura M.; Raible, Annette M.; Jarett, Leonard; Gewirtz, Alan M.

doi:10.1172/jci117860

Cited by 293 publications

(161 citation statements)

References 34 publications

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“…In spite of the ease of reproducibility, the exact mechanism of DNA internalization can only be speculated. Studies on spontaneous DNA internalization in vitro (using viral DNA or oligonucleotides) 25,32,33 or in vivo (using plasmid DNA) 34 exclude the fact that DNA molecules penetrate through pores or areas of partial disruption of the membrane. Thus, two possibilities are considered.…”

Section: Discussionmentioning

confidence: 99%

“…The other is internalization by receptor-mediated endocytosis. Somatic cells have been reported to express a variety of DNA receptor candidates, 25,32,33,[35][36][37][38] none of which showed to be uniquely essential for DNA uptake. Specifically, receptors mediating DNA internalization in human B lymphocytes are poorly understood.…”

Section: Discussionmentioning

confidence: 99%

“…25 Previous studies showed that uptake of exogenous DNA by cells in culture is a function of time and concentration of input DNA, 14 and that only a small fraction persists in the cells 24 h after internalization. [13][14][15] This indicates that the major fraction of internalized DNA is degraded intracellularly.…”

Section: Spontaneous Transfection In Human B Cellsmentioning

confidence: 99%

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Spontaneous transgenesis of human B lymphocytes

et al. 2003

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DNA can cross the cell membrane by natural means, but the functional relevance of this phenomenon has not been fully elucidated. Here, we analyzed spontaneous transgenesis of human B cells using plasmid DNA coding for a functional immunoglobulin (Ig) heavy chain gene under the control of a B-cell-specific promoter. Using polymerase chain reaction (PCR), reverse transcriptase-PCR, and flow cytometry in combination, spontaneous transgenesis was documented in Burkitt's lymphoma cell lines, Epstein-Barr virus-transformed cell lines, and peripheral blood B lymphocytes of the mature naïve phenotype (IgM þ /IgD þ /CD27 À ). By immunoelectron microscopy, the internalized DNA was seen in the lysosomes/late endosomes and in the cytosol proximal to the nucleus. Importantly, spontaneously transgenic B cells processed and presented to major histocompatibility complex (MHC)-restricted T lymphocytes a peptide expressed in the transgenic product. This is the first demonstration that primary B lymphocytes possess a program for the spontaneous internalization of DNA, which in turn imparts the cell with new immunological functions. As spontaneous transgenesis is obtained using a nonviral vector, does not require prior cell activation, and is not associated with chromosomal integration, the findings reported here open new possibilities for genetic manipulations of mature naïve B lymphocytes for therapy and vaccination.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Spontaneous Transfection In Human B Cellsmentioning

confidence: 99%

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Spontaneous transgenesis of human B lymphocytes

et al. 2003

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“…Cells can bind and take up DNA in a receptor-mediated fashion [1,5] but discrimination between active and inactive DNA sequences is not thought to involve differential binding to a cell surface receptor [4]. The ␤ 2 integrin CD11b/CD18 (Mac-1) has been implicated in the uptake of oligonucleotides by neutrophils [6].…”

Section: Recognition Of Cpg Dnamentioning

confidence: 99%

The actions of bacterial DNA on murine macrophages

Sester

Stacey

Sweet

et al. 1999

Journal of Leukocyte Biology

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Murine macrophages are able to distinguish bacterial from mammalian DNA. The response is mimicked by single-stranded oligonucleotides containing unmethylated CG dinucleotides (''CpG'' motifs) in specific sequence contexts. The dose-response curve for activation is influenced by variation in the sequence flanking the core CpG motif. CpG or bacterial DNA activates several signaling pathways in common with bacterial lipopolysaccharide (LPS), leading to induction of cytokine genes such as tumor necrosis factor ␣. Pretreatment with LPS causes desensitization to subsequent activation by CpG DNA. Both stimuli also cause cell cycle arrest in macrophages proliferating in response to the macrophage growth factor colonystimulating factor-1 (CSF-1), but prevent apoptosis caused by growth factor removal. In part, cell cycle arrest by CpG DNA and LPS may be linked to rapid down-modulation of the CSF-1 receptor from the cell surface, a response that occurs in an all-or-nothing manner. The response of macrophages to CpG DNA has aspects in common with the DNA damage response in other cell types, which may provide clues to the underlying mechanism. J. Leukoc. Biol. 66: 542-548; 1999.

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“…At high (micromolar) concentrations, ON are generally reported to be internalised by fluid-phase endocytosis, a process of too low efficiency to guarantee strictly specific effects (20)(21)(22). Indeed, high concentrations lead to multiple non-specific effects, some of which are even sequence dependent (23).…”

Section: Introductionmentioning

confidence: 99%

Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking

Diesbach¹

2002

Nucleic Acids Research

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Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated endocytosis tracer, transferrin (uptake equilibrium, saturation with concentration, specific competition and rapid efflux) and were clearly distinct from those of fluid-phase endocytosis. By analytical subcellular fractionation, particulate ON showed a bimodal distribution after 2 h of uptake, with a low-density peak superimposed on the distribution of endosomes, and a high-density peak overlapping lysosomes. After an overnight chase, only the highdensity peak remained, but it could be dissociated from lysosomes, based on its refractoriness to displacement upon chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate pattern was resolved, by confocal microscopy, from those of transferrin, of a fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3 µM ON-Alexa, its pattern largely overlapped with the fluidphase tracer and LysoTracker. Taken together, these data suggest that ON may be internalised at low concentrations by receptor-mediated endocytosis into unique endosomes, then to dense structures that are distinct from lysosomes. The nature of these two compartments and their significance for ON effect deserve further investigation.

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Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

Cited by 293 publications

References 34 publications

Spontaneous transgenesis of human B lymphocytes

Spontaneous transgenesis of human B lymphocytes

The actions of bacterial DNA on murine macrophages

Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking

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