Related Experiment Videos
Antisense c-myc oligonucleotide cellular uptake and activity
S Wu-Pong1, T L Weiss, C A Hunt
1Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Abstract:
Previously described cell membrane transport mechanisms are unable to account completely for oligodeoxynucleotide cellular uptake. These charged macromolecules enter cells by an incompletely defined mechanism and downregulate gene expression in either the cytoplasm or nucleus. Thus, the goal of this research was to study the mechanism of phosphodiester oligonucleotide cellular uptake in Rauscher Red 5-1.5 erythroleukemia cells. An antisense c-myc oligodeoxynucleotide (21 bases) demonstrated biological activity in these cells using two types of proliferation assays and Northern blot analysis, and was internalized as visualized by confocal laser microscopy. Oligonucleotide uptake appeared to be a complex process consisting of surface binding and internalization. Cellular internalization accounted for up to 40% of total uptake and was partially dependent on both a trypsin-sensitive component and cellular energy. Uptake in these cells was nonspecific and did not appear to be due to receptor-mediated endocytosis. Therefore, because oligonucleotide cellular uptake in other cell types apparently involves an endocytic mechanism, the primary mechanism of oligonucleotide internalization may be cell line dependent.
Insights
Oligodeoxynucleotide cellular uptake is complex, involving surface binding and internalization. This process is partially energy-dependent and may differ between cell types, suggesting a cell line-dependent mechanism for gene regulation.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Existing cell membrane transport models do not fully explain oligodeoxynucleotide cellular uptake.
- Oligodeoxynucleotides are charged macromolecules that enter cells via an undefined mechanism.
- These molecules can modulate gene expression in both the cytoplasm and nucleus.
Purpose of the Study:
- To investigate the cellular uptake mechanism of phosphodiester oligonucleotides in Rauscher Red 5-1.5 erythroleukemia cells.
- To characterize the internalization process and its dependence on cellular factors.
Main Methods:
- Utilized an antisense c-myc oligodeoxynucleotide (21-mer) for biological activity assessment.
- Employed proliferation assays and Northern blot analysis to confirm gene downregulation.
- Visualized oligonucleotide internalization using confocal laser microscopy.
Main Results:
- Demonstrated biological activity and cellular internalization of the c-myc oligodeoxynucleotide.
- Oligonucleotide uptake involves surface binding and internalization, with internalization accounting for up to 40% of total uptake.
- Internalization was partially dependent on a trypsin-sensitive component and cellular energy, indicating a non-specific, non-endocytic mechanism.
Conclusions:
- Oligonucleotide uptake in Rauscher Red 5-1.5 erythroleukemia cells is a complex process involving surface binding and internalization.
- The internalization mechanism is partially energy-dependent and not mediated by receptor-mediated endocytosis.
- The primary mechanism of oligonucleotide internalization may be cell line-dependent, differing from endocytic mechanisms observed in other cell types.