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108 Cell-Penetrating Peptides: Processes and Applications
microinjected into the cytoplasm of living cells localized rapidly into the
nucleus and were specifically inhibited by inhibitors of the NPC import
pathway. 84
3. Utilization of inhibitors of nuclear import. Nuclear import of cargo or
DNA is an active process that is inhibited at 4°C or following energy
depletion, 95 depending on cell culture methods. These methods are dependent
on the cell lines and therefore special care should be taken. Nuclear
import can be blocked by competitive NLS protein coinjection of BSA
or of lectin-specific wheat germ agglutinin (WGA). 96 For example, WGA
has been shown to compete with the α-importin pathway: WGA with
NLS-streptavidine, suggesting that nuclear import of NLS covalently
attached to a plasmid occurs through the α-importin pathway. 86 DNA M9
complex was shown to accumulate in nuclei and to translocate through
NPCs by WGA blocking experiments. 85
4. Control of the cell cycle. Gene expression following microinjection of
DNA in the cytoplasm is generally quite inefficient, in fact, gene expression
is much greater in dividing cells than in nondividing cells. Since the
nuclear membrane is disintegrated during mitosis, the presence of an NLS
is crucial for nuclear transfection of postmitotic cells. Experiments should
also be performed on nondividing cells or in G1 arrested or synchronized
cells and earlier expression (12 h after transfection) should be monitored. 87
Synchronization of cells is cell line-dependent and must be controlled.
Different techniques such as serum deprivation, nocodazole, or thymidine
blocks can be applied. 97
5. Monitoring nuclear accumulation. Accumulation of plasmid in the
nucleus can be determined by quantitative PCR with specific reporter
vectors or by fluorescence microscopy following direct labeling of DNA
or using traceable molecules. 98,99
5.6 CONCLUSIONS
The nucleus constitutes both a major target for drugs and a major barrier for
biologically active molecules. A large number of cell-penetrating peptides have been
designed containing an NLS domain, which allows nuclear targeting. In the case of
cell penetratin peptides or proteins containing an NLS, the role of the NLS is well
established, but its function in nuclear transfer of DNA remains unclear. Several
points need to be clarified to justify the requirement for an NLS and to improve the
design and efficiency of novel NLS-containing cell-penetrating peptides. Standardization
of experiments is essential because cell lines and problems in comparing
different systems require some form of standardization. Also, progress needs to be
made in order to limit toxicity of these novel molecular tools.
In conclusion, NLS-mediated transport is an essential technology or pathway to
improve drug and gene therapies. The design of the next generation of peptide-based
cell delivery systems is likely to combine properties of NLS with other cellular requirements,
such as selectivity, signaling, and the knowledge of cell cycle regulations.