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34 Cell-Penetrating Peptides: Processes and Applications
protein (APP) into neurons developing in vitro. 32 Internalized antisense oligonucleotides
caused a transient inhibition of β-APP synthesis and of neurite elongation at
concentrations in the nM range. A more recent example is the overcoming of radio
resistance in human gliomas by AntpHD-coupled p21 WAF1/CIP1 antisense oligonucleotides.
37 Malignant gliomas are tumors highly resistant against γ-irradiation that
overexpress the cyclin-CDK inhibitor protein p21. This overexpression enhances
survival and suppresses apoptosis after γ-irradiation. The pretreatment of cells with
antisense oligonucleotides, coupled to AntpHD, enhanced γ-irradiation-induced
apoptosis and cytotoxicity in radioresistant glioma cells.
However, the success of strategies based on the internalization of oligonucleotides
is highly dependent on the stability of the oligonucleotides and the half-life
of the target protein. Therefore, we believe that, when possible, internalizing a
peptide with direct target antagonizing activity represents a better approach.
2.4.2.2 AntpHD-Mediated Internalization of Polypeptides
2.4.2.2.1 In Vitro
In a study aimed at understanding the role of small GTP-binding proteins in prolactin
exocytosis, several fusion peptides were constructed linking AntpHD to 30 to 40
amino acids derived from the C-terminal domains of rab1, rab2, and rab3. 33 After
internalization by anterior rat pituitary cells, only rab3 C terminus blocked prolactin
exocytosis. AntpHD-coupled peptides have also been used to inactivate tyrosinekinase
membrane receptors like PDGF-receptor, IGF-I receptor, and insulin receptor.
To that end, Grb10 binding to the activated cytoplasmic domain of the receptors has
been inhibited by internalizing phosphopeptides that bind Grb10 SH2 and SH3
adaptor domains. 38 A similar protocol was used to study the function of another
cellular partner of PDGF, IGF-I, and insulin receptors: PSM (proline-rich, PH SH2
domain-containing signaling mediator). 39 Similarly, to Grb10, PSM possesses SH2
and SH3 adaptor domains and an AntpHD-coupled peptide mimetic of the prolinerich
putative SH3 domain-binding region interfered with PDGF, IGF-I, and insulin,
but not with EGF-induced DNA synthesis. As in the case of Grb10, PSM is a positive
effector for mitogenic signals triggered by PDGF, IGF-I, or insulin and SH2 and
SH3 domains determine the specificity of PSM action in each mitogenic pathway.
2.4.2.2.2 In Vivo
The first in vivo application of AntpHD-mediated vectorization was the induction
of T-cell responses by a peptide derived from the HLA-cw3 cytotoxic-T-cell epitope.
AntpHD-based fusion peptides, expressing the 170–179 amino acids from HLA-
Cw3 or 147–156 amino acids from influenza nucleoprotein with flanking proteasome
recognition sites, were addressed into the cell cytoplasm to allow their presentation
in the MHC-I context. 40 Peptide presentation led to the priming of cytotoxic T cells
in vitro and in vivo (after intraperitoneal injection). In vivo efficiency in antigen
presentation required that the peptide be associated with negative charges under the
form of SDS or polysialic acid. Although this was not investigated, it is believed
that negative charges protect the peptide from degradation and favor its diffusion
within the organism.