Autophosphorylation of polo-like kinase 4 and its role in centriole duplication.

Centrosome duplication occurs once every cell cycle in a strictly controlled manner. Polo-like kinase 4 (PLK4) is a key regulator of this process whose kinase activity is essential for centriole duplication. Here, we show that PLK4 autophosphorylation of serine S305 is a consequence of kinase activation and enables the active fraction to be identified in the cell. Active PLK4 is detectable on the replicating mother centriole in G1/S, with the proportion of active kinase increasing through interphase to reach a maximum in mitosis. Activation of PLK4 at the replicating daughter centriole is delayed until G2, but a level equivalent to the replicating mother centriole is achieved in M phase. Active PLK4 is regulated by the proteasome, because either proteasome inhibition or mutation of the degron motif of PLK4 results in the accumulation of S305-phosphorylated PLK4. Autophosphorylation probably plays a role in the process of centriole duplication, because mimicking S305 phosphorylation enhances the ability of overexpressed PLK4 to induce centriole amplification. Importantly, we show that S305-phosphorylated PLK4 is specifically sequestered at the centrosome contrary to the nonphosphorylated form. These data suggest that PLK4 activity is restricted to the centrosome to prevent aberrant centriole assembly and sustained kinase activity is required for centriole duplication.

Modified poly(propylene imine) dendrimers as effective transfection agents for catalytic DNA enzymes (DNAzymes).

The major bottleneck in gene therapy remains the issue of delivery. In this work, various modified poly(propylene imine) (PPI) dendrimers are introduced as gene transfection agents. Commercially available PPI-dendrimers have been modified (i) at the exterior primary amines with acetyl groups or glycol gallate (PEG-like) groups, and (ii) at the interior tertiary amines with methyl iodide (MeI) or MeCl to produce multiple quaternized cationic sites in the core of the dendrimer. The prepared materials have been tested with respect to their binding capabilities to DNA, their toxicity in cell cultures, their in vitro transfection efficiency and their in vivo delivery possibilities. In all cases, a 33-mer oligonucleotide (DNAzyme) was used. Polyacrylamide gel electrophoresis (PAGE) studies have demonstrated strong but reversible binding, where the quarternized and higher generation dendrimer species have shown more potent binding. Typically, for the modified fourth PPI-dendrimers, binding is observed at a concentration of about 4 microM DNA and a dendrimer-DNA charge ratio of around 2:1-1:1. All the tested PPI-dendrimers display a low cellular toxicity, especially when higher serum contents are used in the culture medium. For example, most of the prepared fourth generation PPI-dendrimers are not or hardly toxic up to at least 20 microM in 20% serum. An in vitro characterization has revealed a high dendrimer-mediated intracellular uptake of the DNAzyme: all the tested fourth generation PPI-dendrimers display transfection efficiencies close to or exceeding 80%, even when the concentration of serum in the medium is increased from 10 to 40%. Finally, the potential of using modified PPI-dendrimers for in vivo gene therapy experiments is demonstrated. Injecting a G4-PEG(MeI)-ssDNA complex intravenously into Nude mice has resulted in a high nuclear uptake as confirmed by co-localization studies.

Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles.

Short nucleic acid sequences specific to oncogene targets such as bcl-2, bcr-abl, and c-myc have been shown to exhibit specific anti-cancer activity in vitro through antigene or antisense activity. Efficient in vivo delivery of oligonucleotides remains a major limitation for the therapeutic application of these molecules. We report herein on the preparation of transferrin-modified nanoparticles containing DNAzymes (short catalytic single-stranded DNA molecules) for tumor targeting as well as their biodistribution using various methods of administration in the mouse. Linear, beta-cyclodextrin-based polymers are complexed with DNAyzme molecules to form sub-50 nm particles termed "polyplexes". The surface properties of the cyclodextrin-containing polyplexes are modified by exploiting the ability of the beta-cyclodextrin substructure and adamantane to form inclusion complexes. Accordingly, conjugates of adamantane with poly(ethylene glycol) (PEG) are prepared and combined with the polyplexes. The adamantane form inclusion complexes with the surface cyclodextrins of the polyplexes to provide a sterically stabilizing layer of PEG. The stabilized polyplexes are also modified with transferrin for increasing targeting to tumor cells expressing transferrin receptors. The preparation, characterization, and in vitro application of these nanoparticles are discussed. The transferrin-polyplexes containing fluorescently-labeled DNAzyme molecules are administered to tumor-bearing nude mice and their biodistribution and clearance kinetics are monitored using a fluorescence imaging system. Four methods of administration are studied: intraperitoneal bolus and infusion, intravenous bolus, and subcutaneous injection. DNAzymes packaged in polyplex formulations are concentrated and retained in tumor tissue and other organs, whereas unformulated DNAzyme is eliminated from the body within 24 hours post-injection. Intravenous and intraperitoneal bolus injections result in the highest fluorescent signal (DNAzyme) at the tumor site. Tumor cell uptake is observed with intravenous bolus injection only, and intracellular delivery requires transferrin targeting.