Multiscale kinetic modeling of liposomal Doxorubicin delivery quantifies the role of tumor and drug-specific parameters in local delivery to tumors.

Nanoparticle encapsulation has been used as a means to manipulate the pharmacokinetic (PK) and safety profile of drugs in oncology. Using pegylated liposomal doxorubicin (PLD) vs. conventional doxorubicin as a model system, we developed and experimentally validated a multiscale computational model of liposomal drug delivery. We demonstrated that, for varying tumor transport properties, there is a regimen where liposomal and conventional doxorubicin deliver identical amounts of doxorubicin to tumor cell nuclei. In mice, typical tumor properties consistently favor improved delivery via liposomes relative to free drug. However, in humans, we predict that some tumors will have properties wherein liposomal delivery delivers the identical amount of drug to its target relative to dosing with free drug. The ability to identify tumor types and/or individual patient tumors with high degree of liposome deposition may be critical for optimizing the success of nanoparticle and liposomal anticancer therapeutics.CPT: Pharmacometrics & Systems Pharmacology (2012) 1, e15; doi:10.1038/psp.2012.16; advance online publication 21 November 2012.

Molecular structures and interactions in the yeast kinetochore.

Kinetochores are the elaborate protein assemblies that attach chromosomes to spindle microtubules in mitosis and meiosis. The kinetochores of point-centromere yeast appear to represent an elementary module, which repeats a number of times in kinetochores assembled on regional centromeres. Structural analyses of the discrete protein subcomplexes that make up the budding-yeast kinetochore have begun to reveal principles of kinetochore architecture and to uncover molecular mechanisms underlying functions such as transmission of tension and establishment and maintenance of bipolar attachment. The centromeric DNA is probably wrapped into a compact organization, not only by a conserved, centromeric nucleosome, but also by interactions among various other DNA-bound kinetochore components. The rod-like, heterotetrameric Ndc80 complex, roughly 600 Å long, appears to extend from the DNA-proximal assembly to the plus end of a microtubule, to which one end of the complex is known to bind. Ongoing structural studies will clarify the roles of a number of other well-defined complexes.

Structural and functional dissection of Mif2p, a conserved DNA-binding kinetochore protein.

Mif2p is the budding-yeast orthologue of the mammalian centromere-binding protein CENP-C. We have mapped domains of Saccharomyces cerevisiae Mif2p and studied the phenotyptic consequences of their deletion. Using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays, we have further shown that Mif2p binds in the CDEIII region of the budding-yeast centromere, probably in close spatial association with Ndc10p. Moreover, ChIP experiments show that Mif2p recruits to yeast kinetochores a substantial subset of inner and outer kinetochore proteins, but not the Ndc80 or Spc105 complexes. We have determined the crystal structure of the C-terminal, dimerization domain of Mif2p. It has a "cupin" fold, extremely similar both in polypeptide chain conformation and in dimer geometry to the dimerization domain of a bacterial transcription factor. The Mif2p dimer seems to be part of an enhanceosome-like structure that nucleates kinetochore assembly in budding yeast.

Molecular analysis of kinetochore-microtubule attachment in budding yeast.

The complex series of movements that mediates chromosome segregation during mitosis is dependent on the attachment of microtubules to kinetochores, DNA-protein complexes that assemble on centromeric DNA. We describe the use of live-cell imaging and chromatin immunoprecipitation in S. cerevisiae to identify ten kinetochore subunits, among which are yeast homologs of microtubule binding proteins in animal cells. By analyzing conditional mutations in several of these proteins, we show that they are required for the imposition of tension on paired sister kinetochores and for correct chromosome movement. The proteins include both molecular motors and microtubule associated proteins (MAPs), implying that motors and MAPs function together in binding chromosomes to spindle microtubules.

Probing the architecture of a simple kinetochore using DNA-protein crosslinking.

In budding yeast, accurate chromosome segregation requires that one and only one kinetochore assemble per chromosome. In this paper, we report the use of DNA-protein crosslinking and nondenaturing gel analysis to study the structure of CBF3, a four-protein complex that binds to the essential CDEIII region of Saccharomyces cerevisiae centromeres. We find that three subunits of CBF3 are in direct contact with CDEIII over a region of DNA that spans 80 bp. A highly asymmetric core complex containing p58(CTF13) p64(CEP3) and p110(NDC10) in direct contact with DNA forms at the genetically defined center of CDEIII. This core complex spans approximately 56 bp of CEN3. An extended complex comprising the core complex and additional DNA-bound p110(NDC10) also forms. It spans approximately 80 bp of DNA. CBF3 makes sequence-specific and -nonspecific contacts with DNA. Both contribute significantly to the energy of CBF3-DNA interaction. Moreover, important sequence-specific contacts are made with bases that are not conserved among yeast centromeres. These findings provide a foundation for understanding the organization of the CBF3-centromere complex, a structure that appears to initiate the formation of microtubule attachment sites at yeast kinetochores. These results also have implications for understanding centromere-binding proteins in higher cells.