Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

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Polo-like kinase 1 (Plk1) is a key regulator of mitotic progression and cell division in eukaryotes. It is highly expressed in tumor cells and considered a potential target for cancer therapy. Here, we report the discovery and application of a novel potent small-molecule inhibitor of mammalian Plk1, ZK-Thiazolidinone (TAL). We have extensively characterized TAL in vitro and addressed TAL specificity within cells by studying Plk1 functions in sister chromatid separation, centrosome maturation, and spindle assembly. Moreover, we have used TAL for a detailed analysis of Plk1 in relation to PICH and PRC1, two prominent interaction partners implicated in spindle assembly checkpoint function and cytokinesis, respectively. Specifically, we show that Plk1, when inactivated by TAL, spreads over the arms of chromosomes, resembling the localization of its binding partner PICH, and that both proteins are mutually dependent on each other for correct localization. Finally, we show that Plk1 activity is essential for cleavage furrow formation and ingression, leading to successful cytokinesis. INTRODUCTIONThe error-free segregation of chromosomes during cell division is necessary for the maintenance of correct ploidy and genomic integrity, and errors in cell division are presumed to lead to aneuploidy and cancer (Rajagopalan and Lengauer, 2004). To ensure that daughter cells receive the correct complement of chromosomes, two key events need to be coordinated. First, chromosomes must be equally segregated, a process that depends on the mitotic spindle. Second, cytokinesis, the process dividing the cell into two, must occur between the two sets of segregated chromosomes. Both of these processes require the activity of a key cell cycle regulator, the Polo-like kinase 1 (Plk1). Plks form a conserved subfamily of serine/threonine protein kinases. The first member to be identified was Polo in Drosophila melanogaster (Llamazares et al., 1991) and, subsequently, four Plk family members have been identified in mammals Barr et al., 2004).Plk1 contains an N-terminal kinase domain and a phosphopeptide-binding C-terminal regulatory polo-box domain (PBD; Leung et al., 2002;Elia et al., 2003b). In vertebrates Plk1 has been implicated in the activation of Cdk1-cyclin B upon entry into mitosis, centrosome maturation via the recruitment of the ␥-tubulin ring complex (␥-TuRC), spindle formation, sister chromatid separation by cohesin removal from the chromosome arms, promotion of anaphase onset through direct phosphorylation of the APC/C complex as well as the inhibition of the APC/C inhibitor Emi1, and finally, mitotic exit and cytokinesis (reviewed in Barr et al., 2004). Fitting with these diverse functions, Plk1 localizes to the centrosomes, spindle poles, and kinetochores in prophase and metaphase, the central spindle in anaphase, and the midbody during cytokinesis. These localizations require the function of the PBD (Jang et al., 2002;Seong et al., 2002) and priming-kinases to generate phosphorylated docking sites that are subsequently recog...

Interferon (IFN) regulatory factor (IRF)-2 was originally described as an antagonist of IRF-1–mediated transcriptional regulation of IFN-inducible genes. IRF-1−/− mice exhibit defective T helper type 1 (Th1) cell differentiation. We have used experimental leishmaniasis to show that, like IRF-1−/− mice, IRF-2−/− mice are susceptible to Leishmania major infection due to a defect in Th1 differentiation. Natural killer (NK) cell development is compromised in both IRF-1−/− and IRF-2−/− mice, but the underlying mechanism differs. NK (but not NK+ T) cell numbers are decreased in IRF-2−/− mice, and the NK cells that are present are immature in phenotype. Therefore, like IRF-1, IRF-2 is required for normal generation of Th1 responses and for NK cell development in vivo. In this particular circumstance the absence of IRF-2 cannot be compensated for by the presence of IRF-1 alone. Mechanistically, IRF-2 may act as a functional agonist rather than antagonist of IRF-1 for some, but not all, IFN-stimulated regulatory element (ISRE)-responsive genes.

,5,6,11,12,13 Monopolar spindle 1 (MPS1), a mitotic kinase that is overexpressed in several human cancers, contributes to the alignment of chromosomes to the metaphase plate as well as to the execution of the spindle assembly checkpoint (SAC). Here, we report the identification and functional characterization of three novel inhibitors of MPS1 of two independent structural classes, N-(4-{2- [(2-cyanophenyl) and N-cyclopropyl-4-{8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl}benzamide (Mps-BAY2b) (two imidazopyrazines). By selectively inactivating MPS1, these small inhibitors can arrest the proliferation of cancer cells, causing their polyploidization and/or their demise. Cancer cells treated with Mps-BAY1 or Mps-BAY2a manifested multiple signs of mitotic perturbation including inefficient chromosomal congression during metaphase, unscheduled SAC inactivation and severe anaphase defects. Videomicroscopic cell fate profiling of histone 2B-green fluorescent protein-expressing cells revealed the capacity of MPS1 inhibitors to subvert the correct timing of mitosis as they induce a premature anaphase entry in the context of misaligned metaphase plates. Hence, in the presence of MPS1 inhibitors, cells either divided in a bipolar (but often asymmetric) manner or entered one or more rounds of abortive mitoses, generating gross aneuploidy and polyploidy, respectively. In both cases, cells ultimately succumbed to the mitotic catastrophe-induced activation of the mitochondrial pathway of apoptosis. Of note, low doses of MPS1 inhibitors and paclitaxel (a microtubular poison) synergized at increasing the frequency of chromosome misalignments and missegregations in the context of SAC inactivation. This resulted in massive polyploidization followed by the activation of mitotic catastrophe. A synergistic interaction between paclitaxel and MPS1 inhibitors could also be demonstrated in vivo, as the combination of these agents efficiently reduced the growth of tumor xenografts and exerted superior antineoplastic effects compared with either compound employed alone. Altogether, these results suggest that MPS1 inhibitors may exert robust anticancer activity, either as standalone therapeutic interventions or combined with microtubule-targeting chemicals. Mitosis is a sophisticated process that ensures the faithful inheritance of the genetic material by the cellular progeny while preventing aneuploidy and chromosomal instability (CIN), two established hallmarks of cancer. 1-3 A set of protein kinases that are collectively known as mitotic kinases, including Aurora kinases (AURKs), mitotic cyclin-dependent kinases (CDKs), Polo-like kinases and monopolar spindle 1 (MPS1), regulates and coordinates multiple aspects of chromosome segregation during mitosis. 4 MPS1 (also known as TTK) is a dual-specificity kinase (meaning that it phosphorylates both serine/threonine and tyrosine residues) with an established role in the proper alignment and orientation of chromosomes on the metaphase plate. 5 MPS1 is a core component of the spindle assembl...