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1.
Cell Cycle ; 12(12): 1822-3, 2013 Jun 15.
Article in English | MEDLINE | ID: mdl-23759568
2.
Assay Drug Dev Technol ; 10(2): 161-78, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22084958

ABSTRACT

High content screening (HCS) has emerged an important tool for drug discovery because it combines rich readouts of cellular responses in a single experiment. Inclusion of cell cycle analysis into HCS is essential to identify clinically suitable anticancer drugs that disrupt the aberrant mitotic activity of cells. One challenge for integration of cell cycle analysis into HCS is that cells must be chemically synchronized to specific phases, adding experimental complexity to high content screens. To address this issue, we have developed a rules-based method that utilizes mitotic phosphoprotein monoclonal 2 (MPM-2) marker and works consistently in different experimental conditions and in asynchronous populations. Further, the performance of the rules-based method is comparable to established machine learning approaches for classifying cell cycle data, indicating the robustness of the features we use in the framework. As such, we suggest the use of MPM-2 analysis and its associated expressive features for integration into HCS approaches.


Subject(s)
High-Throughput Screening Assays/methods , Image Processing, Computer-Assisted/methods , Mitosis/physiology , Algorithms , Anaphase/physiology , Aurora Kinases , Automation , Cell Cycle , Cell Nucleus/ultrastructure , Cells/classification , Coloring Agents , Cytokinesis/physiology , Cytological Techniques , DNA/chemistry , Humans , Immunochemistry , Microscopy , Protein Serine-Threonine Kinases/metabolism , Reproducibility of Results , Support Vector Machine , Tissue Fixation
3.
Cell Cycle ; 8(18): 2984-94, 2009 Sep 15.
Article in English | MEDLINE | ID: mdl-19713763

ABSTRACT

Aurora family kinases regulate numerous mitotic processes, and their dysfunction or overexpression can cause aneuploidy, a contributing factor for tumorigenesis. In vertebrates, the Aurora-B kinase regulates kinetochore maturation, destabilization of improper kinetochore-microtubule attachments, the spindle assembly checkpoint, central spindle organization and cytokinesis. A gene duplication event created the related Aurora-C kinase in mammals. While Aurora-C function is unclear, it has similar structural and localization properties as Aurora-B. Inhibition of either Aurora-B or Aurora-C function causes aneuploidy, while simultaneous inhibition of both causes a higher frequency of aneuploidy. To determine if Aurora-C and -B have overlapping or unique complementary functions during mitosis, we created a system where Aurora-B is replaced by wild-type or kinase-defective mutant Aurora-C in HeLa cells. In this model, Aurora-B protein levels and mitotic functions were suppressed including the regulation of kinetochore-microtubule attachments, the spindle assembly checkpoint, and cytokinesis. Wild-type, but not kinase-defective Aurora-C expression, was able to rescue these functions. Therefore, Aurora-C can perform the same essential functions as Aurora-B in mitosis.


Subject(s)
Mitosis , Protein Serine-Threonine Kinases/physiology , Aurora Kinase B , Aurora Kinase C , Aurora Kinases , Cytokinesis , HeLa Cells , Humans , Kinetochores , Microtubules , Protein Serine-Threonine Kinases/deficiency , Spindle Apparatus
4.
Cell Cycle ; 7(17): 2691-704, 2008 Sep 01.
Article in English | MEDLINE | ID: mdl-18773538

ABSTRACT

Microtubule nucleation and formation from the kinetochore/chromatin have been proposed to contribute to bipolar spindle assembly facilitating equal segregation of chromosomes in mitosis. Although two independent pathways involving the small Ran GTPase-TPX2 proteins and the chromosomal passenger complex proteins have been implicated in the formation of microtubules from the kinetochore/chromatin, detailed molecular mechanisms integrating the pathways and regulating the process have not been well elucidated. This study demonstrates that Aurora kinase-A plays a central role in the kinetochore/chromatin associated microtubule assembly in human cells by integrating the two pathways regulating the process. Silencing by siRNA and over expression of a kinase inactive mutant revealed involvement of Aurora-A at two critical steps. These include accumulation of g-tubulin in the vicinity of kinetochore/chromatin to create microtubule nucleation sites as well as INCENP and TPX2 mediated activation of Aurora-A facilitating formation and stabilization of microtubules. The findings provide the first evidence of Aurora-A, in association with INCENP and TPX2, being a key regulator of kinetochore/chromatin associated microtubule formation in human cells.


Subject(s)
Chromatin/enzymology , Kinetochores/enzymology , Microtubules/enzymology , Protein Serine-Threonine Kinases/metabolism , Aurora Kinase A , Aurora Kinases , Chromatin/ultrastructure , Chromosomal Proteins, Non-Histone/metabolism , Enzyme Activation , Feedback, Physiological , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins , Kinesins/metabolism , Kinetochores/ultrastructure , Microtubule-Associated Proteins/metabolism , Microtubules/ultrastructure , Models, Biological , Neoplasm Proteins/metabolism , Phosphorylation , Protein Binding , Spindle Apparatus/enzymology , Survivin , Tubulin/metabolism
5.
Cell Cycle ; 7(6): 787-95, 2008 Mar 15.
Article in English | MEDLINE | ID: mdl-18239465

ABSTRACT

Aurora-B and -C kinases are members of the Aurora serine/threonine kinase family of mitotic regulators. Aurora-B kinase is evolutionarily conserved from yeast to humans and has multiple functions in chromosome condensation, cohesion, biorientation and in cytokinesis. In contrast, Aurora-C kinase has only been found in mammals, is upregulated in some tumor cell lines and tissues, and has a unique physiological role in spermiogenesis. Despite these known functions, little is known about the function of Aurora-C in mitosis. We have found that Aurora-C interacts with Borealin in addition to the other known members of the Aurora-B chromosomal passenger complex (CPC). We have also found that Aurora-C, like Aurora-B, phosphorylates the centromeric histone Centromere Protein-A (CENP-A) and Borealin in vitro. These molecular mechanisms are consistent with our observation that in the absence of Aurora-B, Aurora-C is sufficient for proper mitotic phosphorylation of CENP-A and centromeric localization of the CPC proteins. Thus, Aurora-C shares Aurora-B substrates and is capable of performing mitotic functions previously attributed only to Aurora-B.


Subject(s)
Autoantigens/metabolism , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Mitosis/physiology , Protein Serine-Threonine Kinases/metabolism , Aurora Kinase B , Aurora Kinase C , Aurora Kinases , Cell Cycle/physiology , Centromere/physiology , Centromere Protein A , Chromosome Segregation/physiology , HeLa Cells , Humans , Phosphorylation , Protein Binding
6.
J Clin Invest ; 117(7): 1893-901, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17607363

ABSTRACT

Mutations in ras and p53 are the most prevalent mutations found in human nonmelanoma skin cancers. Although some p53 mutations cause a loss of function, most result in expression of altered forms of p53, which may exhibit gain-of-function properties. Therefore, understanding the consequences of acquiring p53 gain-of-function versus loss-of-function mutations is critical for the generation of effective therapies for tumors harboring p53 mutations. Here we describe an inducible mouse model in which skin tumor formation is initiated by activation of an endogenous K-ras(G12D) allele. Using this model we compared the consequences of activating the p53 gain-of-function mutation p53(R172H) and of deleting the p53 gene. Activation of the p53(R172H) allele resulted in increased skin tumor formation, accelerated tumor progression, and induction of metastasis compared with deletion of p53. Consistent with these observations, the p53(R172H) tumors exhibited aneuploidy associated with centrosome amplification, which may underlie the mechanism by which p53(R172H) exerts its oncogenic properties. These results clearly demonstrate that p53 gain-of-function mutations confer poorer prognosis than loss of p53 during skin carcinogenesis and have important implications for the future design of therapies for tumors that exhibit p53 gain-of-function mutations.


Subject(s)
Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Alleles , Aneuploidy , Animals , Arginine/genetics , Arginine/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Centrosome , Disease Models, Animal , Enzyme Activation , Gene Expression Regulation, Neoplastic , Glycine/genetics , Glycine/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Lymphatic Metastasis/pathology , Mice , Mice, Inbred C57BL , Mutation/genetics , Skin Neoplasms/genetics , Tumor Suppressor Protein p53/genetics , ras Proteins/genetics , ras Proteins/metabolism
7.
Curr Biol ; 16(14): 1414-21, 2006 Jul 25.
Article in English | MEDLINE | ID: mdl-16860740

ABSTRACT

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , Kinetochores/metabolism , Metaphase/genetics , Nuclear Proteins/physiology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Cell Cycle Proteins/analysis , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosome Positioning , Humans , Kinetochores/ultrastructure , Microtubules/metabolism , Models, Biological , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/genetics , Protein Transport , Proto-Oncogene Proteins/analysis , Proto-Oncogene Proteins/genetics , RNA Interference , Polo-Like Kinase 1
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