Browsing by Author "Kreklau, Emiko L."

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    Aurora A–Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy
    (AACR, 2019-12) Du, Jian; Yan, Lei; Torres, Raquel; Gong, Xueqian; Bian, Huimin; Marugán, Carlos; Boehnke, Karsten; Baquero, Carmen; Hui, Yu-Hua; Chapman, Sonya C.; Yang, Yanzhu; Zeng, Yi; Bogner, Sarah M.; Foreman, Robert T.; Capen, Andrew; Donoho, Gregory P.; Van Horn, Robert D.; Barnard, Darlene S.; Dempsey, Jack A.; Beckmann, Richard P.; Marshall, Mark S.; Chio, Li-Chun; Qian, Yuewei; Webster, Yue W.; Aggarwal, Amit; Chu, Shaoyou; Bhattachar, Shobha; Stancato, Louis F.; Dowless, Michele S.; Iversen, Phillip W.; Manro, Jason R.; Walgren, Jennie L.; Halstead, Bartley W.; Dieter, Matthew Z.; Martinez, Ricardo; Bhagwat, Shripad V.; Kreklau, Emiko L.; Lallena, Maria Jose; Ye, Xiang S.; Patel, Bharvin K. R.; Reinhard, Christoph; Plowman, Gregory D.; Barda, David A.; Henry, James R.; Buchanan, Sean G.; Campbell, Robert M.; Pediatrics, School of Medicine
    Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform–selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A–selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition–associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A–selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.
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    A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase
    (2001-04) Kreklau, Emiko L.; Limp-Foster, Melissa; Liu, Naili; Xu, Yi; Kelley, Mark R.; Erickson, Leonard C.
    DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O6‐methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.