Helen Piwnica-Worms - US grants

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand how the cell division cycle is regulated during a normal cell cycle (cell cycle control) and how cell cycle progression is prevented when unreplicated or damaged DNA is detected (checkpoint control). The passage of cells from one stage of the cell cycle to the next is regulated by several distinct controls that act on the transcription of cyclin genes; the degradation of cyclin proteins; the modification of the cyclin-dependent protein kinases (Cdks) by both reversible phosphorylation and by association with regulatory subunits; and finally by intracellular compartmentalization of cyclin/Cdk components and their regulators. The Cdc25 protein phosphatases positively regulate the cell division cycle by activating CDKs and they are also targets of checkpoint control. In humans and rodents, there are three members of the Cdc25 family, designated Cdc25A, B and C. Studies aimed at elucidating how the Cdc25A protein phosphatase is regulated throughout the cell division cycle and in response to checkpoint activation are proposed. In addition, studies aimed at distinguishing the individual contributions made by Cdc25A, Cdc25B and Cdc25C to cell cycle progression and checkpoint control in both mouse and human cells are proposed. Finally, studies will be performed to elucidate how the checkpoint kinases, Chkl and Chk2 are activated by unreplicated DNA and/or genotoxic stress and how these kinases interface with the cell cycle machinery to cause cell cycle delays in the presence of unreplicated or damaged DNA. Because many cancers are neither curable using existing strategies nor readily detectable at early stages there is a need to identify new targets that can be used both as diagnostic probes and as therapeutic targets. The studies outlined in this proposal investigate basic mechanisms of cell cycle control and checkpoint control. Proteins involved in these pathways may one day be used as diagnostic markers or as targets for designing anti-proliferative drugs.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We employed data-dependent analysis of protein phosphorylation using rapid acquisition nano-LC-linear-quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometry (nano-LC-FT-MS). The accurate m/z values of singly, doubly and triply-charged species calculated from the theoretical protonated masses of peptides phosphorylated at all Ser, Thr or Tyr residues of the human checkpoint 2 (Chk2) protein kinase were used for selected ion extraction and chromatographic analysis. Using a kinase-inactive Chk2 mutant as a control, accurate mass measurements from FT-MS and collision-induced dissociation spectra, 11 Chk2 auto-phosphorylation sites were assigned. Additionally, the presence of additional novel Chk2 phosphorylation sites in two unique peptides was deduced from accurate mass measurements. Selected ion chromatograms of all Chk2 phosphopeptides gave single peaks except in three cases in which two closely eluting species were observed. These pairs of phosphopeptides were determined to be positional isomers from MS/MS analysis. In this study, it was also found that ions due to the neutral loss of phosphoric acid from the parent peptide ion were not prominent in 18 of 36 MS/MS spectra of O-linked Chk2 phosphopeptides. Thus, accurate mass-driven analysis and rapid parallel MS/MS acquisition is a useful method for the discovery of new phosphorylation sites that is independent of the signature losses from phosphorylated amino acid residues.

In 2002, in response to the NIH Roadmap to Molecular Libraries and Imaging, we conceived the process and now have fully established a P50 High Throughput Screening Robotics Core. The new Core represents a multi-departmental effort with fiscal support for establishing the Core coming from P50 funds in combination with the Department of Radiology, Department of Molecular Biology & Pharmacology, and Department of Cell Biology as well as the Howard Hughes Medical Institute. The Dean of Washington University School of Medicine as well as the Director of the Washington University Siteman Cancer Center joined our effort with the commitment of additional funds for further development of the resource. Overall, nearly $2M has been invested in the establishment of this resource. This Core serves as an outstanding on-going example of how our P50 program has leveraged university resources and continues to stimulate interdisciplinary molecular imaging activity throughout the WU campus. Functionally and symbolically, Drs. Helen Piwnica-Worms (Cell Biology), Rafi Kopan (Molecular Biology &Pharmacology), and David Piwnica-Worms (Radiology ) serve as Core Co-Directors with shared administration and scientific oversight of Core activities. This Core is housed in the McDonnell Sciences Building, Room 316, across the street from the East Building Imaging Annex, site of many of the Molecular Imaging Center collaborative activities. Construction of the room and instrument requirements was finished in 2005 and installations of the Beckman Coulter BioMek FX liquid handler as well as an ORCA robot on a 6 ft rail were completed in the summer/fall of 2005. A bio-barrier entryway was constructed and positive-pressure, HEPA filtered air circulation has been installed. Integrated readout and cell culture stations are included with the imaging and analysis instruments. This innovative Core was founded on molecular imaging platforms for readout of a wide range of bio-assays and underpins three of the Research Projects in this application. This Core assures that the personnel and resources for high throughput screening are available to all our ICMIC investigators. Jayne Marasa, senior Core lab manager, directs day-to-day experiments and has optimized several protocols and logistical requirements for our first projects in high throughput screening. Exciting data are now coming on-line during the summer 2006. The Core allows for automated screening of cells cultured in 96 or 384 plate formats and can be applied to multiple investigator-initiated molecular imaging applications arising from P50 investigators and throughout the university community. The applications include: 1) small molecule screens applied to cells engineered by WU investigators for imaging and therapeutic drug leads. 2) small molecule or peptide screens to identify enzyme inhibitors (directed against high priority proteases, kinases, phosphatases, etc.) or modulators of protein-protein interactions. 3) genome-wide siRNA library screens against kinases, phosphatases and E3-ligases in human cells targeting signal transduction, cell growth or cell death responses.

Our broad goals include delineating how the cell division cycle is regulated, determining how cancer cells derail cell cycle regulatory pathways, and ultimately using this information for diagnosing and treating human cancers. We propose to focus our efforts on the Cdc25A protein phosphatase as this enzyme is a key regulator of the cell division cycle in mammals and is overproduced in a wide range of human tumors. Stable cell lines and mouse colonies will be generated that enable Cdc25A regulation to be studied in cells and living mice using molecular imaging technologies. In particular, cell lines that inducibly express a fusion protein between human Cdc25A and firefly luciferase (Cdc25A-FLuc) will be used in a high throughput screen to identify the serine/threonine protein phosphatase holoenzymes that regulate the stability of Cdc25A in vivo. In principal the proteins identified in this study could provide novel targets for imaging agents and therapeutic intervention in cancer treatment. In addition, knock-in mice will be generated that express a fusion protein between endogenous Cdc25A and click beetle red luciferase (Cdc25A-CBRLuc) from the Cdc25A locus. In this way endogenous Cdc25A protein levels can be monitored non-invasively and repetitively in living mice under steady state conditions and in response to DMAdamaging agents and drugs targeting cell cycle checkpoints. In addition, molecular imaging strategies will be applied to mouse models of breast cancer to validate target specificity of rational anti-cancer therapeutic regimens in combination therapies. The reagents and results obtained in these studies will be useful in future clinical trials that combine DNA damaging agents with novel Chk1 inhibitors or novel checkpoint abrogators. The proposed studies are expected to enhance our understanding of basic principles of cell cycle control in mammals and may impact future therapeutic strategies for cancer treatment.

[unreadable] DESCRIPTION (provided by applicant): The Cdc25 phosphatases positively regulate the cell cycle by activating Cdks. In mammals, three family members, denoted Cdc25A, Cdc25B, and Cdc25C, exist. It is unclear why higher eukaryotic organisms encode three Cdc25s while yeast cells survive with one. Mice that can be deleted for Cdc25A were generated. In addition, mice lacking Cdc25B, Cdc25C or both and that can also be conditionally deleted for Cdc25A were generated. These mice will be used to determine the contributions made by Cdc25 family members to adult cell cycles. Another goal of this proposal is to utilize high throughput and molecular imaging strategies to identify novel regulators of Cdc25A. Cdc25A functions throughout the cell cycle to regulate cell cycle transitions and Cdc25A is overproduced in several cancers. Cdc25A activity, stability and interactions with other proteins are regulated by reversible phosphorylation. Thus, identifying the protein kinases that regulate Cdc25A will provide insight into how Cdc25A is regulated under normal and stressed conditions and what pathways leading to Cdc25A overproduction are derailed in human cancers. Finally, experiments are proposed to study a novel regulatory pathway involving ATR, Chk1 and PP2A and to investigate interactions between Chk1 and Cdc25A in mice. In addition to advancing our understanding of basic cell cycle principles, these studies have clinical impact as well. For example, Cdc25A and Cdc25B are overproduced in many human cancers and efforts are underway to develop Cdc25 inhibitors that can be used to treat human cancers. The cytotoxicity of Cdc25 inhibition needs to be assessed before Cdc25 inhibitors can proceed to the clinic. The gene knockout studies proposed in this grant will assess effects of loss of Cdc25 family members in adult mice and may predict how patients will respond to global versus individual Cdc25 inhibition. Another strategy that is currently being used to treat cancer patients is to combine DNA damaging agents with drugs that induce Cdc25A accumulation. In preclinical models, this strategy induces checkpoint bypass and preferential killing of p53-deficient cells. The Chk1 inhibitor UCN-01 is being tested with DNA damaging agents in Phase I and II clinical trials. Thus, the novel kinases and/or regulators of the PP2A/Chk1 pathway identified in the course of our studies are potential druggable targets. Inhibitors that target components of these pathways could substitute for Chk1 inhibitors in the combination therapy described above. The hope is that these inhibitors may induce less genome instability than Chk1 inhibitors. PUBLIC HEALTH RELEVANCE: Experiments are proposed to fully dissect the molecular underpinnings of the PP2A/Chk1/Cdc25A regulatory pathway and to functionally characterize the Cdc25 phosphatases in mice. The information gained from the proposed studies is expected not only to enhance our understanding of basic principles of cell cycle control in mammals but is also expected to directly impact future therapeutic strategies for cancer treatment. [unreadable] [unreadable] [unreadable]

Animal Model; Apoptosis; Area; Biological Process; Biology; Caenorhabditis elegans; cancer cell; Cancer Center; Cancer Center Support Grant; Cell Cycle; Cell Cycle Checkpoint; Cell Death; Cell physiology; Cell Proliferation; Cells; Collaborations; design; Development; Diagnosis; Diagnostic; DNA Repair; Drosophila melanogaster; Educational process of instructing; Environment; Evolution; Faculty; Goals; Human; Internet; Laboratories; Malignant Neoplasms; medical schools; member; Mutation; Normal Cell; novel; Pathway interactions; Process; programs; Protein Deregulation; Proteins; Regulatory Pathway; Research; Research Activity; Research Personnel; Saccharomycetales; Schools; Signal Transduction; Signal Transduction Pathway; Staging; System; telomere; therapeutic target; tool; Training; Translations; Universities; Washington; Xenopus