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University of Maryland Baltimore
Experimental Therapeutics Symposium 2009

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  1. 1. 1<br />Carey Lab InterestsE.T. 10/02/09<br />Gregory B. Carey, Ph.D.<br />Asst. Prof. Microbiology and Immunology<br />Center For Vascular And Inflammatory Diseases<br />Rm 313, Biopark1, 800W Baltimore St.<br />, x68191<br />
  2. 2. Carey Lab interests<br />RedOxBiology: <br />ROS: NO, O2-, H2O2<br />ROS no longer metabolic waste byproducts<br />Homeostatic<br />Act as 2ndmessengers<br />Switching functions??<br />Mechanism of Action of Manumycin-A (Man-A) natural tumoricide<br />Antimicrobial agent ca 1960<br />Ras Inhibitor ca 1990<br />Natural compounds generally well tolerated<br />Broader spectrum of benefits<br />Use to understand cell death resulting from RedOxchanges<br />
  3. 3. Collaborative Interests<br />Tumor biology and Immunobiology: <br />Role PI-3K/Akt pathway in tumor development, survival, progression etc.<br />Dr. Achsah Keegan (UMB)<br />Metabolomics<br />Switching or skewing of pathways in cancer? <br />Unique metabolites, markers etc?<br />IL-4 conditioning causes exclusive dependence on glycolytic respiration<br />Intervene w/ 3-Br Pyruvate and 2-DOG<br />Drs. Keegan UMB) and Wendy Davidson (NIH)<br />Gld mouse work<br />Why does incidence and number of autoreactive immune cells increase w/ aging? (   geriatric lymphomatosis)<br />= deletion failure? <br />Metabolic advantage? Both? <br />What are persistent antigens?<br />RedOx Biology and Proteomics<br />Superoxide lethal for tumors and normal cells (not NO and H2O2)<br />Compartmental ROS overload promotes death in tumors<br />Preceded by biochemical changes on key proteins<br />Are changes cause or effect or unrelated?<br />
  4. 4. Lab InterestsMechanism of Action of Manumycin-A<br />Manumycin-A (Man-A)<br />Stimulates death via ROS/PPase/Caspase cascade<br />PP1 activated, CDK2 deactivated<br />Have also seen potent anti-tumor activity in Nigerian cola nut fractions<br />Dr. Chris Okunje (Formely Walter Reed. Presently U. Penn)<br />Manumycin A<br />Potent anti-tumor and pleiotropic effects in animals<br />Reduced trypanosome infection<br />Reduced cholesterolemia and arterial plaque formation in mice<br />Tumor rejection<br />Broad spectrum, plieotropic effects of Natural cmpds.<br />HOW IS IT WORKING?<br />Usable or adaptable for potential use in humans?<br />
  5. 5. Loss pm<br />Cyt<br />-<br />C Ox<br />NO<br />*, <br />â<br />Dephos<br />Casp<br />DY<br />integrity<br />H<br />O<br />H<br />O<br />·<br />m<br />, <br />&lt;2N<br />â<br />-<br />O<br />PPase<br />2<br />2<br />2<br />2<br />2<br />Response<br />0.5h<br />4h<br />1h<br />Time<br />Figure1. <br />Manumycin<br />-<br />A<br />-<br />Stimulated Cascade of Events. <br />Research Summary I <br />Akt cleavage<br />CDK2 Activity <br />Loss?<br />H2O2 good? O2- bad?<br />5<br />
  6. 6. H2O2<br />X<br />Research Summary II<br />PP1a stimulates O2- generating system?<br />PP1a suppresses H2O2 generator?<br />PP1a<br />Man-A<br />?<br />O2-<br />CDK2 modulates PP1a (Thr320)<br />What is Role of CDK2 in survival pathways?<br />6<br />
  7. 7. Research Summary III<br />?<br />O2-<br />X<br />?<br />Man-A<br />CDK2<br />?<br />H2O2<br />CDK2 stimulates H2O2 generator?<br />CDK2 inhibits O2- generator?<br />7<br />
  8. 8. Research Summary IV<br />CDK2 and PP1 antagonism determines cell fate?<br />Cal-A<br />Thr320PP1a<br />O2-<br />X<br />Thr160-CDK2<br />Roscovitine<br />H2O2<br />8<br />
  9. 9. Research Summary IV<br />SOD regulated by CDK2?<br />X<br />Thr320PP1a<br />Cal-A<br />O2-<br />Rosc.<br />Growth<br />Factors<br />Thr160-CDK2<br />SOD<br />H2O2<br />Tonic H2O2 Includes: <br />-Activation of JAK/STATs<br />-Inhibition of caspases<br />-Inhibition of Tyr and Ser/Thr PPase<br />9<br />
  10. 10. P-Akt, P-MEK<br />Working Model Of Man-A Action<br />Man-A<br />Cal-A<br />NAC<br />CDK2<br />PP1a-POFF<br />PP1a ON<br />ROS<br />Akt, MEK<br />Caspases<br />Akt and MEK<br />cleavage<br />Apoptosis, Quiescence etc.<br />10<br />
  11. 11. Central Hypotheses<br />Controlled RedOx changes <br />dephosphorylation of select, critical targets?<br />RedOx change without dephosphorylation<br /> Uqsticking and proteasomaltargeting?<br />(CDK2 major protagonist)<br />Redox damage + dephosphorylation<br /> caspase targeting?<br />PP1 protagonist?<br />11<br />
  12. 12. Future Directions and Questions<br />R01 CA-138994-A1<br />Role of ROS in Signal Protein Targeting During Apoptosis<br />How are MEK and Akt targeted and not ERK (and others)?<br />ROS Sources?<br />Probing Proteins for Thioreactive Sites<br />DMPO and IAA labeling<br />Spin trap and thiol label<br />Western blot, IP and Mass Spec.<br />12<br />
  13. 13. Future Directions: Questions<br />Is Man-A a trigger or participant in process?<br />Spectral analyses will identify Man-A metabolites<br />Proteomics to examine Man-A adduction to proteins<br />IF HAPPENS!!<br />Anti-Man-A Antibody?<br />Or, Conjugate Man-A to agarose or sepharose<br />Purify targets then subject to Mass Spec. Analysis<br />Man-A important if CDK2 inhibitor or PPase activator<br />13<br />
  14. 14. Acknowledgements<br /><ul><li>Ms. Khandra Sears
  15. 15. Ph.D. candidate, U-MD, Dept. Microbiology and Immunology (’10)
  16. 16. Mr. Allan Carey
  17. 17. Humanities Scholar UMBC
  18. 18. Siddiq al Wahhab
  19. 19. MHHS, Ellicott City
  20. 20. Ms. Kateila Miles
  21. 21. Medical School ‘10
  22. 22. Dr. Hanako Daino
  23. 23. Postdoctoral Fellow, Carey lab 06-08
  24. 24. Dr. Mark Williams
  25. 25. Micro. And Immunol
  26. 26. Dr. Jaeyul Kwon
  27. 27. NIH
  28. 28. Mr. Brian Hampton,
  29. 29. Protein Core CVID
  30. 30. Dr. Austin Yang
  31. 31. Proteomic Core, Greenebaum
  32. 32. Dr. Len Neckers
  33. 33. NIH
  34. 34. Dr. Alexandra Newton
  35. 35. UCSD
  36. 36. Dr. Brian Polster
  37. 37. UMB</li></ul>Funding:<br /> NCI<br /> -CA94027 (’01-’07)<br /> -CA12882 (’07-’11)<br /> Dean’s Office<br />-Sm. Intramural grant ‘05-’07)<br />14<br />
  38. 38. Mechanism of Damaged Protein Processing?<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />Tonic ROS? + Kinase<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-Ser473-P<br />-Ser473<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />Ppase<br />And Trx?<br />-<br />-<br />-<br />-<br />-<br />-<br />-Cys460<br />-<br />-<br />-<br />-<br />-<br />-<br />-Cys460-O<br />-<br />-<br />-<br />-<br />ROS<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-Ser473-P<br />-<br />-<br />-<br />Ppases and <br />Caspases<br />-<br />-<br />-<br />-<br />-<br />-<br />-Cys460-O<br />-<br />-O<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />-<br />