Presentation4

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Presentation4

  1. 1. Acute Myeloid Leukemia<br />CXCR4<br />Oxygen partial pressure<br />1<br />
  2. 2. Acute Myeloid Leukemia<br />2<br />
  3. 3. Chemokine and its receptor<br />Receptor<br />Receptor = CXCR4<br />Chemokine<br />Chemokine = SDF1<br />3<br />
  4. 4. CXCR4/SDF-1<br />Stromal cell<br />SDF-1<br />CXCR4<br />4<br />
  5. 5. In vitro vs In vivo?<br />Physiological Oxygen level<br />5<br />O2 level within bone marrow is ~6.8%.<br />
  6. 6. “CXCR4 expression and biologic activity in acute myeloid leukemia are dependent on oxygen partial pressure”<br /> - Michael Fiegi et al; Blood ; 2009<br />6<br />
  7. 7. 1.What is the Oxygen level in bone marrow of AML patients?<br />19 AML patients<br />6.06% (+1.7%)<br /><10 min<br />Physiological level of O2 within bone marrow of AML patients is 6%.<br />7<br />
  8. 8. 2. Does Oxygen level influence CXCR4 expression in AML?<br />AML<br />cell<br />AML<br />cell<br />?<br />Hypoxic stress<br />CXCR4<br />8<br />
  9. 9. Surface expression of CXCR4 increases at 6% 02<br />AML<br />cell<br />AML<br />cell<br />AML<br />cell<br />6% O2<br />21% O2<br />Hypoxic stress<br />CXCR4<br />9<br />
  10. 10. 3. Does hypoxic AML cells migrate better to stromal factor (SDF-1)?<br />10<br />
  11. 11. 3. Does hypoxic AML cells migrate better to stromal factor (SDF-1)?<br />AML<br />cell<br />Hypoxic<br />Stromal cell<br />11<br />
  12. 12. 4.What are downstream signaling effects of CXCR4-SDF1?<br />Downstream signals are constitutively upregulated under “physiological O2 level” of 6%.<br />12<br />
  13. 13. 5.What are the kinetics of CXCR4 regulation?<br />21% 02<br />6% 02<br />AML<br />AML<br />Increased Transcription?<br />CXCR4<br />m RNA<br />13<br />
  14. 14. 5.What are the kinetics of CXCR4 regulation?<br />6% 02<br />21% 02<br />AML<br />AML<br />5 min<br />CXCR4 regulation to O2 levels is NOT regulated transcriptionaly.<br />CXCR4<br />14<br />
  15. 15. Lipid rafts as sites for regulation of protein expression <br />21% 02<br />21% 02<br />6% 02<br />AML<br />AML<br />AML<br />15<br />
  16. 16. The experiment<br />6% 02<br />AML<br />6%->21 02<br />AML<br />21% 02<br />AML<br />16<br />
  17. 17. 21% 02<br />6% 02<br />21% 02<br />AML<br />AML<br />AML<br />Lipid rafts are regulators of chemokine expression.<br />?<br />CXCR4<br />Lipid Raft<br />17<br />
  18. 18. Shedding of CXCR4 as microparticles<br />6% 02<br />6% -> 21% 02<br />Protein Supernatant<br />Cellular component<br />Western Blot of Sup<br />18<br />
  19. 19. Points to consider<br />19<br />21% ->6% -> 21% 6.8% -> 6% ->21%<br />
  20. 20. Summary (1)<br /> “ Always perform your experiment at physiological Oxygen level so that you don’t miss out on the 3P’s.”<br />20<br />Protein Regulation<br />Protein Shedding<br />Publication in Blood<br />
  21. 21. Summary (2)<br />AML<br />cell<br />AML<br />cell<br />6% Oxygen<br />Stromal cell<br />21<br />
  22. 22. 22<br />Subhashis Sarkar<br />PhD student, Tumor Immunology<br />

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