5. In vitro vs In vivo? Physiological Oxygen level 5 O2 level within bone marrow is ~6.8%.
6. “CXCR4 expression and biologic activity in acute myeloid leukemia are dependent on oxygen partial pressure” - Michael Fiegi et al; Blood ; 2009 6
7. 1.What is the Oxygen level in bone marrow of AML patients? 19 AML patients 6.06% (+1.7%) <10 min Physiological level of O2 within bone marrow of AML patients is 6%. 7
8. 2. Does Oxygen level influence CXCR4 expression in AML? AML cell AML cell ? Hypoxic stress CXCR4 8
9. Surface expression of CXCR4 increases at 6% 02 AML cell AML cell AML cell 6% O2 21% O2 Hypoxic stress CXCR4 9
10. 3. Does hypoxic AML cells migrate better to stromal factor (SDF-1)? 10
11. 3. Does hypoxic AML cells migrate better to stromal factor (SDF-1)? AML cell Hypoxic Stromal cell 11
12. 4.What are downstream signaling effects of CXCR4-SDF1? Downstream signals are constitutively upregulated under “physiological O2 level” of 6%. 12
13. 5.What are the kinetics of CXCR4 regulation? 21% 02 6% 02 AML AML Increased Transcription? CXCR4 m RNA 13
14. 5.What are the kinetics of CXCR4 regulation? 6% 02 21% 02 AML AML 5 min CXCR4 regulation to O2 levels is NOT regulated transcriptionaly. CXCR4 14
15. Lipid rafts as sites for regulation of protein expression 21% 02 21% 02 6% 02 AML AML AML 15
20. Summary (1) “ Always perform your experiment at physiological Oxygen level so that you don’t miss out on the 3P’s.” 20 Protein Regulation Protein Shedding Publication in Blood