1. ROLE OF INTRACELLULAR CALCIUM IN GLUCOCORTICOID-EVOKED LYMPHOID CELL APOPTOSIS Devin Morris California State University, Northridge

2. Apoptosis Release of cyctochrome c Effector Caspases Apoptosis Stimuli/ Death signals Apoptosome formation Activation of caspase cascade Apaf-1 Caspase 9

3. Proliferation Cell Population Cell Death Development Homeostasis Disease Physiological roles of apoptosis

4. R IPC ABC IPC R R RR RR ligand receptor interacting proteins coactivator complex basal trans. factors 1 2 3 4 Glucocorticoid Pathway

5. Susceptibility of CEM Cell Clones to GC-Evoked Apoptosis

6. GC-induced Increase in Intracellular Calcium in GC-Susceptible CEM cells

7. Time- & GC-Dose-Dependent Increase in [Ca +2 ] i in CEM-C7-14 Cells EtOH100 nM Dex 1  M Dex 24 hours 48 hours 4.4%6%14.1% 5%24.8%62.1% ABC DEF

8. Modulators of [Ca +2 ] i Levels Influence GC-Evoked Death of CEM Cells

9. EGTA Suppresses GC-Evoked Increase in [Ca +2 ] i Levels in CEM-C7-14 Cells EtOH100 nM Dex 1  M Dex Ethanol 20  M EGTA ABC DEF 4.4%16.4%35% 2%12.4%23.1%

10. Glucocorticoid Signaling Pathway Pathway Inhibitors Normal Pathway

11. Inhibition of Calmodulin Protects CEM-C7-14 Cells from GC-evoked Death

12. Inhibition of Calmodulin Kinase II Protects CEM- C7-14 Cells from GC-evoked Death

13. Inhibition of Calcineurin Protects CEM-C7-14 Cells from GC-evoked Death

14. Conclusions GCs increase [Ca +2 ] i levels only in the GC-susceptible CEM-C7-14 cell line in a dose dependent manner; not in the GC-resistant sister cell line, CEM-C1-15. Calcium chelation by either BAPTA or EGTA protected CEM-C7-14 cells from GC-evoked apoptosis, in conjunction with a reduction in the amount of free [Ca +2 ] i. The calcium ionophore A23187 causes sensitization of CEM-C1-15 cells to GC-evoked apoptosis. Inhibition of calmodulin, calmodulin kinase II or calcineurin, all intermediates in the calcium signaling pathway, impart varying degrees of protection to CEM-C7-14 cells from GC-evoked apoptosis. Our data demonstrate a clear correlation between calcium signaling and GC-evoked apoptosis

15. Future Goals Further studies will aim to understand Ca +2 -dependent changes in gene regulation that contribute to apoptosis. Candidate genes such as the transcriptional repressor E4BP4, and its downstream targets are being studied. Our ultimate goal is to understand the molecular pathway for apoptosis in T-lymphoid cells as well as in other physiologically relevant models for apoptosis, such as osteoblasts, keratinocytes and macrophages.

16. Acknowledgements Funded by grants from the NIH MBRS-SCORE Program, the CSUN Office of Graduate Studies, Research and International Programs, and the CSUN College of Science & Mathematics. Dr. Rheem Medh Saul Priceman Dr. Carol Shubin NASA CSUN/JPL Pair Program