Enhancement of stress-induced apoptosis in B-lineage cells by caspase-9 inhibitor

1. Enhancement of stress-induced apoptosis in B-lineage cells by caspase-9 inhibitor Nisha Shah et al., BLOOD.2004;104:2873-2878 生科四甲 王彥閔 91390063

2. Introduction • B-lineage (BLIN) Acute lymphoblastic leukemia (ALL) cell line : BLIN-3, BLIN-4L • Caspase-9 inhibitor (C9i) • Pan-caspase inhibitor (PCi) • Mitochondria transmembrane potential (Δψm ) • TMRE : 螢光染劑，偵測粒線體膜電位下降 • YO-PRO :螢光染劑，偵測細胞滲透力改變

3. Propidium Iodide (PI): 染劑，染凋亡晚期細胞 • Annexin V (AV) : 染劑，與細胞內膜的 Phosphatidylserine (PS)結合，主要染早期凋亡的細胞 • BID : 促使細胞凋亡的蛋白 • PARP : caspase substrate • Staurosporine (STSP) : apoptosis inducer • Etoposide (ETOP): apoptosis inducer

4. Apoptosis process • 接受凋亡信號 • 凋亡調控分子間的交互作用 • 蛋白水解酶(caspase)的活化 • 細胞凋亡的連鎖反應 (ex. Substrate cleavage)

5. Caspase 的分類 • Initiator caspase : Caspase8、Caspase2、Caspase 9、Caspase10 • Executioner caspase : Caspase3、Caspase6、Caspase7 Initiator executioner substrate cleavage DNA fragmentation apoptosis

6. initiator executioner initiator ex. PARP Apoptosis Two main pathways : The extrinsic pathway: cell surface death receptors such as CD95/Fas. The intrinsic or mitochondrial pathway : stress, genotoxic agents, or cytokine deprivation.

7. Results Figure 1. C9i enhances apoptosis within 4 hours following removal of BLIN-3 and BLIN-4L cells from fibroblast monolayers.

8. Figure 2. C9i induces changes in cell permeability and loss of Δψm in a dose-dependent manner. Result: reflects a concomitant change in cell membrane permeability and loss of Δψm.

9. Figure 3. PARP cleavage occurs following treatment of BLIN-3 and BLIN-4L with C9i. Result: The degree of PARP cleavage in the presence of C9i (greater in BLIN-3 cells than BLIN-4L cells) correlated with the percentage of annexin V cells.

10. Figure 4. BLIN leukemic cells are sensitive to C9i independent of culture conditions. Result:This result suggested that C9i could also promote apoptosis in nonstressed, healthy cells.

11. Figure 5. Leukemic cell lines vary in their sensitivity to C9i. Result: the leukemic cell lines were generally responsive to both C9i and staurosporine, or responsive to neither.

12. 71% 78 % 86% 91% Figure 6. Reduction of caspase-9 in BLIN-4L.

13. Figure 7. Caspase-9–deficient BLIN-4L cells show increased apoptotic sensitivity to staurosporine. Result: It is noteworthy that caspase-9–deficient cells incubated in medium alone also underwent a significantly greater degree of apoptosis than control siRNA-treated cells.

14. Discussion • Enhancement of apoptosis was unique to treatment with C9i. • C9i is not simply promoting stress-induced apoptosis. • It is conceivable that loss of caspase-9 triggers a compensatory pathway involving another caspase (eg, caspase-2).

15. Conclusion • The sensitivity of B-lineage ALL cell lines and some other leukemia/lymphoma cell lines to C9i alone suggests that it transduces a proapoptotic signal in some cells. • The apoptotic enhancement induced by C9i and/or reduction in caspase-9 could form the foundation for a new approach to enhancing apoptosis in leukemic lymphoid cells.

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