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

IntroductionIntroduction B-lineage (BLIN) Acute lymphoblastic leB-lineage (BLIN) Acute lymphoblastic leukemia (ALL) cell line : BLIN-3, BLIN-4ukemia (ALL) cell line : BLIN-3, BLIN-4LL

Caspase-9 inhibitor (C9i) Caspase-9 inhibitor (C9i) Pan-caspase inhibitor (PCi)Pan-caspase inhibitor (PCi) Mitochondria transmembrane potential Mitochondria transmembrane potential (Δψm )(Δψm )

TMRE : TMRE : 螢光染劑，偵測粒線體膜電位下降螢光染劑，偵測粒線體膜電位下降 YO-PRO :YO-PRO : 螢光染劑，偵測細胞滲透力改變螢光染劑，偵測細胞滲透力改變

Propidium Iodide (PI): Propidium Iodide (PI): 染劑，染劑，染凋亡晚染凋亡晚期細胞期細胞

Annexin V (AV) : Annexin V (AV) : 染劑，染劑，與細胞內膜的 與細胞內膜的 Phosphatidylserine (PS) 結合，主要染早期結合，主要染早期凋亡的細胞凋亡的細胞

BID : BID : 促使細胞凋亡的蛋白促使細胞凋亡的蛋白 PARP : caspase substrate PARP : caspase substrate Staurosporine (STSP) : apoptosis induStaurosporine (STSP) : apoptosis inducercer

Etoposide (ETOP): apoptosis inducerEtoposide (ETOP): apoptosis inducer

接受凋亡信號接受凋亡信號

凋亡調控分子間的交互作用凋亡調控分子間的交互作用

蛋白水解蛋白水解酶酶 (caspase)(caspase) 的活化的活化

細胞凋亡的連鎖反應 細胞凋亡的連鎖反應 (ex. Substrate cleavage)(ex. Substrate cleavage)

Apoptosis process

Caspase Caspase 的分類的分類 Initiator caspase : Initiator caspase : Caspase8Caspase8 、、

Caspase2Caspase2 、、 Caspase Caspase 99 、、 Caspase10Caspase10

Executioner caspase : Caspase3Executioner caspase : Caspase3 、、 CasCaspase6pase6 、、 Caspase7Caspase7

Initiator executioner substrate cleavage DNA fragmentation apoptosis

ex. PARPinitiator

initiator

executioner

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.

ResultsResults

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

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

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.

Figure 4. BLIN leukemic cells are sensitive to C9i independent of culture conditions.

Result:This result suggestedthat C9i could also promote apoptosis in nonstressed,healthy cells.

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

Figure 6. Reduction of caspase-9 in BLIN-4L.

71%

78 %

86%

91%

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.

DiscussionDiscussion

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).

ConclusionConclusion 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.

THE ENDTHE END