Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members.
Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.
Gil-Parrado
Shirley
S
Abteilung für Klinische Chemie und Klinische Biochemie, Chirurgische Klinik Innenstadt, Klinikum der LMU München, Nussbaumstrasse 20, D-80336 Münich, Germany. shirgilpa@web.de
Fernández-Montalván
Amaury
A
Assfalg-Machleidt
Irmgard
I
Popp
Oliver
O
Bestvater
Felix
F
Holloschi
Andreas
A
Knoch
Tobias A
TA
Auerswald
Ennes A
EA
Welsh
Katherine
K
Reed
John C
JC
Fritz
Hans
H
Fuentes-Prior
Pablo
P
Spiess
Eberhard
E
Salvesen
Guy S
GS
Machleidt
Werner
W
D017209:Apoptosis; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000494:pharmacology*; D018287:Carcinoma, Large Cell / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology; D008175:Lung Neoplasms / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D007476:Ionophores; D009842:Oligopeptides; D019253:Proto-Oncogene Proteins c-bcl-2; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; D015759:Ionomycin; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D002154:Calpain; D002118:Calcium