Caspase-independent apoptosis is activated by diazepam-induced mitotic failure in HeLa cells, but not in human primary fibroblasts.
DZ, a benzodiazepine known to affect centrosome separation at prophase, leads to a higher degree of mitotic arrest in HeLa cells than in primary human fibroblasts. In fact, differently from fibroblasts, which undergo a transient block in prophase-to-prometaphase transition, a high proportion of tumor cells attempt to escape from the DZ-imposed mitotic block, fail to undergo complete mitosis and die by mitotic failure. DZ-treated samples showed certain biochemical hallmarks of apoptosis, such as induction of the proapototic Bax protein, mitochondrial alterations assessed by JC-1 staining and TEM analysis, PARP cleavage, and DNA fragmentation. However, in DZ-treated cells, we observed a very low or absent caspase activation as shown by immunofluorescence and immunoblot experiments with antibodies directed to activated caspases and by staining with the pancaspase inhibitor FITC-VAD-FMK. Experiments on mitochondrial depolymerization and apoptosis induction carried out in the presence of specific inhibitors of caspase-2 and caspase-3/7 indicated a caspase-independent apoptotic process induced by DZ. Accordingly, TEM analysis of treated cells revealed ultrastructural features resembling those reported for caspase-independent apoptosis. In conclusion, we hypothesize that HeLa cells override the prophase block imposed by DZ, producing a high rate of aberrant pro-metaphases, which, in turn, activates caspase-independent, apoptosis-like mitotic catastrophe.
Vitale
I
I
Department of Biology, University Roma Tre, V.le Marconi 446, Rome, 00146, Italy.
Antoccia
A
A
Crateri
P
P
Leone
S
S
Arancia
G
G
Tanzarella
C
C
D017209:Apoptosis / Q000187:drug effects*; D020169:Caspases / Q000378:metabolism; D002457:Cell Extracts; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D046529:Microscopy, Electron, Transmission; D008938:Mitosis / Q000187:drug effects*; D008940:Mitotic Index; D009687:Nuclear Proteins / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D049468:Prometaphase / Q000187:drug effects; D019311:Staurosporine / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D002457:Cell Extracts; D009687:Nuclear Proteins; D051028:bcl-2-Associated X Protein; D011065:Poly(ADP-ribose) Polymerases; D020169:Caspases; D019311:Staurosporine; D003975:Diazepam; D003078:Colchicine