Caspase-dependent cleavage of 170-kDa P-glycoprotein during apoptosis of human T-lymphoblastoid CEM cells.
Multidrug resistance (MDR) mediated by the drug efflux protein, 170-kDa P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape cell death induced by chemotherapeutic drugs. Moreover, evidence suggests that cell lines expressing high levels of 170-kDa P-gp are less sensitive to caspase-mediated apoptosis induced by a wide range of death stimuli, including Fas ligand, tumor necrosis factor, and ultraviolet irradiation. However, the fate of 170-kDa P-gp during apoptosis is unknown. In this study, we demonstrate for the first time that 170-kDa P-gp is cleaved during apoptosis of VBL100 human T-lymphoblastoid CEM cells. Apoptotic cell death was induced by LY294002 (a pharmacological inhibitor of the phosphoinositide 3-kinase/Akt survival pathway), H2O2, and Z-LEHD-FMK (a caspase-9 inhibitor which has been recently reported to induce apoptosis in CEM cells). Using an antibody to a common epitope present in both the third and the sixth extracellular loop of P-gp, two cleavage products were detected, with an apparent molecular weight of 80 and 85 kDa. DEVD-FMK (a caspase-3 inhibitor), but not VEID-CHO (a caspase-6 inhibitor), blocked 170-kDa P-gp cleavage. Recombinant caspase-3 was able to cleave in vitro 170-kDa P-gp yielding two fragments of equal size to those generated in vivo. Considering the size of the cleaved fragments and their reactivity with antibodies, which recognize either the N-half or the C-half region of the protein, it is conceivable that the cleavage occurs intracytoplasmically. Since 170-kDa P-gp has been reported to counteract apoptosis, its cleavage may be a mechanism aimed at blocking an important cell survival component.
Mantovani
Irina
I
Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Cell Signalling Laboratory, Università di Bologna, via Irnerio 48, Bologna, Italy.
Cappellini
Alessandra
A
Tazzari
Pier Luigi
PL
Papa
Veronica
V
Cocco
Lucio
L
Martelli
Alberto M
AM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000276:immunology / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology; D006801:Humans; D008970:Molecular Weight; D009025:Morpholines / Q000494:pharmacology; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002867:Chromones; D000939:Epitopes; D009025:Morpholines; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases