Caspase-9 is the upstream caspase activated by 8-methoxypsoralen and ultraviolet-A radiation treatment of Jurkat T leukemia cells and normal T lymphocytes.
OBJECTIVE
A combination of 8-methoxypsoralen and ultraviolet-A radiation (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however scarce information is available concerning the apoptotic pathways activated by PUVA.


METHODS
We used Jurkat human T leukemia cells and normal T lymphocytes to analyze the PUVA-triggered caspase activation pattern by means of immunoblot analysis, in vitro caspase activity assays, and selective caspase inhibitors coupled to flow cytometric analysis.


RESULTS
PUVA treatment induced activation of apical caspases-9 and -8, and of effector caspases-3 and -7 in Jurkat cells and human T lymphocytes. While activation of caspase-9 occurred as early as 1 h after PUVA treatment of Jurkat cells, procaspase-8 cleavage was delayed and was detected 6 h after the exposure. Also in normal T lymphocytes, cleavage of caspase-8 was subsequent to activation of caspase-9. PUVA-dependent proteolytic cleavage of procaspase-8 was blocked by inhibitors selective for either caspase-9 or -3. Moreover, procaspase-8 was cleaved in vitro by activated caspase-3, which gave rise to proteolytic fragments equivalent to those generated in vivo.


CONCLUSIONS
Activation of caspase-8 in PUVA-treated Jurkat cells and normal T lymphocytes is secondary to up-regulation of caspase-9. Overall, our results identify caspase-9 as the critical upstream caspase initiating apoptosis by PUVA in Jurkat T-cells and human T lymphocytes.
Martelli
Alberto M
AM
Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Sezione di Anatomia, Cell Signaling Laboratory, Università di Bologna, via Irnerio 48, 40126 Bologna, Italy. amartell@biocfarm.unibo.it
Cappellini
Alessandra
A
Tazzari
Pier Luigi
PL
Billi
Anna Maria
AM
Tassi
Cristina
C
Ricci
Francesca
F
Falà
Federica
F
Conte
Roberto
R
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D019169:Jurkat Cells; D015458:Leukemia, T-Cell / Q000473:pathology / Q000628:therapy; D008730:Methoxsalen / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000528:radiation effects; D014467:Ultraviolet Therapy / Q000379:methods*
C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D008730:Methoxsalen