title: Caspase-9 is the upstream caspase activated by 8-methoxypsoralen and ultraviolet-A radiation treatment of Jurkat T leukemia cells and normal T lymphocytes.
abstract: 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.
authors.affiliation: 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
authors.forename: Alberto M
authors.initials: AM
authors.lastname: Martelli
authors.forename: Alessandra
authors.initials: A
authors.lastname: Cappellini
authors.forename: Pier Luigi
authors.initials: PL
authors.lastname: Tazzari
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Billi
authors.forename: Cristina
authors.initials: C
authors.lastname: Tassi
authors.forename: Francesca
authors.initials: F
authors.lastname: Ricci
authors.forename: Federica
authors.initials: F
authors.lastname: Falà
authors.forename: Roberto
authors.initials: R
authors.lastname: Conte
mesh_terms: 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*
chemical_list: C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D008730:Methoxsalen