IMMUNOSUPPRESSION - PRE-CLINICAL AGENTS

S.Y. Chung, H.K. Lee, W.Y. Shin, S.J.N. Choi
Department Of Surgery, Chonnam National University Hospital, Gwangju/KOREA

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Introduction Two major apoptotic pathways are dead receptor pathway and mitochondrial pathway. And third apoptotic pathway is endoplasmic reticulum(ER) mediated apoptosis. Methods We examined the effects of FK506 on ER mediated apoptosis of Jurkat cells. We observed cell viability, measurement of H₂O₂ generation, intracellular accumulations of Ca²⁺ and NO, and western blottings of ER stress mediated apoptotic pathway proteins, such as phospho-PERK, PERK, CHOP, Grp78, Grp94, Bcl-2, and Bak proteins. Mitochondrial membrane potential determination by JC-1 staining was performed. Cells were cultured with the presence or absence of FK506. Flow cytometric analysis was performed after PI stain. Results Viability of Jurkat cells were decreased by the addition of FK506 in a dose-dependent manner. FK506 induced cytotoxicity was characterized by sub G0/G1 phase arrest. FK506 induced cell death was confirmed as apoptosis characterized by nuclear fragmentation and caspase-3 protease activation. Intracellular accumulations of Ca²⁺ and NO production were identified in FK506 treated Jurkat cells after 24 hours. Expression of iNOS protein was also noted. Generation of H₂O₂ was identified. Deceased activation of procaspase-12 protease confirmed activation of caspase-12 after 48 hours. Activation of phospho-PERK protein peaked at 36 hours after FK506 treatment. Expressions of CHOP/GADD153, Grp78 and Grp94/BiP proteins were also identified after 36 hours. Expression of Bak protein was also noted. Change of mitochondrial membrane potential transition (MPT) was also noted. Conclusion FK506 treated Jurkat T cells increased the sub G0/G1 phase, nuclear fragmentation and activations of 3 and 12 caspases. FK506 also increased NO production through induction of iNOS and H₂O₂. Reactive oxygen species induced by FK506 resulted in the modulation of Bak protein expression and mitochondrial dysfunction through ER stress mediated pathway. FK506 induced ER mediated apoptosis in Juekat T cells were identified.

Disclosure: All authors have declared no conflicts of interest.