Introduction:To elucidate the mechanism of cytotoxicity in FK506-treated Jurkat T cells, signal transduction pathway of TNF-related events was studied. We will further evaluate the roles of TNF-related death receptors and endoplasmic reticulum-related proteins on the death of Jurkat cells after treatment with FK506.
Methods: Viability of Jurkat T cells was measure by MTT assay. The catalytic activation of caspase-3 and caspase-9 proteases were determined by digestion of fluorogenic biosubstrates and Western blot with anti-caspase-3 and anti-caspase-9 antibodies. The levels of mRNA and proteins for p53, Bax, PUMA, Proline oxidase, TRAIL(TNF related apoptosis inducing ligand), TRAIL-R1(DR4), TRAIL-R2(DR5), Fas, FasL, TNF-α, IL-6, and NFκB were measured by RT-PCR and Western blot with specific antibodies. Also we further examined the localization of TRAIL family proteins using by fluorescent microscope with specific TRAIL family antibodies
Results: FK506 decreased the viability of Jurkat T cells concentration- and time-dependently along with catalytic activation of caspase-3 and caspase-9, p53 phosphorylation, and changes in expression levels of Bax, PUMA, and Proline oxidase protein. It caused an increase in expression of TRAIL, TRAIL-R1(DR4), TRAIL-R2(DR5), Fas, and FasL in the levels of mRNA and proteins of Jurkat T cells. Furthermore, FK506 increased extracellular release of TNF-α and IL-6 cytokines in Jurkat T cells. It also induced the transactivation of NFκB through the dephosphrylation of Ser486 residues in Jurkat t cells.
Conclusion: These results suggest that FK506 induces apoptotic death of Jurkat cells through activation of caspase family protease, Bcl2 family protein related mitochondrial dysfunction, activation of death-receptor and endoplasmic reticulum mediated signaling pathways.