Analysis of transcription factors regulating induction of indoleamine 2,3-dioxygenase by IFN-gamma

IFN-gamma treatment of the human carcinoma cell line ME180 causes cell deat h due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starv ation for tryptophan. A mutant cell line 3B6A derived from ME180 was resist ant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO pro moter-chloramphenicol acetyl transferase (CAT) construct with IFN regulator y factor-1 (IRF-1) resulted in induction of CAT activity in both ME180 and 3B6A cells even in the absence of IFN-gamma This induction was reduced by c otransfection with IRF-2, However, IRF-1 was not able to restore IDO activi ty, suggesting a possible repressor site outside the IDO promoter region. S tat1 alpha (p91) restored both CAT and IDO activities in 3B6A cells followi ng IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-al pha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO, Western blot analysis showed that both constitutive expr ession and induction of Stat1 alpha by IFN-gamma were reduced in 3B6A cells , and double treatment of IFN-gamma with IFN-alpha or IFN-beta restored the expression level of Stat1 alpha, Electrophoretic mobility shift assays ind icated that Stat1 binds to the IFN-gamma-activated sequence (GAS) region in the IDO promoter in ME180 cells following IFN-gamma treatment. Our results indicated that the defect in 3B6A cells was reduced expression of Stat1 al pha and that IRF-1, NF-kappa B, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.