RESUMEN
We have previously reported the isolation of mutant cell lines from the human carcinoma line ME180 that are resistant to the antiproliferative effect of interferon-gamma (IFN-gamma). These cell lines were defective in the induction of indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan catabolism. One of these cell lines, 3B6A, was chosen for further study. This cell line was also defective in the ability of IFN-gamma to protect against vesicular stomatitis virus (VSV) infection. However it maintained a normal antiviral response to IFN-alpha. A promoter-chloramphenicol acetyltransferase (CAT) construct containing the promoter region of IDO, which includes IFN-gamma activation site (GAS), IFN-stimulated response element-1 (ISRE-1), and ISRE-2 regions, was not expressed in 3B6A in the presence of IFN-gamma, indicating that the defect was likely to be in either Stat1 or IFN regulatory factor-1 (IRF-1), transcription factors known to bind to these cis-acting sequences. The induction of other IFN-gamma-inducible genes, such as tryptophanyl-tRNA synthetase (hWRS), was also affected. Electrophoretic mobility shift assays (EMSA) comparing nuclear extracts from parental and mutant cells indicated that Stat1 from the mutant did not bind to GAS sequences. However, Western blot analysis indicated that Stat1 protein was present. This IDO-negative phenotype can be reversed by transfection with a Stat1 expression vector. DNA sequencing of the Stat1 cDNA from wild-type and 3B6A cells indicated that an amino acid change occurred in the Stat1 protein of the mutant at W573, a tryptophan conserved in all known Stat proteins. We hypothesize that a change in this region of the Stat protein affects the response to IFN-gamma but not to IFN-alpha.
Asunto(s)
Proteínas de Unión al ADN/genética , Interferón-alfa/farmacología , Interferón gamma/farmacología , Transactivadores/genética , Triptófano Oxigenasa/genética , Virus de la Estomatitis Vesicular Indiana/fisiología , Cloranfenicol O-Acetiltransferasa/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Múltiples Medicamentos , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa , Interferón alfa-2 , Mutación , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Factor de Transcripción STAT1 , Transactivadores/metabolismo , Transfección , Triptófano Oxigenasa/deficiencia , Células Tumorales Cultivadas , Neoplasias del Cuello UterinoRESUMEN
IFN-gamma treatment of the human carcinoma cell line ME180 causes cell death due to induction of indoleamine 2,3-dioxygenase (IDO) and resulting starvation for tryptophan. A mutant cell line 3B6A derived from ME180 was resistant to IFN-gamma because of loss of IDO activity. Cotransfecting an IDO promoter-chloramphenicol acetyl transferase (CAT) construct with IFN regulatory 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 cotransfection with IRF-2. However, IRF-1 was not able to restore IDO activity, suggesting a possible repressor site outside the IDO promoter region. Stat1alpha (p91) restored both CAT and IDO activities in 3B6A cells following IFN-gamma treatment. 3B6A cells doubly treated with IFN-gamma and IFN-alpha or IFN-beta restored IDO activity, although neither cytokine on its own could induce IDO. Western blot analysis showed that both constitutive expression and induction of Stat1alpha 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 Statla. Electrophoretic mobility shift assays indicated 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 Stat1alpha and that IRF-1, NF-kappaB, and PKR were all involved to some extent in the induction of IDO following IFN-gamma treatment.
