The relationship between transcription of alpha and beta interferon (IFN-alpha and IFN-beta) genes and the interaction of IFN promoter-binding transcription factors has been examined in monoblastoid U937 cells following priming with recombinant IFN-alpha 2 (rIFN-alpha 2) and Sendai virus induction. Pretreatment of U937 cells with rIFN-alpha 2 prior to Sendai virus infection increased the mRNA levels of IFN-alpha 1, IFN-alpha 2, and IFN-beta as well as the final yield of biologically active IFN. Analysis of nuclear protein-IFN promoter DNA interactions by electrophoretic mobility-shift assays demonstrated increased factor binding to IFN-alpha 1 and IFN-beta regulatory domains, although no new induction-specific complexes were identified. On the basis of competition electrophoretic mobility-shift assay results, factors interacting with the IFN-alpha 1 and IFN-beta promoters appear to be distinct DNA-binding proteins. U937 factor binding was localized to the P2 domain (-64 to -55) of the IFN-beta regulatory element, a sequence motif with 80% homology to the recognition site of transcription factor NF-kappa B. Protein-DNA interactions within the IFN-beta P2 domain were, in fact, specifically competed by either excess homologous P2 fragment or the human immunodeficiency virus enhancer element which contains two duplicated NF-kappa B recognition sites. Hybrid promoter-chloramphenicol acetyltransferase fusion plasmids, containing either the IFN-beta regulatory element or the human immunodeficiency virus enhancer element linked to the simian virus 40 promoter, were analyzed for virus and phorbol ester inducibility in epithelial and lymphoid cells, respectively. In the 293 cell line, both plasmids were constitutively expressed but not virus inducible, while in Jurkat cells, chloramphenicol acetyltransferase activity from these plasmids was induced by tumor-promoting agent treatment. These experiments suggest that induction of IFN gene expression may be controlled in part by transcription regulatory proteins binding to an NF-kappa B-like site within the IFN-beta promoter.

A human transient expression assay was used to examine the inducible transcriptional activation of beta interferon (IFN-beta) and IFN-alpha 1 promoters in a homologous cellular environment. Use of 293 cells, an adenovirus DNA-transformed human embryonic kidney cell line, permitted Sendai virus-inducible expression of IFN-beta-CAT hybrid gene. Introduction of the simian virus 40 (SV40) enhancer 5' or 3' to the IFN-CAT gene increased basal (uninduced) levels of chloramphenicol acetyltransferase (CAT) activity; in one construct the SV40 enhancer--IFN-beta regulatory region combination increased the induced CAT activity 50- to 100-fold, suggesting that this may be a generally useful inducible enhancer-promoter combination. No expression from the IFN-alpha-CAT hybrid gene was detected in 293 cells, indicating that human epithelioid cells lack a factor required for expression of the IFN-alpha promoter. However, when the IFN-alpha regulatory region was combined with the SV40 enhancer, a low level of inducible CAT activity was detected in the human transient system.

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus Ela proteins on the activation of alpha interferon subtype 1 (IFN-a,) and IFN-1 promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-, promoter 5-to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-ae promoter in T-antigen-expressing cells displayed a level of inducibility similar to (8,15,20,33,37). The IFN-P regulatory sequences between positions -77 and -37 relative to the mRNA CAP site comprise an inducible enhancer element, with overlapping positive and negative regulatory domains (13-15); the 5' boundary of sequences required for induction may however vary depending on the cell type (8,15). A hexanucleotide repeat (consensus AAGTGA), permutations of which are present throughout the -65 to -109 region of the IFN-P promoter, has been shown to confer virus-induced transcriptional activation to homologous or heterologous promoters (9). Similarly, an IFN-a1 promoter fragment from positions -109 to -64 conferred virus inducibility to a rabbit P-globin promoter segment (39); in addition, a GAAAGPy hexameric repeat possessed the ability to suppress the activity of constitutive enhancer elements (27). tions -68 and -38 and positions -167 to -94. After virus induction, the interferon-regulatory element-binding proteins were at least partially dissociated and replaced by a factor binding to sequences located between positions -77 and -64 (51). Similarly, a gel electrophoresis DNA-binding assay identified an IFN-specific protein in uninduced myeloid KG-1 cells interacting with the upstream region of the IFN-,B promoter (50).We previously described a human transient expression system that accurately reflected the virus-induced activation of the IFN-P promoter (49). Expression of IFN-,-Bchloramphenicol acetyltransferase (CAT) hybrid genes was dependent on induction by virus; however, no expression from the IFN-a-CAT hybrid gene was detectable, indicating that human epithelioid cells lack a factor required for the expression of the IFN-a promoter. In the present study, we demonstrate that both the IFN-P and the previously silent

A human transient expression assay was used to examine the inducible transcriptional activation of beta interferon (IFN-beta) and IFN-alpha 1 promoters in a homologous cellular environment. Use of 293 cells, an adenovirus DNA-transformed human embryonic kidney cell line, permitted Sendai virus-inducible expression of IFN-beta-CAT hybrid gene. Introduction of the simian virus 40 (SV40) enhancer 5' or 3' to the IFN-CAT gene increased basal (uninduced) levels of chloramphenicol acetyltransferase (CAT) activity; in one construct the SV40 enhancer--IFN-beta regulatory region combination increased the induced CAT activity 50- to 100-fold, suggesting that this may be a generally useful inducible enhancer-promoter combination. No expression from the IFN-alpha-CAT hybrid gene was detected in 293 cells, indicating that human epithelioid cells lack a factor required for expression of the IFN-alpha promoter. However, when the IFN-alpha regulatory region was combined with the SV40 enhancer, a low level of inducible CAT activity was detected in the human transient system.

To address the mechanisms that regulate expression of specific growth-related nuclear proto-oncogenes, the transcript levels of the c-fos, c-myc, (2'5')-oligoadenylate synthetase, IFN-alpha 1, and IFN-beta 1 genes have been measured in the human leukemic cell lines KG-1, U937, and HL-60 following growth stimulation by serum, induction of differentiation by tumor-promoting agents, and/or treatment of cells with exogenously supplied alpha interferon (rIFN-alpha 2). Production of fos and myc RNA was measured by S1 mapping, using fos DNA probes which identified either primary unspliced transcripts or steady-state-spliced mRNA levels, and using a myc probe which spanned the two major c-myc start sites, P1 and P2. Pretreatment of a quiescent KG-1 cell population with IFN for 18 hours before serum addition decreased the stimulation of both fos and myc RNA production. In HL-60 and U937 cells, IFN pretreatment had no inhibitory effect on serum-induced fos or myc transcription; however, in U937, rIFN-alpha 2 treatment alone stimulated fos mRNA 11-fold. Expression of 2'5'oligoadenylate synthetase was induced in IFN-treated cultures but not in cells stimulated with serum alone. No serum-induced IFN-alpha 1 or IFN-beta 1 gene expression was observed in KG-1 or U937 cells. These results demonstrate that exogenous rIFN-alpha 2 treatment of quiescent KG-1 cells can antagonize the effect of growth factors by altering expression of nuclear proto-oncogenes, but in general growth inhibition is not obligatorily coupled to inhibition of proto-oncogene transcription.