This laboratory has been involved in studying the interferon (IFN) receptors and the early events in IFN action. Receptors recognized by IFN-alpha and IFN-beta (type I IFN) and IFN- gamma (type II IFN) have been identified and some of their characteristics have been studied. Studies are planned to determine whether the receptors for human IFN (HuIFN)- alpha/beta consist of subunits (or other factors) associated through non-covalent bonds, a possibility suggested by some of our results, by their chromatographic behavior and radiation inactivation analysis to determine their target size, and if indicated, to determine their subunit composition. Role of IFN receptors in differential induction of cellular gene expression by type I and type II IFNs will be studied. We have isolated, from a cDNA library, two cDNA clones that are complementary to mRNAs induced by HuIFN-gamma. One of the cDNA clones is complementary to a 2.2 kb mRNA that is induced very strongly by HuIFN-gamma (- 100-fold) but less so by HuIFN-alpha 2 ( -10- fold). Whereas the induction by HuIFN-gamma is sustained for days, the induction by HuIFN-alpha 2 is transient and is followed by a shut-off, indicating differential regulation by the two types of IFNs. The isolated cDNA clones will be further characterized by restriction mapping and by Southern blotting analysis of genomic DNA to determine whether the corresponding gene exists as a single copy gene or in multiple copies in the genome. The cognate mRNA will be isolated by hybridization selection and translated in vitro to identify the protein product. Nuclear run- off transcription experiments will be carried out to determine whether this induction is a transcriptional effect or a post- transcriptional effect. Such cDNA clones will be used as probes to study whether the differential induction of cellular genes by type I and type II IFNs is due to events at the receptor level or at post-receptor level. For example, we will determine whether the transient induction by HuIFN-alpha 2 and sustained induction by HuIFN-gamma is due to receptor down regulation and recycling, respectively, or due to the appearance of a negative control mechanism in the case of HuIFN-alpha 2 and not with HuIFN- gamma, whether inhibitors of receptor recycling (e.g. monensin) would affect the persistant nature of induction in the case of HuIFN-gamma, and test whether amino acid analogs would prevent the shut-off observed with HuIFN-alpha 2. Finally, we will make attempts to develop a cell-free system which may allow an investigation of the nature of the signal (mediators) generated upon IFN receptor interaction leading to an activation of specific cellular genes.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA029991-11
Application #
3168939
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1987-07-01
Project End
1991-04-30
Budget Start
1990-05-01
Budget End
1991-04-30
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Cancer Prevention Institute
Department
Type
DUNS #
City
Dayton
State
OH
Country
United States
Zip Code
45439
Chon, S Y; Hassanain, H H; Gupta, S L (1996) Cooperative role of interferon regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene. J Biol Chem 271:17247-52
Chon, S Y; Hassanain, H H; Pine, R et al. (1995) Involvement of two regulatory elements in interferon-gamma-regulated expression of human indoleamine 2,3-dioxygenase gene. J Interferon Cytokine Res 15:517-26
Najfeld, V; Menninger, J; Muhleman, D et al. (1993) Localization of indoleamine 2,3-dioxygenase gene (INDO) to chromosome 8p12-->p11 by fluorescent in situ hybridization. Cytogenet Cell Genet 64:231-2
Hassanain, H H; Dai, W; Gupta, S L (1993) Enhanced gel mobility shift assay for DNA-binding factors. Anal Biochem 213:162-7
Burkin, D J; Kimbro, K S; Barr, B L et al. (1993) Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8. Genomics 17:262-3
Hassanain, H H; Chon, S Y; Gupta, S L (1993) Differential regulation of human indoleamine 2,3-dioxygenase gene expression by interferons-gamma and -alpha. Analysis of the regulatory region of the gene and identification of an interferon-gamma-inducible DNA-binding factor. J Biol Chem 268:5077-84
Feng, G S; Dai, W; Gupta, S L et al. (1991) Analysis of interferon-gamma resistant mutants that are possibly defective in their signaling mechanism. Mol Gen Genet 230:91-6
Dai, W; Gupta, S L (1990) Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA. Biochem Biophys Res Commun 168:1-8
Dai, W; Gupta, S L (1990) Regulation of indoleamine 2,3-dioxygenase gene expression in human fibroblasts by interferon-gamma. Upstream control region discriminates between interferon-gamma and interferon-alpha. J Biol Chem 265:19871-7
Gupta, S L (1990) Regulation of cellular gene expression by interferon-gamma: involvement of multiple pathways. Int J Cell Cloning 8 Suppl 1:92-102

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