Interleukin-3 (IL-3) is a colony-stimulating factor which supports the growth land development of hematopoietic cells. Its expression is limited to activated but not resting T lymphocytes, NK cells, mast cells, and thymic epithelial cells. We propose to continue the characterization of the expression and repression in HTLV-uninfected and -infected T-cells and non T-cells by molecular biology techniques. Those sequences important for IL-3 expression will be determined by transient transfection of IL-3 promoter-reporter gene constructs in HTLV-uninfected and infected T cells and in non-T-cell lines. IL-3 promoter constructs will consist !of 5' upstream sequences subcloned in front of the CAT reporter gene, and oligonucleotide site-directed mutants of promoter sequences of these constructs. Enhancer and repressor sequences will be identified by subcloning suspected regulatory regions into a vector containing a heterologous promoter linked to a ,reporter gene. To look for promoter-protein interactions electrophoretic mobility shift assays (EMSA) will be performed using nuclear extracts from T cells, unstimulated and stimulated T cells, HTLV-infected T cells and non-T cells. To differentiate the protein species by molecular weight, UV cross-linking will be performed on retarded bands followed by SDS-PAGE analysis. In order to obtain more 'purified protein fractions, column chromatography using heparin-agarose or oligonucleotide affinity columns will be used, and these fractions will be tested for activity by EMSA. We will continue to characterize IL-3 expression in T cells activated by elevated intracellular cyclic AMP. We have previously shown that increases in intracellular cyclic AMP induce expression of IL-3 in T cells. Using the cAMP analogue, 8-Br-cAMP, dibutyryl cAMP as well as the adenylate cyclase activators PGE(2) and forskolin a dose response curve and a time course of cyclic AMP will on T cells will be developed. IL-3 promoter-reporter gene constructs will be transfected into T cells and stimulated with the cAMP analogue to map the cyclic AMP responsive regions of the IL-3 promoter. In this manner we will characterize the expression of IL-3 in cells in response to conventional T-cell activators and to increases in the intracellular second messenger, cyclic AMP.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08HL002779-02
Application #
3083192
Study Section
Research Training Review Committee (RTR)
Project Start
1992-07-01
Project End
1994-01-01
Budget Start
1993-07-01
Budget End
1994-01-01
Support Year
2
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095