Abstract
Selected clones of Syrian hamster DDT1-MF2 cells are responsive to testosterone for growth. Heparin binding growth factor 1 (HBGF-1) or acidic fibroblast growth factor (aFGF) can replace testosterone (T) in the stimulation of growth in these cells. This phenomena is correlated with testosterone's ability to elevate aFGF mRNA two- to threefold in DDT1 cells. To better understand the possible mechanisms of regulation of aFGF mRNA by steroids and other growth factors, we isolated the aFGF 5′ non-coding exon and its flanking region from a EMBL3 DDT1 genomic library, using a 5′ non-coding exon 69 bp DDT1 aFGF cDNA probe. Clones spanning 30 kb of genomic DNA were isolated. After restriction mapping and DNA sequence analysis, the clones were shown to contain all of the 5′ non-coding exon included in the cDNA and approximately 10 kb of 5′ flanking region. RNase protection and primer extension assays confirmed that the 5′ non-coding exon is included in the DDT1 aFGF mRNA and that a major transcription start site is approximately 136 bp upstream of the 5′ non-coding splice junction of this exon. The 5′ flanking region DNA was inserted into pBLCAT3 reporter gene and transfected into DDT1 cells. Chloramphenicol acetyltransferase (CAT) assays demonstrated that there are promoter elements in the −1645/−392 and −392/+131 regions of the aFGF gene in the context of DDT1 cells. NIH 3T3 cells, on the other hand, show no CAT activity with these aFGF-CAT plasmids. CAT assays also demonstrated that addition of testosterone (T) or aFGF to DDT1 cells increased CAT activity threefold. This activity was mapped to −1645 to −4 bp region of this DDT1 aFGF gene promoter.