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J. Biol. Chem., Vol. 266, Issue 24, 15850-15854, Aug, 1991
F Borellini, YF He, A Aquino, G Yu, SF Josephs and RI Glazer
Myeloblast cell line K562, when stably transfected with the human genomic
c-fes sequence encoding a proto-oncogene tyrosine-protein kinase, acquires
the characteristics of more mature granulocytic cells (WS-1 cells) and the
ability to undergo differentiation (Yu, G., Smithgall, T. E., and Glazer,
R. I. (1989) J. Biol. Chem. 264, 10276- 10281). To explore the role of
transcription factors in the differentiation process, WS-1 cells were
analyzed for the presence of DNA-binding proteins capable of interacting
with the 5'-long terminal repeat (LTR) region of human immunodeficiency
virus (HIV)-1, that contains the binding sequences for transcription
factors Sp1 and NFKB. Southwestern blotting and mobility shift assays
revealed the presence of Sp1 in K562 and WS-1 cells. The DNA-binding
activity of Sp1 was significantly greater in WS-1 cells than in K562 cells,
despite the detection by immuno-blotting of equivalent quantities and
degrees of heterogeneity of Sp1 in both cell lines. DNA footprinting of the
HIV-1 5'-LTR demonstrated that two of the three Sp1-binding sites and both
NFKB binding sequences were protected by nuclear extracts from WS-1 cells,
while no protection was afforded by nuclear extracts from K562 cells.
Analysis of transcription in vitro by primer extension revealed enhanced
initiation of transcription from the HIV-1 5'-LTR by nuclear extracts from
WS-1 cells, but not from K562 cells. These data indicate that the response
evoked by the c-fes tyrosine-protein kinase leads to enhanced DNA binding
activity of Sp1 and NFKB, that results in the activation of transcription
from the HIV-1 5'-LTR.
Increased DNA binding and transcriptional activity associated with transcription factor Sp1 in K562 cells transfected with the myeloid- specific c-fes tyrosine kinase gene
Department of Pharmacology, Georgetown University Medical Center, Washington, D.C. 20007.
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