Mithramycin Inhibits SP 1 Binding and Selectively Inhibits Transcriptional Activity of thq Dihydrofolate Reductase Gene In Vitro and In Vivo

The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Spl. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Spl to both decanucleotide Spl binding sequences which are located 49 and 14 base pairs upstream of the transcription start site. The more distal of the two binding sites exhibits a somewhat higher affinity for Spl. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Spl binding. Promoter-dependent in vitro transcription ofa DHFR template is selectively inhibited by mithramycin when compared to the human H2b histone gene. A similar effect is also noted in vivo. Mithramycin treatment of MCF-7 human breast carcinoma cells containing an amplified DHFR gene induces selective inhibition of DHFR transcription initiation, resulting in a decline in DHFR mRNA level and enzyme activity. This selective inhibition ofDHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells. (J. Clin. Invest. 1991. 88:1613-1621.)