The promoter of the human dihydrofolate reductase (DHFR) gene contains two consensus binding sites for the DNA binding protein Sp1. DNAse protection and gel mobility shift assays demonstrate binding of recombinant Sp1 to both decanucleotide Sp1 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 Sp1. The G-C specific DNA binding drug, mithramycin, binds to both consensus sequences and prevents subsequent Sp1 binding. Promoter-dependent in vitro transcription of a 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 of DHFR expression suggests that it is possible to modulate the overexpression of the DHFR gene in methotrexate resistant cells.

During the past decade there have been remarkable strides in the understanding of the basic mechanism of cancer. It is now clear that there is a set of genes, known as oncogenes, that can cause cells to become malignant if their expression is altered, either by mutation or overexpression. The products of these genes include growth factors, growth factor receptors, signal tranduction proteins, and DNA binding proteins. The normal cellular counterparts of these genes play very important roles in the regulation of growth and proliferation by normal cells. Another set of genes, anti-oncogenes, also play an important role in preventing abnormal cell proliferation. The remarkable explosion of understanding of the pathophysiology of malignancy has led to a common unifying concept of malignant transformation that applies to all tumors. It is likely that these new insights will lead to improved and more specific treatments for malignant disease in the next decade.

Specific interactions between DNA and transcription factors are necessary for transcription initiation. These interactions provide a potential target for the selective inhibition of eukaryotic gene expression. Mithramycin is a DNA binding antibiotic which, in the presence of Mg2+, binds G-C containing sequences in the minor groove. The SV40 early promoter contains six G-C decanucleotide sequences, which are binding sites for the transcriptional activating factor, Sp1. Each of the six Sp1 binding sites of this promoter is protected from DNAse 1 digestion by mithramycin binding. Mithramycin binding to the G-C rich sequences in the SV40 early promoter prevents subsequent protein binding to these sequences. The gel retardation of the SV40 early promoter fragment incubated with a HeLa cell extract is completely abrogated by pretreatment of the DNA fragment with mithramycin. The functional significance of mithramycin binding is reflected in the ability of mithramycin to block promoter function. Mithramycin inhibits promoter dependent transcription in an in vitro runoff transcription system in a concentration dependent manner. This suggests that mithramycin prevents transcriptional activation of the SV40 early promoter by blocking binding of transcriptional activating proteins to G-C rich promoter regions.

Buttercup extract (BE), an extract the buttercup plant (Zanthoriza simplicissima), inhibits RNA and DNA synthesis by HL-60 promyelocytic leukemia cells. Exposure of these cells to 3% BE for 48 hours results in dramatic Inhibition of RNA synthesis without loss of cell viability. The effect of BE is partially reversible over 12–24 hours with the level of RNA synthesis returning nearly to control levels during this time period. INST A synthesis is also reversibly inhibited by exposure to BE. Despite the inhibition of RNA synthesis in HL-60 cells, there is no decrease in the level of c-myc mRNA, even at high BE concentrations. The level of gene-specific mRNA for the c-Ha-ras, cfms, and c-mos genes in these cells also remained constant during exposure to BE. Ribosomal RNA is not degraded during 24 hours of BE treatment in vitro, suggesting that BE does not maintain the relative mRNA level for these genes by selective degradation of other RNA species. The inhibition of RNA and DNA synthesis by BE without a corresponding alteration in the level of expression of the C-MYC gene suggests that this agent dissociates C-MYC expression and cellular proliferation in these cells.