The aureolic acid antitumor antibiotic mithramycin (MTM) inhibits both cancer growth and bone resorption by cross-linking GC-rich DNA, thus blocking binding of Sp-family transcription factors to gene regulatory elements. Transcription of c-src, a gene implicated in many human cancers and required for osteoclast-dependent bone resorption, is regulated by the binding of Sp factors to specific elements in its promoter. Therefore, this gene represents an important anticancer target and a potential lead target through which MTM displays its so far uncharacterized action against osteoclastic bone resorption. Here we demonstrate, using DNA binding studies, promoter reporter assays, and RT-PCR, that MTM inhibits Sp binding to the c-src promoter region, thereby decreasing its expression in human cancer cells. Furthermore, selected mithramycin analogues, namely, premithramycin B, mithramycin SK, 7-demethylmithramycin, 4E-ketomithramycin, and 4C-ketodemycarosylmithramycin, generated through combinatorial biosynthesis, were compared with MTM for their ability to block Sp binding to the c-src promoter. Although most of the tested compounds lost their ability to bind to the DNA, alteration of the MTM 3-pentyl side chain led to a compound (mithramycin SK) with the same DNA binding specificity but with lower binding affinity than MTM. While this compound was comparable to MTM in promoter reporter, gene expression, and anticancer assays, given its weaker interaction with the DNA, it may be much less toxic than MTM. The results presented here supplement recent findings and, moreover, allow new conclusions to be made regarding both the structure-activity relationships, particularly with respect to the alkyl side chains, and the mechanism of action of aureolic acid drugs.
Production of the ADP-ribosylating enzyme exoenzyme S (ExoS) by Pseudomonas aeruginosa has been associated with increased virulence. Previous studies, however, have been unable to confirm an effect of soluble ExoS in cell culture or animal model systems. To determine if bacteria must come in contact with target cells in order for an effect of ExoS to be observed, coculture systems were developed to compare the effects of ExoSand non-ExoS-producing bacteria on eukaryotic cell function. The two P. aeruginosa strains used in these studies, 388 and 388⌬exoS, maintained genetic identity, with the exception that strain 388⌬exoS lacked production of the 49-kDa form of ExoS. When bacteria were cocultured with Detroit 532 fibroblastic cells, ExoS-producing 388 bacteria caused a significant decrease in DNA synthesis and viability compared to the decrease caused by non-ExoS-producing 388⌬exoS bacteria. Maximal differences between the two strains were observed when 10 4 to 10 7 CFU of bacteria/ml were cocultured with Detroit cells for 4 or 6 h. Both strains were effective in eliminating Detroit cell DNA synthesis after a 20-h coculture period. Secreted ExoS had no effect on Detroit cell growth and viability, indicating that bacteria must have contact with target cells for the effect of ExoS on cellular function to be observed. Similar effects on cell proliferation and viability were observed when the two strains were cocultured with the KB epithelioid cell line. ExoS-associated decreases in eukaryotic cell viability were not found to be mediated by an inhibition of protein synthesis. These studies confirm that the 49-kDa ExoS contributes to the cellular pathogenesis of P. aeruginosa by interfering with eukaryotic cell growth and viability. In addition, the coculture system developed which recognizes this effect should provide a means for defining the function of ExoS in vivo.
Triplex-forming oligonucleotides (TFO) that bind DNA in a sequence-specific manner might be used as selective repressors of gene expression and gene-targeted therapeutics. However, many factors, including instability of triple helical complexes in cells, limit the efficacy of this approach. In the present study, we tested whether covalent linkage of a TFO to daunomycin, which is a potent DNA-intercalating agent and anticancer drug, could increase stability of the triple helix and activity of the oligonucleotide in cells. The 11mer daunomycin-conjugated GT (dauno-GT11) TFO targeted a sequence upstream of the P2 promoter, a site known to be critical for transcription of the c-myc gene. Band-shift assays showed that the dauno-GT11 formed triplex DNA with enhanced stability compared to the unmodified TFO. Band shift and footprinting experiments demonstrated that binding of dauno-GT11 was highly sequence-specific with exclusive binding to the 11 bp target site in the c-myc promoter. The daunomycin-conjugated TFO inhibited transcription in vitro and reduced c-myc promoter activity in prostate and breast cancer cells. The daunomycin-conjugated TFO was taken up by cells with a distinctive intracellular distribution compared to free daunomycin. However, cationic lipid-mediated delivery was required for enhanced cellular uptake, nuclear localization and biological activity of the TFO in cells. Dauno-GT11 reduced transcription of the endogenous c-myc gene in cells, but did not affect expression of non-target genes, such as ets-1 and ets-2, which contained very similar target sequences in their promoters. Daunomycin-conjugated control oligonucleotides unable to form triplex DNA with the target sequence did not have any effect in these assays, indicating that daunomycin was not directly responsible for the activity of daunomycin-conjugated TFO. Thus, attachment of daunomycin resulted in increased triplex stability and biological activity of the 11mer GT-rich TFO without compromising its specificity. These results encourage further testing of this approach to develop novel antigene therapeutics.
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