The radiomimetic enediyne C-1027 induces almost exclusively DNA double-strand breaks (DSB) and is extremely cytotoxic. Unique among radiomimetics, ataxia-telangiectasia mutated (ATM) is dispensable for cellular responses to C-1027-induced DNA damage. This study explores the biological activity of three recently bioengineered C-1027 analogues: 7 00

The hypoxic nature of cells within solid tumors limits the efficacy of anticancer therapies such as ionizing radiation and conventional radiomimetics because their mechanisms require oxygen to induce lethal DNA breaks. For example, the conventional radiomimetic enediyne neocarzinostatin is 4-fold less cytotoxic to cells maintained in low oxygen (hypoxic) compared with normoxic conditions. By contrast, the enediyne C-1027 was nearly 3-fold more cytotoxic to hypoxic than to normoxic cells. Like other radiomimetics, C-1027 induced DNA breaks to a lesser extent in cell-free, or cellular hypoxic, compared with normoxic environments. However, the unique DNA interstrand cross-linking ability of C-1027 was markedly enhanced under the same hypoxic conditions that reduced its DNA break induction. Although the unique chemistry of C-1027 allows it to concurrently generate both DNA breaks and cross-links in normoxic cells, a low oxygen environment represses the former and promotes the latter. Thus, treatment with C-1027 offers a facile approach for overcoming the radioresistance associated with poorly oxygenated cells.

Bizelesin is a bifunctional covalent minor groove binding agent which forms adducts with 3'-adenines on opposite DNA strands. DNA lesions induced by bizelesin in genomic DNA of BSC-1 cells, as well as intracellular and purified simian virus 40 (SV40) DNA, were examined. Alkaline sucrose sedimentation analysis indicated a nonrandom distribution of heat-labile damage in BSC-1 cell genomic DNA with frequencies of 1-60 lesions/10(6) base pairs (bp) for bizelesin concentrations from 10 to 400 nM, respectively. Extrapolation of these data suggested that, at 0.15 nM bizelesin, approximately 10(2) adducts per cell may be sufficient to inhibit cell growth by 90% (D10). While the frequency of bizelesin adducts in intracellular SV40 DNA was comparable to that in genomic DNA, higher levels of lesion formation are observed with purified SV40 DNA. Chromatin structure has little effect on localization of bizelesin adducts since treatment of either infected cells or purified SV40 DNA reveals a similar pattern of drug-induced damage. Bizelesin adduction sites (mapped on the SV40 genome as thermally-induced strand breaks at 50-100 bp resolution) are found in regions centered at 4200, 3900, 4700, and approximately 5200. The location of these regions of intense bizelesin bonding coincides with the sites of potential cross-links predicted using the 5'-T-(A/T)4-A-3' sequence. The analysis of bizelesin adducts at the sequence level in the 3943-4451 SV40 DNA fragment indicated that 40% of total damage was in potential cross-linking sites and an additional 35% in the 5'-A-(A/T)4-A-3' monoalkylating sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Three DNA binding polyamides (1-3) were synthesized that bind with high affinity (K a ‫؍‬ 8.7 ⅐ 10 9 M ؊1 to 1.4 ⅐ 10 10 M ؊1 ) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream (1) or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nM ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nM ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of proteinpromoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX-and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.

CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand breaks [Reynolds, R. L., Molineux, I. J., Kaplan, D.J., Swenson, D.H., & Hurley, L.H. (1985) Biochemistry 24, 6228-6237]. We have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, we observe thermally induced breakage of [14C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [3H]thymidine-labeled untreated cells with the [14C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10(6) base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The induction of heat-labile sites in cellular DNA was confirmed by gel electrophoretic analyses of the damage to intracellular simian virus 40 (SV40) DNA in SV40-infected BSC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)