No abstract

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)

The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesintreated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.The cyclopropylpyrroloindole (CPI) 1 drugs are a group of DNA sequence-specific minor groove binders that alkylate the N-3 of adenine at the 3Ј end of the binding sites. CPI drugs are currently in clinical trials for several types of solid tumors (1, 2). The CPI drug, adozelesin, carries a single cyclopropyl group and alkylates a single adenine (3-6). CPI adduct formation on naked DNA is able to block progression of DNA polymerases and helicases (7-9). Previous studies have shown that two different CPI drugs, adozelesin and bizelesin, inhibit both the initiation and elongation stages of cellular and viral DNA replication in cultured cells (10 -12). However, the concentrations of these drugs required to cause S phase arrest are 2-4 orders of magnitude lower than levels of drug required to cause detectable adducts and to block polymerase or helicase progression. These results suggest that inhibition of DNA replication and cell cycle progression in cultured cells upon treatment with CPI drugs occurs via a trans-acting mechanism rather than by directly blocking DNA replication fork progression. The most likely explanation for this trans-inhibition of DNA replication is through cellular DNA damage response pathways or checkpoints.Since both viral, simian virus 40 (SV40), and cellular DNA replication are inhibited at similar CPI levels, it is possible to use the more easily studied viral system to elucidate how CPI treatment results in DNA replication arrest. SV40 DNA replication is the most well studied model for eukaryotic DNA replication. An in vitro system was developed that requires only one viral protein, SV40 large T antigen, an exogenous plasmid DNA template containing the SV40 origin sequence, and primate...

Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).