Terry A. Beerman

DNA strand scission by the antitumor protein neocarzinostatin.

Abstract The antibiotic protein, neocarzinostatin, induces the scission of DNA strands in vivo and in vitro . HeLa cell DNA prelabelled with [ 14 C] thymidine is cut into large pieces with a peak at 80–90S when cells are incubated with 0.5 to 5.0 μg/ml of highly purified neocarzinostatin. Incubation of the antibiotic (0.5 μg/ml) with [ 3 H] SV40 DNA in the presence of 2-mercaptoethanol results in the conversion of superhelical DNA I to nicked circular duplex DNA II. At high levels of drug, smaller fragments of linear DNA are produced. Strand breaks are detected in both neutral and alkaline sucrose gradients, indicating that drug susceptibility is not due to alkali-labile bonds.

The relationship between DNA strand-scission and DNA synthesis inhibition in HeLa cells treated with neocarzinostatin

Neocarzinostatin inhibits DNA synthesis in HeLa S3 cells and induces the rapid limited breakage of cellular DNA. The fragmentation of cellular DNA appears to precede the inhibition of DNA synthesis. Cells treated with drug at 37 degrees C for 10 min and then washed free of drug show similar levels of inhibition of DNA synthesis or cell growth, or of strand-scission of DNA as when cells were not washed. If cells are preincubated with neocarzinostatin at 0 degrees C before washing, the subsequent incubation of 37 degrees C results in no inhibition of DNA synthesis or cell growth, or cutting of DNA. Isolated nuclei or cell lysates derived from neocarzinostatin-treated HeLa S3 cells are inhibited in DNA synthesis but this can be overcome in cell lysates by adding activated DNA. A cytoplasmic fraction from drug-treated cells can stimulate DNA synthesis by nuclei isolated from untreated cells, whereas nuclei from drug-treated cells are not stimulated by the cytoplasmic fraction from untreated cells. By contrast, neocarzinostatin does not inhibit DNA synthesis when incubated with isolated nuclei, but it can be shown that under these conditions the DNA is already degraded and is not further fragmented by the drug. These data suggest that the drugs ability to induce breakage of cellular DNA in HeLa S3 cells is an essential aspect of its inhibition of DNA replication and may be responsible for the cytotoxic and growth-inhibiting actions of neocarzinostatin.

Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drugs cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.

Effect of minor groove binding drugs on mammalian topoisomerase I activity

Three minor groove binding drugs, distamycin A, bisbenzimide (Hoechst 33258) and 4,6-diamidino-2-phenylindole (DAPI), were examined for their abilities to modulate the activity of topoisomerase I purified from L1210 cells. At 0.5 and 1.0 microM, distamycin stimulated topoisomerase I relaxation of supercoiled DNA by 38 and 13%, respectively, while increasing the drug concentration above 2.0 microM resulted in inhibition. Inhibition was reversible. Complete relaxation could be achieved even in the presence of inhibitory concentrations of distamycin if the incubation time with topoisomerase I was increased from 7.5 to 120 min. The velocity of topoisomerase I mediated relaxation was reduced by 2 microM distamycin at DNA levels ranging from 350 to 2000 ng/reaction. Hoechst 33258 and DAPI inhibited topoisomerase I relaxation in a concentration-dependent manner. Hoechst 33258 and distamycin were equivalent in their abilities to inhibit topoisomerase I, whereas DAPI had a lesser effect (e.g. relaxation was reduced by 50% with 2.7 microM distamycin and 2.8 microM Hoechst 33258 compared to 5 microM DAPI). This study suggests that ligand binding in the minor groove can be a factor in the regulation of topoisomerase I activity.

DNA strand scission activity of metalloporphyrins.

Abstract The iron porphyrin derivatives, iron (III) meso-tetra(4-N-methylpyridyl)-porphine (Fe(III)T4MPyP), aceto-iron (III) meso-tetra(3-N-methylpyridyl)porporphine (AcO-Fe(III)T3MPyP), and iron (III) meso-tetra(p-sulfonatophenyl)-porphine (Fe(III)TSPP), have been shown to induce strand scissions in DNA. Incubation of these porphyrins with PM2 DNA results in the conversion of circular supercoiled DNA to the nicked circular duplex form. The presence of dithiothreitol increases the extent of the nicking reaction. Fe(III)TSPP, which, unlike Fe(III)T4MPyP and AcO-Fe(III)T3MPyP, does not bind to DNA, is the least effective of the three porphyrins in inducing strand scissions in PM2. Both Fe(III)T4MPyP and AcO-Fe(III)T3MPyP induce strand scissions in cellular DNA of pre-labeled HeLa S 3 cells while Fe(III)TSPP has a very limited effect.

Topoisomerase-II-mediated lesions in nascent DNA: Comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA

This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.

INHIBITION OF INITIATION OF SIMIAN VIRUS 40 DNA REPLICATION IN INFECTED BSC-1 CELLS BY THE DNA ALKYLATING DRUG ADOZELESIN

Adozelesin is a member of a family of extraordinarily cytotoxic DNA damaging agents that bind to the DNA minor groove in a sequence-specific manner and form covalent adducts with adenines. Previous studies employing purified enzymes and adozelesin-modified template DNAs suggested that adozelesin-DNA adducts inhibit DNA replication at the level of nascent DNA chain elongation. In this study, neutral/neutral two-dimensional agarose gel electrophoresis was employed to analyze simian virus 40 (SV40) DNA replication intermediates recovered from adozelesin-treated SV40 virus-infected cells. SV40 replication intermediates rapidly disappeared from infected cells when they were treated with adozelesin, but not when the cells were also treated with aphidicolin to block maturation of replicating SV40 DNA. We conclude that the disappearance of SV40 replication intermediates induced by adozelesin treatment was a consequence of maturation of these intermediates in the absence of new initiation events. Adozelesin inhibition of nascent chain elongation is first observed at concentrations above those needed to block initiation. Adozelesin treatment inhibits SV40 DNA replication at concentrations that produce adducts on just a small fraction of the intracellular population of SV40 DNA molecules.

Effects of minor groove binding drugs on camptothecin-induced DNA lesions in L1210 nuclei

Topoisomerase I inhibition detected in mammalian cells can be correlated with reduced tumor growth. Camptothecin specifically inhibits topoisomerase I by stabilization of a covalently linked DNA-enzyme complex and associated DNA single-strand breaks. Whether perturbations in nuclear DNA structure can alter camptothecin-induced DNA damage was examined using the non-intercalative DNA minor groove binders distamycin, Hoechst 33258 and DAPI (4,6-diamidino-2-phenylindole). L1210 nuclei were treated with camptothecin alone or in the presence of single minor groove binders. DNA-protein crosslinks and single-strand breaks were determined using potassium-sodium dodecyl sulfate precipitation and alkaline elution respectively. Distamycin produced a dose-dependent decrease in DNA-protein crosslinks and strand breaks. This effect was reduced if nuclei were treated with camptothecin prior to distamycin addition. Distamycin was unable to reverse lesions once induced or to prevent repair of damage upon camptothecin removal. Hoechst 33258 and DAPI also decreased camptothecin-induced DNA damage. The order of inhibitory potency was: distamycin greater than Hoechst greater than DAPI. This order corresponded to the molecular weights as well as to the size of the nucleotide binding sites of the drugs. Identifying agents which alter such DNA lesions should provide better understanding of the chemotherapeutic activity of camptothecin as well as help elucidate new leads for drug combinations of improved therapeutic benefit.

The Antitumor Enediyne C-1027 Alters Cell Cycle Progression and Induces Chromosomal Aberrations and Telomere Dysfunction

This study examined the extent of chromosome instability induced in cultured human colon carcinoma HCT116 cells by the antitumor radiomimetic enediyne antibiotic C-1027. Spectral karyotype analysis showed frequent intrachromosomal fusions and fragmentations 26 hours after addition of as little as 0.035 nmol/L C-1027. When the concentration was increased to 0.14 nmol/L C-1027, 92% of cells showed chromosomal aberrations compared with only 2.9% after treatment with an equivalent growth inhibitory dose of ionizing radiation (20 Gy). Thus, chromosome misrejoining was associated to a much greater extent with C-1027-induced than with ionizing radiation-induced cell growth inhibition. Despite these aberrations, a large fraction of C-1027-treated cells progressed into G1. Comet analysis showed that these extensive chromosomal anomalies were not due to increased induction or reduced repair of C-1027-induced compared with ionizing radiation-induced strand breaks. Fluorescence in situ hybridization analysis showed that misrejoining of telomere repeats (i.e., chromosomes joined end to end at their telomeres or fused together after complete loss of telomere sequences) was observed within 26 hours of C-1027 addition. The extreme cytotoxicity of C-1027 may reflect both induction and erroneous repair of DNA double-strand break in the whole genome and/or in subgenomic targets such as telomere sequences.

Inhibition of transcription factor-DNA complexes and gene expression by a microgonotropen

Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug development studies. Equipping minor groove-binding agents with positively charged, major groove-contacting side chains yields microgonotropens (MGTs). Previously, we demonstrated that MGTs were superior inhibitors of TF/DNA complexes in cell-free assays compared with “classical” groove binders, but MGTs showed limited ability to inhibit gene expression. To determine what chemical characteristics contribute to or improve activity, we evaluate five MGTs for their effectiveness in inhibiting TF complex formation and resultant transcription by using the c-fos serum response element (SRE) as a target. MGT L1 binds DNA via a bisbenzimidazole equipped with a tripyrrole moiety. It is compared with analog L2, which has been functionalized with propylamines on each of the three pyrroles. L2, which binds DNA at subpicomolar concentrations, was at least three orders of magnitude more potent than L1 at inhibiting TF binding to the c-fos SRE in cell-free assays. Unlike L1 and previous MGTs, L2 also inhibited endogenous c-fos expression in NIH 3T3 cells at micromolar levels. Structure/activity relationships suggest that, although the tripyrrole/polyamine functional group of L2 may be largely responsible for its inhibition of TF complexes in cell-free assays, its bisbenzimidazole moiety appears to impart improved cellular uptake and activity. These findings make L2 a promising lead candidate for future, rational MGT design.