Sensitivity and permeability of the anthramycin producing organism Streptomyces refuineus to anthramycin and structurally related antibiotics.
(17/25)Streptomyces refuineus, the microorganism which produces the DNA reactive antibiotic anthramycin, has shown to possess a quite specific mechanism to survive and grow in the presence of this antibiotic. Stationary phase cells are insensitive to anthramycin since the antibiotic is prevented form entering these cells. However, cells in early log phase are inhibited by concentrations of anthramycin that are later produced by these same cells. Significantly, sibiromycin, a closely related antibiotic, is taken up by cells of S. refuineus independent of the age of the culture. Anthramycin reacts in vitro equally as well as DNA isolated from S. refuineus and other procaryotic and eucaryotic cells. When S. refuineus has reached the production phase the anthramycin is probably biosynthesized outside the cell membrane which also becomes specifically impermeable to anthramycin. (+info)
Reaction of anthramycin with DNA. Biological consequences of DNA damage in normal and xeroderma pigmentosum cell.
(18/25)Anthramycin, an antitumor antibiotic produced by Streptomyces refuineus, produces a well defined covalent adduct with DNA and lies within the narrow groove of DNA, attached through a thermal-labile covalent animal linkage to the exocyclic amino group of guanine, without detectable distortion of the helix (Petrusek, R. L., Anderson, G. L., Garner, T. F., Fannin, Q. L., Kaplan, D. J. Zimmer, S. G., and Hurley, L. H. (1981) Biochemistry 20, 1111-1119). This paper described results in which the biological consequences of DNA damage and repair by repair-proficient and a repair-deficient xeroderma pigmentosum (XP 12RO) cell line are presented. Anthramycin has been shown to produce excision-dependent single and double strand breaks in DNA, both of which appear to persist many hours after removal of the drug from the media. The lower ability of the xeroderma pigmentosum cell line to remove ability of the xeroderma pigmentosum cell line to remove anthramycin lesions from DNA is correlated with a decreased cell survival. The biological consequences of DNA damage (genetic effects, DNA strand breakage, and cytotoxicity) are discussed with respect to the defined structure and stability of the anthramycin-deoxyguanosine adduct. (+info)
Mechanism of interaction of CC-1065 (NSC 298223) with DNA.
(19/25)CC-1065 (NSC 298223), a potent new antitumor antibiotic produced by Streptomyces zelensis, interacts strongly with double-stranded DNA and appears to exert its cytotoxic effects through disruption of DNA synthesis. We undertook this study to elucidate the sites and mechanisms of CC-1065 interaction with DNA. The binding of CC-1065 to synthetic and native DNA was examined by differential circular dichroism or by Sephadex chromatography with photometric detection. The binding of CC-1065 with calf thymus DNA was rapid, being complete within 2 hr, and saturated at 1 drug per 7 to 11 base pairs. The interaction of CC-1065 with synthetic DNA polymers indicated a specificity for adenine- and thymine-rich sites. Agarose gel electrophoresis of CC-1065-treated supercoiled DNA showed that CC-1065 did not intercalate. Site exclusion studies using substitutions in the DNA grooves showed CC-1065 to bind primarily in the minor groove. CC-1065 did not cause DNA breaks; it inhibited susceptibility of DNA to nuclease S1 digestion. It raised the thermal melting temperature of DNA, and it inhibited the ethidium-induced unwinding of DNA. Thus, in contrast to many antitumor agents, CC-1065 stabilized the DNA helix. DNA helix overstabilization may be relevant to the mechanism of action of CC-1065. (+info)
Streptomyces spadicogriseus, a new species producing anthramycin.
(20/25)The taxonomic description of Streptomyces spadicogriseus, a new species belonging to the Gray Series of streptomycetes as classified by Pridham and Tresner, is presented. This new species is distinguishable from the known members of the Gray Series. Streptomyces spadicogriseus produces anthramycin but bears no taxonomic relation to the known producer of the antibiotic: S. refuineus var. thermotolerans. (+info)
Regulation of tyrosine biosynthesis by phenylalanine in anthramycin-producing Streptomyces refuineus.
(21/25)The regulation of tyrosine production in the anthramycin-producing organism Streptomyces refuineus var. thermotolerans has been studied with wild-type and tyrosine auxotrophic organisms. Growth of the auxotroph on minimal medium plus phenylalanine suggested that phenylalanine may increase the supply of tyrosine. In incubation with whole cells, tyrosine levels increased in response to added phenylalanine. However, no radiolabeled tyrosine was detected after incubation with 14C-phenylalanine. Thus, no phenylalanine hydroxylase is present. Phenylalanine was found to feedback inhibit prephenate dehydratase, resulting in an increase in NAD-dependent prephenate dehydrogenase activity, thus channeling prephenic acid toward tyrosine. (+info)
Proof for the biosynthetic conversion of L-[indole-15N]tryptophan to [10-15N]anthramycin using (13C, 15N) labelling in conjunction with 13C-NMR and mass spectral analysis.
(22/25)Using 13C-NMR and mass spectral analysis we have demonstrated that the N-10 nitrogen of anthramycin is biosynthetically derived from the indole-nitrogen of tryptophan. Our experimental approach was to bring a 15N atom, which is derived from L-[indole-15N]tryptophan, and a 13C atom which is derived from DL-[1-13C]tyrosine, into adjacent positions of anthramycin. From resonance intensities and 13C-15N spin-spin coupling in the 13C-NMR spectrum of didehydroanhydroanthramycin, a derivative of anthramycin, we could then determine the 13C enrichment at C-11 and the proportion of 13C bonded to 15N at N-10. These results when combined with mass spectral analysis and isotopic dilution measurements proved that the indole nitrogen of tryptophan was completely retained at N-10 of anthramycin. (+info)
DNA binding, induction of unscheduled DNA synthesis, and excision of anthramycin from DNA in normal and repair-deficient human fibroblasts.
(23/25)The reaction of the antitumor antibiotic anthramycin with cellular DNA and the ability of normal human fibroblasts cells and xeroderma pigmentosum (XP) cells to respond to this injury has been evaluated. The binding of [15-3H]anthramycin to cellular DNA in human skin fibroblasts occurred in a linear manner up to 6 h. Treatment with unlabeled antibiotic resulted in unscheduled (repair) DNA synthesis in human skin fibroblasts maintained in hydroxyurea, whereas negligible unscheduled DNA synthesis was observed in cells of an excision-defective strain of XP. Confluent nondividing normal skin fibroblast cells were able to remove 86% of the bound anthramycin within 72 h, however XP cells were only able to remove 49% during the same incubation period. These results are discussed in terms of the types of DNA damage produced by anthramycin in vitro and the likely repair pathways involved in removing lesions produced on DNA by anthramycin. (+info)
Cellular pharmacology of novel C8-linked anthramycin-based sequence-selective DNA minor groove cross-linking agents.
(24/25)The cellular pharmacology of a series of C8-linked pyrrolobenzodiazepine dimers with polymethylene linkers of n = 3-6 (compounds 1-4) has been studied in a range of human tumour cell lines. The four compounds showed the same pattern of relative activity in five ovarian carcinoma cell lines and one cervical carcinoma cell line with the order of IC50 values of 1 < or = 3 < 4 < 2, which correlated with the previously demonstrated DNA interstrand cross-linking ability of the compounds in plasmid DNA. In human leukaemic K562 cells the agents produced a block in the G2/M phase of the cell cycle characteristic of cross-linking drugs, and extensive interstrand cross-linking was observed in cells by alkaline elution with no evidence of single-strand breaks. Cross-links continued to increase up to 24 h following a 1 h exposure to drug, and no repair was evident by 48 h. In a series of ovarian and cervical carcinoma cell lines with acquired resistance to cisplatin no cross-resistance to the most potent compound 1 was observed in two lines whose major mechanism of resistance to cisplatin was reduced platinum transport. Cross-resistance to 1 was observed in a cell line (A2780cisR) possessing elevated glutathione, and depletion of intracellular glutathione using D,L-buthionine-S,R-sulphoximine (BSO) from 10.25 nmol to 2.8 nmol 10(-6) cells reduced the level of resistance from 11-fold to 2-fold compared with sensitive cells. Cross-linking in the resistant cells was restored to 80% of the level in the parent line by BSO pretreatment. There was also a correlation between glutathione levels and sensitivity to 1 measured in several other ovarian cell lines. Compound 1 also showed cross-resistance in the doxorubicin-resistant cell line 41MdoxR and partial cross-resistance in CH1doxR cells. Both these lines possess elevated levels of p170 glycoprotein. Following treatment with 6 microM verapamil, the resistance in these lines decreased almost 2-fold and 8-fold respectively. (+info)