The structure of the sulfur-containing chromophore of phleomycin, and chemical transformation of phleomycin to bleomycin.
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Antibiotics, Antineoplastic↗
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Control of in vivo (cellular) phleomycin sensitivity by nuclear genotype, growth phase, and metal ions.
Nuclear genotype, growth phase, and the presence of metal ions all proved to be important in controlling the lethal effects of phleomycin in eukaryotic Saccharomyces cerevisiae. Among 120 normal and radiation-sensitive strains compared for their sensitivities to lethal effects of phleomycin, all mutant strains exhibiting enhanced sensitivities to phleomycin killing were also sensitive to killing by ionizing radiation. Mutants exhibiting sensitivities to phleomycin similar to normal strains of the same ploidy were sensitive to ultraviolet radiation. We conclude that cellular recovery from phleomycin-induced damage in yeast depends upon the function of some or all of 13 independent genes and upon at least some of the same steps in cellular pathways for the biological repair of damage by ionizing radiation. In this respect, the action of phleomycin is similar to the action of its structurally similar analog, bleomycin, even though phleomycin was substantially more cytotoxic. Stationary-phase haploid yeast cells were more sensitive than exponentially growing cells to killing by phleomycin. Survival of stationary-phase yeast was reduced to 0.3 +/- 0.07% (S.E.) after 20-min exposures to phleomycin (1 microgram/ml; approximately 6.7 x 10(-7) M), but lethal effects of phleomycin were completely eradicated (98% survival) by the presence of 0.05 M ethylenediaminetetraacetate during the treatment period. The inactivation indicates an important role for one or more metal ion(s) in the in vivo toxicity of the phleomycin-bleomycin group of anticancer antibiotics.
Bleomycin↗
Effects of coumarin, thiopurines, and pyronin Y on amplification of phleomycin-induced death and deoxyribonucleic acid breakdown in Escherichia coli.
Phleomycin (</=2 mug/ml) induces neither deoxyribonucleic acid (DNA) breakdown nor cell death in stationary-phase Escherichia coli B cells, but the addition of 8 mm caffeine immediately initiates these changes in the same way as increasing the phleomycin concentration 10-fold. This phenomenon is termed "amplification" (6). Pyronin Y, a number of nontoxic thio- and mercaptopurines (of which the most active were 6,7- and 6,9-dimethyl-2-methylthiopurine), and coumarin have been found to be considerably more efficient amplifiers of phleomycin activity than caffeine. Thus 2 mm 6,7- and 6,9-dimethyl-2-methylthiopurine, 0.16 mm pyronin, and 4 mm coumarin killed 10 to 100 times more phleomycin-treated bacteria within 2 hr than 8 mm caffeine. As with caffeine, amplification of cell death by these compounds was accompanied by degradation of DNA to acid-soluble fragments. A number of compounds including 2,6-dichloropurine, 6-hydroxy-2-methylthiopurine, alpha-naphthol, beta-naphthol, naphthionic acid, and alpha-naphthol-4,8-disulphonic acid inhibited the action of phleomycin, if they were present in the cell suspension during phleomycin treatment, but some caused amplification if added subsequent to the phleomycin. Although no mutants resistant to >/=10 mug of phleomycin per ml were observed among 10(11)E. coli B cells screened, such mutants occurred with a frequency of 10(-6) to 10(-7) among cultures resistant to 1 to 2 mug of phleomycin per ml. These double mutants were cross-resistant to phleomycin plus caffeine. The amplifying compounds, though structurally dissimilar, shared the common characteristic of binding selectively to denatured DNA as measured by equilibrium dialysis methods. The implications of these observations in supporting a model of phleomycin amplification proposed previously (6) and their utility in providing a logic for developing a new class of antibiotics are discussed.
Antibiotics, Antineoplastic↗
Genetic changes and bioassays in bleomycin- and phleomycin-treated cells, and their relationship to chromosomal breaks.
The recombinogenicity of damaged chromosomes in diploid Saccharomyces cerevisiae cells treated with bleomycin and structurally related phleomycin was measured, along with aneuploidy and mutation events. Phleomycin was substantially (up to 26-fold) more effective than bleomycin in producing genetic changes at all concentrations, even when colony-forming abilities of cells growing in the presence of bleomycin or phleomycin were similar. These results suggest that the DNA lesions produced by the two structurally related analogs could differ in their nature or frequency, or could be processed differently by the cells. Bioassays were developed and used to compare the cytotoxicities of freshly dissolved bleomycin and phleomycin with the cytotoxicities of lysates prepared from bleomycin- and phleomycin-treated cells. Unexpectedly, lysates prepared from bleomycin-treated cells were 1.5-3.5 times more cytotoxic than freshly dissolved bleomycin after 45-min treatments (3-33 x 10(-6) M). In contrast, lysates prepared from phleomycin-treated cells were 3-38 times less cytotoxic than freshly dissolved phleomycin (0.5-6.4 x 10(-6) M). Cytotoxicities of all lysates were higher after 36-h treatments than after 45-min treatments. At 3.3 x 10(-6) M, this increase was eightfold for bleomycin and 15-fold for phleomycin. Nevertheless, lysates from phleomycin-treated cells were considerably more cytotoxic than lysates from bleomycin-treated cells or freshly prepared bleomycin, consistent with the higher effectiveness of phleomycin than bleomycin in producing chromosomal breaks, genetic changes, and cell killing.
Aneuploidy↗
Amplification of the antibiotic effects of the bleomycins, phleomycins and tallysomycins: its dependence on the nature of the variable basic groups.
The bleomycins, phleomycins and tallysomycins are structurally similar glycopeptide antibiotics. Within each class, individual members differ only in the structure of a basic group. The antibiotic effect of phleomycin (Bristol batch A9331-648) against Escherichia coli is amplified substantially by a number of simple heterocyclic and aromatic compounds. In this paper a sample of 26 such compounds were tested for this property with 25 different phleomycins, bleomycins and tallysomycins. The nature of the variable basic group of the phleomycins, bleomycins and tallysomycins determined the response obtained with all amplifiers, although variation of response was much less marked with caffeine which potentiated the cytotoxic effects of all the phleomycins, bleomycins and tallysomycins tested. Phleomycins and bleomycins having two or three guanidino groups in the variable basic group, or phleomycins having a secondary amino group within a methylene chain and a terminal 2-phenylethyl substituent, were amplified by most compounds, whereas the cytotoxicity of others was enhanced little or not at all. Similar phleomycins, having a secondary amino and a terminal guanidino group and no 2-phenylethyl substituent showed little enhancement, and in these cases the inclusion of a 2-phenylethyl substituent had a major influence in determining amplifiability. Bleomycins and phleomycins having identical basic groups were amplified to similar extents by the sample of 26 amplifying agents used.
Bleomycin↗
Internucleosomal cleavage and chromosomal degradation by bleomycin and phleomycin in yeast.
Native chromosomal structure, breakage, and overall degradation were studied following the reaction of whole cells with the anticancer drug bleomycin and structurally related phleomycin. Electrophoretic analyses of cellular DNA established that phleomycin was more reactive with DNA than equimolar bleomycin in the range of 0.67-33 x 10(-6) M, produced an optimally visible, though less-extended, oligonucleosomal series at concentrations 12 to 35 times lower than bleomycin, and degraded DNA within nucleosomes. Chromosomes were cleaved into nucleosomes and degraded by phleomycin over substantially narrower dose ranges (1 to 2 x 10(-6) M) than by bleomycin (about 1 to 17 x 10(-5) M). Bleomycin exhibited higher specificity for internucleosomal cleavage than phleomycin, and trimmed but did not degrade nucleosomes at less than or equal to 3 x 10(-5) M. Identical nucleosomal repeat sizes (166 +/- 3.8 base pairs) were produced by the analogues. The higher reactivity of phleomycin does not result solely from its higher rate of internucleosomal and intranucleosomal chromatin cleavage, since short phleomycin reactions always resulted in more extensive chromatin cleavage than long bleomycin reactions at low concentrations. In vivo (cellular) repair of chromatin damage was comparable (approximately 90% in 1 h) after cells were exposed to low drug concentrations which produced similar numbers of chromatin breaks, and thus also does not account for the higher chromosomal breakage caused by phleomycin than bleomycin at low doses. At high doses, unrepaired breaks are substantially higher after phleomycin treatments than after bleomycin treatments, and thus contribute to the higher lethal effects of phleomycin than bleomycin.
Bleomycin↗
A Saccharomyces cerevisiae phleomycin-sensitive mutant, ph140, is defective in the RAD6 DNA repair gene.
The antibiotic bleomycin is used as an anticancer agent for treating a variety of tumours. The antitumour effect of bleomycin is related to its ability to produce lesions such as apurinic/apyrimidinic sites and single- and double-strand breaks in the cellular DNA. Phleomycin is a structurally related form of bleomycin, but it is not used as an anticancer agent. While phleomycin can also damage DNA, neither the exact nature of these DNA lesions nor the cellular process that repairs phleomycin-induced DNA lesions is known. As a first step to understand how eukaryotic cells provide resistance to phleomycin, we used the yeast Saccharomyces cerevisiae as a model system. Several phleomycin-sensitive mutants were generated following gamma-radiation treatment and among these mutants, ph140 was found to be the most sensitive to phleomycin. Molecular analysis revealed that the mutant ph140 harbored a mutation in the DNA repair gene RAD6. Moreover, a functional copy of the RAD6 gene restored full phleomycin resistance to strain ph140. Our findings indicate that the RAD6 protein is essential for yeast cellular resistance to phleomycin.
4-Nitroquinoline-1-oxide↗
Cleavage of cellular and extracellular Saccharomyces cerevisiae DNA by bleomycin and phleomycin.
Low-molecular-weight phleomycin (Mr approximately 1500-1600) is considerably less active on a per mol basis than structurally related bleomycin in degrading purified Saccharomyces cerevisiae DNA. Phleomycin also exhibits a substantially higher requirement than bleomycin for ferrous ions. However, phleomycin (0.13 to 3.3 x 10(-6) M) produced 7 to 350 times more breaks than bleomycin in prelabeled intracellular [2-14C]DNA and [6-3H]DNA and is considerably more cytotoxic than bleomycin. Phleomycin and bleomycin produced equivalent numbers of DNA breaks at equivalent, physiologically meaningful levels of survival, indicating that DNA breaks are related to lethal properties of the anticancer glycopeptides. Phleomycin degradation of extracellular DNA was only detectable at greater than or equal to 1.7 x 10(-4) M, approximately two orders of magnitude higher than the concentrations of phleomycin which yielded equivalent fragmentation of intracellular DNA, indicating that phleomycin causes substantially more degradation of intracellular DNA than extracellular DNA. In contrast, bleomycin (greater than or equal to 1.7 x 10(-5) M) degradation of purified DNA is quite extensive and considerably greater than the degradation of DNA in cells incubated with the same or higher concentrations of bleomycin. Neither phleomycin nor bleomycin cleaved extracellular DNA in the absence of ferrous ions, although both chemical analogues cleaved intracellular DNA without adding iron. Therefore, the requirement for metal ion in stimulating DNA degradation by the two structural families of glycopeptidic antibiotics is met by the cell itself.
Bleomycin↗
The ATM-related Tel1 protein of Saccharomyces cerevisiae controls a checkpoint response following phleomycin treatment.
MEC1 and TEL1 encode ATR- and ATM-related proteins in the budding yeast Saccharomyces cerevisiae, respectively. Phleomycin is an agent that catalyzes double-strand breaks in DNA. We show here that both Mec1 and Tel1 regulate the checkpoint response following phleomycin treatment. MEC1 is required for Rad53 phosphorylation and cell-cycle progression delay following phleomycin treatment in G1, S or G2/M phases. The tel1Delta mutation confers a defect in the checkpoint responses to phleomycin treatment in S phase. In addition, the tel1Delta mutation enhances the mec1 defect in activation of the phleomycin-induced checkpoint pathway in S phase. In contrast, the tel1Delta mutation confers only a minor defect in the checkpoint responses in G1 phase and no apparent defect in G2/M phase. Methyl methanesulfonate (MMS) treatment also activates checkpoints, inducing Rad53 phosphorylation in S phase. MMS-induced Rad53 phosphorylation is not detected in mec1Delta mutants during S phase, but occurs in tel1Delta mutants similar to wild-type cells. Finally, Xrs2 is phosphorylated after phleomycin treatment in a TEL1-dependent manner during S phase, whereas no significant Xrs2 phosphorylation is detected after MMS treatment. Together, our results support a model in which Tel1 contributes to checkpoint control in response to phleomycin-induced DNA damage in S phase.
Cell Cycle↗
An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.
A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain.
Bleomycin↗
Effect of phleomycin on polyoma virus synthesis in mouse embryo cells.
The addition of phleomycin (25 mug) to primary mouse embryo cells infected with polyoma virus was found to cause 96% inhibition of the synthesis of infectious virus. When ribonucleic acid and protein synthesis was investigated in these cells by use of isotope incorporation, it was found that neither was inhibited drastically. Immunofluorescent staining studies with the use of antibody directed to the viral structural proteins showed that proteins were synthesized in the presence of the antibiotic. However, when deoxyribonucleic acid (DNA) synthesis was investigated, it was found that DNA synthesis in uninfected cells was completely inhibited within the initial 10 hr of phleomycin addition, whereas DNA synthesis in infected cells proceeded at a reduced rate. Selective DNA extraction (Hirt method) of phleomycin-treated infected cells demonstrated that synthesized viral DNA was salt-extractable, similar to that in infected control cells lacking phleomycin. This extracted DNA was further fractionated by ethidium bromide-cesium chloride density gradient equilibrium centrifugation. The phleomycin-treated preparations revealed twice as much component II (circular nicked and linear) as component I (supercoiled) DNA, whereas the DNA from normally infected control cells showed the reverse picture. It was also demonstrated that viral particles synthesized in the presence of phleomycin did not contain component I DNA. This packaged DNA was found to consist of fragments of both the host and viral types. Cells that were prelabeled with (3)H-thymidine and then treated with phleomycin demonstrated host DNA degradation. However, fragments formed from prelabeled host DNA were not encapsidated into viral particles.
Anti-Bacterial Agents↗
Identification of a Tn5 determinant conferring resistance to phleomycins, bleomycins, and tallysomycins.
Tn5 conferred resistance to the related antibiotics, phleomycins, bleomycins, and tallysomycins in Escherichia coli and Salmonella typhimurium. For pure phleomycins the level of resistance was influenced by the structure of the terminal basic group. Deletion derivatives of a pBR322::Tn5 plasmid were used to show that the phleomycin resistance determinant is located between the previously identified neomycin and streptomycin resistance determinants. The pattern of expression of phleomycin and neomycin resistance in the deletion derivatives suggests that the phleomycin resistance gene is transcribed from the same promoter, PL, which is essential for expression of neomycin and streptomycin resistance. The location of the phleomycin resistance determinant correlates with the location of an open reading frame in the Tn5 sequence, which codes for a polypeptide of 126 amino acids.
Bleomycin↗
Differential effect of phleomycin on the infectivity of poliovirus and poliovirus-induced ribonucleic acids.
The infectivity of intact poliovirus was not affected by exposure to the antibiotic phleomycin at concentrations as high as 200 mug/ml, whereas that of the singlestranded poliovirus ribonucleic acid (RNA) was inactivated to 99% by pretreatment of the RNA with phleomycin at a concentration of 2 mug/ml. The infectivity of double and multistranded RNA was 10 times less sensitive than that of singlestranded RNA to the action of this antibiotic. Preincubation of HeLa cells for 30 min with 10 to 50 mug of phleomycin reduced the sensitivity of the cells to infection by viral RNA and intact virus, indicating that phleomycin interferes with cellular functions necessary for virus replication. When phleomycin was added to cells at different times after infection with single- or double-stranded RNA, the highest inactivation of infective centers was observed immediately after infection. With time of incubation at 37 C, the infective centers became more resistant to the action of phleomycin.
Anti-Bacterial Agents↗
The mechanism of DNA breakage by phleomycin in vitro.
Phleomycin induces DNA breakage in vitro in the presence of the sulphydryl compound dithiothreitol. The reaction appears to be free radical-mediated, and requires oxygen and metal ions. Reaction rate is limited by the concentration of oxygen, which is converted to hydrogen peroxide during DNA breakage. However there is no net change in the sulphydryl compound. The proposed reaction mechanism involves metal ion/oxygen-catalysed oxidation of dithiothreitol to its free radical form, which reacts with phleomycin, leading to formation of activated phleomycin and regeneration of free sulphydryl. Free phleomycin is converted to an inactive form, but activation of phleomycin bound to DNA leads to DNA breakage.
Anti-Bacterial Agents↗
Effects of phleomycin-induced DNA damage on the fission yeast Schizosaccharomyces pombe cell cycle.
The effect of phleomycin, a bleomycin-like antibiotic, has been investigated in the fission yeast, Schizosaccharomyces pombe. We report that in response to phleomycin-induced DNA damage, growth was inhibited and S. pombe cells arrested in the G2-phase of the cell cycle. DNA repair mutants rad9 and rad17 did not arrest and were hypersensitive to phleomycin. Cell cycle mutants that entered mitosis without monitoring the completion of DNA replication also displayed an increased sensitivity to this DNA-damaging agent. Thus, phleomycin could be used as a tool in the fission yeast S. pombe model system for the study of DNA damage and cell cycle checkpoints, or as a new selective agent.
Cell Cycle↗
Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae.
Phleomycin, a water-soluble antibiotic of the bleomycin family is as effective against Saccharomyces cerevisiae cells as against Escherichia coli cells. The ble gene of transposon Tn5, which confers resistance to phleomycin, was inserted in place of the iso-1-cytochrome C (CYC1) gene on an autonomously replicative multicopy E. coli-yeast shuttle plasmid. Higher resistance levels are obtained in S. cerevisiae when the region immediately upstream from the initiation codon conforms to the nucleotide sequence stringencies observed in almost every yeast gene. The expected regulation pattern of the whole CYC1 promoter confers different phleomycin resistance levels to the cell under varying physiological conditions. Partial deletions in the CYC1 promoter lead to changes in the resistance level of cells which are mostly accounted for by the removal of known positive and negative regulatory elements. Some of the vector constructions allow direct selection of phleomycin-resistant transformants on rich media.
Bleomycin↗
Phleomycin resistance as a dominant selectable marker in CHO cells.
The Tn5 and the Streptoalloteichus hindustanus (Sh) ble genes conferring resistance to bleomycin-phleomycin antibiotics have been cloned into a mammalian vector under the RSV-LTR promoter. The resulting plasmids, pUT506 and pUT507 respectively, were used to transfect CHO cells by either the calcium phosphate or the recently described polybrene-DMSO method. Phleomycin- or bleomycin-resistant clones arose with a higher frequency after transfection with pUT507, and pUT507 transfectants were more resistant to both antibiotics than pUT506 transfectants. Phleomycin resistance in pUT507 transfectants was stable and associated with integration of plasmid sequences in genomic DNA. The Sh ble gene, which confers a dominant phleomycin-resistance phenotype, should provide a useful transferable selectable marker in CHO cells as well as in other animal cell lines.
Animals↗
Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding.
The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.
Animals↗