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PubMed1993

EPR spectroscopic investigation of the lability of oxygen in activated bleomycin: implications for the mechanism of bleomycin-mediated DNA degradation.

Bleomycin (BLM), an antitumor antibiotic, is capable of degrading DNA through the formation of activated BLM, an activated iron-oxygen complex of the drug with a unique EPR spectrum. A recent study [Rabow, L. E., McGall, G. H., Stubbe, J., & Kozarich, J. W. (1990) J. Am. Chem. Soc. 112, 3203-3208] has cast doubt onto the "hydroxyl-radical-rebound" mechanism, commonly accepted for cytochrome P-450 [McMurray, T.J., & Groves, J.T. (1985) in Cytochrome P-450: Structure, Mechanism, and Biochemistry (Ortiz de Montellano, P., Ed.) pp 1-28, Plenum, New York] and proposed for the anaerobic nucleic base release reaction of BLM, by demonstrating that the source of oxygen in the products of this reaction is solvent molecules and not molecular oxygen. A central issue in this debate is whether the oxygen of activated BLM is available for exchange with that of solvent. The lability of oxygen in activated BLM has been investigated through the use of EPR spectroscopy to measure the exchange of 17O (I = 5/2) between activated BLM and solvent. Evidence for the lack of oxygen exchange between activated BLM and solvent is presented, and the implications of this result for the mechanism of BLM-mediated DNA degradation are discussed.

Bleomycin↗
PubMed2001

Solution structure of Co(III)-bleomycin-OOH bound to a phosphoglycolate lesion containing oligonucleotide: implications for bleomycin-induced double-strand DNA cleavage.

Bleomycin (BLM) is an antitumor antibiotic that is used clinically. Its major cause of cytotoxicity is thought to be related to BLM's ability to cause double-strand (ds) DNA cleavage. A single molecule of BLM appears to cleave both strands of DNA in the presence of its required cofactors Fe(2+) and oxygen without dissociating from the helix. A mechanism for this process has been proposed based on a model structure of the hydroperoxide of Co(III)-BLM (CoBLM) bound sequence-specifically to an intact duplex containing a GTAC site, a hot spot for ds cleavage [Vanderwall, D. E., Lui, S. M., Wu, W., Turner, C. J., Kozarich, J. W., and Stubbe, J. (1997) Chem. Biol. 4, 373-387]. In this paper, we present a structural model for the second cleavage event. Two-dimensional NMR spectroscopy and molecular modeling were carried out to study CoBLM bound to d(CCAAAGXACTGGG).d(CCCAGTACTTTGG), where X represents a 3'-phosphoglycolate lesion next to a 5'-phosphate. Assignments of 729 NOEs, including 51 between the drug and the DNA and 126 within the BLM molecule, have been made. These NOEs in addition to 96 dihedral angle constraints have been used to obtain a well-defined structural model for this complex. The model reveals that the bithiazole tail is partially intercalated between the T19 and the A20 of the duplex and that the metal binding domain is poised for abstraction of the T19 H4' in the minor groove. The modeling further reveals that the predominant conformation of the bithiazole protons is trans. Two cis conformations of these protons are also observed, and ROESY experiments provide evidence for interconversion of all of these forms. The relationship of these observations to the model for ds cleavage is presented.

Antibiotics, Antineoplastic↗
PubMed1982

Cobalt-bleomycin-deoxyribonucleic acid system. Evidence of deoxyribonucleic acid bound superoxo and mu-peroxo cobalt-bleomycin complexes.

Co(II) interacts with bleomycin in aqueous solution, in the presence of air, to give a short-lived mononuclear superoxo Co(III) complex (I). Then, two molecules of complex I react together, with the loss of oxygen, to yield the dinuclear mu-peroxo Co(III) complex (II); the dimerization follows a second-order rate law with k2 = 200 +/- 50 M-1 s-1 at 25 degrees C. The rate of dimerization is lowered by a factor of 2000 when DNA is present at a molar ratio of [nucleotide]/[Co] higher than 16. These results and studies of circular dichroism and electron paramagnetic resonance spectra of complexes strongly suggest the binding of the superoxo complex to DNA (I') as well as that of the mu-peroxo complex (II'); the binding of 1 molecule of complex II for every 2.9 base pairs in DNA has been determined with an apparent equilibrium constant of 8.4 x 10(4) M-1.

Bleomycin↗
PubMed1986

Functional analogues of bleomycin: DNA cleavage by bleomycin and hemin-intercalators.

New hemin-intercalators (Hem-G's) that cleave DNA were synthesized, on the basis of 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole (Glu-P-1) as an intercalator moiety. Hem-G's, which possess an intramolecular ligand of the ferrous ion (a histidine or imidazole moiety), cleave DNA very efficiently and act at guanine-pyrimidine sequences preferentially. Bleomycin (BLM) also cleaved DNA with the same base-sequence selectivity shown by Hem-G's. The 5'-terminus of the DNA fragments cleaved by Hem-G's or by BLM is a phosphoryl group, while the 3'-terminus of the cleaved DNA fragments does not possess a 3'-phosphoryl group. There are more than three kinds of 5'-end 32P-labeled DNA fragments, which can be substrates of terminal deoxynucleotidyl transferase (TdT). One of the 3'-termini of the cleaved DNA fragments is a 3'-hydroxy group. The mobility of the 3'-end 32P-labeled DNA fragment cleaved by Hem-G's or by BLM corresponds to the removal of pyrimidine bases having guanine at the 5'-side. The mobility of one kind of the cleaved 5'-end 32P-labeled DNA fragments corresponds to the removal of guanine having pyrimidine at the 3'-side, followed by 3'-dephosphorylation. We propose that there exist plural mechanisms for DNA cleavage by Hem-G's or by BLM. The deduced structures of the cleaved DNA fragments suggest that one of the mechanisms involves deletion of two nucleotide units from DNA.

Base Sequence↗
PubMed2003

NMR study of the interaction between Zn(II) ligated bleomycin and Streptoalloteichus hindustanus bleomycin resistance protein.

Bleomycin (Bm), a 1.4 kDa glycopeptide excreted by Streptomyces verticillus, is a natural antibacterial compound used in therapy as antineoplastic drug. To counteract its biological activity, cells have developed several resistance mechanisms, one of these based on proteins able to tightly bind Bm. In this paper, the interaction of Zn(2+)-Bm with the Streptoalloteichus hindustanus Bm resistance protein (ShBle) has been investigated by solution state NMR. Sequential nOe and chemical shift index have shown that the fold of the protein (in absence or presence of Bm) is identical to the previously published X-ray structure. The dimeric nature of ShBle is confirmed by the diffusion tensor as determined by NMR relaxation data. Using isotope filtered nOe experiment, intermolecular nOes between Bm and ShBle have been observed as used for modeling. While the interaction of the Bm metal binding site with ShBle appears to be uniquely defined, several conformations of the bithiazole moieties are compatible with the NMR data. Binding of Bm also induces changes of the local dynamics (stretch N85-G91), as shown by (15)N relaxation data. These results are discussed in the context of several Bm analogues able to interact with ShBle and of the recently published X-rays structures.

Acetyltransferases↗
PubMed2000

Crystal structures of the transposon Tn5-carried bleomycin resistance determinant uncomplexed and complexed with bleomycin.

The transposon Tn5 carries a gene designated ble that confers resistance to bleomycin (Bm). In this study, we determined the x-ray crystal structures of the ble gene product, designated BLMT, uncomplexed and complexed with Bm at 1.7 and 2.5 A resolution, respectively. The structure of BLMT is a dimer with two Bm-binding pockets composed of two large concavities and two long grooves. This crystal structure of BLMT complexed with Bm gives a precise mode for binding of the antibiotic to BLMT. The conformational change of BLMT generated by binding to Bm occurs at a beta-turn composed of the residues from Gln(97) to Thr(102). Crystallographic analysis of Bm bound to BLMT shows that two thiazolium rings of the bithiazole moiety are in the trans conformation. The axial ligand, which binds a metal ion, seems to be the primary amine in the beta-aminoalanine moiety. This report, which is the first with regard to the x-ray crystal structure of Bm, shows that the bithiazole moiety of Bm is far from the metal-binding domain. That is, Bm complexed with BLMT takes a more extended form than the drug complexed with DNA.

Acetyltransferases↗
PubMed1998

Protection of mammalian cells from the toxicity of bleomycin by expression of a bleomycin-binding protein gene from Streptomyces verticillus.

A gene, blmA, encodes a bleomycin (Bm)-binding protein, designated BLMA, from Bm-producing Streptomyces verticillus and confers resistance to Bm in Streptomyces and Escherichia coli cells. In the present study, by transfection of the gene into COS-1 cells with a plasmid designated pEF-BOS/blmA, which contains a strong promoter from the human polypeptide chain elongation factor 1alpha, we transiently overproduced BLMA at a high level of approximately 4% of the whole cell protein. Although NIH/3T3 cells transfected with pEF-BOS/blmA, designated NIH/3T3-BR cells, stably expressed BLMA, its expression level was about 0.1% of the total protein. Using an anti-BLMA monoclonal antibody reported previously [Sugiyama et al. (1995) FEBS Lett. 362, 80-84], we revealed that BLMA is localized in the nucleus of pEF-BOS/blmA-transfected COS-1 and NIH/3T3-BR cells. Semi-permeabilized nuclear transport experiments showed that BLMA penetrates the nuclear envelope by energy- and transporter-independent passive diffusion, suggesting that the karyophilic nature of BLMA may be due to the acidic nature of the protein. NIH/3T3-BR cells were 130-fold more resistant to Bm than the host cells. NIH/3T3 cells exhibited a swollen nuclear envelope and a malformed spindle body and overexpressed at least 4 kinds of stress proteins including calreticulin and mitochondrial matrix protein P1 when exposed to 25 microg/ml of Bm, whereas NIH/3T3-BR cells grew without morphological alteration and expressed no stress proteins under the same conditions. Furthermore, reverse transcription-polymerase chain reaction and Northern blot analysis showed that the expression of interleukin-6, an inflammatory cytokine, is activated by addition of Bm in NIH/3T3 cells, but not in the NIH/3T3-BR cells. These results suggest that BLMA contributes to protection of mammalian cells from the inflammatory effect of Bm.

3T3 Cells↗
PubMed1981

Bleomycin therapy and anaesthesia. The possible hazards of oxygen administration to patients after treatment with bleomycin.

Bleomycin is a cytotoxic drug used in the treatment of teratoma of the testis. This drug appears to sensitize the lungs so that acute lung damage occurs with concentrations of oxygen normally considered free from toxic effects. Two anaesthetics administered to the same patient undergoing thoracotomy are reported. No postoperative lung damage was produced on either occasion.

Adult↗
PubMed1987

Activation of bleomycin-Fe(III) by bleomycin-Cu(II) and cysteine.

When bleomycin (BLM) was incubated with DNA at 37 degrees C for 30 minutes in the presence of BLM-Cu(II) and cysteine, the DNA became more acid-soluble compared with the reaction without BLM-Cu(II). Superoxide dismutase did not suppress this increased DNA chain breakage. This combination of BLM-Cu(II) and cysteine did not enhance BLM-Fe(II)-induced DNA chain breakage, but caused the DNA chain breakage by inactive BLM-Fe(III). Combination of BLM-Fe(III) with BLM-Cu(II) and cysteine also caused production of malondialdehyde-like product from DNA. Anaerobic incubation of DNA with BLM-Fe(III) and BLM-Cu(I) followed by aerobic incubation produced malondialdehyde-like product, but the incubation with CuCl instead of BLM-Cu(I) did not. These results suggest that activation of BLM-Fe(III) by BLM-Cu(II) and cysteine seems to be due to the reduction of BLM-Fe(III) to BLM-Fe(II) by BLM-Cu(I) formed in the reaction of BLM-Cu(II) and cysteine.

Animals↗
PubMed1999

A mammalian vector carrying the bleomycin N-acetyltransferase gene from bleomycin-producing Streptomyces verticillus as a selective marker.

A gene, blmB, encodes a bleomycin (Bm) N-acetyltransferase, designated BAT, from Bm-producing Streptomyces verticillus and confers resistance to Bm in Streptomyces and Escherichia coli. COS-1 cells transfected with a plasmid designated pEF-BOS/blmB, in which blmB is under the control of a strong promoter from the human polypeptide chain elongation factor 1alpha, transiently produced BAT. Immuno-cytochemical analysis using an anti-BAT monoclonal antibody revealed that BAT was localized in the nucleus of the blmB-carrying COS-1 cells. NIH/3T3 cells, transfected with pEF-BOS/blmB, stably expressed BAT at least for one month. The stable transformants of blmB showed specific resistance to the Bm family of antibiotics, suggesting that blmB has potential as a selective marker in gene transfer studies with mammalian cells.

Acetyltransferases↗
PubMed1990

Reduction of chromosomal damage by bleomycin in lymphocytes from subjects supplemented with carotenoids. Relevance in bleomycin tumour chemotherapy. Preliminary results.

In a one year-study, 9 healthy human donors were being supplemented with beta-carotene (BC) plus canthaxanthin (CX), to determine the effect of carotenoids on chromosomal damage (micronuclei) induced in the donors' lymphocyte cell cultures by exposure to bleomycin (BLM), an antineoplastic drug that has been shown to produce chromosomal aberrations through the production of free radicals. The first four months monitoring data, including determination of carotenoid blood levels, are here reported. These data show that carotenoid supplementation significantly decrease (up to 50%) the formation of micronuclei induced by BLM in human lymphocyte cell cultures. This decrease is in correlation with carotenoid blood levels.

Adult↗
PubMed1988

Plasma and pulmonary pharmacokinetics of bleomycin in murine strains that are sensitive and resistant to bleomycin-induced pulmonary fibrosis.

Previous studies have shown that C57Bl/6N mice are sensitive and BALB/c mice are resistant to the pulmonary fibrotic effects of bleomycin (BLM). We assessed the plasma elimination and pulmonary content of BLM in C57Bl/6N and BALB/c mice treated with a single dose of [3H]BLM (80 mg/kg i.v.) to determine whether these murine strains show corresponding differences in BLM pharmacokinetics and pulmonary disposition after systemic administration of the drug. Serial blood samples were obtained from each animal and lungs were collected after pulmonary lavage or vascular perfusion with saline. Administration of BLM (80 mg/kg i.v.) produced significant elevations in lung hydroxyproline (35%) in C57Bl/6N but not in BALB/c mice. In contrast, BALB/c mice were more sensitive to pulmonary fibrosis induced with cyclophosphamide (200 mg/kg i.p.) compared to C57Bl/6N mice, indicating that strain sensitivity to pulmonary fibrosis is drug specific in these mice. BLM showed first order plasma elimination kinetics over 30 min in both strains with a shorter half-life in the sensitive strain (9.6 +/- 0.3 min in C57Bl/6N vs. 12.7 +/- 1.9 min in BALB/c). Plasma elimination deviated from first order kinetics after 30 min in both strains and plasma levels of BLM were up to 2-fold higher in the resistant strain over a 3-hr time course. Radioactivity in saline-perfused lungs was also significantly higher (1.5-2-fold) in BALB/c mice for least 1 hr after BLM injection. A similar fraction of the total lung radioactivity (approximately 80%) was recovered from both strains by pulmonary lavage, suggesting that BLM enters the alveolar spaces relatively freely in each strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Acute pulmonary toxicity of bleomycin: DNA scission and matrix protein mRNA levels in bleomycin-sensitive and -resistant strains of mice.

The severity of bleomycin (BLM)-induced pulmonary fibrosis in mice varies markedly among several different murine strains. We have examined the DNA from lungs of sensitive (i.e., C57BL/6N) and resistant (i.e., BALB/c) strains of mice using a nucleoid sedimentation technique to detect early in vivo changes in the integrity of DNA after intravenous BLM. Mice received intravenous injections of BLM (80 mg/kg) or vehicle; lung nucleoids were prepared 15 min to 6 hr later. BLM produced striking decreases in nucleoid sedimentation distance versus paired controls in both strains within 15 min after injection, indicating extensive DNA scission. Repair of DNA strand breaks was complete in the resistant (BALB/c) mice by 5 hr; in contrast, only partial repair occurred in the sensitive (C57BL/6N) strain during that time. We then examined lungs for subsequent changes in steady state poly-(A)+ RNA levels and mRNA levels for lung matrix proteins (type I procollagen, type III procollagen, and fibronectin). Steady state levels of poly-(A)+ RNA were depressed to 50% of control 1 through 6 days after BLM injection in the lungs of sensitive mice. Resistant mice had pulmonary poly-(A)+ RNA levels similar to those of C57BL/6N mice, except for a 2-fold elevation 1 day after BLM injection. BLM injection affected the steady state levels of mRNA encoding lung matrix proteins differently than total poly-(A)+ RNA. Fibronectin mRNA/poly(A)+ RNA was elevated 2-fold 1 day after BLM treatment only in the sensitive strain and remained elevated at 3 and 6 days. In contrast, alpha 2I procollagen mRNA increased in both murine strains and alpha 1III procollagen mRNA decreased in both strains. Thus, a 7-fold or greater increase in the type I: type III procollagen mRNA ratio was seen in both strains 3 to 6 days after BLM injection. These data demonstrate that BLM treatment rapidly produces extensive pulmonary DNA damage in vivo, that persistence of DNA damage rather than the initial level of strand scission is associated with sensitivity to BLM lung disease in these mice, and that changes in the levels of mRNA encoding pulmonary matrix proteins occur in vivo within 1 to 3 days after intravenous BLM treatment.

Animals↗
PubMed1996

Transgenic mice expressing the Sh ble bleomycin resistance gene are protected against bleomycin-induced pulmonary fibrosis.

Despite the high efficiency of bleomycin (BLM) as a chemotherapeutic agent against various carcinomas, the potentially lethal and chronic fibrotic response of the lung is a major dose-limiting side effect. Here, we explore the possibility of a direct inhibition of lung tissue injury by in vivo expression of the actinomycetes BLM resistance protein Sh ble. Transgenic mice expressing the Sh ble gene under the control of a composite viral promoter were produced after introduction of the transgene into D3 ES cells. The protein was detected at high level in lungs, spleen, and kidney. We then assessed its ability to modulate the BLM-induced fibrotic response in the transgenic mice in comparison with C57BL/6 and 129/Sv parental mice. Cumulative doses of 300, 400, or 500 mg/kg BLM were administered either by i.p. or s.c. repeated injections in the different strains. Transgenic mice were shown to be clearly less sensitive to BLM toxicity, as assessed by lung histology. The pulmonary hydroxyproline content in the treated transgenic mice was close to its baseline level, whereas it was up to 50% higher than the control level in C57BL/6 and 129/Sv parental mice. These observations are consistent with the hypothesis that a resistance gene specifically expressed in lungs may prevent the BLM-induced inflammation.

Acetyltransferases↗