Antitumor Drug Bleomycin Research Articles

GSH-Responsive Drug Delivery System for Active Therapy and Reducing the Side Effects of Bleomycin.

The application of drug delivery system (DDS) has achieved breakthroughs in many aspects, especially in the field of tumor treatment. In this work, polyethylene glycol (PEG)-modified hollow mesoporous manganese dioxide (HMnO2@PEG) nanoparticles were used to load the anti-tumor drug bleomycin (BLM). When the DDS reached the tumor site, HMnO2@PEG was degraded and reduced to Mn2+ by the overexpression of glutathione in the tumor microenvironment, and the drug was released simultaneously. BLM coordinated with Mn2+ in situ, thereby greatly improving the therapeutic activity of BLM. The results of in vivo and in vitro treatment experiments showed that the DDS had excellent responsive therapeutic activation ability. In addition, Mn2+ exhibited strong paramagnetism and was used for T1-weighted magnetic resonance imaging in vivo. Furthermore, this therapeutic mode of responsively releasing drugs and activating in situ effectively attenuated pulmonary fibrosis initiated by BLM. In short, this DDS could help in avoiding the side effects of drugs.

TRPM2 channels in alveolar epithelial cells mediate bleomycin-induced lung inflammation

Lung inflammation is a major adverse effect of therapy with the antitumor drug bleomycin (BLM). Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable channel that is activated by oxidative stress through the production of ADP-ribose. We herein investigated whether TRPM2 channels contributed to BLM-induced lung inflammation. The intratracheal instillation of BLM into wild-type (WT) mice increased the number of polymorphonuclear leukocytes (PMNs) and inflammatory cytokine levels in the lung. Increases in inflammatory markers in WT mice were markedly reduced in trpm2 knockout (KO) mice, which demonstrated that the activation of TRPM2 channels was involved in BLM-induced lung inflammation. The expression of TRPM2 mRNA was observed in alveolar macrophages, alveolar epithelial cells, and lung fibroblasts. Actually, TRPM2 protein was expressed in lung tissues. Of these, TRPM2 channels in epithelial cells were activated by the addition of H2O2 following a BLM pretreatment, resulting in the secretion of macrophage inflammatory protein-2 (MIP-2). The H2O2-induced activation of TRPM2 by the BLM pretreatment was blocked by the poly(ADP-ribose) polymerase (PARP) inhibitors PJ34 and 3-aminobenzamide. The accumulation of poly(ADP-ribose) in the nucleus, a marker for ADP-ribose production, was strongly induced by H2O2 following the BLM pretreatment. Furthermore, administration of PRAP inhibitors into WT mice markedly reduced recruitment of inflammatory cells and MIP-2 secretion induced by BLM instillation. These results suggest that the induction of MIP-2 secretion through the activation of TRPM2 channels in alveolar epithelial cells is an important mechanism in BLM-induced lung inflammation, and the TRPM2 activation is likely to be mediated by ADP-ribose production via PARP pathway. TRPM2 channels may be new therapeutic target for BLM-induced lung inflammation.

Ultrasensitive electrogenerated chemiluminescent DNA-based biosensing switch for the determination of bleomycin

An ultrasensitive electrogenerated chemiluminescent (ECL) DNA-based biosensing switch for the determination of bleomycin (BLM) was developed based on Fe(II) ·BLM-mediated hairpin DNA strand cleavage and a structure-switching ECL-dequenching mechanism. A thiolated ss-DNA was used as a substrate for BLMs: one terminus was tethered onto an electrode surface, and the other terminus was labelled with the ECL quencher ferrocene to form a hairpin structure. This thiolated ss-DNA self-assembled on to the tris(2,2′-bipyridine)ruthenium-gold nanoparticle composite modified gold electrode. In the presence of Fe(II) ·BLM, the ECL DNA biosensing switch undergoes an irreversible cleavage event that can trigger a significant increase in ECL intensity. The relationship of ECL intensity and the concentration of BLMs was found to be linear in the range of 5fM – 5000fM with a detection limit of 2fM. This work demonstrates that the design of a highly sensitive ECL DNA-based biosensing switch that uses the sequence selectivity of DNA cleavage mediated by the antitumor drug BLM in combination with a chemical quencher, such as ferrocene, to quench ECL signal(s), offers a promising approach for the determination of ultratrace amounts of antitumor drugs.

Dérivé de la bléomycine générant moins de ROS ? Moins de fibrose ? Une alternative dans le développement d’une thérapie anticancéreuse efficace mais moins toxique

Deglycobleomycin (DBLM), the aglycon of the glycopeptide antitumor drug bleomycin (BLM), was first used since 1980 during comparative studies between BLM and DBLM in order to elucidate the role of the sugar component in the mechanism of action of BLM. In fact, the deglycosylation of BLM reduce the toxicity of this molecule and fails to produce reactive oxygen species, responsible for pulmonary fibrosis, and for anti-neoplastic activity of BLM. This causes toxic DNA lesions and ultimately leads to cell death. The therapeutic use of BLM is limited by a dose-dependent lung toxicity that eventually leads to fibrosis. Testing BLM-derivative molecules and defining their molecular mechanisms involved in tumor cell death may help to design new therapeutic approach with limited toxicity profile while maintaining anti-tumoral properties. The present review was undertaken to determine the effect of carbohydrate moiety in the mechanism of BLM induced cytotoxicity behind a comparative studies between BLM and DBLM.

Tandem Mass Spectrometry-Based Detection of C4′-Oxidized Abasic Sites at Specific Positions in DNA Fragments

Oxidative damage to DNA has been linked to aging, cancer, and other biological processes. Reactive oxygen species and various antitumor agents including bleomycin and ionizing radiation have been shown to cause oxidative DNA sugar damage. Detection of DNA lesions is important for understanding the toxicological or therapeutic consequences associated with such agents. C4'-oxidized abasic sites (C4-AP) are produced by the antitumor drug bleomycin and ionizing radiation. The currently available methods for the detection of C4-AP cannot provide both structural and sequence information. We have developed an LC-ESI-MS-based approach for specific detection and mapping of C4-AP from a mixture of lesions. We show using Fe-bleomycin-damaged DNA that C4-AP can be detected at cytosine and thymine sites by direct MS analysis. Our results reveal that collision-induced dissociation of C4-AP-containing oligonucleotides results in preferential fragmentation at C4-AP sites with the formation of the unique a* ions (18 amu more than the a-B ions) that allow mapping of the C4-AP sites. Various chemical modification strategies (e.g., reduction with NaBH4 and NaBD4 and derivatization with methoxyamine and hydrazine, followed by LC-MS analysis) were also used for unambiguous detection of C4-AP sites. Finally, we show that the methods described here can detect the presence of C4-AP at specific sites in a complex sample such as hydroxyl radical-damaged DNA. The LC-MS approach was also used for the simultaneous detection of the other C4'-oxidation end product, 3'-phosphoglycolate, at a specific site in hydroxyl radical-damaged DNA. Thus, LC-MS provides a rapid and direct approach for the detection and mapping of oxidative DNA lesions.

Deletion of the chromatin remodeling gene SPT10 sensitizes yeast cells to a subclass of DNA‐damaging agents

The Saccharomyces cerevisiae SPT10 protein possesses a DNA-binding domain that is fused to a putative histone acetyltransferase domain. It binds specifically to upstream-activating sequence elements in the core histone promoters and plays a direct role in histone gene regulation. SPT10 is also required for cell-cycle-specific K56 acetylation at histone genes, allowing the recruitment of the nucleosome remodeling factor Snf5 and subsequent regulation of gene transcription. We reisolated the SPT10 gene in a functional genome-wide screen designed to identify haploid yeast mutants that are hypersensitive to the antitumor drug bleomycin, which acts by damaging DNA. In addition to bleomycin, we show that spt10Delta mutants are also hypersensitive to a limited set of genotoxic agents that create DNA strand breaks, but not to 254-nm ultraviolet light or 4-nitroquinoline-1-oxide, which generate helix distortion. The hypersensitivities of the spt10Delta mutant to the genotoxic agents are rescued by a single copy plasmid carrying the SPT10 gene. We further showed that spt10Delta mutants displayed a modest twofold increase spontaneous mutant frequency, as compared to the parent. Following exposure to bleomycin, these mutants accumulate unrepaired lesions, e.g., DNA strand breaks with blocked 3'-ends in the chromosomal DNA. This defect is not due to the altered expression level or the enzymatic activities of a key DNA repair enzyme, APN1, which is known to repair DNA strand breaks with blocked ends. We propose that SPT10 mediates repair of a subset of DNA lesions by acetylating histones to promote recruitment of DNA repair enzymes.

Iron coordination chemistry of N-(bis(2-pyridyl)methyl)pyridine-2-carboxamide

The amidate function participates in the coordination chemistry of iron containing biomolecules such as the anti-tumor drug bleomycin and the enzyme nitrile hydratase. Our interest in amidate coordination prompted an investigation of the iron complexes of the potentially tetradentate ligand N-(bis(2-pyridyl)methyl)pyridine-2-carboxamide (HL). A number of complexes have been isolated and structurally characterized, including [FeII(L)2] (1), [FeIII(LOCH3)Br2(CH3OH)] (3), [FeIII2(μ-OH)2(LOCH3)2Br2] (4), and [FeIII4(μ-OCH3)2(LO)2Br6] (5). In these complexes L acts as a meridional tridentate ligand, as previously observed for the corresponding [Cu(L)Cl(CH3OH)] complex (Inorg. Chem. 39 (2000) 5326). In the cases of 3 and 4, the hydrogen of the tertiary carbon has been replaced by a methoxy group in the course of complex synthesis. In the case of 5, the tertiary hydrogen is replaced by hydroxide, and this oxygen and the dangling pyridine act as a bidentate ligand to a second iron ion. When the reaction of FeBr3 and HL was carried out in acetonitrile in the presence of base but in the absence of air, the ligand was cleaved into two pieces, affording [FeIIBr2(pyridine-2-carboxamide)(di-2-pyridylketone)] (6). It is proposed that the coordination of the amide nitrogen of HL to an iron(III) center as an amidate activates the α-CH bond and results in the oxidation of the α-CNamide bond to an imine.

Cloning and characterization of a phosphopantetheinyl transferase from Streptomyces verticillus ATCC15003, the producer of the hybrid peptide–polyketide antitumor drug bleomycin

Background: Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of carrier proteins by the covalent attachment of the 4′-phosphopantetheine (P-pant) moiety of coenzyme A to a conserved serine residue, a reaction absolutely required for the biosynthesis of natural products including fatty acids, polyketides, and nonribosomal peptides. PPTases have been classified according to their carrier protein specificity. In organisms containing multiple P-pant-requiring pathways, each pathway has been suggested to have its own PPTase activity. However, sequence analysis of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 failed to reveal an associated PPTase gene. Results: A general approach for cloning PPTase genes by PCR was developed and applied to the cloning of the svp gene from S. verticillus. The svp gene is mapped to an independent locus not clustered with any of the known NRPS or PKS clusters. The Svp protein was overproduced in Escherichia coli, purified to homogeneity, and shown to be a monomer in solution. Svp is a PPTase capable of modifying both type I and type II acyl carrier proteins (ACPs) and peptidyl carrier proteins (PCPs) from either S. verticillus or other Streptomyces species. As compared to Sfp, the only ‘promiscuous’ PPTase known previously, Svp displays a similar catalytic efficiency ( k cat/ K m) for the BlmI PCP but a 346-fold increase in catalytic efficiency for the TcmM ACP. Conclusions: PPTases have recently been re-classified on a structural basis into two subfamilies: ACPS-type and Sfp-type. The development of a PCR method for cloning Sfp-type PPTases from actinomycetes, the recognition of the Sfp-type PPTases to be associated with secondary metabolism with a relaxed carrier protein specificity, and the availability of Svp, in addition to Sfp, should facilitate future endeavors in engineered biosynthesis of peptide, polyketide, and, in particular, hybrid peptide–polyketide natural products.

DFT Study of the Metal Coordination Center Domain of Fe(II)−Bleomycin

We present a comprehensive analysis of the geometric and electronic properties of the metal coordination center domain of the iron(II) complex of the antitumor drug bleomycin (BLM) using density functional theory (DFT). The various experimental studies devoted to establishing the nature of the metal coordination center in BLM suggest that the metal is five-coordinated, with a sixth site available for exogenous ligand binding. Among the five ligands, four of them form an equatorial coordination sphere, but the nature of the fifth axial ligand remains controversial. To elucidate the nature of the metal coordination center in Fe(II)−BLM, six structurally different conformers resembling the active site have been investigated using DFT. An optimal geometry has been found for each conformer in the singlet, triplet, and quintet electronic states. The electronic ground state was identified as a four-coordinate species with intermediate spin state. However, when the results of these calculations were compared with the latest 2D NMR data, the best agreement was found between the five-coordinated theoretical model and experiment. Stereoelectronic analysis of four-coordinate conformers has shown that the Fe out-of-plane displacement controls the spin state. The high-spin state potential energy surface intersects the triplet state potential energy surface along the doming-mode coordinate when the iron atom is displaced about 0.4 Å from the equatorial nitrogens average plane. This intersection is the result of different iron d orbital occupancy in quintet and triplet electronic states.

An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003

We have previously proposed that the BlmIV and BlmIII non-ribosomal peptide synthetases are involved in the formation of the bithiazole moiety of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003. We report here the identification and characterization of an oxidation domain in BlmIII. The oxidation domain shows local homology to a family of oxidoreductases and is present in all thiazole-forming non-ribosomal peptide synthetase modules known to date. Both the blmIII-Ox domain and blmIII gene were expressed in Escherichia coli, and the resulting BlmIII-Ox and BlmIII proteins were purified to homogeneity. The oxidation domain contains one molar equivalent of non-covalently bound FMN as a prosthetic group. These results provide experimental evidence for an oxidation domain within non-ribosomal peptide synthetases, suggesting that BlmIII-Ox probably catalyzes the thiazoline to thiazole oxidation in bleomycin biosynthesis.

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

Background: The structural and catalytic similarities between modular nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) inspired us to search for a hybrid NRPS–PKS system. The antitumor drug bleomycin (BLM) is a natural hybrid peptide–polyketide metabolite, the biosynthesis of which provides an excellent opportunity to investigate intermodular communication between NRPS and PKS modules. Here, we report the cloning, sequencing, and characterization of the BLM biosynthetic gene cluster from Streptomyces verticillus ATCC15003. Results: A set of 30 genes clustered with the previously characterized blmAB resistance genes were defined by sequencing a 85-kb contiguous region of DNA from S. verticillus ATCC15003. The sequenced gene cluster consists of 10 NRPS genes encoding nine NRPS modules, a PKS gene encoding one PKS module, five sugar biosynthesis genes, as well as genes encoding other biosynthesis, resistance, and regulatory proteins. The substrate specificities of individual NRPS and PKS modules were predicted based on sequence analysis, and the amino acid specificities of two NRPS modules were confirmed biochemically in vitro. The involvement of the cloned genes in BLM biosynthesis was demonstrated by bioconversion of the BLM aglycones into BLMs in Streptomyces lividans expressing a part of the gene cluster. Conclusion: The blm gene cluster is characterized by a hybrid NRPS–PKS system, supporting the wisdom of combining individual NRPS and PKS modules for combinatorial biosynthesis. The availability of the blm gene cluster has set the stage for engineering novel BLM analogs by genetic manipulation of genes governing BLM biosynthesis and for investigating the molecular basis for intermodular communication between NRPS and PKS in the biosynthesis of hybrid peptide–polyketide metabolites.

An oxidation domain in the BlmIII non-ribosomal peptide synthetase probably catalyzing thiazole formation in the biosynthesis of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003.

We have previously proposed that the BlmIV and BlmIII non-ribosomal peptide synthetases are involved in the formation of the bithiazole moiety of the anti-tumor drug bleomycin in Streptomyces verticillus ATCC15003. We report here the identification and characterization of an oxidation domain in BlmIII. The oxidation domain shows local homology to a family of oxidoreductases and is present in all thiazole-forming non-ribosomal peptide synthetase modules known to date. Both the blmIII-Ox domain and blmIII gene were expressed in Escherichia coli, and the resulting BlmIII-Ox and BlmIII proteins were purified to homogeneity. The oxidation domain contains one molar equivalent of non-covalently bound FMN as a prosthetic group. These results provide experimental evidence for an oxidation domain within non-ribosomal peptide synthetases, suggesting that BlmIII-Ox probably catalyzes the thiazoline to thiazole oxidation in bleomycin biosynthesis.

Catalytic Oxidation with a Non-Heme Iron Complex That Generates a Low-Spin FeIIIOOH Intermediate

The antitumor drug bleomycin (BLM) is proposed to act via a low-spin iron(III) hydroperoxide intermediate called "activated bleomycin". To gain more insight into the mechanistic aspects of catalytic oxidation by these intermediates we have studied the reactivity of [(N4Py)Fe(CH3CN)](ClO4)2 (1) (N4Py = N,N-bis(2-pyridylmethyl)-N-bis(2-pyridyl)methylamine) with excess H2O2. Under these conditions a transient purple species is generated, [(N4Py)FeOOH]2- (2), which has spectroscopic features and reactivity strongly reminiscent of activated bleomycin. The catalytic oxidation of alkanes such as cyclohexane, cyclooctane, and adamantane by 1 with H2O2 gave the corresponding alcohols and ketones in up to 31% yield. It was concluded, from the O2 sensitivity of the oxidation reactions, the formation of brominated products in the presence of methylene bromide, and the nonstereospecificity of the oxidation of cis- or trans-dimethylcyclohexane, that long-lived alkyl radicals were formed during the oxidations. Oxidation of alkenes did not afford the corresponding epoxides in good yields but resulted instead in allylic oxidation products in the case of cyclohexene, and cleavage of the double bond in the case of styrene. Addition of hydroxyl radical traps, such as benzene and acetone, led to only partial quenching of the reactivity. The kinetic isotope effects for cyclohexanol formation, ranging from 1.5 in acetonitrile to 2.7 in acetone with slow addition of H2O2, suggested the involvement of a more selective oxidizing species in addition to hydroxyl radicals. Monitoring the UV/Vis absorption of 2 during the catalytic reaction showed that 2 was the precursor for the active species. On the basis of these results it is proposed that 2 reacts through homolysis of the O-O bond to afford two reactive radical species: [(N4Py)Fe(IV)O]2+ and *OH. The comparable reactivity of 1 and Fe-BLM raises the possibility that they react through similar mechanistic pathways.

An allele of the yeast RPB7 gene, encoding an essential subunit of RNA polymerase II, reduces cellular resistance to the antitumor drug bleomycin

Bleomycin is an antitumor drug that kills cells by introducing lesions in DNA. Thus, normal cells exposed to bleomycin must rely on efficient DNA repair mechanisms to survive. In the yeast Saccharomyces cerevisiae, the transcriptional activator Imp2 is required to fend off the toxic effects of bleomycin. However, it remains unclear whether Imp2 controls the expression of a protein that either repairs bleomycin-induced DNA lesions, or detoxifies the drug, and or both. To gain further insight into the mechanisms by which yeast cells mount a response towards bleomycin, we began to sequentially characterize the genetic defect in a collection of bleomycin-sensitive mutants that were previously isolated by mini-Tn3 transposon mutagenesis. A rescue plasmid designed to integrate at the site of the mini-Tn3 insertion was used to identify the defective gene in one of the mutant strains, HCY53, which was not allelic to IMP2. We showed that in strain HCY53, the mini-Tn3 was inserted at the distal end of an essential gene RPB7, which encodes one of the two subunits, Rpb4-Rbp7, that forms a subcomplex with RNA polymerase II. Since rpb7 null mutants are nonviable, it would appear that the rpb7::mini-Tn3 allele produces a protein that retains partial biological function thus permitting cell viability, but which is unable to provide bleomycin resistance to strain HCY53. The defective phenotype of strain HCY53 could be corrected by a plasmid bearing the entire RPB7 gene. Two dimensional gel analysis revealed that the expression of several proteins were diminished or absent in the rpb7::mini-Tn3 mutant when challenged with bleomycin. These results are in accord with our previous report that bleomycin resistance in yeast is controlled at the transcriptional level.

An allele of the yeast RPB7 gene, encoding an essential subunit of RNA polymerase II, reduces cellular resistance to the antitumor drug bleomycin

Bleomycin is an antitumor drug that kills cells by introducing lesions in DNA. Thus, normal cells exposed to bleomycin must rely on efficient DNA repair mechanisms to survive. In the yeast Saccharomyces cerevisiae, the transcriptional activator Imp2 is required to fend off the toxic effects of bleomycin. However, it remains unclear whether Imp2 controls the expression of a protein that either repairs bleomycin-induced DNA lesions, or detoxifies the drug, and or both. To gain further insight into the mechanisms by which yeast cells mount a response towards bleomycin, we began to sequentially characterize the genetic defect in a collection of bleomycin-sensitive mutants that were previously isolated by mini-Tn3 transposon mutagenesis. A rescue plasmid designed to integrate at the site of the mini-Tn3 insertion was used to identify the defective gene in one of the mutant strains, HCY53, which was not allelic to IMP2. We showed that in strain HCY53, the mini-Tn3 was inserted at the distal end of an essential gene RPB7, which encodes one of the two subunits, Rpb4-Rbp7, that forms a subcomplex with RNA polymerase II. Since rpb7 null mutants are nonviable, it would appear that the rpb7::mini-Tn3 allele produces a protein that retains partial biological function thus permitting cell viability, but which is unable to provide bleomycin resistance to strain HCY53. The defective phenotype of strain HCY53 could be corrected by a plasmid bearing the entire RPB7 gene. Two dimensional gel analysis revealed that the expression of several proteins were diminished or absent in the rpb7::mini-Tn3 mutant when challenged with bleomycin. These results are in accord with our previous report that bleomycin resistance in yeast is controlled at the transcriptional level.

Excision of C-4′-oxidized Deoxyribose Lesions from Double-stranded DNA by Human Apurinic/Apyrimidinic Endonuclease (Ape1 Protein) and DNA Polymerase β

Oxidative damage to DNA deoxyribose generates oxidized abasic sites (OAS) that may constitute one-third of ionizing radiation damage. The antitumor drug bleomycin produces exclusively OAS in the form of C-4-keto-C-1-aldehydes in unbroken DNA strands and 3'-phosphoglycolate esters terminating strand breaks. We investigated whether two human DNA repair enzymes can mediate OAS excision in vitro: Ape1 protein (the main human abasic endonuclease (also called Hap1, Apex, or Ref1)) and DNA polymerase beta, which carries out both the abasic excision and the resynthesis steps. We used a duplex oligonucleotide substrate with one main target for bleomycin-induced damage. Ape1 catalyzed effective incision at the C-4-keto-C-1-aldehyde sites at a rate that may be only a few-fold lower than incision of hydrolytic abasic sites at the same location. Consistent with several previous studies, Ape1 hydrolyzed 3'-phosphoglycolates 25-fold more slowly than C-4-keto-C-1-aldehydes. DNA polymerase beta excised the 5'-terminal OAS formed by Ape1 incision at a rate similar to its removal of unmodified abasic residues. Polymerase beta-mediated excision of 5'-terminal OAS was stimulated by Ape1 as it is for unmodified abasic sites. Escherichia coli Fpg (MutM) protein also excised 5'-terminal OAS, but in our hands, the RecJ protein did not. These observations help define mammalian pathways of OAS repair, point to interactions that might coordinate functional steps, and suggest that still unknown factors may contribute to removal of 3'-phosphoglycolate esters.

Bleomycin-induced arthritis and dermatitis in rats.

We investigated the effect of the antitumor drug bleomycin (BLM) on synovial membrane and periarticular deep dermis in 10-wk-old young adult rats. BLM was found to induce edema, mononuclear cell infiltration and necrosis of the synovial membrane in the knee and tarsal joint, and inflammation in the deep dermis of the plantar hindfoot and digital pulvini in these rats after subcutaneous administration of 20 mg/kg for 3 days. After a 4-wk recovery period, foci of degenerative collagen bundles were observed in the deep dermis of the plantar hindfoot in spite of complete recovery of the lesions in the other dermal and synovial membrane sites. The synovitis was determined to begin with vesiculation of the macrophage-type lining cells, followed by edema and cell infiltration, especially near ligament insertion sites in the knee joint. The early dermal lesion consisted of dissociation of endothelial and subendothelial cells in small blood vessels thought to be postcapillary venules, edema, and monocyte infiltration. The severity of arthritis was greater in young adults than juvenile rats. From these results, BLM was shown to have a toxic effect on synovial lining cells and to induce inflammation in the synovial membrane and periarticular dermis.

NMR studies of the paramagnetic complex Fe(II)-bleomycin.

The coordination chemistry of the iron(II) complex of the antitumor drug bleomycin has been extensively investigated with a number of spectroscopic and chemical techniques. However, the actual structure of this complex is not established. In this report, we present NMR studies of the paramagnetic Fe(II)BLM and use one- and two-dimensional methods to assign the paramagnetically shifted features to particular protons. The data analysis points toward the primary and secondary amines of the beta-aminoalanine fragment, the pyrimidine and imidazole rings, and the amide nitrogen of the beta-hydroxyhistidine fragment as ligands to the metal center. Correlation of the T1 values with the metal-proton distances derived from the NMR-generated solution structure of HOO-Co(III)BLM [Wu, W., Vanderwall, D. E., Lui, S. M., Tang, X.-J., Turner, C. J., Kozarich, J. W., & Stubbe, J. (1996) J. Am. Chem. Soc. 118, 1268-1280] indicates that the two metallobleomycins share similar structures. The chemical shifts as well as the T1 values of the sugar protons indicate that these fragments are close but not bound to the metal in Fe(II)BLM.

In vitro detection of endonuclease IV-specific DNA damage formed by bleomycin in vivo.

Endonuclease IV of Escherichia coli has been implicated by genetic studies in the repair of DNA damage caused by the antitumor drug bleomycin, but the lesion(s) recognized by this enzyme in vivo have not been identified. We used the sensitive primer activation assay, which monitors the formation of 3'-OH groups that support in vitro synthesis by E.coli DNA polymerase I, to determine whether endonuclease IV-specific damage could be detected in the chromosomal DNA of cells lacking the enzyme after in vivo treatment with bleomycin. Chromosomal DNA isolated after a 1 h bleomycin treatment from wild-type, endonuclease IV-deficient (nfo-) and endonuclease IV-overproducing (p-nfo; approximately 10-fold) strains all supported modest polymerase activity. However, in vitro treatment with purified endonuclease IV activated subsequent DNA synthesis with samples from the nfo- strain (an average of 2.6-fold), to a lesser extent for samples from wild-type cells (2.1-fold), and still less for the p-nfo samples (1.5-fold). This pattern is consistent with the presence of unrepaired damage that correlates inversely with the in vivo activity of endonuclease IV. Incubation of the DNA from bleomycin-treated nfo- cells with polymerase and dideoxynucleoside triphosphates lowered the endonuclease IV-independent priming activity, but did not affect the amount of activation seen after endonuclease IV treatment. Primer activation with DNA from the nfo- strain could also be obtained with purified E.coli exonuclease III in vitro, but a quantitative comparison demonstrated that endonuclease IV was > or = 5-fold more active in this assay. Thus, endonuclease IV-specific damage can be detected after in vivo exposure to bleomycin. These may be 2-deoxy-pentos-4-ulose residues, but other possibilities are discussed.

Photoinduced DNA Cleavage Reactions by Designed Analogues of Co(III)-Bleomycin: The Metalated Core Is the Primary Determinant of Sequence Specificity.

A 2,4'-bithiazole group has been covalently attached to the Co(III) complex of a designed ligand PMAH that mimics the metal-binding locus of the antitumor drug bleomycin (BLM). The deprotonated PMA(-) ligand binds Co(III) via five nitrogens located in primary and secondary amines, a pyrimidine and an imidazole ring, and a peptide moiety. The 2,4'-bithiazole group is tethered to the [Co(PMA)](2+) unit via an imidazole that is connected to the bithiazole moiety with a (CH(2))(3) spacer. The structure of this hybrid analogue, namely, [Co(PMA)(Bit)]Cl(2) (7, Bit = 2'-methyl-2,4'-bithiazole-4-carboxamido-N'-(3-propyl)imidazole) has been established by spectroscopic techniques. 7 promotes photocleavage of DNA at micromolar concentrations. Unlike simpler analogues like [Co(PMA)(H(2)O)](2+) and [Co(PMA)Cl)](+) which induce random DNA cleavage upon UV illumination, 7 exhibits sequence specificity in the DNA photocleavage reaction. Intriguing is to note that 7 exhibits the same 5'GG-N3' sequence preference as another hybrid analogue [Co(PMA)(Int-A)]Cl(2) (6, Int-A = acridine-9-carboxamido-N'-(3-propyl)imidazole) that contains an acridine moiety as the DNA-binding group. The observed sequence specificity of 6 and 7 therefore does not reflect the sequence preferences of the DNA-binding groups (acridine and bithiazole). The results indicate that the metalated core of the hybrid analogues, i.e., the [Co(PMA)](2+) unit is the key factor in determining their sequence specificity.