Bleomycin

 Bleomycin (BLM), discovered in 1962, is a family of low-molecular-weight metalloglycopeptides )M_r approximately 1500) produced by Streptomyces verticillus and isolated from the culture media of S. verticillus as copper complexes (1-3). It is widely used as an anticancer antibiotic in patients. BLM acts as a limited endonuclease and produces a variety of lesions in DNAs by a mechanism involving free radical attack on deoxyribose in both DNA strands. BLM damages DNAs in ways that mimic ionizing radiation. The detailed knowledge of the complicated chemistry of BLM and its interactions with DNA in vivo and in vitro drives much of its use in applications to molecular biology. BLM and structurally related analogues are considered radiomimetic and oxidative DNA-cleaving reagents, and as such they are utilized as chemical tools to study and understand the activities of this class of agents. Tools of molecular biology are also employed to understand the chemical, biological, and clinical aspects of the mechanism of action of BLMs. In addition, broadly used cloning strategies in several organisms use a gene conferring resistance to BLM and structurally related analogues as a selectable marker. Phleomycin (PLM) is used as the selective agent and is available in commercial formulations. BLM causes multiple changes to cells and is cytocidal, and its cytotoxicity is high where there is no or only a limited barrier to BLM reaching cellular targets. BLM effectively kills all types of cells tested. Cellular resistance is conferred in several ways, including protection by nucleosomes in chromatin, DNA repair, metabolic inactivation of BLM, BLM-resistance proteins, and restricted entry of BLM. An overview of resistance mechanisms was recently published (4).

1.Structure and Activated Complex

 The structurally complex BLMs contain a metal-binding domain and a DNA-binding domain (Fig.( 1. BLMs require metals and oxygen species for their activity (5-9). The metal-binding domain is also the site of oxygen activation and is attached to a disaccharide group; it binds redox-active transition metals, such as Fe(II), Co(II), Cu(II), Ni(II), and Zn(II). The most stable BLM–O₂–metal complex is formed with cobalt (8). When a BLM-Fe(II)-O2 complex binds to DNA and the Fe(II(oxidizes to Fe(III), the complex attacks the C4′ position of DNA deoxyribose (10-13). The complex thereby behaves as a limited endonuclease.

Figure 1. Structures of bleomycins and phleomycins (16, 117-119).

The coplanar bithiazole moiety partially intercalates into the minor groove between bases of DNA. The cationic C-terminal amines are also involved in interactions with nucleic acids. The terminal amines in BLM A₂ and BLM B₂ are dimethylsulfonium propylamine and agmatine, respectively, and are similar in length and bear one positive charge (Fig. 1). BLM B₂ produces considerably more DNA breaks and killing than BLM A₂ over a wide range of chemical concentrations (14). Without the terminal amine, the BLM molecule no longer cleaves DNA or possesses antitumor activity.

BLM and structurally related PLM (15) differ in the oxidation state of their sulfur heterocycles (Fig.( 1. One of the two conjugated thiazole rings of the BLM bithiazole is modified by hydrogenation to 4,5-dihydrothiazole (thiazoline) in PLM (16, 17). In addition, the C-terminal amines in clinical preparations of BLMs differ chemically and quantitatively from prepared mixtures of PLMs. Tallysomycin is closely related to BLM (18, 19).

2. Anticancer Use

 BLM is an important therapeutic agent that is useful as a single agent in treating several human cancers and is widely used in combination chemotherapy and radiotherapy. The water-soluble product used in cancer treatment, Blenoxane, is a family of 11 metal-free congeners differing in their terminal amines. The clinical mixture (20) is comprised mainly of BLM A₂ [approximately 55% to 70% (usually 68% to 69%)] and BLM B₂ (approximately 25% to 32%). The effectiveness of BLM as an anticancer agent is associated with its ability to produce lesions in DNA (21, 22.(

 BLM is principally used in patients with lymphoma or a variety of solid tumors. It has been included in regimens for treating malignant and peripheral T-cell lymphomas, as well as in combination BEP chemotherapy (BLM, etoposide, cisplatin) for metastatic testicular teratoma and Hodgkins and non-Hodgkins lymphoma. BLM is also used in the chemotherapy and management of Kaposi's sarcoma (eg, pulmonary, gastrointestinal, epidemic, disseminated), as well as with vincristine in combination chemotherapy for epidemic Kaposi's sarcoma. BLM does not cause bone-marrow, hepatic, or renal toxicities, nor does it cause cardiosuppression. Pulmonary fibrosis is a side effect of BLM treatment that limits its use. The molecular mechanism of lung fibrosis is being investigated, but is not fully known. Patients with genetic susceptibility are particularly susceptible to lung fibrosis (23, 24). In animal models, taurine (25) or taurine and niacin (26) counter BLM-induced lung fibrosis. The newer BLM derivatives, peplomycin and liblomycin, were developed because of their lower pulmonary toxicity and broader antitumor spectrum in animal studies (27).

 3.Mechanism of Action on Nucleic Acids

3.1.Chemical Action on DNA: DNA Damage 

The unique chemical action of BLMs in the presence of oxygen and Fe(II) catalytically cleaves double-stranded DNA in vivo and in vitro. Single- and double-stranded breaks are produced, leaving 5′-phosphate- and 3′-phosphoglycolate-termini (11, 28-32). The most genotoxic and lethal lesions for cells are double-stranded breaks. BLM recognizes 5′-phosphoguanylyl(3′,5′)thymidine or 5′-phosphoguanylyl(3′,5′)cytosine sequences most frequently, releasing the pyrimidines when they are located to the 3′ side of guanosine (28-30, 33-36) and leaving DNA alkali-labile (32, 37). While cleavage at the first site is G-Py-specific, the second nucleophilic attack by a BLM–Fe(II)–O₂ complex on the opposite DNA strand is not a sequence-specific cleavage and instead is probably targeted by the structural perturbation of the DNA at the first cleavage site (38, 39). Deoxyribose degradation also produces 3-(pyrimidin-1′-yl)-2-propenal and 3-(purin-9′-yl)-2-propenal (11, 31, 40(and oligonucleotide 3′-(phosphoro-2-O-glycolic acid) derivatives (11, 31, 41). DNA strand breaks are stoichiometric with the production of base propenals (42).

3.2.Preferential Cleavage between Nucleosomes 

BLM preferentially cleaves linker regions of chromatin between nucleosomes (43-47). The enhanced resistance of DNA bound to nucleosomes seems to be related to the necessity of a conformational change for BLM binding and intercalation. In yeast, internucleosomal cleavage and DNA degradation (46) and killing (48) are less pronounced in logarithmic phase cells than in cells that are in stationary phase. The cellular and molecular basis for this may relate to the highly effective use of BLM on particular solid tumors.

3.3.Bleomycin and Phleomycin 

PLM was discovered before bleomycin, but was too toxic in patients to be used as an anticancer drug. PLM is also substantially more effective than BLM on a per mole basis in yeast in producing cell killing (48), DNA breaks in intracellular DNAs (49), release of nucleosomes from chromatin (45) ,and genetic changes (50). Thus, the DNA lesions produced by the BLM and PLM could differ in their nature or frequency, or they could be processed differently by the cells. The mechanisms of BLM and PLM interaction with DNA in vitro also appear to differ (51-54). PLM exhibits a higher requirement than BLM for ferrous ions (49). Bithiazole intercalation in DNA is thought to be necessary for producing double-stranded, but not single-stranded, breaks in vitro (52). Accordingly, BLM produces more double-strand breaks than PLM in PM₂ phage DNA (52, 54). Cu(II)BLM, but not Cu(II) PLM, intercalates (51). BLM, but not PLM, degrades relaxed DNA to a greater extent  than either positively or negatively superhelical DNA (54). On the other hand, BLM and PLM cleave DNA in vitro at similar preferred sites at similar frequencies (55, 56) and produce comparable numbers of DNA breaks under some conditions (57, 58).

3.4. RNA

BLM cleaves a variety of RNAs. The most studied have been transfer RNA and tRNA precursors. In contrast to DNA cleavage, BLM-induced RNA cleavage is usually wholly or predominately at a single site, often at a junction between single- and double-stranded RNA regions (59-62).

4.DNA Repair

4.1.Biochemical and Genetic Evidence for Radiomimetic Properties  The cellular processes that repair BLM-induced DNA lesions are not entirely known. The most important mechanisms for the repair of BLM-induced DNA damage are recombinational repair, base-excision repair, and post-replication repair. BLM and ionizing radiation produce similar lesions in DNA, although BLM produces a narrower spectrum of products than ionizing radiation. DNA breaks are introduced approximately linearly with increasing concentrations of BLM (14) and ionizing radiation (63). Some of the chromosomal lesions produced after either BLM treatments or ionizing irradiation can be ligated immediately and lead to a quick component of DNA rejoining, but other lesions require more time for processing before the termini of the DNA molecules become substrates for ligation (14, 63-65). For example, the unusual phosphoglycolate must be removed by the DNA 3′-repair diesterase activity of apurinic and apyrimidinic endonuclease (66, 67). After extension of the remaining DNA strand by one nucleotide, DNA ligase resynthesizes intact DNA molecules by forming a phosphodiester bond between adjacent 3′-hydroxyl and 5′-phosphoryl termini (14). Rapid and slower components of DNA rejoining have been identified in several laboratories. An ultrarapid phase of cellular recovery accompanies rapid repair in human cells (64,( 68, suggesting that some of the cytotoxic treatment effects of BLM could be counteracted in the clinic and reduce chemotherapeutic effectiveness.

The importance of DNA repair is shown by the many studies in various organisms of mutants hypersusceptible to killing by BLM and defective in repair of DNA lesions. All rad mutations of Saccharomyces cerevisiae (69-72) that confer hypersensitivity to killing by BLM analogues also confer cross-sensitivity to ionizing radiation (73-78), so pathways are shared for the repair of chromosomal damage by bleomycins and ionizing radiation. Moreover, mutant strains with altered resistance to lethal effects of BLM have been isolated and characterized in several laboratories, and direct selection for mutations conferring hypersensitivities to lethal effects of BLM resulted in mutants exhibiting cross-hypersensitivities to ionizing radiation.

4.2.Genetic Recombination and Mutation  Mechanisms of recombination are important for DNA repair and rejoining recombinant DNA. BLM is recombinogenic and mutagenic (38, 50, 79-85). The amount of recombination or mutation caused by BLM depends upon the assay system and treatment conditions. BLM was found to be weakly mutagenic to mitochondrial DNA (86).

5.Additional Cellular Targets

5.1.Membrane 

The plasma membrane restricts BLM internalization in some mammalian cells (87, 88). This could be due to the polar and charged groups on the BLM molecule. Membrane damage by BLM (68, 89(or cell electropermealization (88) circumvents this restriction.

5.2.Cell Wall 

BLM molecules are readily taken up into yeast cells, but are not equally distributed from cell to cell (89). BLM initially localizes to cell walls, causes cell wall and membrane damage, and aids the enzymatic conversion of cells to spheroplasts (75, 89-91). BLM alters the anchorage of several mannoproteins in the cell wall matrix of intact cells or isolated cell walls, and it disrupts essential cell wall polymers. These activities facilitate the entry of BLM into yeast cells.

5.3. BLM Hydrolase 

BLM hydrolase hydrolyzes and inactivates BLM. The enzyme is a thiol proteinase that has DNA-binding and peptide-cleavage domains. It binds DNA and RNA. BLM hydrolase activity protects cells from BLM toxicity, but limits the use of BLM in cancer chemotherapy. Although its normal cellular function is unknown, the enzyme is present in diverse organisms, including humans (92-96(and yeast (97-100). Expression of the yeast BLM hydrolase in mammalian cells results in resistance to BLM (101). A member of a galactose regulatory system (102, 103), yeast BLM hydrolase binds to nicked double-stranded DNA, single-stranded DNA, and RNA, without sequence specificity (104). This enzyme associates with plasma membranes and is in the cytosol (105). Human BLM hydrolase was recently shown to exhibit endopeptidase activity (106). This enzyme is thought to play a role in the development of resistance to BLM during chemotherapy (95, 107-109).

5.4. BLM Resistance Proteins 

Several proteins in microorganisms that produce BLM or structural analogues actually confer resistance to these products. Vectors bearing genes encoding these proteins confer high levels of resistance to BLM and related antibiotics and are used as cloning vehicles. The proteins bind and form stable complexes with BLM with high specificity, thereby preventing BLM from complexing with DNA. One of these proteins is encoded by the Streptoalloteichus hindustanus (Sh) ble gene (110-112) found on the Tn5 bacterial transposon, along with additional genes encoding resistance to other antibiotics. Expression of the Sh ble gene in transgenic mice reduced BLM toxicity in the mice and protected against lung fibrosis (113, 114). High levels of the protein were detected in lungs, kidney, and spleen.

5.5.Multiple Targets and Cytotoxicity 

Multiple cellular targets of BLM are expected because of the chemical mechanism of action of the molecule and because some of the BLM-hypersensitive mutants isolated in different organisms do not exhibit hypersensitivity to lethal effects of radiation. Multiple cellular enzymes are important for surviving the toxicities of BLM, and deficiencies in their function could reduce chances of survival. The relationship and significance of each cellular target of BLM to cellular toxicity are not known. Why BLM causes apoptosis in some types of cells and not in others is also unknown. BLM is not considered apoptotic at low concentrations. Far less is known about targets of BLM in cells than about the chemical mechanism of action of BLM in vitro on defined substrates.

Additional factors modulate BLM activity. Generally, BLM preferentially affects mitotic cells in the

G2-M phase of the cell cycle. BLM is unlikely to be a useful agent to study meiosis because of the abundant lesions the molecule produces in DNA. Intracellular metal ion concentrations and pH also modulate BLM activities (48, 115, 116). Studies elucidating the roles of the multiple targets of BLM and factors that modulate BLM activities will improve our understanding and efficacious uses of the widely studied BLM family of related compounds.

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