Gene/Protein Disease Symptom Drug Enzyme Compound
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The nuclear enzyme DNA topoisomerase II catalyzes the breakage and resealing of duplex DNA and plays an important role in several genetic processes. It also mediates the DNA cleavage activity and cytotoxicity of clinically important anticancer agents such as etoposide. We have examined the activity of topoisomerase II during the first cell cycle of quiescent BALB/c 3T3 cells following serum stimulation. Etoposide-mediated DNA break frequency in vivo was used as a parameter of topoisomerase II activity, and enzyme content was assayed by immunoblotting. Density-arrested A31 cells exhibited a much lower sensitivity to the effects of etoposide than did actively proliferating cells. Upon serum stimulation of the quiescent cells, however, there was a marked increase in drug sensitivity which began during S phase and reached its peak just before mitosis. Maximal drug sensitivity during this period was 2.5 times greater than that of log-phase cells. This increase in drug sensitivity was associated with an increase in intracellular topoisomerase II content as determined by immunoblotting. The induction of topoisomerase II-mediated drug sensitivity was aborted within 1 h of exposure of cells to the protein synthesis inhibitor cycloheximide, but the DNA synthesis inhibitor aphidicolin had no effect. In contrast to the sensitivity of cells to drug-induced DNA cleavage, maximal cytotoxicity occurred during S phase. A 3-h exposure to cycloheximide before etoposide treatment resulted in nearly complete loss of cytotoxicity. Our findings indicate that topoisomerase II activity fluctuates with cell cycle progression, with peak activity occurring during the G2 phase. This increase in topoisomerase II is protein synthesis dependent and may reflect a high rate of enzyme turnover. The dissociation between maximal drug-induced DNA cleavage and cytotoxicity indicates that the topoisomerase-mediated DNA breaks may be necessary but are not sufficient for cytotoxicity and that the other factors which are particularly expressed during S phase may be important as well.
Mol Cell Biol 1987 Sep
PMID:Topoisomerase-specific drug sensitivity in relation to cell cycle progression. 282 20

Apoptosis is characterized by the nonrandom cleavage of DNA. After continuous treatment of MOLT-4 human T lymphoblastoid cells with the topoisomerase II inhibitor etoposide (50 microM) and the nongenotoxic agent N-methylformamide (300 mM), apoptosis was confirmed by electron microscopy. Analysis of DNA integrity by conventional gel electrophoresis failed to detect internucleosomal DNA cleavage. Resolution of DNA by field inversion gel electrophoresis showed fragments of 50 kilobases (kb). Etoposide induced the transient appearance of an additional DNA band of > 600 kb, which was temporally coincident with DNA-protein complex formation and was rapidly reversible upon drug removal. This DNA band was not observed after N-methylformamide treatment. In situ DNA end-labeling showed the incorporation of biotinylated dUTP into 50-kb DNA fragments but not etoposide-induced DNA fragments of > 600 kb. DNA end-labelling with terminal deoxynucleotidyltransferase was therefore not dependent upon intenucleosomal DNA cleavage, and fragments of approximately 50 kb were characterized by free 3'-OH termini that were not occluded by topoisomerase II protein. Although we considered that topoisomerase II potentially played an active role in the fragmentation of higher order chromatin during apoptosis, the results showed that DNA cleavage by topoisomerase II induced reversible, protein-associated fragments of > 600 kb and not irreversible cleavage to 50-kb fragments. The reversible cleavage of DNA to fragments of > 600 kb appears to be a signal for the engagement of apoptosis and is not an initial step in the sequential unwinding of chromatin.
Mol Pharmacol 1995 May
PMID:Investigation of the mechanism of higher order chromatin fragmentation observed in drug-induced apoptosis. 774 85

The genotoxic and cytotoxic effects of etoposide (VP-16), a topoisomerase II inhibitor, on male rat spermatogenic cells were studied by analysing induction of micronuclei during meiosis. Micronuclei (MN) were scored in early spermatids after different time intervals corresponding to exposure of different stages of meiotic prophase. Etoposide had a strong effect on diplotene-diakinesis I cells harvested 1 day after exposure, and a significant effect also on late pachytene cells harvested 3 days after exposure. The effect at 18 days corresponding to exposure of preleptotene stage of meiosis (S-phase) was weaker but also statistically significant. Adriamycin was used as a positive control in this study. The results indicate a different mechanism of action of etoposide compared with adriamycin and other chemicals studied previously with the spermatid micronucleus test. DNA flow cytometry was carried out to assess cytotoxic damage at the same time intervals (1, 3, and 18 days after treatment) at stages I and VII of the seminiferous epithelial cycle allowing a study of cytotoxicity to different spermatogenic cell stages. Damage of differentiating spermatogonia was observed by a decrease in the cell numbers of the 2C peak 1 and 3 days after treatment and by a reduction of the number of 4C cells (primary spermatocytes) 18 d after etoposide treatment. Adriamycin also killed differentiating spermatogonia. Since the cell population which showed a high induction of MN by etoposide was not reduced in number, the genotoxic effect is remarkable. We conclude that etoposide is a potent inducer of genotoxicity and patients treated with this agent during cancer chemotherapy are at a risk of genetic damage.
Environ Mol Mutagen 1994
PMID:Etoposide (VP-16) is a potent inducer of micronuclei in male rat meiosis: spermatid micronucleus test and DNA flow cytometry after etoposide treatment. 795 23

Etoposide (VP-16) is one of several DNA-damaging agents that induce subcellular structural changes associated with apoptosis. VP-16 exerts its DNA-damaging and cytotoxic effects subsequent to interference with DNA topoisomerase II activity. VP-16 also stimulates c-jun and c-fos mRNA expression in some cell lines, including human leukemia K562 and HL-60 cells. To compare the temporal relationship between drug-induced c-jun expression and apoptosis, we examined cell morphology, cell viability, DNA integrity, and c-jun induction during VP-16 treatment of K562 and HL-60 cells. VP-16 (10 microM)-induced internucleosomal DNA damage and nuclear fragmentation were readily apparent within 6 hr in HL-60 cells but were absent in K562 cells treated for up to 24 hr. Some internucleosomal DNA damage was observed in K562 cells but only after treatment with 100 microM VP-16 for 24 hr. In contrast, VP-16-induced DNA single-strand breaks, VP-16-induced topoisomerase II/DNA covalent complex formation, and VP-16-mediated growth inhibition were similar in K562 and HL-60 cells. Also, the time course of VP-16-induced c-jun mRNA expression was comparable for both K562 and HL-60 cell lines. Western blot analysis of whole-cell lysates showed that Bcl-2 protein levels were 13-fold greater in HL-60 cells than in K562 cells. Thus, the resistance of VP-16-treated K562 cells to apoptosis was not attributable to protection by Bcl-2. Furthermore, the relatively high levels of Bcl-2 in HL-60 cells were not sufficient to protect these cells against apoptosis. Together, our results indicate that the temporal coupling of VP-16-induced DNA damage, c-jun expression, and apoptosis is cell type specific and suggest that different signaling pathways for apoptosis are operating in these two human leukemia cell lines.
Mol Pharmacol 1994 Oct
PMID:Differential induction of etoposide-mediated apoptosis in human leukemia HL-60 and K562 cells. 796 39

The mechanism by which etoposide, a topoisomerase II inhibitor, killed replicating mouse L929 fibroblasts was investigated. Etoposide at 10 microM killed 70% of the cells within 4 days, a result that was accompanied by DNA fragmentation. A characteristic "ladder" pattern of DNA fragmentation was confirmed by agarose gel electrophoresis. Simultaneous exposure of the cells to 10 microM etoposide plus 1 microM cycloheximide reduced both the extent of cell killing and the fragmentation of DNA. Delayed addition of cycloheximide protected cells only if cycloheximide was added 1-6 hr after exposure to etoposide. When added 6-24 hr after treatment with etoposide, cycloheximide lost the ability to protect cells. Cell growth was completely inhibited by either etoposide or cycloheximide. Furthermore, DNA synthesis was inhibited by either etoposide or cycloheximide within 6 hr. Protein synthesis, however, was not inhibited by etoposide. Thus, the ability of cycloheximide to protect cells correlated with inhibition of protein synthesis, rather than inhibition of DNA synthesis. A 1-hr exposure to 2.5 mM N-methyl-N-nitrosourea similarly inhibited DNA synthesis within 6 hr. without affecting protein synthesis. However, no loss of viability accompanied N-methyl-N-nitrosourea treatment. Thus, an imbalance between protein synthesis and DNA synthesis cannot explain the cell killing by etoposide. H-7, a protein kinase C inhibitor, prevented the cell killing and DNA fragmentation, whereas aurintricarboxylic acid, an endonuclease inhibitor, reduced the extent of DNA fragmentation but did not have an effect on cell killing. The data document that the killing of replicating mouse fibroblasts by etoposide represents an example of programmed cell death (apoptosis) that depends on protein synthesis. Although protein synthesis is required during the first 24 hr of exposure to etoposide, cell death is delayed until several days later.
Mol Pharmacol 1994 Nov
PMID:Programmed cell death (apoptosis) of mouse fibroblasts is induced by the topoisomerase II inhibitor etoposide. 796 76

The suspect human carcinogen, etoposide, is known to be genotoxic, producing both gene and chromosomal mutations, probably by virtue of its ability to inhibit topoisomerase II activity. The present paper describes assays conducted using the Salmonella assay, the mouse lymphoma tk+/- assay (gene and chromosomal mutation analysis and molecular analysis of tk-/- mutants) and the mouse bone marrow micronucleus assay. Nonreproducible, weak, dose-related increases in mutation frequency in strain TA98 (but not TA1538 or TA1537) of Salmonella typhimurium were observed. Etoposide was highly mutagenic at the heterozygous thymidine kinase (tk+/-) locus of L5178Y mouse lymphoma cells at concentrations below 0.1 micrograms/ml. Mostly small colony mutants were induced, consistent with the potent clastogenicity also observed. Molecular analysis of mutants indicated that 83% and 92% of large and small colony mutants, respectively, had lost the entire target gene sequence. Chromosomally aberrant L5178Y cells were approximately 2 to 600-fold more prevalent than small tk-/- mutant colonies. This suggests that the viable target for etoposide-mediated clastogenesis in the selective assay is approximately one-fifth of chromosome 11b, itself being approximately one-fortieth of the mouse genome. An unusually potent response was observed for etoposide in the mouse bone marrow micronucleus assay (63.1 +/- 18 MPE/1,000 PE 24 hours after an oral dose of 1 mg/kg). The minimum detectable dose level in the assay was between 0.01 and 0.1 mg/kg. At dose levels between 1 and 15 mg/kg, an inverse dose response was observed. This reduction in assay response was not due to the small concommitant decrease in the incidence of polychromatic erythrocytes, a conclusion based on studies with N-methyl-N-nitrosourea. Animals sampled 48 hours after dosing with etoposide (10 mg/kg) had no polychromatic erythrocytes in the bone marrow. These observations for the micronucleus assay await explanation. The chemical structure of etoposide is displayed and discussed within the context of such strong mutagenic activity being associated with a nonelectrophilic agent.
Environ Mol Mutagen 1994
PMID:Potent clastogenicity of the human carcinogen etoposide to the mouse bone marrow and mouse lymphoma L5178Y cells: comparison to Salmonella responses. 773 7

Etoposide (VP 16-213), the epipodophyllotoxin derivative that is widely used in the treatment of cancer, forms complexes with DNA-topoisomerase type II alpha to exert its cytotoxicity. The drug was evaluated in vivo in Swiss albino mouse bone marrow cells for its ability to induce clastogenicity and sister chromatid exchanges (SCEs). Doses of 5, 10, 15, and 20 mg/kg body weight etoposide given intraperitoneally induced a dose-dependent significant increase of clastogenicity (Trend test, alpha < or = 0.05). The aberrations induced were predominantly chromatid types. The drug shows specificity for S-phase cells: cells harvested 6 and 12 hr posttreatment showed a significantly increased number of damaged cells and aberrations per cell. Doses of 0.5, 1.0, 2.5, 5.0, and 10.0 mg etoposide/kg body weight induced a dose-dependent significant induction of SCEs (Trend test, alpha < or = 0.05). The minimal effective concentration was 0.5 mg/kg body weight. Etoposide significantly prolonged the cell cycle time at all concentrations tested: 12-13 hr in treated animals vs. 11 hr in control. The results confirm in vivo cell cycle phase specificity of the drug and further designate etoposide as a potent clastogen and a genotoxic agent in mice.
Environ Mol Mutagen 1994
PMID:Etoposide (VP-16): cytogenetic studies in mice. 816 93

Etoposide, a nonintercalating antitumor drug, is a potent inhibitor of topoisomerase II activity. When Trypanosoma equiperdum is treated with etoposide, cleavable complexes are stabilized between topoisomerase II and kinetoplast DNA minicircles, a component of trypanosome mitochondrial DNA (T. A. Shapiro, V. A. Klein, and P. T. Englund, J. Biol. Chem. 264:4173-4178, 1989). Etoposide also promotes the time-dependent accumulation of small minicircle catenanes. These catenanes are radiolabeled in vivo with [3H]thymidine. Dimers are most abundant, but novel structures containing up to five noncovalently closed minicircles are detectable. Analysis by two-dimensional gel electrophoresis and electron microscopy indicates that dimers joined by up to six interlocks are late replication intermediates that accumulate when topoisomerase II activity is blocked. The requirement for topoisomerase II is particularly interesting because minicircles do not share the features postulated to make this enzyme essential in other systems: for minicircles, the replication fork is unidirectional, access to the DNA is not blocked by nucleosomes, and daughter circles are extensively nicked and (or) gapped.
Mol Cell Biol 1994 Jun
PMID:Mitochondrial topoisomerase II activity is essential for kinetoplast DNA minicircle segregation. 819 10

Etoposide, a topoisomerase II inhibitor used in cancer therapy, has been shown to induce apoptosis in vitro in a variety of cell types. In the present study, we have characterized the effects of etoposide on undifferentiated rat pheochromocytoma PC12 cells. Etoposide killed PC12 cells in a time- and concentration-dependent manner. 20-24 h incubation with 10 micrograms/ml etoposide induced 25-50% cell death. Hoechst 33258 staining revealed apoptotic morphology in dying cells. No evidence was found of either oligonucleosomal DNA fragmentation, as shown by agarose gel electrophoresis, or endonuclease involvement, as shown by the inability of aurintricarboxylic acid to prevent cell death. Cycloheximide and actinomycin-D were unable to prevent etoposide cytotoxicity indicating that the process is not dependent upon de novo protein or mRNA synthesis. NGF (5 ng/ml) prevented etoposide-induced PC12 cell death. These results offer an example of how the morphological features of apoptosis are not necessarily associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis.
Brain Res Mol Brain Res 1997 Sep
PMID:Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis. 933 35

Etoposide (VP-16) is extensively used to treat cancer, yet its efficacy is calamitously associated with an increased risk of secondary acute myelogenous leukemia. The mechanisms for the extremely high susceptibility of myeloid stem cells to the leukemogenic effects of etoposide have not been elucidated. We propose a mechanism to account for the etoposide-induced secondary acute myelogenous leukemia and nutritional strategies to prevent this complication of etoposide therapy. We hypothesize that etoposide phenoxyl radicals (etoposide-O(.)) formed from etoposide by myeloperoxidase are responsible for its genotoxic effects in bone marrow progenitor cells, which contain constitutively high myeloperoxidase activity. Here, we used purified human myeloperoxidase, as well as human leukemia HL60 cells with high myeloperoxidase activity and provide evidence of the following. 1) Etoposide undergoes one-electron oxidation to etoposide-O(.) catalyzed by both purified myeloperoxidase and myeloperoxidase activity in HL60 cells; formation of etoposide-O(.)radicals is completely blocked by myeloperoxidase inhibitors, cyanide and azide. 2) Intracellular reductants, GSH and protein sulfhydryls (but not phospholipids), are involved in myeloperoxidase-catalyzed etoposide redox-cycling that oxidizes endogenous thiols; pretreatment of HL60 cells with a maleimide thiol reagent, ThioGlo1, prevents redox-cycling of etoposide-O(.) radicals and permits their direct electron paramagnetic resonance detection in cell homogenates. VP-16 redox-cycling by purified myeloperoxidase (in the presence of GSH) or by myeloperoxidase activity in HL60 cells is accompanied by generation of thiyl radicals, GS(.), determined by HPLC assay of 5, 5-dimethyl-1-pyrroline glytathionyl N-oxide glytathionyl nitrone adducts. 3) Ascorbate directly reduces etoposide-O(.), thus competitively inhibiting etoposide-O(.)-induced thiol oxidation. Ascorbate also diminishes etoposide-induced topo II-DNA complex formation in myeloperoxidase-rich HL60 cells (but not in HL60 cells with myeloperoxidase activity depleted by pretreatment with succinyl acetone). 4) A vitamin E homolog, 2,2,5,7, 8-pentamethyl-6-hydroxychromane, a hindered phenolic compound whose phenoxyl radicals do not oxidize endogenous thiols, effectively competes with etoposide as a substrate for myeloperoxidase, thus preventing etoposide-O(.)-induced redox-cycling. We conclude that nutritional antioxidant strategies can be targeted at minimizing etoposide conversion to etoposide-O(.), thus minimizing the genotoxic effects of the radicals in bone marrow myelogenous progenitor cells, i.e., chemoprevention of etoposide-induced acute myelogenous leukemia.
Mol Pharmacol 1999 Sep
PMID:Mechanism-based chemopreventive strategies against etoposide-induced acute myeloid leukemia: free radical/antioxidant approach. 1046 37


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