Increased mutant frequencies in the HPRT gene locus of leukemia HL-60 cells treated with succinylacetone.

Succinyl acetone (SA) was initially identified in the urine of patients with tyrosinemia type I, an autosomally recessive inherited disease. SA has been used to downregulate the activity of myeloperoxidase (MPO) through its specific inhibition of heme biosynthesis and to investigate the biological properties of MPO in the human myeloid leukemic (HL-60) cell line. The goal of this study is to evaluate the mutagenic potential of SA by determining the frequencies of somatic mutations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) reporter gene in HL-60 cells following treatment with the chemical. Treatments of HL-60 cells with 500 micromol/L SA for 72 h, a condition generally used to inhibit the MPO activity, resulted in a significantly increased HPRT mutant frequency (HPRT-Mf), compared with the control of untreated cells (47.25 x 10(-6) versus 7.5 x 10(-6), respectively, p <0.01). Treatment of the cells with lower doses of SA also led to an increase in HPRT-Mf but this was significant only with 200 micromol/L (28.67 x 10(-6), p<0.05) and not with doses lower than 100 micromol/L (p0.05), compared with the control of untreated cells (7.5 x 10(-6)). These data show a dose-response increase in HPRT-Mf in HL-60 cells treated with SA, suggesting that this chemical causes mutations in the HPRT locus in these cells either directly or indirectly through its inhibition of the MPO activity.

Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane, by ascorbate/dihydrolipoate in living HL-60 cells.

Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.

Pro-oxidant and antioxidant mechanisms of etoposide in HL-60 cells: role of myeloperoxidase.

Etoposide is an effective anticancer agent whose antitumor activity is associated with its phenolic E-ring, which can participate in intracellular redox cycling reactions. Myeloperoxidase (MPO)-catalyzed one-electron oxidation of the etoposide phenolic ring and/or interaction of this phenolic moiety with reactive radicals yields its phenoxyl radical, whose reactivity may determine the pro- or antioxidant effects of this molecule in cells. Using MPO-rich HL-60 cells, we directly demonstrated that both anti- and pro-oxidant activities of etoposide are realized in cells. Etoposide acted as an effective radical scavenger and antioxidant protector of phosphatidylethanolamine, phosphatidylcholine, and other intracellular phospholipids against H2O2-induced oxidation in HL-60 cells with constitutively high MPO activity and in HL-60 cells depleted of MPO by an inhibitor of heme synthesis, succinyl acetone. MPO-catalyzed production of etoposide phenoxyl radicals observed directly in HL-60 cells by electron paramagnetic resonance (EPR) did not result in oxidation of these membrane phospholipids, suggesting that the radicals were not reactive enough to trigger lipid oxidation. MPO-dependent pro-oxidant activity of etoposide was directly demonstrated by (a) the ability of intracellular reduced glutathione (GSH) to eliminate EPR-detectable etoposide phenoxyl radicals, (b) the ability of etoposide phenoxyl radicals to oxidize GSH and protein thiols (after preliminary depletion of intracellular GSH with a maleimide reagent, ThioGlo-1), and (c) the disappearance of these effects after depletion of MPO by pretreatment of cells with succinyl acetone. In addition, titration of intracellular GSH (in intact cells) using the maleimide reagent ThioGlo-1 resulted in remarkably augmented EPR-detectable etoposide phenoxyl radicals and enhanced etoposide-induced topoisomerase II-DNA covalent complexes. In conclusion, the phenolic moiety of etoposide acts as an effective free radical scavenger, accounting for its antioxidant action. Whereas one-electron oxidation of etoposide by free radical scavenging and/or by MPO results in a phenoxyl radical with low reactivity toward lipids, its high reactivity toward thiols is a determinant of its pro-oxidant effects in HL-60 cells.

Synthesis and biological activity of a photoaffinity etoposide probe.

The epipodophyllotoxin etoposide is a potent and widely used anticancer drug that targets DNA topoisomerase II. The synthesis, photochemical, and biological testing of a photoactivatable aromatic azido analogue of etoposide also containing an iodo group is described. This azido analogue should prove useful for identifying the etoposide interaction site on topoisomerase II. Irradiation of the azido analogue and an aldehyde-containing azido precursor with UV light produced changes in their UV--visible spectra that were consistent with photoactivation. The azido analogue strongly inhibited topoisomerase II and inhibited the growth of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II--DNA covalent complexes were significantly increased subsequent to UV irradiation of drug-treated human leukemia K562 cells as compared to etoposide-treated cells. These results suggest that the photoactivated form of etoposide is a more effective topoisomerase II poison either by interacting directly with the enzyme or with DNA subsequent to topoisomerase II-mediated strand cleavage.

Antioxidant mechanisms of nitric oxide against iron-catalyzed oxidative stress in cells.

Three distinct antioxidant pathways are considered through which iron-catalyzed oxidative stress may be regulated by nitric oxide (NO). The first two pathways involve direct redox interactions of NO with iron catalytic sites and represent a fast response that may be considered an emergency mechanism to protect cells from the consequences of acute and intensive oxidative stress. These are (i) NO-induced nitrosylation at heme and non-heme iron catalytic sites that is capable of directly reducing oxoferryl-associated radicals, (ii) formation of nitrosyl complexes with intracellular "loosely" bound redox-active iron, and (iii) an indirect regulatory pathway that may function as an adaptive mechanism that becomes operational upon long-term exposure of cells to NO. In the latter pathway, NO down-regulates expression of iron-containing proteins to prevent their catalytic prooxidant reactions.

The catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces differentiation and apoptosis in human leukemia K562 cells.

The bisdioxopiperazines ICRF-187 (dexrazoxane), ICRF-193, and ICRF-154 are catalytic noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce protein-linked DNA strand breaks. In this study, we showed that bisdioxopiperazines induced erythroid differentiation, inhibited human leukemia K562 cell growth, and caused a slow induction of apoptosis. Dexrazoxane treatment caused DNA endoreduplication resulting in large highly polyploid cells. This result suggested the lack of a DNA topoisomerase II activity-based cell cycle checkpoint. The percentage of K562 cells that became apoptotic was much larger than the percentage of cells that stained for hemoglobin, suggesting that prior differentiation was not required for induction of apoptosis. Use of the Bcr-Abl tyrosine kinase inhibitor STI-571 resulted in a reduction in Bcl-xL levels and potentiation of dexrazoxane-induced apoptosis related to an earlier onset and more extensive cleavage of caspase-3. These results indicated that dexrazoxane-induced apoptosis is associated with a caspase-3 activation/cleavage pathway. In addition, these results were consistent with the antiapoptotic signaling function of Bcr-Abl to regulate expression of Bcl-xL. The ability of dexrazoxane to induce differentiation and apoptosis suggests that bisdioxopiperazines may be useful in treating some types of leukemia.

The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines.

The differentiating agent and histone deacetylase inhibitor, sodium butyrate (NaB), was shown previously to cause a transient, 3-17-fold induction of human DNA topoisomerase II alpha (topo II alpha) gene promoter activity and a 2-fold increase in topo II alpha protein early in monocytic differentiation of HL-60 cells. This observation has now been extended to other short chain fatty acids and aromatic butyrate analogues, and evidence is presented that human topo II alpha promoter induction correlates closely with histone H4 acetylation status. Because increased topo II alpha expression is associated with enhanced efficacy of topo II-poisoning antitumor drugs such as etoposide, the hypothesis tested in this report was whether NaB pretreatment could sensitize HL-60 myeloid leukemia and K562 erythroleukemia cells to etoposide-triggered DNA damage and cell death. A 24-72 h NaB treatment (0.4-0.5 mM) induced topo II alpha 2-2.5-fold in both HL-60 and K562 cells and caused a dose-dependent enhancement of etoposidestimulated, protein-linked DNA complexes in both cell lines. At concentrations with minimal effects on cell cycle kinetics (0.4 mM in HL-60; 0.5 mM in K562), NaB pretreatment also modestly enhanced etoposidetriggered apoptosis in HL-60 cells, as determined morphologically after acridine orange/ethidium bromide staining, and substantially increased K562 growth inhibition and poly(ADP-ribose)polymerase cleavage after etoposide exposure. Therefore, a temporal window may exist whereby a differentiating agent may sensitize experimental leukemias to a cytotoxic antitumor agent. These results indicate that histone deacetylase inhibitors should be investigated for etoposide sensitization of other butyrate-responsive hematopoietic and nonhematopoietic tumor lines in vitro and in vivo.

The catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces endopolyploidy in Chinese hamster ovary cells.

The bisdioxopiperazines, including dexrazoxane (ICRF-187), are catalytic or noncleavable complex-forming inhibitors of DNA topoisomerase II that do not produce DNA strand breaks. In this study we show that dexrazoxane inhibits the division of Chinese hamster ovary (CHO) cells resulting in marked increases in cell size (up to 80 microm in diameter), volume (up to 150-fold greater), and ploidy (as high as 32N). This last result indicates that the dexrazoxane-induced DNA reduplication was restricted to once per cell cycle. Kinetic analysis of the flow cytometry data indicated that the conversion between successively higher ploidy levels was progressively slowed at longer times of exposure to dexrazoxane. Both the protein and DNA content of dexrazoxane-treated CHO cells increased linearly over time in the same proportion. Light and electron microscopic studies of dexrazoxane-treated cells showed ring-like multilobulated nuclei. Immunohistochemical staining of dexrazoxane-treated cells showed that F-actin and acetylated alpha-tubulin were present in large, highly organized networks. Immunohistochemical staining of the dexrazoxane-treated CHO cells also showed that the topoisomerase II alpha colocalized with the DNA of the multilobulated nuclei. Staining of gamma-tubulin revealed that the dexrazoxane-treated cells contained multiple centrosomes, indicating that dexrazoxane prevents cytokinesis but not centrosome reduplication. It is concluded that dexrazoxane inhibits CHO cytokinesis in cells by virtue of its ability to inhibit topoisomerase II.

Regulation of DNA-dependent protein kinase activity by ionizing radiation-activated abl kinase is an ATM-dependent process.

Ionizing radiation (IR) treatment results in activation of the nonreceptor tyrosine kinase c-Abl because of phosphorylation by ATM. In vitro evidence indicates that DNA-dependent protein kinase (DNA-PK) can also phosphorylate and thus potentially activate Abl kinase activity in response to IR exposure. To unravel the role of ATM and DNA-PK in the activation of Abl, we assayed Abl, ATM, and DNA-PK activity in ATM- and DNA-PKcs-deficient cells after irradiation. Our results show that despite the presence of higher than normal levels of DNA-PK kinase activity, c-Abl fails to become activated after IR exposure in ATM-deficient cells. Conversely, normal activation of both ATM and c-Abl occurs in DNA-PKcs-deficient cells, indicating that ATM but not DNA-PK is required for activation of Abl in response to IR treatment. Moreover, activation of Abl kinase activity by IR correlates well with activation of ATM activity in all phases of the cell cycle. These results indicate that ATM is primarily responsible for activation of Abl in response to IR exposure in a cell cycle-independent fashion. Examination of DNA-PK activity in response to IR treatment in Abl-deficient cells expressing mutant forms of Abl or in normal cells exposed to an inhibitor of Abl suggests an in vivo role for Abl in the down-regulation of DNA-PK activity. Collectively, these results suggest a convergence of the ATM and DNA-PK pathways in the cellular response to IR through c-Abl kinase.

Myeloperoxidase-catalyzed phenoxyl radicals of vitamin E homologue, 2,2,5,7,8-pentamethyl- 6-hydroxychromane, do not induce oxidative stress in live HL-60 cells.

We used myeloperoxidase-containing HL-60 cells to generate phenoxyl radicals from nontoxic concentrations of a vitamin E homologue, 2,2, 5,7,8-pentamethyl-6-hydroxychromane (PMC) to test whether these radicals can induce oxidative stress in a physiological intracellular environment. In the presence of H(2)O(2), we were able to generate steady-state concentrations of PMC phenoxyl radicals readily detectable by EPR in viable HL-60 cells. In HL-60 cells pretreated with succinylacetone, an inhibitor of heme synthesis, a greater than 4-fold decrease in myeloperoxidase activity resulted in a dramatically decreased steady-state concentrations of PMC phenoxyl radicals hardly detectable in EPR spectra. We further conducted sensitive measurements of GSH oxidation and protein sulfhydryl oxidation as well as peroxidation in different classes of membrane phospholipids in HL-60 cells. We found that conditions compatible with the generation and detection of PMC phenoxyl radicals were not associated with either oxidation of GSH, protein SH-groups or phospholipid peroxidation. We conclude that PMC phenoxyl radicals do not induce oxidative stress under physiological conditions in contrast to their ability to cause lipid peroxidation in isolated lipoproteins in vitro.

Mechanism-based chemopreventive strategies against etoposide-induced acute myeloid leukemia: free radical/antioxidant approach.

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.

Mechanisms of nitric oxide protection against tert-butyl hydroperoxide-induced cytotoxicity in iNOS-transduced human erythroleukemia cells.

We studied nitric oxide-mediated protection against tert-butyl hydroperoxide (t-BuOOH)-induced cytotoxicity in a subline of human erythroleukemia K562 cells (K/VP.5) and in K/VP.5 cells transduced with a retroviral vector containing the human iNOS gene (K/VP. 5-iNOS). K/VP.5-iNOS cells were 2-fold less sensitive to the cytotoxic effects of t-BuOOH compared to K/VP.5 cells. A nitric oxide-donor, NOC-15 ((Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1- ium-1, 2-diolate), protected K/VP.5 cells against t-BuOOH-induced cytotoxicity and provided an additional increment of protection in K/VP.5-iNOS cells. Under conditions of excess t-BuOOH and deficiency of iron catalytic sites (hemoglobin, Hb) in K/VP.5-iNOS cells, the increase of intracellular Hb concentration is the main contributor to enhanced sensitivity of the cells to t-BuOOH-induced cytotoxicity (despite the effects of small amounts of endogenously produced nitric oxide). Protection against t-BuOOH-induced cytotoxicity in K/VP.5-iNOS cells was diminished by treatment with an iNOS inhibitor, L-N(G)-monomethylarginine (L-NMA), but was restored upon addition of NOC-15 to L-NMA-treated cells. Incubation of K/VP.5 cells with NOC-15 resulted in the production of dinitrosyl complexes of non-heme iron and hexacoordinated heme iron nitrosyl complexes based on low-temperature EPR spectra. In K/VP.5-iNOS cells, only a weak EPR signal of dinitrosyl complexes of non-heme iron was observed in the absence of NOC-15. In addition, no heme iron nitrosyl complexes were discernible in the EPR spectra from K/VP.5-iNOS cells. Upon addition of NOC-15 to K/VP.5-iNOS cells, the EPR signal of dinitrosyl complexes of non-heme iron was enhanced, and the EPR signal of nitrosylated heme iron became discernible. It was determined that levels of non-heme and heme (hemoglobin) iron were dramatically decreased in K/VP.5-iNOS cells compared to K/VP.5 cells, thus explaining the decreased intensities of EPR signals of nitrosylated species. In addition, t-BuOOH-induced oxoferryl-Hb-associated protein-centered free radical species as well as t-BuO(*) alkoxyl radicals were observed in these two cell lines. These t-BuOOH-induced radical species were greatly reduced in K/VP.5-iNOS cells compared to K/VP.5 cells, consistent with a reduction in heme iron levels in the iNOS-expressing cells. Most importantly, the combined action of NOC-15 and t-BuOOH resulted in complete elimination of both oxoferryl-associated radical EPR signals as well as those from dinitrosyl complexes of non-heme iron and nitrosylated heme iron in both K/VP.5-iNOS cells and K/VP.5 cells. We conclude that two independent pathways operate in erythroleukemia cells for nitric oxide-mediated protection against t-BuOOH-induced cytotoxicity. First, prolonged endogenous production of nitric oxide results in a decreased content of catalytic non-heme iron and heme iron sites through posttranscriptional regulation of iron homeostasis. Second, nitric oxide can chemically reduce t-BuOOH-induced oxoferryl and t-BuO(*) alkoxyl radicals.

The cardioprotective and DNA topoisomerase II inhibitory agent dexrazoxane (ICRF-187) antagonizes camptothecin-mediated growth inhibition of Chinese hamster ovary cells by inhibition of DNA synthesis.

Dexrazoxane (ICRF-187), which is clinically used to reduce doxorubicin-induced cardiotoxicity, has cell growth inhibitory properties through its ability to inhibit the catalytic activity of DNA topoisomerase II. A study was undertaken to investigate whether preincubating Chinese hamster ovary cells (CHO) with dexrazoxane prior to camptothecin treatment resulted in potentiation. Camptothecin is a DNA topoisomerase I poison. It was found that pretreating CHO cells with concentrations of dexrazoxane sufficient to strongly inhibit topoisomerase II for periods from 18 to 96 h resulted in significant antagonism of camptothecin-mediated growth inhibition. Lower concentrations that were sufficient to cause partial inhibition of topoisomerase II and partial dexrazoxane-mediated cell growth inhibition had little effect on camptothecin-mediated growth inhibition. Neither topoisomerase I protein levels nor camptothecin-induced topoisomerase I-DNA covalent complexes were affected by dexrazoxane concentrations that were sufficient to cause antagonism of camptothecin-induced growth inhibition. However, under these experimental conditions, dexrazoxane caused a decrease in DNA synthesis. Therefore, results presented here confirm the importance of the DNA synthesis-dependent replication fork interaction with topoisomerase I-DNA covalent complexes for the expression of camptothecin activity. It is concluded that dexrazoxane and camptothecin analogs should be used with caution in combination chemotherapy.

Induction of human breast cancer cell apoptosis from G2/M preceded by stimulation into the cell cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane.

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, A(II)) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of A(II). MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 microM A(II) when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1-2 Mb fragments appeared after 6 hr, while 30-50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30-50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that A(II) blocks MDA-MB-231 cells in G2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. A(II) demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, "normal" human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that A(II) caused stimulation into S phase, and that G2/M was the phase of the cell cycle from which cells apoptosed. A(II) caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited A(II)-induced cell death, indicating that its toxicity requires de novo protein synthesis.

The one-ring open hydrolysis intermediates of the cardioprotective agent dexrazoxane (ICRF-187) do not inhibit the growth of Chinese hamster ovary cells or the catalytic activity of DNA topoisomerase II.

Dexrazoxane (ICRF-187), which is clinically used to reduce doxorubicin-induced cardiotoxicity, has growth inhibitory properties through its ability to inhibit the catalytic activity of DNA topoisomerase II. Because the bisdioxopiperazine dexrazoxane undergoes significant ring-opening hydrolysis under physiological conditions to form two one-ring open hydrolysis intermediates, a study was undertaken to determine if these two intermediates had either any growth inhibitory or topoisomerase II inhibitory effects. Neither of the one-ring open intermediates exhibited growth inhibitory effects towards Chinese hamster ovary cells nor were they able to inhibit topoisomerase II. Thus, it was concluded that only intact dexrazoxane is able to inhibit the catalytic activity of topoisomerase II.

Mitindomide is a catalytic inhibitor of DNA topoisomerase II that acts at the bisdioxopiperazine binding site.

The antitumor drug mitindomide (NSC 284356) was shown to inhibit the decatenation activity of human and Chinese hamster ovary (CHO) topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.1]. Mitindomide did not induce the formation of topoisomerase II-DNA covalent cleavable complexes in CHO cells. These results taken together indicate that mitindomide is a catalytic/noncleavable complex-forming-type inhibitor of topoisomerase II. The growth inhibitory effects of mitindomide and dexrazoxane toward a sensitive parent CHO cell line and the dexrazoxane-resistant DZR cell line, which is highly (500-fold) resistant to the bisdioxopiperazine dexrazoxane, were measured. The DZR cell line was shown to be 30-fold cross-resistant to mitindomide. Mitindomide, like dexrazoxane, was shown to inhibit cleavable complex formation by the topoisomerase II poison etoposide. The attenuated inhibition of etoposide-induced cleavable complexes in DZR compared with CHO cells was, likewise, very similar for dexrazoxane and mitindomide. Together these results suggest that mitindomide acts at the same site on topoisomerase II as does dexrazoxane and other bisdioxopiperazines. Various molecular parameters obtained by molecular modeling were compared for mitindomide and dexrazoxane. Mitindomide, which is conformationally very rigid, has highly coplanar imide rings, as does dexrazoxane in the solid state. Other molecular parameters, such as the imide nitrogen-to-imide nitrogen bond distances, and polar and nonpolar surface areas were also very similar. Thus, it is concluded that mitindomide exerts its antitumor effects through its inhibition of topoisomerase II by binding to the bisdioxopiperazine binding site.

Characterization of a Chinese hamster ovary cell line with acquired resistance to the bisdioxopiperazine dexrazoxane (ICRF-187) catalytic inhibitor of topoisomerase II.

A Chinese hamster ovary (CHO) cell line highly resistant to the non-cleavable complex-forming topoisomerase II inhibitor dexrazoxane (ICRF-187, Zinecard) was selected. The resistant cell line (DZR) was 1500-fold resistant (IC50 = 2800 vs 1.8 microM) to continuous dexrazoxane exposure. DZR cells were also cross-resistant (8- to 500-fold) to other bisdioxopiperazines (ICRF-193, ICRF-154, and ICRF-186), and somewhat cross-resistant (4- to 14-fold) to anthracyclines (daunorubicin, doxorubicin, epirubicin, and idarubicin) and etoposide (8.5-fold), but not to the other non-cleavable complex-forming topoisomerase II inhibitors suramin and merbarone. The cytotoxicity of dexrazoxane to both cell lines was unchanged in the presence of the membrane-active agent verapamil. DZR cells were 9-fold resistant to dexrazoxane-mediated inhibition of topoisomerase II DNA decatenation activity compared with CHO cells (IC50 = 400 vs 45 microM), but were only 1.4-fold (IC50 = 110 vs 83 microM) resistant to etoposide. DZR cells contained one-half the level of topoisomerase II protein compared with parental CHO cells. However, the specific activity for decatenation using nuclear extract topoisomerase II was unchanged. Etoposide (100 microM)-induced topoisomerase II-DNA complexes in DZR cells and isolated nuclei were similarly one-half the level found in CHO cells and in isolated nuclei. However, the ability of 500 microM dexrazoxane to inhibit etoposide (100 microM)-induced topoisomerase II-DNA covalent complexes was reduced 4- to 6-fold in both DZR cells and nuclei compared with CHO cells and nuclei. In contrast, there was no differential ability of aclarubicin or merbarone to inhibit etoposide-induced topoisomerase II-DNA complexes in CHO compared with DZR cells and isolated nuclei. It was concluded that the DZR cell line acquired its resistance to dexrazoxane mainly through an alteration in the topoisomerase II target.

Nitric oxide prevents oxidative damage produced by tert-butyl hydroperoxide in erythroleukemia cells via nitrosylation of heme and non-heme iron. Electron paramagnetic resonance evidence.

We studied protective effects of NO against tert-butylhydroperoxide (t-BuOOH)-induced oxidations in a subline of human erythroleukemia K562 cells with different intracellular hemoglobin (Hb) concentrations. t-BuOOH-induced formation of oxoferryl-Hb-derived free radical species in cells was demonstrated by low temperature EPR spectroscopy. Intensity of the signals was proportional to Hb concentrations and was correlated with cell viability. Peroxidation of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin metabolically labeled with oxidation-sensitive cis-parinaric acid was induced by t-BuOOH. An NO donor, (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]-diazen-1-iu m-1, 2-diolate], produced non-heme iron dinitrosyl complexes and hexa- and pentacoordinated Hb-nitrosyl complexes in the cells. Nitrosylation of non-heme iron centers and Hb-heme protected against t-BuOOH-induced: (a) formation of oxoferryl-Hb-derived free radical species, (b) peroxidation of cis-parinaric acid-labeled phospholipids, and (c) cytotoxicity. Since NO did not inhibit peroxidation induced by an azo-initiator of peroxyl radicals, 2, 2'-azobis(2,4-dimethylvaleronitrile), protective effects of NO were due to formation of iron-nitrosyl complexes whose redox interactions with t-BuOOH prevented generation of oxoferryl-Hb-derived free radical species.

Chemical synthesis and biological activity of a novel fluorescent etoposide derivative.

The antineoplastic activity of etoposide resides in its ability to poison the nuclear enzyme DNA topoisomerase II (topo II). The factors that control the cellular entry and subcellular distribution of etoposide remain poorly understood. Therefore, we have synthesized a novel fluorescence-labeled etoposide (Bodipyetoposide) by coupling 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionylethylenediamine (Bodipy) to 4'-benzyloxycarbonyl-4'-demethylepipodophyllotoxin beta-D-glucopyranoside, a precursor of etoposide. Bodipy-etoposide retained the ability to stabilize topo II-DNA covalent complexes in isolated nuclei, although it was significantly less potent and efficacious than etoposide. The growth inhibitory activity of Bodipy-etoposide was also approximately 200-fold less than that of etoposide in human leukemia K562 and DU-145 prostatic carcinoma cells. Nonetheless, etoposide-resistant K/VP.5 and K/VP.5-1 leukemia cells were cross-resistant to Bodipy-etoposide compared with parental K562 cells. Analysis by flow cytometry revealed a concentration-dependent Bodipy-etoposide cell association with no significant difference in drug association in the etoposide-resistant cell lines relative to the parental K562 cells. Using confocal laser scanning microscopy, we found significant cytoplasmic perinuclear localization of Bodipy-etoposide. Thus, Bodipy-etoposide displays promise as a tool to probe the factors controlling entry and subcellular distribution of etoposide-like compounds in live cells.

Non-random peroxidation of different classes of membrane phospholipids in live cells detected by metabolically integrated cis-parinaric acid.

Quantitative assays of lipid peroxidation in intact, living cells are essential for evaluating oxidative damage from various sources and for testing the efficacy of antioxidant interventions. We report a novel method based on the use of cis-parinaric acid (PnA) as a reporter molecule for membrane lipid peroxidation in intact mammalian cells. Using four different cell lines (human leukemia HL-60, K562 and K/VP.5 cells, and Chinese hamster ovary (CHO) fibroblasts), we developed a technique to metabolically integrate PnA into all major classes of membrane phospholipids, i.e., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and cardiolipin, that can be quantified by HPLC with fluorescence detection. Integrated PnA constituted less than 1% of lipid fatty acid residues, suggesting that membrane structure and characteristics were not significantly altered. Low concentrations (20-40 microM) of tert-butyl hydroperoxide (t-BuOOH) caused selective oxidation of PnA residues in phosphatidylserine and phosphatidylethanolamine of K562 cells and K/VP.5 cells while cell viability was unaffected. At higher t-BuOOH concentrations (exceeding 100 microM), however, a progressive, random oxidation of all major phospholipid classes occurred and was accompanied by significant cell death. In HL-60 cells, phosphatidylethanolamine, phosphatidylserine and cardiolipin were sensitive to low concentrations of t-BuOOH, while phosphatidylcholine and phosphatidylinositol were not affected. Phosphatidylinositol was the only phospholipid that responded to the low concentrations of t-BuOOH in CHO cells. At high t-BuOOH concentrations, again, all phospholipid classes underwent extensive oxidation. All phospholipids were nearly equally affected by peroxidation induced by a initiator of peroxyl radicals, 2,2'-azobis-(2,4-dimethylvaleronitrile) AMVN), in K562 cells. In gamma-irradiated (4-128 Gy) CHO cells, phosphatidylserine was the most affected phospholipid class (34% peroxidation) followed by phosphatidylinositol (24% peroxidation) while the other three phospholipid classes were apparently unaffected. Since loss of PnA fluorescence is a direct result of irreparable oxidative loss of its conjugated double bond system, the method described allows for selective and sensitive monitoring of oxidative stress in live cells without interference from cell repair mechanisms.