Etoposide Targets Topoisomerase IIα and IIβ in Leukemic Cells: Isoform-Specific Cleavable Complexes Visualized and Quantified<i>In Situ</i>by a Novel Immunofluorescence Technique

Willmore, Elaine; Frank, Adrian; Padget, Kay; Tilby, Michael J.; Austin, Caroline A.

doi:10.1124/mol.54.1.78

Cited by 138 publications

(149 citation statements)

References 28 publications

Supporting

Mentioning

140

Contrasting

Unclassified

Order By: Relevance

“…This effect is reversible and furthermore etoposide has a short elimination half-life (Hsiang and Liu, 1989;Slevin, 1991;Caldecott et al, 1993;Willmore et al, 1998). Together with the slow formation of carboplatin-DNA adducts, the swift reversal of etoposide stabilised topo II cleavable complexes may result in reduced anti-tumour effect in patients.…”

Section: Discussionmentioning

confidence: 99%

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

et al. 2002

View full text Add to dashboard Cite

The growth inhibitory effects of cisplatin and etoposide on neuroblastoma cell lines were investigated in several scheduled combinations. Results were analyzed using median effect and combination index analyses. In all schedules in which cisplatin was administered prior to etoposide a synergistic effect was observed. Conversely, an antagonistic effect was seen in all schedules where etoposide was administered before cisplatin. The design of the widely used OPEC protocol (oncovins, platinum agents, epipodophyllotoxins and cyclophosphamide) for management of the paediatric solid tumour neuroblastoma was based on clinical and in vitro evaluations of sequentially scheduled cisplatin and the epipodophyllotoxin, teniposide. These studies indicated that, for optimal anti-tumour effect, cisplatin should be administered prior to the epipodophyllotoxin (Hayes et al, 1981;Shafford et al, 1984;Pritchard et al, 1985).In several more recent clinical protocols for neuroblastoma, this apparently optimal order of drug administration has not been retained. The order of administration has been reversed in some regimens (Gordon et al, 1992;Tweddle et al, 2001) or the drugs are administered concurrently (reviewed in Pinkerton et al, 2000). If the OPEC rationale was correct, this altered scheduling pattern could result in sub-optimal response. Furthermore, in many of these schedules, cisplatin has been replaced by carboplatin. This will accentuate the change in order of administration because, despite the fact that carboplatin forms essentially the same final DNA adducts as cisplatin, the cytotoxic cross-linked structures develop more slowly than with cisplatin (Knox et al, 1986).The present study was aimed at determining, using contemporary methods of analysis, whether the currently used epipodophyllotoxin, etoposide, in combination with cisplatin, caused effects on neuroblastoma cell lines that were dependent upon the relative timings of the drug exposures. MATERIALS AND METHODS Cell culture and drug solutionsThe neuroblastoma cell lines SHSY5Y (Ciccarone et al, 1989) and NGP (Brodeur et al, 1977) were kindly provided by Drs P Lovat and D Tweddle (University of Newcastle upon Tyne). Cells were grown in RPMI 1640 (Dutch modification, supplemented with 10% v/v foetal bovine serum, and antibiotics (Gibco BRL)) at 378C/5% CO 2 . Frequent tests for mycoplasma infection were always negative. Etoposide was dissolved in methanol and stored at 7208C. Cisplatin was freshly dissolved in DMSO just prior to each experiment and immediately diluted into medium. During drug exposures, the concentrations of methanol and DMSO were kept below 1% and 0.001% respectively in treated and control wells. Sulphorhodamine B (SRB) assayFor each experiment, cells were inoculated into three 96-well tissue culture plates and incubated for 24 h prior to starting drug exposures (time zero). Plates were exposed to graded concentrations of either: the first drug in the schedule only followed by the diluent for the second drug; the diluent for the first drug in the s...

show abstract

Section: Discussionmentioning

confidence: 99%

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

et al. 2002

View full text Add to dashboard Cite

show abstract

“…16-Bit images were then analyzed to quantify the levels of blue and green fluorescence. All images were corrected for stray light and camera background and were subjected to blue and green shade correction to compensate for variations in intensity of illumination and non-uniformities in light transmission (34). Graphing and statistical analysis was carried out using GraphPad Prism software (Cherwell Scientific, Oxford, UK).…”

Section: Methodsmentioning

confidence: 99%

“…The slides were placed in lysis buffer containing 1% SDS, 80 mM potassium phosphate (pH 6.8), 10 mM EDTA, and protease inhibitors for 30 min, followed by incubation for 30 min in 1 M NaCl plus protease inhibitors. The slides were then washed in PBS and incubated with anti-Top2␤ (18513␤) (34) or anti-Top2␣ and -␤ (4882; raised against the N-terminal 140 kDa of bovine Top2) antibody for 1 h in PBS-T (PBS containing 0.1% Tween 20) with 1% (w/v) bovine serum albumin. The slides were washed three times in PBS-T and incubated for 1 h with rabbit fluorescein isothiocyanate-conjugated secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

NK314, a Topoisomerase II Inhibitor That Specifically Targets the α Isoform

Toyoda

Kagaya

Cowell

et al. 2008

Journal of Biological Chemistry

108

View full text Add to dashboard Cite

Topoisomerase II (Top2) is a ubiquitous nuclear enzyme that relieves torsional stress in chromosomal DNA during various cellular processes. Agents that target Top2, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective anticancer drugs used in the clinic. Mammalian cells possess two genetically distinct Top2 isoforms, both of which are the target of these agents. Top2␣ is essential for cell proliferation and is highly expressed in vigorously growing cells, whereas Top2␤ is nonessential for growth and has recently been implicated in treatment-associated secondary malignancies, highlighting the validity of a Top2␣-specific drug for future cancer treatment; however, no such agent has been hitherto reported. Here we show that NK314, a novel synthetic benzo[c]phenanthridine alkaloid, targets Top2␣ and not Top2␤ in vivo. Unlike other Top2 inhibitors, NK314 induces Top2-DNA complexes and double-strand breaks (DSBs) in an ␣ isoform-specific manner. Heterozygous disruption of the human TOP2␣ gene confers increased NK314 resistance, whereas TOP2␤ homozygous knock-out cells display increased NK314 sensitivity, indicating that the ␣ isoform is the cellular target. We further show that the absence of Top2␤ does not alleviate NK314 hypersensitivity of cells deficient in non-homologous end-joining, a critical pathway for repairing Top2-mediated DSBs. Our results indicate that NK314 acts as a Top2␣-specific poison in mammalian cells, with excellent potential as an efficacious and safe chemotherapeutic agent. We also suggest that a series of human knock-out cell lines are useful in assessing DNA damage and repair induced by potential topoisomerase-targeting agents. DNA topoisomerase II (Top2)2 is a ubiquitous nuclear enzyme that alters the topological structure of DNA and chromosomes through a transient DNA double-strand break (DSB) and subsequent religation of the DSB (1, 2). The enzyme has been implicated in many aspects of DNA metabolism, including DNA replication, repair, transcription, and chromosome condensation/segregation (1, 3). Top2 has been of considerable interest to human medicine, because it is an important target for cancer chemotherapy (4). Top2-targeting agents, involving etoposide, doxorubicin, and mitoxantrone, are among the most effective and widely used anticancer drugs in cancer chemotherapy (5, 6). These agents are referred to as "Top2 poisons," because they convert the essential enzyme into a highly cytotoxic DNA-damaging agent through the formation of "cleavage complex" (also called "cleavable complex"), in which a Top2-linked DNA strand-passing intermediate is stabilized, allowing the generation of a DSB (7,8).Mammalian cells possess two genetically distinct Top2 isoforms (9, 10). Despite their similar structural features (ϳ70% identity at the amino acid level) and biological properties, the two isoforms are differentially regulated and play different roles in living cells. Top2␣ is most abundantly expressed in rapidly growing tissues and its expression is cell cycle-regulated...

show abstract

“…19 Briefly, HL-60 cells (5 3 10 5 per each well) were incubated with SH-7 for 6 h in 6-well plates. Then, cells are embedded in agarose onto a microscope slide.…”

Section: Trapped In Agarose Dna Immunostaining (Tardis) Assaymentioning

confidence: 99%

SH‐7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor

Yang

Chen

Duan

et al. 2006

Intl Journal of Cancer

View full text Add to dashboard Cite

1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate (SH-7), a new naphthoquinone compound, derived from shikonin, exhibited obvious inhibitory actions on topoisomerase II (Topo II) and topoisomerase I (Topo I), which were stronger than its mother compound shikonin. Notably, the SH-7's inhibitory potency on Topo II was much stronger than that on Topo I. In addition, SH-7 significantly stabilized Topo II-DNA cleavable complex and elevated the expression of phosphorylated-H2AX. The in vitro cell-based investigation demonstrated that SH-7 displayed wide cytotoxicity in diversified cancer cell lines with the mean IC 50 value of 7.75 lM. One important finding is SH-7 displayed significant cytotoxicity in the 3 MDR cell lines, with an average IC 50 value nearly equivalent to that of the corresponding parental cell lines. The average resistance factor (RF) of SH-7 was 1.74, which was much lower than those of reference drugs VP-16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39). Further studies illustrated that SH-7 had the marked apoptosis-inducing function on leukemia HL-60 cells, which was validated to be of mitochondria-dependence. The in vivo experiments showed that SH-7 had inhibitory effects on S-180 sarcoma implanted to mice, SMMC-7721, BEL-7402 human hepatocellular carcinoma and PC-3 human prostate cancer implanted to nude mice. Taken together, these results suggest that SH-7 induces DSBs as a Topo II inhibitor, which was crucial to activate the apoptotic process, and subsequently accounts for its both in vitro and in vivo antitumor activities. The well-defined Topo II inhibitory activity, antitumor effects particularly with its obvious anti-MDR action, better solubility and less toxicity make SH-7 as a potential antitumor drug candidate for further research and development.

show abstract

Etoposide Targets Topoisomerase IIα and IIβ in Leukemic Cells: Isoform-Specific Cleavable Complexes Visualized and QuantifiedIn Situby a Novel Immunofluorescence Technique

Cited by 138 publications

References 28 publications

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

NK314, a Topoisomerase II Inhibitor That Specifically Targets the α Isoform

SH‐7, a new synthesized shikonin derivative, exerting its potent antitumor activities as a topoisomerase inhibitor

Contact Info

Product

Resources

About