| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics |
cDNA
UMR 8532 Centre National de la Recherche Scientifique, Physicochimie et Pharmacologie des Macromolécules Biologiques, Institut Gustave Roussy, 94805 Villejuif Cedex [T. K., B. R., S. L., B. L., J-M. S., J. M., A. J-S.], and UPR 9044 Centre National de la Recherche Scientifique, Génétique Moléculaire et Intégration des Fonctions Cellulaires, BP 8, 94801 Villejuif Cedex [H. J-S.], France
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
is 4–5-fold lower than in the parental DC-3F cells, whereas topoisomerase IIß is undetectable. Cloning and sequencing of topoisomerase II cDNAs from DC-3F and DC-3F/9-OH-E cells revealed an allele polymorphism, one allele differing from the other by the presence of seven silent mutations and three mutations in the noncoding region. In addition, the mutated allele contains three missense mutations located close to the ATP binding site (Thr371Ser) or to the catalytic site (Ala751Gly; Ile863Thr). To analyze the contribution of these topoisomerase II alterations to their resistance phenotype, DC-3F/9-OH-E cells were transfected with an eukaryotic expression vector containing the human topoisomerase II cDNA. In one transfected clone, the amount of topoisomerase II isoform and the catalytic activity were similar to that in the parental DC-3F cells. These cells, which contain only topoisomerase II, are then a unique mammalian cell line to analyze the physiological and pharmacological properties of this enzyme. However, the restoration of a nearly normal topoisomerase II activity in the DC-3F/9-OH-E cells did not have the same effect on their sensitivity to different enzyme inhibitors; a 75% reversion of the resistance, associated with a 2–3-fold increased stabilization of the cleavable complex, was observed with both etoposide and m-AMSA, two drugs that inhibit the DNA religation step in the enzyme catalytic cycle; in contrast, the transfected cells remained fully resistant to ellipticine derivatives that did not induce the stabilization of the cleavable complex. We hypothesized that a trans-acting factor, inhibiting the induction of cleavable complex formation by drugs that are not religation inhibitors, might be present in the resistant cells. However, such a factor was not detected in in vitro experiments, and other hypotheses are discussed. |
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Two forms of topoisomerase II, encoded by separate genes, exist in mammals: topoisomerase II (Mr 170,000 form) and topoisomerase IIß (Mr 180,000 form; Ref. 6, 7, 8, 9
). The two enzymes differ by some of their biochemical and pharmacological properties and in particular by their sensitivity to different inhibitors (7)
. Quantitative and/or qualitative topoisomerase II alterations have been observed in cell lines resistant to various topoisomerase II inhibitors (reviewed in Refs. 10
and 11
). Reduction in the amount of one isoform may account for the resistance to drugs such as etoposide (12)
or mitoxanthrone (13)
. We described previously a subline of the Chinese hamster lung cell line DC-3F that was selected for the highest possible resistance to the DNA-intercalating agent 9-OH-E (14)
. This resistance was associated with reduced topoisomerase II activity and decreased capacity of the topoisomerase II inhibitor to stabilize the cleavable complex (15)
. In previous studies, we showed by Northern and immunoblot analyses that, in the parental DC-3F cells, the enzyme was about 20-fold more abundant than the ß enzyme. In the DC-3F/9-OH-E resistant cells, the amount of enzyme was about 4–5-fold smaller than that in the sensitive cells, whereas the ß enzyme was undetectable (16)
. Cloning and sequencing of the topoisomerase IIß cDNAs from the sensitive and 9-OH-E resistant cells revealed the presence of a mutation at position 1710 that converts a Trp codon (TGG) to a stop codon (TGA) and thus accounts for the loss of the ß isoform in DC-3F/9-OH-E cells (17)
.
The relative contribution of a decreased expression of either topoisomerase II or ß to the resistance phenotype of the DC-3F/9-OH-E cells remains difficult to ascertain. For example, Gudkov et al. (18)
, using genetic suppressor elements, induced in human cells a decrease of the topoisomerase II activity comparable with that observed in DC-3F/9-OH-E cells and demonstrated that it resulted in resistance to topoisomerase II-interactive drugs. However, in that system, the resistance to the tested inhibitors was only about 3–5-fold, compared with several hundred-fold in DC-3F/9-OH-E cells. In an attempt to evaluate the role of or ß enzyme modifications to their resistance phenotype, DC-3F/9-OH-E cells were transfected with a eukaryotic expression vector containing either the topoisomerase II or ß cDNA. In cells where a normal topoisomerase IIß activity was restored, different levels of cleavable complex formation and resistance reversion were observed with each topoisomerase II inhibitor examined. Most striking was the almost complete reversion of the resistance to m-AMSA, indicating that topoisomerase IIß is the preferential target for this compound (19)
.
As reported in this study, in addition to an allele polymorphism at the topoisomerase II locus in DC-3F cells, three missense mutations were identified in the topoisomerase II cDNA from DC-3F/9-OH-E cells. DC-3F/9-OH-E cells were then transfected with a plasmid carrying the human topoisomerase II cDNA (hTOP2; Ref. 20
), and the phenotypic properties of transfected cells in which a normal topoisomerase II activity had been restored are described. These cells, in which only topoisomerase II is expressed, are a remarkable mammalian system to analyze the physiological functions of this enzyme and its pharmacological role as a target for some essential antitumor agents.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-32P]dCTP and [-32P]dUTP were obtained from Amersham-Pharmacia-Biotech (Orsay, France). 9-OH-E was kindly provided by Dr. E. Lescot (Institut Gustave Roussy, Villejuif, France). Restriction enzymes were from New England Biolabs (Beverly, MA) and Appligène (Illkirch, France). Probes were labeled with Nonaprimer labeling kit from Appligène. T4 DNA ligase and calf intestinal phosphatase were purchased from Roche-Diagnostics (Meylan, France). Etoposide and m-AMSA, obtained from Sigma-Aldrich Chimie (Saint-Quentin-Fallavier, France), were dissolved in DMSO. NMHE was purchased from Pasteur-Mérieux (Lyon, France) and dissolved in water. 9-OH-E was dissolved in HCl 10-3 M. All stock solutions were 10-2 M.
Cell Lines and Culture
The Chinese hamster lung cell lines DC-3F and DC-3F/9-OH-E (14)
were maintained as described previously (16)
. The 9-OH-E resistant cells, DC-3F/9-OH-E, were selected, by adding stepwise, increasing concentrations of the drug to the cell growth medium (14)
.
Cell Survival
The in vitro colony formation assay was used to determine survival fractions after 3 h of drug exposure. Exponentially growing cells (2 x 105) were plated into 60-mm-diameter Petri dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ) for 18 h at 37°C before drug treatment. After a 3-h drug exposure, the cells were trypsinized, and appropriate dilutions were made to seed 300 treated cells into 60-mm-diameter Petri dishes. Colonies were stained and counted 8–10 days later.
Cloning of Topoisomerase II cDNA from DC-3F and DC-3F/9-OH-E Cells
Polyadenylated RNAs were purified from total RNA as described previously (16)
. The cDNA libraries were constructed using the Zap cDNA synthesis kit from Stratagene (La Jolla, CA), following the manufacturer’s instructions. cDNAs were cloned in the Uni-ZAP XR vector and packaged in 
phagemid as described previously (17)
. Approximately 4 x 105 phages from each library were plated out and screened by plaque hybridization with the human topoisomerase II SP1' probe (16)
, derived from the SP1 probe (8)
, kindly provided by Dr. K. B. Tan (Smith Kline and French Laboratories, King of Prussia, PA). Forty-eight plaques picked up from the DC-3F library and 27 from the DC-3F/9-OH-E library were purified by tertiary screening. cDNA fragments from single positive plaques were excised by digestion with XhoI and EcoRI; two clones from the DC-3F and eight from the DC-3F/9-OH-E libraries were found to contain inserts with sizes higher than 4.6 kb, the approximate size expected for a complete topoisomerase II cDNA. From each library, one pBluescript phagemid, containing an insert of 
5.1 kb, was then excised from the Uni-ZAP vector and transfected in XL1-Blue bacteria, following the Stratagene protocol. Restriction analysis showed that these fragments span the entire coding region of the topoisomerase II cDNA, with 90–95 bp upstream of the initiation codon.
DNA sequencing was performed and analyzed at ACT Gene-Euro Sequence Gene Service (Génopole, Evry, France) on an ABI 377 sequencer using ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit with Amplitaq DNA polymerase FS (Perkin-Elmer/Applied Biosystems Division, Foster City, CA).
PCR Amplification
PCR reaction mixtures (100 µl) contained 10 mM Tris-HCl (pH 9), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 0.01% (w/v) gelatin, 0.2 mM of each deoxynucleotide triphosphate, 1.2 µM of each primer, 1 unit of Taq polymerase (ATGC), and 1 µg of genomic DNA. PCR was carried out using the thermocycler Hybaid Omnigene (Teddington, United Kingdom). An initial 3 min denaturation at 94°C was followed by 30 PCR cycles: 15 s at 55°C, 30 s at 72°C, and 15 s at 94°C. The sense primer was 5'-CCCCTGGTCTGATTCTGAATCA-3', and the antisense primer was 5'-CCTTTCCATCAGAACCTGAG-3'. After digestion with BamHI or AcsI, PCR products were fractionated by electrophoresis in 1.2% agarose.
For RT-PCR, RNAs were prepared using the RNeasy Midi kit (Qiagen, S.A. Courtaboeuf, France), and mRNAs were purified from total RNAs using an mRNA purification kit (Amersham-Pharmacia-Biotech). Single-stranded cDNA was generated by reverse transcription of 100 ng of polyadenylated RNA from each cell line using random primers (RT-PCR kit; Stratagene), following the manufacturer’s instructions. The cDNAs were then amplified as described above. After digestion with BamHI or AcsI, PCR products were fractionated by electrophoresis in 10% polyacrylamide TBE (89 mM Tris base, 89 mM boric acid, and 2.5 mM EDTA, pH 8.3) gel.
Vectors
The plasmid pBS-hTOP2 containing the human topoisomerase II cDNA (hTOP2; Ref. 20
) was a gift from Dr. J.C. Wang (Harvard University, Cambridge, MA). It was propagated in E. coli strain XL1-Blue. The plasmid was purified and digested with restriction endonucleases NotI and HindIII. Two inserts, a NotI-HindIII fragment (1517 bp) and a HindIII-HindIII fragment (4083 bp) were purified from agarose gels. The eukaryotic expression vector, pSV-Sport1 (Life Technologies, Rockville, MD) was digested at the NotI and HindIII sites within the polylinker, treated with calf intestinal phosphatase and ligated to the NotI-HindIII fragment to give the intermediate plasmid pSV-hTOP2. This plasmid was then digested with restriction endonuclease HindIII, treated with calf intestinal phosphatase, and ligated to the HindIII-HindIII fragment from pBS-hTOP2 to give the final pSV-hTOP2 plasmid (Fig. 1)
. From this second step, both sense (pSV-hTOP2) and antisense (pSV-hTOP2') constructs were selected.
|
cDNA into DC-3F/9-OH-E CellsTransient expression of pSV-hTOP2 was determined in cells transfected with the calcium phosphate precipitated plasmid as described above. The transfection medium was replaced after 24 h with fresh medium, and the gene expression was analyzed 48 h later.
cDNA Probes
The probe SP1', a EcoRI-XbaI fragment (1504 bp) of the human hTOP2 cDNA isolated from plasmid pUC13-SP1, has been described previously in detail (16)
. The ß-actin cDNA probe was kindly provided by Dr. F. Dautry (Institut Gustave Roussy, Villejuif, France). These probes were labeled by random priming with [-32P]dCTP using Nonaprimer labeling kit (Appligène).
Antihuman DNA Topoisomerase II Antibodies
The rabbit polyclonal antibody (designated A6), recognizing both topoisomerase II and ß, was described previously by Khélifa et al. (16)
. The murine antihuman topoisomerase II monoclonal antibody was purchased from TopoGEN, Inc. (Columbus, OH).
Northern Blot Analysis
RNAs were extracted by the guanidine thiocyanate technique (23)
, fractionated by electrophoresis on 1.2% (w/v) agarose gels containing 7% formaldehyde, and transferred to Hybond N-nylon membrane (RPN 3050; Amersham-Pharmacia-Biotech) in 150 mM ammonium acetate. Prehybridization was performed for 2 h at 42°C in 40% formamide, 5x SSC (1x SSC is 0.15 M NaCl and 15 mM sodium citrate), 50 mM phosphate buffer (pH 6.8), 5x Denhardt’s solution, and 0.1% SDS. Hybridization was then performed for 20 h in the same buffer containing the 32P-labeled probe. The membrane was washed twice at room temperature for 15 min in 0.1% SDS, 2x SSPE (1x SSPE is 10 mM NaH2PO4, 0.13 M NaCl, and 1 mM EDTA) and twice at 50°C for 30 min in 0.1% SDS, 1x SSPE. After autoradiography at -70°C, the autoradiograms were obtained on Fuji RX film with a DuPont Cronex Lightning-Plus screen.
RNase Protection Assay
The plasmid pBS-hTOP2 was digested with NotI and HincII. The resulting 108-bp fragment was then inserted in the pSV-Sport1 plasmid between the NotI and SnaBI sites of the polylinker. In this construction, named pSV-hTOP2R, the NotI-HincII fragment is located downstream of the T7 RNA polymerase promoter and in the reverse orientation. The plasmid pSV-hTOP2R was then linearized at the AvrII site in the SV40 promoter, downstream of the inserted fragment. The plasmid was then transcribed from the T7 promoter, with [-32P]UTP (Amersham-Pharmacia-Biotech) to generate a 230-bp probe. The template was removed by treatment with the DNase RQ1 from Promega (Charbonnières, France), and the labeled probe was hybridized in solution (80% formamide at 42°C) with total cellular RNA. After treatment with a mixture of RNase A and RNase T1 (Promega) to remove the single-stranded RNAs, the hybrids were analyzed on a 6% denaturing PAGE.
DNA Extraction and Southern Blot Analysis
Genomic DNAs were extracted from cultured cells as described by Laird et al. (24)
. After digestion with restriction enzymes, DNA fragments (10 µg/lane) were fractionated by electrophoresis on 0.8% (w/v) agarose gels. After transfer to Hybond-N nylon membranes (Amersham-Pharmacia-Biotech) by vacuum blotting (Pharmacia-Amersham-Biotech), prehybridization, hybridization, and washing were carried out as described above.
Preparation of Nuclear Extracts
Nuclear extracts were prepared from 108 exponentially growing cells as described by Riou et al. (25)
. Freshly prepared protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µg/ml aprotinin, and 10 µg/ml soybean trypsin inhibitor) were added to all buffers. Protein concentration in the nuclear extracts was determined as described previously (26)
. Before use, the extracts could be stored at -70°C for a period of time not exceeding 48 h.
Western Blot Analysis
Freshly prepared nuclear extracts (40 µg) were loaded onto a 7.5% SDS-polyacrylamide gel according to the method of Laemmli (27)
. Proteins were transferred (4 mA/cm2) to nitrocellulose membranes (Gelman Sciences, Ann Arbor, MI) and then probed with the polyclonal antibody A6 as described previously (16)
. For detection with the monoclonal antibody, the membranes were reacted for 4 h at room temperature in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO40.7H2O, 1.4 mM KH2PO4, at pH 7.3), 2% nonfat milk containing a 1:1000 dilution of the monoclonal antibody. After three washes (10 min each) in PBS, 0.2% Tween 20, the membranes were incubated for 1 h at room temperature in PBS, 2% nonfat milk containing 1:500 dilution of an antimouse immunoglobulin labeled with horseradish peroxidase (Amersham-Pharmacia-Biotech). After three washes (10 min each) at room temperature in PBS, 0.2% Tween 20, immunoreative bands were detected by light emission using an enhanced chemiluminescence Western-blotting detection system (Amersham-Pharmacia-Biotech).
Topoisomerase II Reactions
Decatenation of Kinetoplast DNA.
Approximately 0.25 µg of 3H-labeled Trypanosoma cruzi kinetoplast DNA was incubated with nuclear extracts (10 µl; 3.5 µg/ml) in topoisomerase II reaction buffer [40 mM Tris-HCl (pH 8), 50 mM KCl, 10 mM MgCl2, 5 mM EDTA, 1 mM ATP, and 0.5 mM DTT] for different periods of time at 37°C. The reaction was terminated by the addition of 1 M NaCl and 3.3 mM EDTA. The decatenated DNA was separated by filtration through a Millipore filter, and a 100-µl aliquot was counted in scintillation buffer Instagel (Packard, Meriden, CT).
Topoisomerase II-mediated Cleavage Reaction.
pSP65 DNA (0.2 µg) was incubated with nuclear extracts (2 µl; 2.5 µg/µl), in the presence or absence of drug, in topoisomerase II reaction buffer for 15 min at 37°C. The cleavage reaction was terminated by the addition of SDS and proteinase K to a final concentration of 0.4% and 0.1 mg/ml, respectively, and the mixture was incubated for an additional 30 min at 50°C. After proteinase K digestion, 5 µl of loading buffer (0.25% bromphenol blue, 30% glycerol) were added to each sample (15 µl). The products of cleavage reactions were separated on 0.8% agarose gels for about 15 h (2.5 V/cm) in TBE containing 0.25 mg/ml of ethidium bromide. DNA bands were visualized by transillumination with UV light (312 nm) and were photographed. To quantify the circular pSP65 DNA double-strand cleavage, negative films were scanned with a densitometer (Chromoscan 3; Joyce-Loebl, Gateshead, United Kingdom). The densitometer was connected to a computer, which stored and analyzed the data. The peak areas of linearized DNA (form III) were calculated.
Measurements of DNA-Protein Cross-Links by Alkaline Elution
DPCs were assayed by DNA denaturing alkaline elution (pH 12.1) carried out under nondeproteinizing conditions using protein adsorbing filter (polyvinyl chloride; Gelman Sciences, Ann Arbor, MI) as described previously (28)
. Both 14C-labeled cells and 3H-labeled cells (internal standard) were X-ray irradiated with 3000 rads on ice just prior to elution. DPC frequencies were calculated as described by Ross et al. (29)
.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
cDNAs from DC-3F and DC-3F/9-OH-E Cells. and ß in the parental and resistant cells were cloned and sequenced.4
Analysis of the ß cDNA was reported previously (17)
. In DC-3F cells, based on a group of seven silent mutations spread all over the coding region and three mutations in the noncoding region, an allele polymorphism was identified at the topoisomerase II locus.5
One of these silent mutations, at position 3933, creates a BamHI site. From the cDNA sequences, PCR primers were designed to generate a 151-bp fragment containing both this BamHI site, specific of the mutated allele, and an AcsI site, common to both alleles. Using these primers, one 815-bp fragment was amplified from genomic DNA, indicating the presence of intronic sequences in this region (Fig. 2A
, Lanes 1 and 4). The fragments amplified from DC-3F and DC-3F/9-OH-E DNAs were only partially digested by BamHI, yielding two fragments of 716 and 99 bp, whereas 50% of the PCR product remained at the original size of 815 bp (Fig. 2A
, Lanes 2 and 5). As expected, the DNAs from both cell lines were completely digested by AcsI (Fig. 2A
, Lanes 3 and 6). These data indicated that indeed two topoisomerase II alleles are present in DC-3F cells, one allele differing from the other by the presence of the silent mutations, and this polymorphism is conserved in DC-3F/9-OH-E cells. However, in these cells, we found that the mutated allele contains three additional point mutations, resulting in amino acid substitutions at positions 1112 (Thr371Ser), 2252 (Ala751Gly), and 2588 (Ile863Thr).
|
, Lanes 1 and 4). As shown on Fig. 2B
, Lane 2, the DC-3F fragment was partially digested by BamHI, yielding two fragments of 82 and 69 bp. More than 80% of the fragment amplified from the DC-3F cells was resistant to BamHI digestion, whereas only a small fraction of the DC-3F/9-OH-E fragment was resistant to BamHI digestion (Fig. 2B
, Lane 5). These data indicated that topoisomerase II transcripts are predominantly produced from the nonmutated allele in DC-3F cells and from the mutated allele in DC-3F/9-OH-E cells.
Transfection of the hTOP2 cDNA in DC-3F/9-OH-E Cells.
To restore a normal topoisomerase II activity in the DC-3F/9-OH-E subline, the cells were transfected with the plasmid pSV-hTOP2 containing the human topoisomerase II cDNA. The Chinese hamster topoisomerase II cDNA was not available when this study was initiated. The hTOP2 cDNA was first excised from the pBS-hTOP2 plasmid and transferred to the eucaryotic expression vector pSV-Sport1. The pSV-Sport1 vector was selected among several others tested as the one which in our cells yielded the best expression of the transfected gene, both in transient and permanent transfection experiments.
DC-3F/9-OH-E cells were cotransfected with two plasmids: pSV-hTOP2 and pSVtk-neo-ß, which confers the resistance to the antibiotic G418. Among 186 clones growing in the presence of G418, five were found to contain the transfected pSV-hTOP2, as shown by Southern blot hybridization. Northern blot analysis revealed in all of them an increased amount of topoisomerase II mRNA as compared with the untransfected DC-3F/9-OH-E cells. Clone 24, which contained the highest amount of this mRNA, and clone 14, which is representative of the others, were selected for further characterization. In agreement with our previous data (16)
, Fig. 3A
shows that in the DC-3F/9-OH-E cell line, the 5.7-kb endogenous transcript was about 4–5-fold less abundant than in the DC-3F cells, as estimated by scanning densitometry. In the transfected clones, a transcript corresponding to the same size was expressed at a higher level than in the untransfected DC-3F/9-OH-E cells. In clone 14, the amount of this transcript was approximately the same as in the DC-3F cells, whereas it was 5–6-fold higher in clone 24. Additional transcripts (about 8.4, 7.8, and 4 kb) were also detected in clone 24. Integrity of the transfected hTOP2 gene was examined by Southern blot analysis of SmaI and NheI-digested genomic DNA from clones 14 and 24 (Fig. 3C)
. The topoisomerase II-specific probe SP1' essentially hybridized with one fragment corresponding to the expected transgene size (5.6 kb), indicating that both clones contain the full-length hTOP2 cDNA. Clone 24 contains a few additional bands, which may explain the various Top2 transcripts detected in these cells. Gene copies in clone 14 were about 5–6-fold less abundant than in clone 24, which is consistent with the data from the Northern blot analysis.
|
shows that a fragment of 190 bp was detected in clone 24 (a faint band, not visible on the photograph, was also observed on the autoradiogram with clone 14), thus showing the presence of the SV40 early promoter sequence in the initial part of the hTOP2 RNA. This indicates that the hTOP2 cDNA is transcribed in the transfected clones. Furthermore, the amounts of this fragment in clones 14 and 24 were approximately in proportion of the hTOP2 expression level. There was no hybrid detection in the control lanes (tRNA, DC-3F, and untransfected DC-3F/9-OH-E).
|
present in the nuclear extracts from the transfected cells was first analyzed by immunoblot using two antibodies. These analyses (Fig. 5)
showed that in the resistant cells the amount of topoisomerase II was 4–5-fold smaller than in the sensitive cells, in agreement with our previous reports (16)
. Immunoblot analysis with the A6 antibody (Fig. 5A)
showed that the expression of topoisomerase II was 3-fold increased in clone 24 as compared with that in the DC-3F/9-OH-E cells. In clone 14, the amount of the enzyme was about the same as in the resistant cells. Comparable results were obtained with the anti-topoisomerase II monoclonal antibody (Fig. 5B)
.
|
. There was a 4-fold decrease in catalytic activity of DC-3F/9-OH-E extracts as compared with DC-3F extracts. In clone 24, the expression of the hTOP2 gene restored a normal catalytic activity. In clone 14, the catalytic activity remained approximately at the same level as in the resistant cells. These results show that topoisomerase II activity in these cell lines parallels the amounts of topoisomerase II proteins determined by Western blot analysis in the previous experiment (Fig. 5)
.
|
, shows that DNA cleavage of pSP65 DNA by nuclear extracts from DC-3F cells was stimulated either in the presence of 9-OH-E or NMHE at concentrations ranging from 0 to 12 µM, following a biphasic process as usually observed with DNA intercalators (30)
. When pSP65 DNA was incubated in identical conditions with nuclear extract from the resistant cells, DNA cleavage induced by 9-OH-E or NMHE was reduced to <10% of that induced with DC-3F nuclear extract. In clone 24, expression of the transfected hTOP2 cDNA that restored a nearly normal topoisomerase II activity had no effect on 9-OH-E or NMHE induced DNA cleavage. Fig. 7, C and D
, shows that the stimulation of DNA cleavage by etoposide (0–100 µM) or m-AMSA (0–64 µM) was about three times higher with the nuclear extract from DC-3F cells than with the nuclear extracts from either the DC-3F/9-OH-E cells or clone 14. A biphasic process was again observed with m-AMSA. With the extract from clone 24, the DNA cleavage stimulation by etoposide was close to that observed with the nuclear extract from DC-3F, whereas the stimulation induced by m-AMSA reached 60% of the level observed with DC-3F nuclear extract.
|
. In the same range of concentrations (0–5 µM), this drug did not induce any detectable DPC in either resistant or transfected cells. Different results were obtained when the cells were treated with etoposide (Fig. 8B)
: the amount of DNA damage was about 3-fold higher in the DC-3F than in the DC-3F/9-OH-E cells; in clone 24, the amount of DPC was about 2-fold higher than in the untransfected DC-3F/9-OH-E cells.
|
activity in the DC-3F/9-OH-E cells is associated with an increased induction of DNA lesions in the presence of etoposide but not in the presence of 9-OH-E.
Drug Resistance.
The sensitivity of the transfected cells to the topoisomerase II inhibitors 9-OH-E, NMHE, etoposide, and m-AMSA was determined. Fig. 9A
shows that the DC-3F/9-OH-E cells and the transfected clones 14 and 24 display the same resistance level to 9-OH-E. A similar result was obtained with the other ellipticine derivative, NMHE (Fig. 9B)
. The results were different with the other drugs: both the untransfected DC-3F/9-OH-E cells and clone 14 were about 44-fold resistant to etoposide (Fig. 9C)
; in contrast, in clone 24, the resistance was reduced 4-fold. As reported previously (30)
, the resistance of the DC-3F/9-OH-E cells to m-AMSA was very high, 500-fold. This resistance remained at the same level in clone 14 but again was about 4-fold decreased in clone 24 (Fig. 9D)
.
|
activity in clone 24 does not affect the cellular resistance to ellipticine derivatives but restores a large part of the sensitivity to other topoisomerase II inhibitors, such as etoposide and m-AMSA. The above results suggested the presence in DC-3F/9-OH-E cells of a factor inhibiting the capacity of ellipticine derivatives to increase the number of cleavable complexes. We then hypothesized that, in in vitro experiments, this factor might inhibit the induction of cleavable complex formation on plasmid DNA incubated with nuclear extracts from DC-3F cells in the presence of NMHE. Various amounts of nuclear extracts from DC-3F and DC-3F/9-OH-E cells were mixed in a final volume of 5 µl (total proteins, 5 µg), and these mixtures were assayed for stimulation of pSP65 DNA cleavage in the presence of NMHE (6 µM). Fig. 10
shows that the amount of cleavable complex decreased linearly as the percentage of nuclear extract from resistant cells increased from 0 to 100%, thus indicating that the complex-forming activity in the extract from sensitive cells was simply diluted by the extract from the resistant cells. This experiment rules out the hypothesis of a factor present in excess in DC-3F/9-OH-E cells that would inhibit the induction of cleavable complex formation by ellipticines.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2 years, a several hundred-fold resistance to topoisomerase II inhibitors was reached in DC-3F/9-OH-E cells. Biochemical characterization of these cells first revealed a 4–5-fold decreased topoisomerase II activity as compared with the parental DC-3F cells. This was markedly different from other cell lines in which a similar reduction of the enzyme activity resulted in a 4–5-fold resistance to the same molecules (18
, 31)
. It was then suggested that multiple genetic changes, which accumulated during the long selection process of DC-3F/9-OH-E cells, might cooperate to reach this very high resistance level. The goal of this work was to analyze the contribution of topoisomerase II alterations to the resistance phenotype of DC-3F/9-OH-E cells.
Chinese hamster topoisomerase II cDNA was first cloned and sequenced in CHO cells sensitive or resistant to etoposide (32)
. Sequencing of topoisomerase II cDNA in DC-3F cells revealed an allele polymorphism: one allele carrying the same sequence as that found in CHO cells, the other differing from the first one by the presence of seven silent mutations and three point mutations in the noncoding region.5
In DC-3F/9-OH-E cells, three amino acid substitutions, also carried by the mutated allele, were identified at positions 371, 751, and 863. One of these mutations (371) is located close to the ATP binding site, and the others are located in the vicinity of the catalytic site, on each side of tyrosine 804 involved in the covalent enzyme-DNA complex. All are located in, or close to, regions identified previously as mutational hotspots of topoisomerase II (11)
. However, the effects of these mutations on the catalytic properties of the enzyme and its sensitivity to the various inhibitors remain to be established. Because one of the silent mutations (position 3933) created a BamHI site in the mutated allele, it was possible to identify each allele and to analyze their relative expression in the sensitive and resistant cells. Both alleles are present in DC-3F and DC-3F/9-OH-E cells. However, transcription of the wild-type allele was strongly predominant in DC-3F cells, whereas it was the opposite for the resistant cells. Furthermore, in another cell line selected from DC-3F cells for resistance to a new olivacine derivative, named S16020-2, only the mutated allele was detected in the cell genome, and the cDNA library as well,5
thus confirming that the mutated allele is preferentially expressed in DC-3F-resistant variants.
One approach to analyze the role of topoisomerase II alterations in the DC-3F/9-OH-E phenotype was to complement these cells with the wild-type enzyme. After cotransfection of the DC-3F/9-OH-E cells with the plasmids pSVtk-neo-ß and pSV-hTOP2, about 190 clones were selected on the basis of their resistance to G418, but only five of them were found to contain the hTOP2 gene. By Northern blot analysis, an increased transcription of the topoisomerase II gene was observed in all of the five clones, although in four of these clones, the amount of enzyme detected by immunobloting remained very close to that in the untransfected resistant cells. However, in one of them, clone 24, the amount of topoisomerase II protein and the enzyme catalytic activity were similar to those in the parental DC-3F cells. The low expression level of the topoisomerase gene in the majority of the clones is not due to a defect in the construction because, in transient transfection experiments in both NIH3T3 and DC-3F/9-OH-E cells, a high expression of the hTOP2 gene was observed (data not shown). In previous cotransfection experiments of the DC-3F/9-OH-E cells, using the pSVtk-neo-ß plasmid associated with either the mouse c-myc or the human MDR3 gene, expression of the cotransfected gene was observed in >50% of the G418-resistant clones (33)
. Both the low efficiency of cotransfection with the hTOP2 gene and its low expression level in the transfected clones indicated that the overexpression of this gene was toxic for the transfected cells, thus preventing the selection of cells expressing hTOP2 at a high level. Such a toxicity was observed previously in other systems, including yeast transfected with the pYEPTOP2-PGAL1 plasmid (34)
, CHO cells (35)
, P388 murine leukemia cells (36)
, and DC-3F/9-OH-E cells transfected with pSV-hamTOP2ß, a plasmid containing the hamster topoisomerase IIß cDNA (19)
. Toxicity resulting from topoisomerase II overexpression in the transfected cells might be due to the stimulation of abnormal genetic rearrangements that are potentially lethal (37)
and induction of apoptosis (38)
.
Mammalian topoisomerases II and ß share a high structural homology and similar enzymatic properties. They both efficiently complement temperature-sensitive topoisomerase II mutations in yeast (39)
. However, a number of studies indicate that in mammalian cells, the two isoforms serve unique functions, as suggested in particular by their distinct cellular distribution and chromatin binding at mitosis. Recently, it was shown that a high affinity binding of the enzyme to chromatin is responsible for the essential role of topoisomerase II in chromosome condensation/disjunction at mitosis. Furthermore, topoisomerase IIß does not substitute the isoform in its mitotic functions (40)
. These data show that the properties of the topoisomerase II isoforms in their native environment are clearly different from those observed in yeast, which uses either one isoform at mitosis without distinction. Therefore, drug testing in yeast systems might not be as relevant as initally envisaged (41
, 42)
. A mutation introducing a stop codon in topoisomerase IIß cDNA completely inhibits its expression in DC-3F/9-OH-E cells. Restoration of a normal topoisomerase IIß activity in these cells revealed a highly preferential interaction of m-AMSA with this isoform (19)
, which was confirmed by other studies on a human leukemia cell line (43)
but not detected in yeast (42)
. Clone 24, in which both the amount of topoisomerase II protein and catalytic activity were restored at the same level as in DC-3F cells, is then a unique mammalian cell line to study the physiological function of this isoform and its role as a target for antitumor topoisomerase II inhibitors. Indeed, the effects of various molecules tested on these cells turned out to be quite different from those observed either in vitro with purified enzyme or in yeast.
The recovery of a normal enzyme activity in clone 24 did not have the same effect on the cellular sensitivity to different topoisomerase II inhibitors. Whereas 75% of the sensitivity to m-AMSA and etoposide was restored, the resistance to 9-OH-E and NMHE was not modified. The ability of these drugs to increase the number of cleavable complexes present on the genome parallels their cytotoxicity; the ellipticine derivatives did not increase the number of cleavable complexes either in the untransfected DC-3F/9-OH-E cells or in clone 24, whereas the amount of these complexes induced by etoposide or m-AMSA in clone 24 was increased up to 60% of the amount measured in DC-3F cells. This observation confirms that an increased number of cleavable complexes is an essential step in the toxicity pathway of these molecules. However, restoration of the amount of topoisomerase II to the DC-3F level is not sufficient to restore the capacity of certain drugs to increase the complex formation in DC-3F/9-OH-E cells.
As pointed out earlier, DC-3F/9-OH-E cells, and clone 24 as well, are devoid of any topoisomerase II ß activity (17)
. If the toxicity of the ellipticine derivatives used in these experiments was specifically mediated by topoisomerase IIß, then the loss of this enzyme isoform would explain the absence of cleavable complex formation in DC-3F/9-OH-E cells and the nonreversion of the resistance phenotype in cells transfected with the enzyme cDNA. However, although the and ß isoforms display different sensitivities to different inhibitors (7
, 44)
, such a high specificity toward ellipticine derivatives has never been demonstrated either in in vitro or in vivo experiments. Furthermore, in DC-3F/9-OH-E cells complemented with a normal topoisomerase IIß activity, the resistance to etoposide and m-AMSA was approximately 40 and 90%, decreased respectively, whereas the resistance to ellipticines remained unchanged (19)
. The similarity between the phenotypes of the ß transfected cells and clone 24 also strongly argues against the possibility that clone 24 properties might be due to some mutation in the transfected gene that would have been selected by chance.
Several studies (5)
have shown that different drugs can increase the number of cleavable complexes through distinct mechanisms; some drugs, like etoposide or m-AMSA, impair the ability of the enzyme to religate the cleaved DNA, whereas others, like ellipticines, quino-lones, and genistein, which do not inhibit the religation step, are presumed to accelerate the forward rate of complex formation. Restoration in DC-3F/9-OH-E of either topoisomerase II (clone 24) or topoisomerase IIß (19)
activities at the initial DC-3F level resulted in a partial reversion of the resistance to drugs that inhibit the religation step but had nearly no effect on drugs that do not, like ellipticines, S16020-2,5
and genistein (data not shown). In other words, in this system, the resistance to religation inhibitors appears to be recessive, whereas the resistance to compounds enhancing the forward rate of complex formation would be dominant.
Several hypotheses can be considered to explain the absence of cleavable complex formation in clone 24 treated with ellipticine derivatives: (a) In the parental DC-3F cells, the complex formation induced by compounds that do not inhibit the religation step requires a cofactor that is absent in the resistant cells. However, this hypothesis seems unlikely because these compounds are able to induce the complex formation in vitro with purified enzyme, and, in hybrids between sensitive and resistant cells, the resistance phenotype should be recessive, whereas it is partially dominant (45)
; (b) in DC-3F/9-OH-E cells, a trans-acting factor inhibits the complex formation induced by ellipticine derivatives. If such a factor were present in excess, nuclear extracts from resistant cells should inhibit the cleavable complex formation with nuclear extracts from sensitive cells in the presence of ellipticines but not in the presence of etoposide. The experiments in Fig. 10
, which show results with NMHE identical to those reported previously with etoposide (46)
, rule out this hypothesis. However, it does not exclude the possibility of an inhibiting factor stoichiometrically interacting with either topoisomerase II or ß. Other experimental approaches, based for example on the immunoprecipitation of these complexes or a yeast two-hybrid system (47)
, would be required to detect such a factor. Another possibility would be the formation of heterodimers associating enzyme subunits from the mutated endogenous gene and from the transfected gene. If the mutations in the endogenous gene make the enzyme resistant to certain inhibitors like ellipticines, then one may assume that heterodimers of this type might also be resistant to the same drugs. However, this hypothesis is highly unlikely because the transfected enzyme is in large excess more than the endogenous enzyme. Therefore, a large fraction of the enzyme coded by the transgene should constitute drug sensitive homodimers; (c) a posttranslational modification of the enzyme might be required for cleavable complex induction by drugs that are not religation inhibitors. Although posttranslational phosphorylation of topoisomerase II has been studied extensively, its consequences on the enzyme functions are not fully understood. Topoisomerase II can be phosphorylated on multiple sites by several protein kinases, including a proline-directed kinase (48)
, protein kinase C (49)
, and casein kinase II (50)
. It was reported recently that, after abrogation of intracellular Ca2+ transients that lead to site-specific hypophosphorylation of topoisomerase II, a significant decrease in etoposide- or m-AMSA-induced cleavable complex formation and a corresponding decrease in cytotoxicity was observed (51)
. Whether the activity of ellipticine derivatives, as well as that of the other drugs that share the same mechanism of DNA-topoisomerase II complex induction, is controlled by such a reaction or another as yet unidentified posttraductional modification remains to be established.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported in part by Association pour la Recherche sur le Cancer (ARC), Villejuif, France, and Ligue Nationale Française contre le Cancer (LNC). S. L. received fellowships from ARC, LNCC, and Société Française du Cancer. 
2 To whom requests for reprints should be addressed, at CNRS UMR 8532, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif, France.
3 The abbreviations used are: m-AMSA, 4'-(9-acridinylamino)methanesulfon-m-anisidide; 9-OH-E, 9-hydroxyellipticine; NMHE, 2-N-methyl-9-hydroxy-ellipticinium; DPC, DNA-protein cross-link.
4 Accession numbers at the EMBL/EBI database to the sequence data reported in this study are Y16592, Y16593, and Y16594.
5 S. Le Mée, F. Chaminade, C. Delaporte, J. Markovits, J-M. Saucier, and A. Jacquemin-Sablon. Importance of the N-[2(dimethylamino)ethyl]carbamoyl side chain in the mechanism of action and cellular resistance to the antitumor DNA topoisomerase II inhibitor S16020-2, submitted for publication.
Received 3/29/99. Accepted 8/ 4/99.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and topoisomerase IIß genes: characterization and mapping to human chromosomes 17 and 3, respectively. Cancer Res., 52: 231-234, 1992. and ß in Chinese hamster lung cells resistant to topoisomerase II inhibitors. Mol. Pharmacol., 46: 323-328, 1994.[Abstract]
. Anti-Cancer Res., 17: 4243-4252, 1997.[Medline]
. Cancer Res., 58: 4519-4524, 1998. and IIß can functionally substitute for yeast TOP2 in chromosome segregation and recombination. Mol. Genet. Genet., 252: 79-86, 1996.
are not adopted by topoisomerase IIß in human H69 cells. J. Biol. Chem., 273: 33660-33666, 1998.. Cancer Res., 58: 5298-5300, 1998. is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase. Eur. J. Biochem., 231: 491-497, 1995.[Medline]
. Evidence of a role for protein kinase C. J. Biol. Chem., 270: 28357-28363, 1995. in a phosphorylation-independent manner. J. Biol. Chem., 273: 3635-3642, 1998.. Biochem. J., 336: 727-733, 1998.
This article has been cited by other articles:
|
|
 |
|
  A. Skladanowski, M.-G. Come, M. Sabisz, A. E. Escargueil, and A. K. Larsen Down-Regulation of DNA Topoisomerase II{alpha} Leads to Prolonged Cell Cycle Transit in G2 and Early M Phases and Increased Survival to Microtubule-Interacting Agents Mol. Pharmacol., September 1, 2005; 68(3): 625 - 634. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Gros, C. Delaporte, S. Frey, J. Decesse, B. R. de Saint-Vincent, L. Cavarec, A. Dubart, A. V. Gudkov, and A. Jacquemin-Sablon Identification of New Drug Sensitivity Genes Using Genetic Suppressor Elements: Protein Arginine N-Methyltransferase Mediates Cell Sensitivity to DNA-damaging Agents Cancer Res., January 1, 2003; 63(1): 164 - 171. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Le Mée, F. Chaminade, C. Delaporte, J. Markovits, J.-M. Saucier, and A. Jacquemin-Sablon Cellular Resistance to the Antitumor DNA Topoisomerase II Inhibitor S16020-2: Importance of the N-[2(Dimethylamino)ethyl]carbamoyl Side Chain Mol. Pharmacol., October 1, 2000; 58(4): 709 - 718. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), and clone 24 (
) were incubated with 0.25 µg of Trypanosoma cruzi kinetoplast [3H]DNA. At the indicated times, aliquots were taken, and the reaction was stopped. Values are the means of at least two independent determinations; bars, SE.
), DC-3F/9-OH-E (•), clone 14 (