Cancer Research Landon Prizes for Basic and Translational Cancer Research Cancer Health Disparities Conference 2009
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
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Urasaki, Y.
Right arrow Articles by Pommier, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Urasaki, Y.
Right arrow Articles by Pommier, Y.
[Cancer Research 61, 1964-1969, March 1, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Characterization of a Novel Topoisomerase I Mutation from a Camptothecin-resistant Human Prostate Cancer Cell Line

Yoshimasa Urasaki, Gary S. Laco, Phillipe Pourquier, Yuji Takebayashi, Glenda Kohlhagen, Christopher Gioffre, Hongliang Zhang, Devasis Chatterjee, Panayotis Pantazis and Yves Pommier1

Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255 [Y. U., G. S. L., P. Po., G. K., C. G., H. Z., Y. P.]; Institute of Development, Aging, and Cancer, Tohoku University, Sendai 980-8575, Japan [Y. T.]; and Department of Molecular Biology, Cell Biology, and Biochemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 [D. C., P. Pa.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the past several decades, new cancer chemotherapies have been developed at a remarkable rate, and consequently some malignancies are now curable, such as leukemias (1) , lymphomas (2) , and testicular tumors (3) . However, even in these cases, drug resistance is a major limitation of chemotherapy, and there are still numerous primary, and recurrent, refractory cases (4) . DNA top12 has recently been investigated as a new target for chemotherapy (5 , 6) . In higher eukaryotes, top1 is an essential enzyme that relaxes DNA supercoiling and relieves torsional strain during DNA processing, including replication, transcription, and repair. The natural product CPT targets the top1/DNA covalent complex, a catalytic intermediate, by reversibly blocking top1-mediated religation of the DNA and release of free top1 (7) . In the presence of CPT, top1 remains covalently linked to one strand of the DNA, and thus leaves the DNA with a protein-linked DNA single-stranded break, which triggers cytotoxic lesions in metabolizing DNA (8 , 9) .

Several CPT derivatives have recently been introduced for cancer therapy, including irinotecan and topotecan. Therefore, defining the mechanisms of CPT resistance is important in developing more efficacious derivatives. One approach to define the cellular resistance mechanisms and the top1/CPT interactions is by selecting cell lines for CPT resistance, and then determining putative top1 mutations that may confer resistance. A number of top1 mutations have been identified in this way (10) . Such mutations generally confer high-level resistance to CPT and its derivatives both in vivo and in vitro (10 , 11) . The recently determined X-ray crystal structure of human top1 complexed with DNA (12 , 13) allows the resistance mutations to be understood in terms of their interaction with DNA, and potentially with CPT. However, the top1 interactions with CPT proposed to date are based on two hypothetical models of CPT binding in the top1 active site (12 , 14) . Without a crystal structure of the top1/DNA/CPT complex, the existing mutations that confer CPT resistance can only suggest potential top1/CPT interactions. In this study, we identified a novel mutation in the top1 gene of CPT-resistant cell lines, and showed that this mutation results in a CPT-resistant enzyme in vitro using a recombinant mutant top1. This new CPT-resistance mutation allows further definition of the top1/CPT interactions and is consistent with the existing top1/CPT models.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Chemicals, and Enzymes.
The human prostate carcinoma cell line DU-145 (15) was cultured in RPMI 1640 (Life Technologies, Gaithersburg, MD) containing 10% FCS at 5% CO2 and 37°C. The CPT-resistant DU-145 cell sublines 145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively) were established by continuous exposure of DU-145 cells to 9-nitro-CPT (16) . The RC0.1 and RC1 cell lines grow in the presence of 0.1 µM and 1 µM drug concentrations, respectively. [{alpha}-32P]dGTP and [{alpha}-2P]cordycepin were purchased from New England Nuclear (Boston, MA). Human top1 was expressed in Sf9 insect cells using a recombinant baculovirus and purified as described previously (17) .

Cytotoxicity Assays.
The MTT assay was used to determine the cellular sensitivity to drugs. Three thousand cells were seeded per well in 96-well microtiter plates. The cells were incubated at 37°C in the continuous presence of drug for 3 days. Growth inhibition was then assayed by adding 50 µg of MTT dye per well. After 4-h incubation, 100 µl of DMSO were added and absorbances were measured at 550 nm using an E-max microtiter plate reader. Results are expressed as percentage of growth inhibition, mean ± SD, of at least three independent experiments.

Preparation of Nuclear Extracts.
Nuclear extracts were made as described previously (18) with minor modifications. Briefly, 107 exponentially grown cells were washed twice in 1x ice-cold nucleus buffer (150 mM NaCl, 1 mM KH2PO4, 5 mM MgCl2, and 1 mM EGTA) and recovered by centrifugation at 200 x g for 10 min. Cell pellets were resuspended in fresh nucleus buffer containing 0.03% Triton X-100 and incubated at 4°C for 10 min. After centrifugation at 200 x g for 10 min, pelleted nuclei were washed twice in nucleus buffer. Proteins were then salt-extracted by adjusting the final NaCl concentration to 0.35 M and gentle mixing at 4°C for 30 min. After centrifugation at 12,000 x g for 30 min, supernatants were collected and protein concentrations evaluated using the Bradford assay (19) .

top1-induced DNA Cleavage Assay.
top1-mediated cleavage activities were measured using the 161-bp PvuII-HindIII fragment of pSK plasmid (Stratagene, La Jolla, CA) and a 22-mer oligonucleotide containing a single top1 cleavage site. In the experiments shown in Fig. 8Citation , the 22-mer oligonucleotide contained a trans-opened BaP at the N6-amino group of a central 2'-deoxyadenosine in the scissile strand (20) . End labeling and preparation of the DNA fragments were performed as described previously (21) . Labeled DNA (~50 fmol per reaction) was incubated with either purified top1, or nuclear extracts, for 30 min at 25°C in the presence or absence of drug in 1x top1 reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 µg/ml BSA]. Reactions were stopped by adding SDS (0.5% final concentration). After ethanol precipitation, samples were resuspended in gel electrophoresis loading buffer [80% formamide, 45 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue (pH 8)]. Reaction products were separated on either a 7% or a 20% denaturing polyacrylamide gel (7 M urea) in 1x TBE for 2 h at 40 V x cm at 50°C for the pSK fragment and the 22-mer oligonucleotide, respectively. Imaging and quantitation were performed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).



View larger version (62K):
[in this window]
[in a new window]
  		Fig. 8. Determination of top1 cleavage activity by recombinant R364H top1. Various amounts of WT top1 (WT) and of the mutant top1 (R364H) were reacted with either unmodified 22-mer double-stranded oligonucleotide (left), or a suicide oligonucleotide (right), containing benzo[a]pyrene 7,8-diol 9,10-epoxide adduct (BaP) at position +1. In this case G+1 had to be changed to an A+1 (20) . Arrowhead, the top1 cleavage site. Lanes C, no top1 added; Lanes 1–3, WT 14, 7, and 3.5 ng, respectively; Lanes 4–6, R364H 14, 7, and 3.5 ng; Lanes 1–6, left panel, with 10 µM CPT. Lower panels, quantitations of the gels shown at the top of the figure.

 
top1-mediated DNA Relaxation Assays.
Reaction mixtures (10 µl) contained 0.3 µg of supercoiled SV40 DNA per top1 assay. Reactions were performed at room temperature for the indicated times and were terminated by the addition of SDS (0.5% final concentration). An amount of 1.1 µl of 10x loading buffer [20% Ficoll 400, 0.1 M sodium EDTA (pH 8), 1.0% SDS, and 0.25% bromphenol blue] was then added, and reactions were loaded onto 1% agarose gels. After electrophoresis, the gels were stained with 1X buffer solution containing 10 µg/ml of ethidium bromide and were visualized by transillumination with UV light (300 nm). Quantitations were performed using a FluoroImager (Molecular Dynamics).

Immunoblot Analysis of top1.
For top1 analysis, ~107 cells were lysed in radioimmunoprecipitation assay buffer (1x PBS, 1% NP40, 0.5% sodium deoxycholate, and 0.1% SDS); then an equal volume of 1x Tris-glycine SDS sample buffer (126 mM Tris-HCl, 20% glycerol, 4% SDS, and 0.005% bromphenol blue) was added. The lysates were placed in boiling water for 5 min, loaded onto the 8% polyacrylamide/SDS gels, electrophoresed for 3 h at 150 V, and transferred onto a nitrocellulose membrane using a Bio-Rad semidry blotter (Bio-Rad, Hercules, CA) according to the manufacturer’s protocol. Imumunoblotting was performed using the top1 monoclonal antibody (a kind gift from Dr. Y-C. Cheng, Yale University, New Haven, CT) as described previously (22) .

Detection of top1 Mutation.
RNA was extracted from the parental and drug-resistant cell lines using a RNeasy Mini kit (Qiagen, Valenica, CA) according to the manufacturer’s protocol. RNA was reverse transcribed using the SuperScript preamplification system (Life Technologies) according to the manufacturer’s protocol. Four regions of the top1 cDNA tat were already known to bear mutations responsible for CPT resistance (11) were amplified using the following pairs of primers (sense and antisense, respectively): for region A, 5'-CTTTAAAGACTGGAGAAAGGAAATGACTAATG-3' and 5'-TCTTTGCTACAGTTGATGATTATATCCTC-3'; for region B, 5'-GAAGACTGGAAGTCCAAAGAGATGAAAGT-3' and 5'-GTCAAGCCCTCCATGAGATCCTGAAGATG-3'; for region C, 5'-GGGAAGGACTCCATCAGATACTATAACAA-3' and 5'-GGGGCTGTCAGTTCTTTTAGCTGCTGCTGT-3'; and for region D, 5'-CAGTTGATGAAGCTGGAAGTTCAAGCCACAGA-3' and 5'-CTGGGTTTTGTTGTAAATCTTCTCAATTGGGAC-3'.

PCR amplification was performed using the PCR reagent system (Life Technologies, Inc.) under the following conditions: preincubation was at 95°C for 2 min followed by 30 cycles at 95°C (1 min); 55°C (1 min); 72°C (1 min), and a final extension of 10 min at 72°C. Cloning of the PCR fragments was performed using the TOPO TA cloning system (Invitrogen, Carlsbad, CA). Transformation of competent cells and DNA minipreps were performed according to standard procedures. Sequencing was performed using the BigDye primer cycle sequencing reaction kit (Applied Biosystems, Foster City, CA). Analysis of the top1 sequences was performed using Sequencher software (Genes Code Corporation, Ann Arbor, MI).

Expression of Mutant top1 and Purification.
WT and mutant R364H top1 expression were expressed and purified as follows. The WT top1 construct used is essentially the same as reported previously (23) . Briefly, either the WT or the mutant R364H top1 gene was cloned into a baculovirus transfer vector (pBacGus-1; Novagen, Madison, WI) and was used to make a recombinant baculovirus following the manufacturer’s recommendations (BD-PharMingen, San Diego, CA). The WT and R364H mutant top1 proteins were then expressed in TN5 insect cells (HighFive; Invitrogen) by the recombinant baculovirus, and purified via the NH2-terminal His tag as described previously (17 , 24) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The human prostate carcinoma cell lines RC0.1 and RC1 were developed from the parental DU-145 cell line by continuous exposure to 9-nitro-CPT. Although the parental DU-145 cells are sensitive to 9-nitro-CPT at 20–40 nM concentrations, the resistant cells (RC0.1 and RC1) can grow in the presence of 0.1 and 1 µM 9-nitro-CPT, respectively (16) . DU-145, RC0.1 and RC1 cells were characterized for resistance to other top1 inhibitors and for quantitative and/or qualitative modifications in top1.

Cross-Resistance to top1 Inhibitors.
We initially examined the RC0.1 and RC1 cell lines for resistance to other CPT derivatives, as well as other chemotherapeutic agents that are not active against top1 (i.e., top2, DNA and RNA synthesis and tubulin inhibitors). Both the RC0.1 and RC1 cell lines exhibited resistance to CPT and to the clinically relevant CPT derivatives, SN-38 and topotecan (~100- to 1000-fold; Fig. 1Citation and Table 1Citation ). There was a limited resistance (<10-fold) to NB-506, an intercalating top1 inhibitor (25) . Interestingly, neither of the cell lines was cross-resistant to the tubulin inhibitor vincristine, and the antimetabolites 5-fluorouracil or 1-ß-D-arabinofuranosylcytosine. Both the RC0.1 and RC1 cell lines were highly sensitive to the top2 inhibitors, VP-16 and ADR (Table 1)Citation .



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1. Determination of CPT sensitivity of DU-145 and resistant cell lines. The CPT-sensitive parental DU-145 cell line and resistant cell lines RC0.1 and RC1 were cultured in the presence of the indicated CPT concentrations for 3 days. Cell viability was determined using the MTT assay. Error bars, SDs (for five independent experiments).

 

View this table:
[in this window]
[in a new window]
  		Table 1 Sensitivity of DU-145, RC0.1, and RC1 cells to various anticancer drugs

 
top1 Protein Levels in the Resistant Cell Lines.
Reduction of top1 is a common mechanism that can account for cellular resistance to CPT, because it has been observed in other CPT-resistant cell lines (26, 27, 28, 29, 30, 31, 32) . Therefore, we analyzed the parental DU-145 cell line and both of the resistant cell lines RC0.1 and RC1 for top1 expression by the Western-blot methodology. top1 protein levels were comparable in the RC0.1 and RC1 and in the parental DU-145 cells (Fig. 2)Citation .



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 2. Immunoblot analysis for top1 in parental and CPT-resistant DU-145 cells. Cell extracts (amounts of protein, amounts loaded) were separated by SDS-PAGE. Immunoblotting was performed using monoclonal antibody against top1. kDa, Mr, in thousands.

 
top1 Catalytic Activity in SV40 DNA Relaxation Assay.
top1 activity was assayed in the nuclear extracts of DU-145 and RC1 cells by their ability to relax supercoiled SV40 DNA. The results in Fig. 3Citation demonstrate that both DU-145 and RC1 nuclear extracts were catalytically active in the SV40 DNA relaxation assays. However, whereas the top1 catalytic activity in the DU-145 extract was sensitive to CPT, it was resistant to CPT in the RC1 extract (Fig. 3Citation , bottom). These results demonstrate that this RC1 nuclear extract had no detectable defect in top1 activity but was resistant to CPT in the DNA relaxation assay.



View larger version (64K):
[in this window]
[in a new window]
  		Fig. 3. top1 relaxation activity assay in parental and CPT-resistant DU-145 cells. Supercoiled SV40 DNA was incubated with the indicated amounts of nuclear extract protein from the parental DU-145 and the RC1 cell lines. Incubation was for 30 min at room temperature in the absence or presence of 0.3 µM CPT. Reaction products were separated by agarose gel electrophoresis and stained with ethidium bromide, and supercoiled DNA was quantitated. NE, nuclear extract; Sc, supercoiled DNA; R, relaxed DNA.

 
CPT Resistance in the Cleavage Complex Assay.
Using the defined high-affinity top1 oligonucleotide substrate (Fig. 4ACitation ; Refs. 24 , 33 , 34) , the DU-145, RC0.1, and RC1 nuclear extracts were tested for the ability of CPT treatment to cause the accumulation of cleavage complexes, by the inhibition of top1 religation (32) . The32 oligonucleotide was cleaved by DU-145 extract in the presence of CPT. Cleavage was markedly reduced with the RC0.1 and RC1 extracts (Fig. 4, B and C)Citation .



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4. CPT-induced cleavage complexes induced by nuclear extracts from DU-145, RC0.1, and RC1 cells. In A, cleavage assays were performed using 32P-end-labeled oligonucleotide DNA; *A-3', the 32P-labeled cordycepin. In B, DNA substrates were incubated with 1.0 µg nuclear extract at room temperature in the absence (-) or presence (+) of 10 µM CPT. Reactions were terminated at 5 min (Lanes 3, 9, and 15); 15 min (Lanes 4, 10, and 16); 30 min (Lanes 5, 11, and 17); 60 min (Lanes 6, 12, and 18); and 90 min (Lanes 7, 13, and 19). In C, cleavage products were electrophoresed on 20% denaturing polyacrylamide gels and quantitated.

 
The pSK fragment, which contains several preferred sequences for top1 cleavage (35) was also tested to determine whether reduction of CPT-induced top1-mediated DNA cleavage was global or site specific. Fig. 5Citation shows that the RC0.1 and RC1 extracts were resistant to CPT-induced cleavage complex accumulation at all site examined. These results suggest that the cellular top1 in the RC1 and RC0.1 cell is likely resistant to CPT-mediated DNA cleavage, whereas top1 in the DU-145 cells is sensitive.



View larger version (64K):
[in this window]
[in a new window]
  		Fig. 5. CPT-induced cleavage complex accumulation in parental and CPT-resistant DU-145 cells at different top1 sites. Cleavage assays were performed using a 32P-labeled pSK substrate. DNA was incubated with nuclear extracts (each right triangle, 0.5, 1.0, and 2.0 µg of nuclear extract protein) at room temperature for 30 min in the absence (-) or presence (+) of 10 µM CPT. Reactions were terminated by adding SDS, and the cleavage products were electrophoresed on 7% denaturing polyacrylamide gels.

 
top1 Mutation in Resistant Cell Lines.
To test whether the CPT-resistant cell lines carried a mutant top1, mRNA was isolated from the parental and resistant cell lines, and subjected to reverse transcription-PCR. The resulting PCR products were cloned and sequenced, and revealed the same single-point mutation in both the RC0.1 and the RC1 top1 cDNA. This mutation, a G to A transition, results in an Arginine to Histidine mutation at amino acid residue 364 (R364H; Fig. 6Citation ). The WT top1 allele was not detected in either the RC0.1 or the RC1 cells (five individual clones were analyzed for each cell line), whereas the parental DU-145 cells contained only the WT allele.



View larger version (42K):
[in this window]
[in a new window]
  		Fig. 6. top1 mutation in the RC0.1 and RC1 cells. RNA was extracted from parental, CPT-sensitive DU-145, and CPT-resistant RC0.1 and RC1 cells. Sequencing of top1 was performed by standard reverse transcription-PCR amplification. Arrow, the point mutation (G to A transition).

 
Recombinant top1 Is Catalytically Active and CPT Resistant.
To confirm that the R364H mutation is responsible for the CPT resistance of the RC0.1 and RC1 cells, we constructed a recombinant top1 gene vector that contained the R364H mutation in the expressed top1. The R364H top1 protein was expressed in insect cells using recombinant baculovirus and was purified to >95% purity. The R364H top1 was next tested for catalytic activity and resistance to CPT using the SV40 relaxation assays (Fig. 7)Citation and the cleavage complex assay with oligonucleotide substrates as described previously (Fig. 8)Citation . In the SV40 relaxation assay, the R364H top1 protein was catalytically active and resistant to CPT (Fig. 7)Citation . For the oligonucleotide assays, two different substrates were used: (a) the high-affinity 22-mer oligonucleotide; and (b) an oligonucleotide with a BaP adduct inserted at the +1A, position relative to the top1 site (20) . This modified oligonucleotide has been shown to be a CPT-independent suicide substrate for top1 (36) . The R364H top1 had similar activity as the WT top1 on the suicide oligonucleotide substrate (Fig. 8)Citation , which is consistent with normal catalytic activity (Fig. 7)Citation . However, in the unmodified 22-mer oligonucleotide, the R364H top1 was highly resistant to CPT-induced cleavage complex accumulation as compared with WT top1 (Fig. 8)Citation . We conclude that the R364H top1 mutation renders top1 resistant to CPT.



View larger version (77K):
[in this window]
[in a new window]
  		Fig. 7. top1 relaxation activity and CPT resistance of recombinant R364H top1. WT top1 (WT) and R364H mutant top1 (R364H) were reacted with SV40 DNA for various periods of time and samples were analyzed. Lane C, control without top1; Lanes 1, 2, 3, 4, and 5, samples at 0, 5, 10, 30, and 60 min, respectively. All of the reactions contained 0.5 ng of the respective top1, and 0.5 µg SV40 DNA. A, WT without CPT; B, WT + 10 µM CPT; C, R364H without CPT; and D, R364H + 10 µM CPT. Sc, supercoiled DNA; R, relaxed DNA.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we have characterized the top1-mediated resistance to CPT of human prostate carcinoma cell lines derived from the parental, CPT-sensitive DU-145 cell line. A number of CPT-resistant cell lines have been previously isolated and characterized (reviewed in Ref. 10 ), and from them, the mechanisms of CPT resistance can be grouped into three categories. In the first category, resistance is related to alterations in cellular drug accumulation/distribution and metabolism; the second category includes quantitative and qualitative differences in top1; and the third category includes resistance factors downstream from cleavage complex resolution.

In the drug sensitivity assay, the RC0.1 and RC1 cell lines showed cross-resistance to CPT and its derivatives but were not resistant to drugs that do not target top1 (Table 1)Citation . The low cross-resistance of the RC0.1 and RC1 cell lines to NB-506 (an intercalating top1 inhibitor) is consistent with the possibility that NB-506 targets other cellular factors in addition to top1 (37) . The enhanced sensitivity of the RC0.1 and RC1 cells to VP-16 and ADR might be conferred by the increased level of top2 in RC0.1 and RC1 cells (data not shown).

The high level of CPT resistance of the RC0.1 and RC1 cell lines as compared with that of the parental cell line (Fig. 1)Citation , suggested that a specific alteration in the cellular top1 might be responsible for the resistance. We tested the RC0.1 and RC1 nuclear extracts for top1 activity and resistance to CPT, using the SV40 DNA relaxation assay as well as labeled DNA fragments (see Figs. 3Citation 4Citation 5Citation ). Nuclear extracts from RC0.1 and RC1 cells clearly demonstrated top1 catalytic activity in the SV40 relaxation assay, which was resistant to CPT treatment (Fig. 3)Citation . Analysis with the labeled DNA fragments showed that CPT trapped the top1 cleavage complex in the parental nuclear extracts, but not in either the RC0.1 or the RC1 nuclear extracts (Figs. 4Citation and 5Citation ). CPT stabilization of the top1 cleavage complex is the most important factor in CPT-mediated cytotoxicity (10) . Consequently, the RC0.1 and RC1 cell lines have acquired resistance to CPT, apparently because of the lack of CPT-mediated stabilization of the top1/DNA cleavage complexes.

Alterations in the top1 protein have been reported to result in resistance to CPT. The alterations include lower levels of cellular top1 protein, decreased top1 catalytic activity because of mutations, as well as top1 mutations that effect CPT binding [reviewed in (10) ]. Decreasing the levels of top1 protein in CPT-resistant cells is a strategic approach used by the cell (26, 27, 28, 29, 30, 31, 32) , thereby reducing the number of CPT-stabilized top1/DNA cleavage complexes that cause DNA damage. However, in our case, the top1 protein levels were not decreased in the resistant cell lines (Fig. 2)Citation . These results implicated a top1 mutation in the resistant cell lines, which results in CPT resistance. As shown in Fig. 6Citation , we, in fact, found a new top1 mutation, which changes the Arginine 364 codon to Histidine (R364H) in the CPT-resistant cell lines RC0.1 and RC1. To confirm that the CPT resistance was due to the R364H mutation, we constructed a recombinant top1 with the R364H mutation (top1/R364H), and examined the corresponding CPT sensitivity and catalytic activity. As shown in Fig. 7Citation and 8Citation , the recombinant top1/R364H is catalytically active in the SV40 DNA relaxation assays and with the suicide oligonucleotide substrate. Consistent with the results obtained with nuclear extracts, the top1/R364H enzyme was highly resistance to CPT in both the SV40 DNA relaxation, and in the oligonucleotide DNA cleavage assays (Fig. 7Citation and 8Citation ).

The R364H point mutation is located in the highly conserved core A region of top1 (see Fig. 3Citation in Ref. 10 ), and is within the top1 amino acid residues 361–364 that have been recently reported to be critical for CPT resistance (38, 39, 40) . Moreover, this mutation site is close to the catalytic tyrosine, and other mutations that confer CPT resistance as judged by analysis of the top1 crystal structure (Fig. 9Citation Refs. 12 , 13 , 33 ). Interestingly, in this scheme, the previously reported top1 CPT resistance mutations [G503S (41 , 42) ; D533G (26 , 43) , D533N (44) ; G717V+T729I (45) ; L721R+N722A (46) ; N722S (32 , 47) ; and T729A (48) ] are assembled in close proximity to the catalytic tyrosine (Tyr723 in human) and the bound DNA (Fig. 9)Citation . The resistance of the top1/R364H is probably attributable to the loss of a critical H-bond between R364 and CPT. In fact, such an interaction has been predicted to exist between R364 and the CPT E-ring lactone moiety based on a hypothetical CPT binding model (12) .



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 9. Structure of human top1 and clustering of residues associated with CPT resistance. A and B, the top1 protein structure in a front and side view relative to the DNA linear axis, respectively. C and D, the DNA backbone in the same orientation as in A and B, respectively, as well as the proximity of the residues involved in CPT resistance (in red). C and D, (black), the catalytic tyrosine (Y723).

 
We did not detect the WT top1 RNA and genomic DNA (data are not shown) in the RC0.1 and RC1 cell lines. This likely means that lack of the WT top1 allele is required for the cell to acquire CPT resistance, because CPT would turn the WT top1 into a cellular poison. We previously observed a similar situation in the CPT-resistant human leukemia CEM/C2 cells, which had both a CPT-resistant top1 mutation (N722S; Ref. 32 ) and lack of expression of normal top1 allele (47) . These observations suggest that CPT resistance results from a 2-step genetic process: (a) mutation of one of the alleles leading to a catalytically active and CPT-resistant top1; and (b) silencing or loss of the WT, CPT-sensitive top1 allele.

From these results, we conclude that the CPT resistance of the RC0.1 and RC1 cells is primarily attributable to the reduced formation of CPT-induced top1 cleavage complexes. The R364H mutation together with previously defined top1 resistance mutations provide new insight in the CPT molecular interactions and should be useful to model interactions between CPT and top1/DNA complexes and in the development of new CPT derivatives.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Laboratory of Molecular Pharmacology, National Cancer Institute, NIH, Building 37, Room 4E28, 37 Convent Drive MSC 4255, Bethesda, MD 20892-4255. Phone: (301) 496-5944; Fax: (301) 402-0752; E-mail: pommier{at}nih.gov Back

2 The abbreviations used are: top1, topoisomerase I; CPT, camptothecin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; pSK, pBluescript; WT, wild-type; VP-16, etoposide; ADR, doxorubicin; BaP, benzo[a]pyrene. Back

Received . Accepted 12/28/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Brenner M. K., Pinkel D. Cure of leukemia. Semin. Hematol., 36: 73-83, 1999.[Medline]
  2. DeVita V. T., Jr., Canellos G. P. The lymphomas. Semin. Hematol., 36: 84-94, 1999.[Medline]
  3. Milosevic M. F., Gospodarowicz M., Warde P. Management of testicular seminoma. Semin. Surg. Oncol., 17: 240-249, 1999.[Medline]
  4. Ueda T., Miyawaki S., Asou N., Kuraishi Y., Hiraoka A., Kuriyama K., Minami S., Ohshima T., Ino T., Tamura J., et al Response-oriented individualized induction therapy with six drugs followed by four courses of intensive consolidation, 1 year maintenance and intensification therapy: the ALL90 study of the Japan Adult Leukemia Study Group[published erratum appears in Int. J. Hematol., 68: Dec., i-ii, 1998]. Int. J. Hematol., 68: 279-289, 1998.[Medline]
  5. Cabanillas F. The role of topoisomerase-I inhibitors in the treatment of non-Hodgkin’s lymphoma. Semin. Hematol., 36: 11-15, 1999.[Medline]
  6. Kantarjian H. New developments in the treatment of acute myeloid leukemia: focus on topotecan. Semin. Hematol., 36: 16-25, 1999.[Medline]
  7. Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem., 260: 14873-14878, 1985.[Abstract/Free Full Text]
  8. Shao R. G., Cao C. X., Zhang H., Kohn K. W., Wold M. S., Pommier Y. Replication-mediated, DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA:DNA-PK complexes. EMBO J., 18: 1397-1406, 1999.[Medline]
  9. Strumberg D., Pilon A. A., Smith M., Hickey R., Malkas L., Pommier Y. Conversion of topoisomerase I cleavage complexes on the leading strand of ribosomal DNA into 5'-phosphorylated DNA double-strand breaks by replication runoff. Mol. Cell Biol., 20: 3977-3987, 2000.[Abstract/Free Full Text]
  10. Pommier Y., Pourquier P., Urasaki Y., Wu J., Laco G. S. Topoisomerase, I. inhibitors: selectivity and cellular resistance. Drug Resistance Update, 2: 307-318, 1999.[Medline]
  11. Gupta M., Fujimori A., Pommier Y. Eukaryotic DNA topoisomerases I. Biochim. Biophys. Acta, 1262: 1-14, 1995.[Medline]
  12. Redinbo M. R., Stewart L., Kuhn P., Champoux J. J., Hol W. G. J. Crystal structure of human topoisomerase I in covalent and noncovalent complexes with DNA. Science (Washington DC), 279: 1504-1513, 1998.[Abstract/Free Full Text]
  13. Stewart L., Redinbo M. R., Qiu X., Hol W. G. J., Champoux J. J. A model for the mechanism of human topoisomerase I. Science (Washington DC), 279: 1534-1541, 1998.[Abstract/Free Full Text]
  14. Fan Y., Weinstein J. N., Kohn K. W., Shi L. M., Pommier Y. Molecular modeling studies of the DNA-topoisomerase I ternary cleavable complex with camptothecin. J. Med. Chem., 41: 2216-2226, 1998.[Medline]
  15. Stone K. R., Mickey D. D., Wunderli H., Mickey G. H., Paulson D. F. Isolation of a human prostate carcinoma cell line (DU 145). Int. J. Cancer, 21: 274-281, 1978.[Medline]
  16. Chatterjee D., Wyche J. H., Pantazis P. Induction of apoptosis in malignant and camptothecin-resistant human cells. Ann. NY Acad. Sci., 803: 143-156, 1996.[Abstract]
  17. Zhelkovsky A. M., Moore C. L., Rattner J. B., Hendzel M. J., Furbee C. S., Muller M. T., Bazett-Jones D. P. Overexpression of human DNA topoisomerase I in insect cells using a baculovirus vector. Protein Expr. Purif., 5: 364-370, 1994.[Medline]
  18. Minford J., Pommier Y., Filipski J., Kohn K. W., Kerrigan D., Mattern M., Michaels S., Schwartz R., Zwelling L. A. Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II. Biochemistry, 25: 9-16, 1986.[Medline]
  19. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254, 1976.[Medline]
  20. Pommier Y., Kohlhagen G., Laco G. S., Sayer J. M., Kroth H., Jerina D. M. Position-specific trapping of topoisomerase I-DNA cleavage complexes by the intercalated benzo[a]pyrene diol epoxide adducts at the 6-amino group of adenine. Proc. Natl. Acad. Sci. USA, 97: 10739-10744, 2000.[Abstract/Free Full Text]
  21. Pourquier P., Kohlhagen G., Ueng L-M., Pommier Y. Topoisomerase I and II activity assays Brown R. Böger-Brown U. eds. . Methods in Molecular Medicine: Cytototoxic Drug Resistance Mechanisms, 28: 95-110, Humana Press Totowa, NJ 1999.
  22. Takebayashi Y., Pourquier P., Yoshida A., Kohlhagen G., Pommier Y. Poisoning of human DNA topoisomerase I by ecteinascidin 743, an anticancer drug that selectively alkylates DNA in the minor groove. Proc. Natl. Acad. Sci. USA, 96: 7196-7201, 1999.[Abstract/Free Full Text]
  23. Pourquier P., Takebayashi Y., Urasaki Y., Gioffre C., Kohlhagen G., Pommier Y. Induction of topoisomerase I cleavage complexes by 1-ß-D-arabinofuranosylcytosine (ara-C) in vitro and in ara-C-treated cells. Proc. Natl. Acad. Sci. USA, 97: 1885-1890, 2000.[Abstract/Free Full Text]
  24. Pourquier P., Pilon A. A., Kohlhagen G., Mazumder A., Sharma A., Pommier Y. Trapping of mammalian topoisomerase I and recombinations induced by damaged DNA containing nicks or gaps: importance of DNA end phosphorylation and camptothecin effects. J. Biol. Chem., 272: 26441-26447, 1997.[Abstract/Free Full Text]
  25. Yoshinari T., Matsumoto M., Arakawa H., Okada H., Noguchi K., Suda H., Okura A., Nishimura S. Novel antitumor indolocarbazole compound 6-N-formylamino-12,13-dihydro-1,11- dihydroxy-13-(ß-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (NB-506): induction of topoisomerase I-mediated DNA cleavage and mechanisms of cell line-selective cytotoxicity. Cancer Res., 55: 1310-1315, 1995.[Abstract/Free Full Text]
  26. Andoh T., Ishii K., Suzuki Y., Ikegami Y., Kusunki Y., Takemoto Y., Okada K. Characterization of a mammalian mutant with a camptothecin resistant DNA topoisomerase I. Proc. Natl. Acad. Sci. USA, 84: 5565-5569, 1987.[Abstract/Free Full Text]
  27. Eng W. K., McCabe F. L., Tan K. B., Mattern M. R., Hofmann G. A., Woessner R. D., Hertzberg R. P., Johnson R. K. Development of a stable camptothecin-resistant subline of P388 leukemia with reduced topoisomerase I content. Mol. Pharmacol., 38: 471-480, 1990.[Abstract]
  28. Sugimoto Y., Tsukahara S., Oh-hara T., Liu L. F., Tsuruo T. Elevated expression of DNA topoisomerase II in camptothecin-resistant human tumor cell lines. Cancer Res., 50: 7962-7965, 1990.[Abstract/Free Full Text]
  29. Tan K. B., Mattern M. R., Eng W. K., McCabe F. L., Johnson R. K. Nonproductive rearrangement of DNA topoisomerase I and II genes: correlation with resistance to topoisomerase inhibitors. J. Natl. Cancer Inst. (Bethesda), 81: 1732-1735, 1989.[Abstract/Free Full Text]
  30. Chang J. Y., Dethlefsen L. A., Barley L. R., Zhou B. S., Cheng Y. C. Characterization of camptothecin-resistant Chinese hamster lung cells [published erratum appears in Biochem. Pharmacol., 45:787, 1993]. Biochem. Pharmacol., 43: 2443-2452, 1992.[Medline]
  31. Woessner R. D., Eng W. K., Hofmann G. A., Rieman D. J., McCabe F. L., Hertzberg R. P., Mattern M. R., Tan K. B., Johnson R. K. Camptothecin hyper-resistant P388 cells: drug-dependent reduction in topoisomerase I content. Oncol. Res., 4: 481-488, 1992.[Medline]
  32. Fujimori A., Harker W. G., Kohlhagen G., Hoki Y., Pommier Y. Mutation at the catalytic site of topoisomerase I in CEM/C2, a human leukemia cell line resistant to camptothecin. Cancer Res., 55: 1339-1346, 1995.[Abstract/Free Full Text]
  33. Redinbo M. R., Champoux J. J., Hol W. G. Structural insights into the function of type IB topoisomerases. Curr. Opin. Struct. Biol., 9: 29-36, 1999.[Medline]
  34. Bonven B. J., Gocke E., Westergaard O. A high affinity topoisomerase I binding sequence is clustered at DNase I hypersensitive sites in Tetrahymena R-chromatin. Cell, 41: 541-551, 1985.[Medline]
  35. Strumberg D., Pommier Y., Paull K., Jayaraman M., Nagafuji P., Cushman M. Synthesis of cytotoxic indenoisoquinoline topoisomerase I poisons. J. Med. Chem., 42: 446-457, 1999.[Medline]
  36. Pommier Y., Kohlhagen G., Pourquier P., Sayer J. M., Kroth H., Jerina D. M. Benzo[a]pyrene diol epoxide adducts in DNA are potent suppressors of a normal topoisomerase I cleavage site and powerful inducers of other topoisomerase I cleavages. Proc. Natl. Acad. Sci. USA, 97: 2040-2045, 2000.[Abstract/Free Full Text]
  37. Urasaki Y., Laco G., Takebayashi Y., Bailly C., Kohlhagen G., Pommier Y. Use of camptothecin-resistant mammalian cell lines to evaluate the role of topoisomerase I in the antiproliferative activity of the indolocarbazole, NB-506, and its topoisomerase I binding site. Cancer Res., 61: 504-508, 2001.[Abstract/Free Full Text]
  38. Rubin E., Pantazis P., Toppmeyer D., Giovanella B., Kufe D. Identification of a mutant human topoisomerase I with intact catalytic and resistance to 9-nitro-camptothecin. J. Biol. Chem., 269: 2433-2439, 1994.[Abstract/Free Full Text]
  39. Li X-G., Haluska P., Hsiang Y-H., Bharti A. K., Kufe D. W., Liu L. F., Rubin E. H. Involvement of amino acids 361 to 364 of human topoisomerase I in camptothecin resistance and enzyme catalysis. Biochem. Pharmacol., 53: 1019-1027, 1997.[Medline]
  40. Benedetti P., Fioranti P., Capuani L., Wang J. C. Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine. Cancer Res., 53: 4343-4348, 1993.[Abstract/Free Full Text]
  41. Tanizawa A., Pommier Y. Topoisomerase I alteration in a camptothecin-resistant cell line derived from Chinese hamster DC3F cells in culture. Cancer Res., 52: 1848-1854, 1992.[Abstract/Free Full Text]
  42. Tanizawa A., Beitrand R., Kohlhagen G., Tabuchi A., Jenkins J., Pommier Y. Cloning of Chinese hamster DNA topoisomerase I cDNA and identification of a single point mutation responsible for camptothecin resistance. J. Biol. Chem., 268: 25463-25468, 1993.[Abstract/Free Full Text]
  43. Tamura H-O., Kohchi C., Yamada R., Ikeda T., Koiwa O., Patterson E., Keene J. D., Okada K., Kjeldsen E., Nishikawa K., Andoh T. Molecular cloning of a cDNA of a camptothecin-resistant human DNA topoisomerase I and identification of mutation sites. Nucleic Acids Res., 19: 69-75, 1991.[Abstract/Free Full Text]
  44. Saleem A., Ibrahim N., Patel M., Li X-G., Gupta E., Mendoza J., Pantazis P., Rubin E. H. Mechanism of resistance in a human cell line exposed to sequential topoisomerase poisoning. Cancer Res., 57: 5100-5106, 1997.[Abstract/Free Full Text]
  45. Wang L-F., Ting C-Y., Lo C-K., Su J-S., Mickley L. A., Fojo A. T., Whang-Peng J., Hwang J. Identification of mutations of DNA topoisomerase I response for camptothecin resistance. Cancer Res., 57: 1516-1522, 1997.[Abstract/Free Full Text]
  46. Knab A. M., Fertala J., Bjornsti M-A. Mechanisms of camptothecin resistance in yeast DNA topoisomerase I mutants. J. Biol. Chem., 268: 22322-22330, 1993.[Abstract/Free Full Text]
  47. Fujimori A., Hoki Y., Popescu N. C., Pommier Y. Silencing and selective methylation of the normal topoisomerase I gene in camptothecin-resistant CEM/C2 human leukemia cells. Oncol. Res., 8: 295-301, 1996.[Medline]
  48. Kubota N., Kanzawa F., Nishio K., Takeda Y., Ohmori T., Fujiwara Y., Terashima Y., Saijo N. Detection of topoisomerase I gene point mutation in CPT-11 resistant lung cancer cell line. Biochem. Biophys. Res. Commun., 188: 571-577, 1992.[Medline]



This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
L.-C. Ma, C.-C. Kuo, J.-F. Liu, L.-T. Chen, and J.-Y. Chang
Transcriptional Repression of O6-Methylguanine DNA Methyltransferase Gene Rendering Cells Hypersensitive to N,N'-Bis(2-chloroethyl)-N-nitrosurea in Camptothecin-Resistant Cells
Mol. Pharmacol., August 1, 2008; 74(2): 517 - 526.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. Sirikantaramas, M. Yamazaki, and K. Saito
From the Cover: Mutations in topoisomerase I as a self-resistance mechanism coevolved with the production of the anticancer alkaloid camptothecin in plants
PNAS, May 6, 2008; 105(18): 6782 - 6786.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
Z. Liao, R. W. Robey, J. Guirouilh-Barbat, K. K. W. To, O. Polgar, S. E. Bates, and Y. Pommier
Reduced Expression of DNA Topoisomerase I in SF295 Human Glioblastoma Cells Selected for Resistance to Homocamptothecin and Diflomotecan
Mol. Pharmacol., February 1, 2008; 73(2): 490 - 497.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
K. Takagi, T. S. Dexheimer, C. Redon, O. Sordet, K. Agama, G. Lavielle, A. Pierre, S. E. Bates, and Y. Pommier
Novel E-ring camptothecin keto analogues (S38809 and S39625) are stable, potent, and selective topoisomerase I inhibitors without being substrates of drug efflux transporters
Mol. Cancer Ther., December 1, 2007; 6(12): 3229 - 3238.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
R. D. Dinnen, L. Drew, D. P. Petrylak, Y. Mao, N. Cassai, J. Szmulewicz, P. Brandt-Rauf, and R. L. Fine
Activation of Targeted Necrosis by a p53 Peptide: A NOVEL DEATH PATHWAY THAT CIRCUMVENTS APOPTOTIC RESISTANCE
J. Biol. Chem., September 14, 2007; 282(37): 26675 - 26686.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
I. V. Lebedeva, I. Washington, D. Sarkar, J. A. Clark, R. L. Fine, P. Dent, D. T. Curiel, N. J. Turro, and P. B. Fisher
Strategy for reversing resistance to a single anticancer agent in human prostate and pancreatic carcinomas
PNAS, February 27, 2007; 104(9): 3484 - 3489.
[Abstract] [Full Text] [PDF]

Home page
 Molecular Cancer TherapeuticsHome page
Y. Arakawa, H. Suzuki, S. Saito, and H. Yamada
Novel missense mutation of the DNA topoisomerase I gene in SN-38-resistant DLD-1 cells.
Mol. Cancer Ther., March 1, 2006; 5(3): 502 - 508.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. A. Shah, N. Kemeny, A. Hummer, M. Drobnjak, M. Motwani, C. Cordon-Cardo, M. Gonen, and G. K. Schwartz
Drg1 Expression in 131 Colorectal Liver Metastases: Correlation with Clinical Variables and Patient Outcomes
Clin. Cancer Res., May 1, 2005; 11(9): 3296 - 3302.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Yu, E. Khan, M. A. Khaleque, J. Lee, G. Laco, G. Kohlhagen, S. Kharbanda, Y.-C. Cheng, Y. Pommier, and A. Bharti
Phosphorylation of DNA Topoisomerase I by the c-Abl Tyrosine Kinase Confers Camptothecin Sensitivity
J. Biol. Chem., December 10, 2004; 279(50): 51851 - 51861.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
J.-P. Annereau, G. Szakacs, C. J. Tucker, A. Arciello, C. Cardarelli, J. Collins, S. Grissom, B. R. Zeeberg, W. Reinhold, J. N. Weinstein, et al.
Analysis of ATP-Binding Cassette Transporter Expression in Drug-Selected Cell Lines by a Microarray Dedicated to Multidrug Resistance
Mol. Pharmacol., December 1, 2004; 66(6): 1397 - 1405.
[Abstract] [Full Text] [PDF]

Home page
 Biophys. JHome page
G. Chillemi, M. Redinbo, A. Bruselles, and A. Desideri
Role of the Linker Domain and the 203-214 N-Terminal Residues in the Human Topoisomerase I DNA Complex Dynamics
Biophys. J., December 1, 2004; 87(6): 4087 - 4097.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Fiorani, R. J. D. Reid, A. Schepis, H. R. Jacquiau, H. Guo, P. Thimmaiah, P. Benedetti, and M.-A. Bjornsti
The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons
J. Biol. Chem., May 14, 2004; 279(20): 21271 - 21281.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Chatterjee, Y. Bai, Z. Wang, S. Beach, S. Mott, R. Roy, C. Braastad, Y. Sun, A. Mukhopadhyay, B. B. Aggarwal, et al.
RKIP Sensitizes Prostate and Breast Cancer Cells to Drug-induced Apoptosis
J. Biol. Chem., April 23, 2004; 279(17): 17515 - 17523.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
S. Antony, M. Jayaraman, G. Laco, G. Kohlhagen, K. W. Kohn, M. Cushman, and Y. Pommier
Differential Induction of Topoisomerase I-DNA Cleavage Complexes by the Indenoisoquinoline MJ-III-65 (NSC 706744) and Camptothecin: Base Sequence Analysis and Activity against Camptothecin- Resistant Topoisomerases I
Cancer Res., November 1, 2003; 63(21): 7428 - 7435.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
G. Chillemi, P. Fiorani, P. Benedetti, and A. Desideri
Protein concerted motions in the DNA-human topoisomerase I complex
Nucleic Acids Res., March 1, 2003; 31(5): 1525 - 1535.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
W. C. Reinhold, H. Kouros-Mehr, K. W. Kohn, A. K. Maunakea, S. Lababidi, A. Roschke, K. Stover, J. Alexander, P. Pantazis, L. Miller, et al.
Apoptotic Susceptibility of Cancer Cells Selected for Camptothecin Resistance: Gene Expression Profiling, Functional Analysis, and Molecular Interaction Mapping
Cancer Res., March 1, 2003; 63(5): 1000 - 1011.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
R. Danesi, F. De Braud, S. Fogli, T. M. De Pas, A. Di Paolo, G. Curigliano, and M. Del Tacca
Pharmacogenetics of Anticancer Drug Sensitivity in Non-Small Cell Lung Cancer
Pharmacol. Rev., March 1, 2003; 55(1): 57 - 103.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin Jr., and L. Stewart
The mechanism of topoisomerase I poisoning by a camptothecin analog
PNAS, November 26