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[Cancer Research 62, 3716-3721, July 1, 2002]
© 2002 American Association for Cancer Research

Experimental Therapeutics

Novel Mutation of Topoisomerase I in Rendering Cells Resistant to Camptothecin

Jang-Yang Chang1, Jin-Fen Liu, Shin-Hun Juang, Tsang-Wu Liu and Li-Tzong Chen

Division of Cancer Research, National Health Research Institutes, 100 Taipei, Taiwan [J-Y. C., J-F. L., S-H. J., T-W. L., L-T. C.], and Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan [L-T. C.], Republic of China


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To identify mechanisms of camptothecin (CPT) resistance and the relationship between CPT-resistant cells and other anticancer agents, a CPT-resistant cell line (CPT30) and its partial revertant cell line (CPT30R) were established from a human nasopharyngeal carcinoma cell line (HONE-1). CPT30 and CPT30R cells displayed a 14- and 3.5-fold resistance to CPT compared with HONE-1 cells, respectively. The resistant and partial revertant cell lines showed cross-resistance to topotecan and increased sensitivity to cisplatin, carboplatin, and 1,3-bis(chloroethyl)-1-nitrosurea. The topoisomerase (Top) I catalytic activity of CPT30 and CPT30R cells was 30% and 200%, respectively, compared with that of HONE-1 cells. The expression of Top I protein and mRNA levels in CPT30 cells was 40% and 30% less than that in HONE-1 cells, respectively, whereas in CPT30R cells, the levels of Top I protein and mRNA were 50% and 20% higher, respectively, than that in HONE-1 cells. Both the resistant and revertant cell line whole-cell lysates demonstrated different levels of sensitivity to CPT in in vitro assays in comparison with that of HONE-1 cells. Furthermore, CPT exhibited 15- and 7-fold better binding affinity in stabilizing protein-linked DNA breaks in HONE-1 cells than in CPT30 and CPT30R cells, respectively. Direct DNA sequencing of the reverse transcription-PCR product and genomic DNA revealed a point mutation resulting in E418K mutation in the Top I of both CPT30 and CPT30R cells. Wild-type Top I RNA and genomic DNA were also detected in these two cell lines. A yeast system was used to examine whether this mutation could be responsible for CPT resistance. Our results showed that a single amino acid change (E418K) resulted in CPT resistance. Therefore, quantitative and qualitative changes in Top I were responsible for CPT resistance in CPT30 cells. CPT resistance in CPT30R cells was caused by mutation of Top I.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eukaryotic DNA Top2 I is an essential enzyme that regulates the topological changes of DNA that accompany DNA replication, transcription, recombination, and chromosome segregation during mitosis (1, 2, 3) . Top I introduces transient single-stranded DNA breaks in one of the phosphodiester backbones of the duplex DNA and results in a reversible Top I/DNA covalent complex (4, 5, 6) . CPT, a plant alkaloid isolated from Camptotheca acuminata, has been shown to stabilize the Top I/DNA covalent complex, which leads to DNA damage through collision with replication forks when replication occurs and subsequent cell death (7 , 8) . However, no direct correlation between the levels of Top I/DNA cleavable complex and CPT cytotoxicity has been observed. It has been demonstrated that CPT is an S-phase-specific DNA-damaging agent (9) . However, CPT, at high concentrations, can kill both S-phase and non-S-phase cells (10) . The mechanism of cytotoxicity of CPT in non-S-phase tumor cells may be through apoptosis (11) . In addition, yeast lines deficient in the RAD52 repair gene were hypersensitive to CPT, yet CPT produced equal amount of PLDBs (12 , 13) . Furthermore, several lines of evidence have shown that a variety of DNA alterations originating from endogenous and exogenous sources induce the formation of Top I/DNA cleavable complexes (reviewed in Ref. 14 ). An 8–10-fold enhancement of Top I cleavage complex is observed when O6-methylguanine is incorporated in oligonucleotide at a (+) position relative to a unique Top I cleave site (15) . Moreover, an enhancement of cytotoxicity of Top I inhibitor by depletion of methylguanine-DNA methyltransferase with O6-benzylguanine was observed (16) . Thus, repair of Top I-mediated DNA lesion may play an important role for cellular response to CPT.

Although several CPT analogue compounds, including topotecan and irinotecan, have recently been introduced into clinical practice for the treatment of colorectal and ovarian cancers, the response rates remain low, and the overall survival rate has not improved substantially (17 , 18) . Intrinsic or acquired tumor-mediated drug resistance is the major obstacle resulting in lack of tumor responsiveness in patients undergoing therapy. Thus, defining the cellular resistant mechanisms to rationally design new derivatives to overcome drug resistance is crucial. Most of the reported CPT-resistant cell lines were developed by continuous exposure to either CPT or CPT plus other mutagenic agents, and these studies have identified a number of Top I mutations (19, 20, 21) . Such mutations generally confer high-level resistance to CPT, which may not be clinically relevant. Furthermore, mutation of both alleles of Top I cDNA without expression of wt Top I was found in these CPT-resistant cell lines. In addition, it has been demonstrated previously that CPT-resistant cells were collaterally sensitive to Top II inhibitors, due to either compensatory elevation of the Top II level or increased sensitivity of Top II to its inhibitors (22 , 23) . Little is known about the relationship between CPT-resistant cells and anticancer agents other than Top II inhibitors, especially those drugs involved in the DNA repair process.

In this study, a CPT-resistant cell line (CPT30) was established from a human nasopharyngeal carcinoma cell line (HONE-1), and a partial revertant cell line (CPT30R) was established from this CPT-resistant cell line. A novel mutation in the Top I gene of both CPT30 and CPT30R cells was identified. wt Top I RNA and gene were also expressed in these two cell lines. In addition, enhancement of sensitivity to cisplatin, carboplatin, and BCNU was observed in both CPT30 and CPT30R cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
A human nasopharyngeal carcinoma cell line (HONE-1) was provided by Prof. Ching-Hwa Tsai (School of Medicine, National Taiwan University). The characteristics of this cell line have been described previously (24) . As supplied to us, this cell line was negative for EBV. Cells were maintained in RPMI 1640 supplemented with 5% fetal bovine serum and 1% kanamycin in a humidified 5% CO2 incubator at 37°C. CPT, VP-16, cisplatin, carboplatin, BCNU, and other chemicals were purchased from Sigma Chemical Co. Stock solutions of these drugs, with the exception of water-soluble drugs, were dissolved in DMSO at 20 mM, stored at -20°C, and diluted in water immediately before use. [{alpha}-32P]dCTP was purchased from Amersham. Saccharomyces cerevisiae strain JN2-134, plasmids YCpGAL1-hTOP1 and YCpGAL1-hTOP1G363C, and polyclonal Top II antibody were kindly provided by Dr. Jawlang Hwang (Institute of Biomedical Science, Academia Sinica, Taipei, Taiwan). Human Top I cDNA and anti-Top I antibody were kindly provided by Dr. Yung-Chi Cheng (Yale University, New Haven, CT).

Growth Inhibition Assay.
Cells in logarithmic phase were cultured at a density of 5000 cells/ml/well in a 24-well plate. The resistant cells were maintained in drug-free medium for 3 days before use. Cells were exposed to various concentrations of drugs for 72 h. The methylene blue dye assay was used to evaluate the effects of the drugs on cell growth, as described previously (25) , and the concentration of drug that inhibited 50% of cell growth (IC50) was determined.

Top I Catalytic Activity Assay.
Top I DNA catalytic activity using whole-cell lysates was measured by the relaxation of supercoiled pBR322 plasmid DNA in vitro, as described previously (26) . One unit of enzyme activity was defined as the relaxation of 50% of the supercoiled DNA substrate. Photographs of the resulting DNA/agarose gels were taken under UV light, and the band intensities were quantitated by using a densitometric scan. The specific activity was determined by calculating the number of units of catalytic DNA activity/µg of whole-cell lysate.

Western Blot Analysis.
Crude cellular extracts were prepared for immunoblot analysis as described previously (22) . Detection of immunoreactive signals was accomplished with Western Blot Chemiluminescent Reagent Plus (Perkins Elmer LifeSciences, Inc.). The intensity of the specific band was quantitated using an AlphaImager 2000 system.

Northern Blot Analysis.
The isolation and analysis of Top I mRNA levels were performed as described previously (22) . Human glyceraldehyde-3-phosphate dehydrogenase cDNA was used as an internal control. The filter was scanned, and the specific band was quantitated by using the AlphaImager 2000 system. The expression level of Top I was calculated as the ratio of the radioactivity in the Top I band relative to that of the glyceraldehyde-3-phosphate dehydrogenase band.

Measurement of PLDBs in Vivo.
A potassium-SDS coprecipitation (K-SDS) assay was used for measurement of PLDBs in vivo, as described previously (27) . The apparent Kd values for CPT with each cell were determined graphically from double reciprocal plots of the saturation curves.

Assessment of Cellular CPT Accumulation.
CPT accumulation was examined by flow fluorocytometer. In brief, cells suspended at a concentration of 1 x 106 cells/ml in a buffer containing RPMI 1640 and 10 mM HEPES (pH 7.4) were treated with different concentrations of CPT for 30 min at 37°C and then analyzed immediately in a FACSVantage flow cytometer (Becton Dickinson, Mountain View, CA). Detection was performed using an Enterprise ion laser with a 370 nm excitation filter and a 430 nm emission filter. Ten thousand events were collected from each sample. The mean fluorescence intensity was determined using software supplied by the manufacturer. The accumulation of CPT was determined by the fold increase in mean fluorescence units of CPT-treated cells (mean fluorescence units of CPT-treated cells - mean fluorescence units of control cells)/mean fluorescence units of control cells.

Detection of Top I Mutation.
Total RNA was extracted from the parental, drug-resistant, and revertant cells using a RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. RNA was reverse-transcribed by using Superscript II RNase H reverse transcriptase (Life Technologies, Inc.) according to the manufacturer’s protocol. Five mutation hot spot regions of Top I cDNA were amplified using the following primer pairs: (a) region A, 5'-GACATGATTGGGGACCACCT-3' and 5'-TTGTTATCTGGCTCAGGAAC-3'; (b) region B, 5'-AGACCTCGAGATGAGGATGA-3' and 5'-AAATACTGGCTCATCTGGGT-3'; (c) region C, 5'-ACTACCAAGGAAATATTTAG-3' and 5'-CTCTGTCCAGGAAACCAGCC-3'; (d) region D, 5'-GAAGTCCGGCATGATAACAA-3' and 5'-TTCTCATCCGGGGCTGTCAG-3'; and (e) region E, 5'-TACAGCAGCAGCTAAAAGAA-3' and 5'-GTTTGTTAAGACTTGCTGCC-3'.

PCR amplification was performed under the following conditions: preincubation was performed at 48°C for 45 min and at 94°C for 2 min followed by 40 cycles of 94°C (30 s), 58°C (1 min), and 68°C (2 min) and a final extension at 68°C (10 min). The PCR product was then split into two fractions: (a) one-half of the reaction product was purified by agarose gel, and purified DNA was sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA); and (b) the other half of the PCR product was subcloned into the expression vector pT-Adv using Advan TAgeTM PCR cloning kit (CLONTECH). DNA sequence analysis of Top I was performed using ABI PRISM 310 Genetic Analyzer (Applied Biosystems).

Determination of CPT Sensitivity of Yeast Cells Expressing wt or mut Human DNA Top I.
The method of determination of CPT sensitivity of yeast cells expressing wt or mut human DNA Top I was as described previously (21) . Briefly, yeast strain JN2-134, which lacks yeast DNA Top I, was individually transformed with YCpGAL1-hTOP1, YCpGAL1-hTOP1E418k, and YCpGAL1-hTOP1G363C by treatment with lithium acetate (28) . Individual transformants were grown in minimal glucose media lacking uracil to A595 nm = 0.3. Cells were suspended in minimal media containing galactose in the presence of CPT at a concentration of 75 µg/ml. At the indicated times, cells were serially diluted and plated on minimal uracil-minus plates. The number of cells forming colonies were scored after 4 days.

TaqMan Real-time PCR.
Reverse transcription was performed in a final volume of 20 µl containing 1x first-strand buffer, 4 units of RNaseOUT, 10 mM DTT, 200 units of Superscript II RNase H- reverse transcriptase (Life Technologies, Inc.), 1.5 µM oligo(dT)12–18 primer, and 2 µg of total RNA. The samples were incubated at 42°C for 50 min. All primers and probes were designed using Primer Express version 2.0 (PE Application Bioscience, Foster City, CA), and oligonucleotide probes for TaqMan PCR reaction were labeled with 6-carboxyfluorescein and 3' prime quencher 6-carboxy-N,N,N,N'-tetramethylrhodamine. The following primers and probe sequences were used for Top I gene amplification: 5'-TCCAGGACATAAGTGGAAAGAAGTC-3', forward primer; 5'-CGGTGAACTAGGGTTAAGCATGATGTAT-3', reverse primer. The sequence of the TaqMan fluorogenic probe for the wt Top I gene was 5'-ACCAAAGGACCTGTCTCTTGTAGGTTCC-3', and the sequence of the probe for mut Top I was 5'-ACCAAAGGACCTGTTTCTTGTAGGTTCC-3'.

All real-time PCR reactions were performed in the ABI PRISM 5700 Sequence Detector in a 50-µl final volume. The PCR mixture contains 1x TaqMan Universal PCR Master Mix, 400 nM each forward and reverse primer, 200 nM TaqMan probe, 1.5% DMSO, and 1.25 units of AmpliTaq Gold. The thermal cycling condition comprised an enzyme activation step at 50°C for 2 min and a denaturation step at 95°C for 10 min, with 40 cycles of 95°C for 15 s and 64°C for 1 min. Experiments were performed in triplicates. For quantification of PCR results, fluorescence signal intensities were plotted against the number of PCR cycles on a semilogarithmic scale. The amplification cycle at which the first significant increase of fluorescence occurred was designated the threshold cycle (CT). This was done for all samples to be tested in parallel with a standardization series, with known concentrations of human ß-actin cDNA plasmid as template. The CT value of each sample was then compared with those in the standardization series, and the corresponding concentration of human ß-actin was read as the expression level of the tested gene. As a measure of the relative gene expression of wt versus mut Top I in each cell line, the value was derived from the ratio of the copy number of wt or mut Top I mRNA to the copy number of endogenous human ß-actin.

Cell Cycle Analysis.
Flow cytometric analysis of propidium iodide-stained cells was performed with a FACS IV flow cytometer (Becton Dickinson). Cell cycle analysis was performed according to the mathematical model of Jett (29) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of CPT-resistant and Partial Revertant Cell Lines.
From the parental HONE-1 cell line, a CPT-resistant cell line was established by continuous exposure to stepwise increasing concentrations of CPT from 7.5 to 30 nM over a period of 1 year. The cells were grown stably in the medium in the presence of 30 nM CPT and designated CPT30. Then, CPT30 cells were cloned, and these single-cell clones were found to possess similar IC50s. A representative clone was used for all subsequent assays. The stability of CPT resistance in CPT30 cells was determined by assessing the sensitivity after a period of CPT-free culturing. After 8 months of CPT-free incubation, the drug-free line displayed significantly decreased IC50 to CPT compared to its resistant counterpart but remained 3.5-fold more resistant than the parental HONE-1 cells. As a result, this cell line was termed partial revertant (CPT30R).

Growth Inhibition Assay.
The cell doubling time for HONE-1, CPT30, and CPT30R cells was similar, on the order of 21 h. The asynchronous cell population in these three cell lines displayed an identical distribution pattern based on DNA content analysis (data not shown). The sensitivity of HONE-1, CPT30, and CPT30R cells to various anticancer agents is shown in Table 1Citation . Both CPT30 and CPT30R cells exhibited resistance to CPT and to a clinically relevant derivative, topotecan (~3.5–14-fold). No cross-resistance or collateral sensitivity to the Top II inhibitor VP-16 or the antimicrotubule agent vincristine was observed. Interestingly, both CPT30 and CPT30R cells were highly sensitive to DNA-alkylating agents, cisplatin, carboplatin, and BCNU (Table 1)Citation . Verapamil, which is able to reverse the P170-associated multidrug-resistant phenotype, did not alter the cytotoxicity of CPT toward parental, revertant, and resistant cells.


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  		Table 1 Cytotoxicity of various agents against parental, resistant, and revertant cell lines

 
DNA Top I Catalytic Activity.
The ATP-independent DNA Top I catalytic activity in each cell line was assessed by adding whole-cell lysates to a reaction mixture containing pBR322 DNA as a substrate, and the resulting specific activities were compared in Table 2Citation . The resistant and partial revertant cells expressed 30% and 200% of the specific activity levels of HONE-1 cells, respectively.


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  		Table 2 specific activity of Top I in cell extracts and the relative amount of Top I protein and mRNAa

 
The sensitivity of Top I to CPT was determined by using two units of Top I from each cell line, 0.5 µg of pBR322 DNA, and various concentrations of CPT. As seen in Fig. 1Citation , the dose to inhibit 50% of Top I catalytic activity for HONE-1, CPT30, and CPT30R cells was 20 µM, >100 µM, and 37 µM, respectively. At a dose of 100 µM, CPT inhibited 100%, 40%, and 75% of Top I catalytic activity for HONE-1, CPT30, and CPT30R cells, respectively.



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  		Fig. 1. Effect of CPT on Top I activity of whole-cell lysates isolated from HONE-1, CPT30, and CPT30R cells. Supercoiled pBR322 DNA was incubated with 2 units of Top I of HONE-1, CPT30, and CPT30R cells, respectively, and various concentrations of CPT in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 60 mM KCl, 2.5 mM MgCl2, 0.5 mM EDTA, and 50 µg/ml BSA at 37°C for 30 min. A, the DNA was analyzed by electrophoresis in 1% agarose gel. B, supercoiled DNA was quantitated using a densitometry, and the fraction of relaxed DNA in the whole substrate DNA was plotted as a function of the concentrations of CPT (HONE-1, {diamondsuit}; CPT30, {blacktriangleup}; CPT30R, {square}).

 
DNA Top I Protein and Gene Expression.
Western blot analysis was used to assess the levels of Top I and II protein expression in these cells. As seen in Fig. 2ACitation , levels of Top I protein were 60% and 150% in CPT30 and CPT30R cells, respectively, when compared with those in HONE-1 cells. The level of Top II protein expression in these three cell lines was identical. In Fig. 2BCitation , a Northern blot of HONE-1, CPT30, and CPT30R total RNA was probed with a 1.8-kb Top I cDNA. The relative levels of Top I mRNA in CPT30 and CPT30R cells were 70% and 120%, respectively, when compared with the level of expression of Top I mRNA in parental cells.



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  		Fig. 2. Analysis of Top I and II protein (A) and mRNA (B) levels in HONE-1, CPT30, and CPT30R cells. Whole-cell lysates (100 µg) isolated from each cell line were probed with monoclonal Top I and polyclonal Top II antibodies. Total RNA (30 µg) was isolated from each cell line and probed with 32P-labeled Top I cDNA.

 
Measurement of PLDB Level.
After a 30-min exposure to increasing concentrations of CPT, the steady-state level of PLDB increased in a dose-dependent manner. At 25 µM, the number of CPT-induced PLDBs in CPT30 and CPT30R cells was about one-fourth and one-half of that in HONE-1 cells (Fig. 3)Citation . The apparent Kd, which reflects the binding affinity of CPT in stabilizing PLDB association with Top I from each cell line, was determined by double reciprocal plots of the concentration-response relationship for CPT. The apparent Kd for CPT-induced PLDBs in HONE-1, CPT30, and CPT30R cells was 2.2 ± 0.3, 34 ± 2, and 15 ± 2.5 µM, respectively.



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  		Fig. 3. CPT-induced PLDBs in HONE-1 ({diamondsuit}), CPT30 ({blacktriangleup}), and CPT30R ({blacksquare}) cells. The experiment was performed as described previously (26) . Data represent the means from at least three independent experiments. Bars, SD.

 
Cellular Accumulation of CPT.
The decrease in the number of CPT-induced PLDBs in these CPT-resistant and revertant cells could be due to a decrease in the cellular accumulation of drug. Thus, CPT accumulation was determined by using flow cytometry. As seen in Fig. 4Citation , the intracellular accumulation of CPT was increased in a dose-dependent manner. No difference in the level of intracellular accumulation of CPT was observed in these three cell lines.



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  		Fig. 4. Measurement of cellular accumulation of CPT in HONE-1 ({diamondsuit}), CPT30 ({blacktriangleup}), and CPT30R ({blacksquare}) cells. Cells were incubated with different concentrations of CPT at 37°C for 30 min and analyzed immediately in a FACSVantage flow cytometer. Each value is the average of two independent experiments.

 
Top I Mutation in Resistant and Partial Revertant Cell Lines.
To clarify whether the CPT-resistant and partial revertant cells expressed a mut Top I protein, total RNA was isolated from the parental, resistant, and partial revertant cells and subjected to reverse transcription-PCR. The resulting PCR products were sequenced directly, and this analysis revealed an identical point mutation in Top I at nucleotide residue 1463 of both CPT30 and CPT30R cells. This mutation, a G to A transition, results in a glutamate to lysine mutation at amino acid residue 418 (Fig. 5)Citation . Furthermore, individual clones were analyzed for each cell line; the mut Top I was expressed in 7 of 11 and 5 of 10 clones in the CPT30 and CPT30R cell lines, respectively, whereas the parental cells expressed only wt Top I. We next used genomic DNA isolated from these cells as PCR template to further characterize the Top I gene. Both CPT30 and CPT30R cells expressed a wt and a mut Top I gene, whereas HONE-1 cells expressed only the wt Top I gene (data not shown).



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  		Fig. 5. Detection of Top I mutation in CPT30 and CPT30R cells. RNA was isolated from HONE-1, CPT30, and CPT30R cells. Sequencing of Top I was performed by standard reverse transcription-PCR amplification. Arrow, the point mutation.

 
G to A Mutation Is Responsible for CPT Resistance.
To investigate whether the G to A mutation at amino acid residue 418 is responsible for CPT resistance, recombinant methods were used to express the mut Top I enzyme with the single mutation for analysis using a yeast system. Plasmids containing both the wt and individual mutation were constructed and designated YCpGAL1-hTOPI (wt), YCpGAL1-hTOPIG363C, and YCpGAL1-hTOPIE418K. Yeast strain JN2-134, which is devoid of yeast Top I, was then transfected with these plasmids, and their growth in media with or without CPT was measured. As seen in Fig. 6Citation , YCpGAL1-hTOPIE418K displayed resistance to CPT compared with JN2-134 cells transfected with wt YCpGAL1-hTOPI, and the degree of resistance to CPT was similar to that in YCpGAL1-hTOPIG363C.



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  		Fig. 6. Determination of CPT sensitivity of yeast cells expressing wt and mutated human DNA Top I. The methods were as described previously (21) . Each data point was the average of two experiments. JN2-134, {bullet}; YCpGAL1-hTOP1G363C, {blacktriangleup}; YCpGAL1-hTOP1E418K, {square}; YCpGAL1-hTOP1, {circ}.

 
Levels of Expression of wt and mut Top I Gene in HONE-1, CPT30, and CPT30R Cells Using Real-time PCR.
To extrapolate the gene expression of cell samples and validate the dynamic range of real-time PCR, a standard curve with a 10-fold serial dilution of ß-actin cDNA plasmid was used. The results demonstrated a strong linear relationship between the CT and the log of the starting copy number (R2 >= BORDER="0"> 0.99; data not shown). The expression levels of wt and mut Top I gene in HONE-1, CPT30, and CPT30R cells are presented in Table 3Citation . Only wt Top I gene was expressed in HONE-1 cells, whereas mut Top I gene was expressed predominantly in CPT30 and CPT30R cells. The ratio of expression of wt:mut Top I gene was 0.13 and 0.26 in CPT30 and CPT30R cells, respectively.


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  		Table 3 Comparing the expression levels of wt and mut Top I gene in HONE-1, CPT30, and CPT30R cells by real-time PCR

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we characterized the Top I-mediated resistance mechanisms to CPT in a human nasopharyngeal carcinoma cell line derived from the parental HONE-1 cell line. This CPT-resistant clone was 14-fold more resistant to CPT when compared with parental cells. After 8 months of maintaining these resistant cells in drug-free media, a partial revertant cell line was established, which displayed a 4-fold reduced level of resistance to CPT compared with CPT-resistant cells. These partial revertant cells provide valuable information with regard to the relevant resistance phenotype because the key resistance factors should be partially lost.

Using the growth inhibition assay, the CPT30 and CPT30R cell lines were resistant to CPT and its derivative, topotecan, but were not cross-resistant to drugs that did not target Top I (Table 1)Citation . Interestingly, both CPT30 and CPT30R cells were more sensitive to cisplatin, carboplatin, and BCNU but not VP-16 as compared with HONE-1 cells. Although this may not be the general phenomenon for all CPT-resistant cells, it provides a unique model to further study the relationship between CPT-resistant cells and these DNA-alkylating agents.

CPT-resistant cell lines have been established previously that resulted from either reduced Top I level, mutation in Top I, or reduced cellular accumulation of CPT, which resulted in production of a reduced level of PLDBs (19, 20, 21, 22 , 30 , 31) . To study the mechanisms responsible for the development of resistance in CPT30 and CPT30R cells, a series of quantitative and qualitative assays of Top I and CPT-Top I interactions were performed. By using Western blot analysis and the pBR322 DNA relaxation assay, the expression and catalytic activity of Top I in CPT30 and CPT30R cells were shown to be significantly different from those in HONE-1 cells (Table 2Citation ; Fig. 2Citation ). Despite the fact that the cellular content and catalytic activity of Top I in CPT30R cells were higher than those in HONE-1 cells, CPT30R cells still showed more resistance to CPT than HONE-1 cells. In addition, Top I catalytic activity of CPT30R cells was 6-fold higher than that in CPT30 cells, whereas CPT30 cells were 4-fold more resistant to CPT than CPT30R cells. These observations suggested that factors other than a reduced level of Top I protein expression were responsible for CPT resistance in CPT30R cells. Analysis with supercoiled DNA showed that Top I of HONE-1, CPT30, and CPT30R cells exhibited different levels of sensitivity to CPT (Fig. 2)Citation , which implicated the involvement of Top I mutations that affect CPT binding in CPT resistance in both CPT30 and CPT30R cells. Additionally, the number of CPT-induced PLDBs in CPT30 and CPT30R cells was much lower than that in HONE-1 cells, which did not result from a reduction in the cellular concentration of CPT because no difference in cellular accumulation of CPT was observed among these three cell lines (Fig. 4)Citation . Furthermore, by calculating the apparent Kd, CPT exhibited 15- and 7-fold better binding affinity in stabilizing PLDBs in HONE-1 cells than it did in CPT30 and CPT30R cells, respectively, which further suggested a Top I mutation in both CPT30 and CPT30R cells. As shown in Fig. 5Citation , we found a novel Top I mutation that changes the glutamate 418 codon to lysine (E418K) in CPT30 and CPT30R cells. To confirm that the E418K mutation could result in CPT resistance, a recombinant Top I with the E418K mutation was constructed, and we examined the corresponding CPT sensitivity in a yeast system. As shown in Fig. 6Citation , JN2-134 yeast transfected with YCpGAL1-hTOPI E418K exhibited resistance to CPT, and furthermore, the level of resistance to CPT in Top I with the E418K mutation and Top I with the G363C mutation was identical. These results indicated that a Top I mutation at codon 418 results in resistance to CPT. It has been proposed that the interaction of Top I and DNA involving subdomain II of the core domain and the COOH-terminal of Top I is quite limited but that the interaction of those involving subdomain I and III is very extensive. Furthermore, protein-DNA contacts do occur, including residues 410–429 from subdomain I, which position into the major groove roughly opposite the cleavage site (32) . Mutation of Top I amino acid residues 361–364, which is located in subdomain I of the core domain, has been recently reported to be critical for CPT resistance (19 , 33 , 34) . In our study, the E418K point mutation is also located in the highly conserved subdomain I of core domain. Thus, we hypothesized that the resistance of the Top I/E418K is probably attributable to the reduction in Top I-DNA interactions. Additional studies are needed to prove it.

It has been reported previously that the Top I CPT resistance mutation always displayed a very high level of resistance to CPT and lacked expression of wt Top I (19, 20, 21 , 33 , 34) . Also, several lines of evidence showed that mut Top I fully preserved enzymatic activity (19, 20, 21) . In our study, despite the fact that quantitative and qualitative changes of Top I in CPT30 cells were observed, the cells showed a relatively low level of resistance to CPT compared with previously established CPT-resistant cell lines. Furthermore, the Top I catalytic activity in CPT30R cells was 2- and 6-fold higher than that in HONE-1 and CPT30 cells, respectively. Despite the fact that the mutation in Top I was confirmed in both CPT30 and CPT30R cells, 40% and 75% of Top I in CPT30 and CPT30R cells, respectively, was still sensitive to CPT (Fig. 1)Citation . Therefore, we proposed that wt Top I existed in both CPT30 and CPT30R cells. By using genomic DNA and RNA from HONE-1, CPT30, and CPT30R cells as PCR template to characterize the Top I gene, our results clearly demonstrated that both CPT30 and CPT30R cells expressed wt and mut Top I gene (Fig. 5)Citation . Furthermore, the expression levels of wt and mut Top I gene in HONE-1, CPT30, and CPT30R cells were measured by real-time PCR. mut Top I gene was expressed predominantly in both CPT30 and CPT30R cells (Table 3)Citation . In addition, the ratio of wt:mut Top I in CPT30R cells was 2-fold higher than that in CPT30 cells. This implied that the increasing content and enzymatic activity of Top I in CPT30R cells, which resulted in decreasing in resistance to CPT, could be due to the enhancement of expression of wt Top I gene. Top I gene down-regulation can be due to gene rearrangement, promoter mutation, or gene methylation. It was suggested that specific methylation contributes to the silencing of the normal Top I alleles in a CPT-resistant human leukemia cell line (35) . In the present study, down-regulation of the Top I gene in CPT30 cells was documented. However, after withdrawing the selection pressure, the expression level of wt Top I was increased predominantly in CPT30R cells. These results suggested that during the selection process, mutation of one of the alleles that leads to CPT-resistant Top I and wt Top I gene is transiently suppressed, but wt Top I gene restores its function partially after removing the selection pressure and before the second step of mutation occurs. The mechanisms involved in these changes are under investigation.

Taken together, we demonstrate that both quantitative and qualitative changes in Top I are involved in the development of resistance to CPT in CPT30 cells. A mutation in Top I was responsible for CPT resistance in CPT30R cells. A novel mutation of TopI/E418K was identified in both resistant and partial revertant cells. This provides a model to help further understand the molecular interactions between DNA and Top I.


   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 Cancer Cooperative Ward, National Taiwan University Hospital, Division of Cancer Research, National Health Research Institutes, 7 Chung-Shan South Road, Taipei, Taiwan. Phone: 886-2-23562879; Fax: 886-2-23892737; E-mail: jychang{at}nhri.org.tw Back

2 The abbreviations used are: Top, topoisomerase; CPT, camptothecin; PLDB, protein-linked DNA break; wt, wild-type; BCNU, 1,3-bis(chloroethyl)-1-nitrosurea; mut, mutant. Back

Received 1/22/02. Accepted 5/ 1/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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