ARTICLES

The ras Oncogene-Mediated Sensitization of Human Cells to Topoisomerase II Inhibitor-Induced Apoptosis

Han-Mo Koo, Marcia Gray-Goodrich, Glenda Kohlhagen, Mary Jane McWilliams, Michael Jeffers, Anne Vaigro-Wolff, W. Gregory Alvord, Anne Monks, Kenneth D. Paull, Yves Pommier, George F. Vande Woude

Affiliations of authors: H.-M. Koo, M. J. McWilliams, M.Jeffers (ABL—Basic Research Program), M. Gray-Goodrich, A. Vaigro-Wolff, A. Monks (Science Applications International Corporation—Frederick), W. G. Alvord (Data Management Services, Inc.), National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD; G. Kohlhagen, Y. Pommier (Laboratory of Molecular Pharmacology, Division of Cancer Treatment), K. D. Paull (deceased) (Information Technology Branch, Developmental Therapeutics Program, Division of Cancer Treatment), G. F. Vande Woude (Division of Basic Sciences), National Cancer Institute, Bethesda, MD.

Correspondence to: George F. Vande Woude, Division of Basic Sciences, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Bldg. 469, Frederick, MD 21702 (e-mail: woude{at}ncifcrf.gov).


    ABSTRACT
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
BACKGROUND: Among the inhibitors of the enzyme topoisomerase II (an important target for chemotherapeutic drugs) tested in the National Cancer Institute's In Vitro Antineoplastic Drug Screen, NSC 284682 (3'-hydroxydaunorubicin) and NSC 659687 [9-hydroxy-5,6-dimethyl-1-(N-{2(dimethylamino)ethyl}carbamoyl)-6H-pyrido-(4,3-b)carbazole] were the only compounds that were more cytotoxic to tumor cells harboring an activated ras oncogene than to tumor cells bearing wild-type ras alleles. Expression of the multidrug resistance proteins P-glycoprotein and MRP (multidrug resistance-associated protein) facilitates tumor cell resistance to topoisomerase II inhibitors. We investigated whether tumor cells with activated ras oncogenes showed enhanced sensitivity to other topoisomerase II inhibitors in the absence of the multidrug-resistant phenotype. METHODS: We studied 20 topoisomerase II inhibitors and individual cell lines with or without activated ras oncogenes and with varying degrees of multidrug resistance. RESULTS: In the absence of multidrug resistance, human tumor cell lines with activated ras oncogenes were uniformly more sensitive to most topoisomerase II inhibitors than were cell lines containing wild-type ras alleles. The compounds NSC 284682 and NSC 659687 were especially effective irrespective of the multidrug resistant phenotype. The ras oncogene-mediated sensitization to topoisomerase II inhibitors was far more prominent with the non-DNA-intercalating epipodophyllotoxins than with the DNA-intercalating inhibitors. This difference in sensitization appears to be related to a difference in apoptotic sensitivity, since the level of DNA damage generated by etoposide (an epipodophyllotoxin derivative) in immortalized human kidney epithelial cells expressing an activated ras oncogene was similar to that in the parental cells, but apoptosis was enhanced only in the former cells. CONCLUSIONS: Activated ras oncogenes appear to enhance the sensitivity of human tumor cells to topoisomerase II inhibitors by potentiating an apoptotic response. Epipodophyllotoxin-derived topoisomerase II inhibitors should be more effective than the DNA-intercalating inhibitors against tumor cells with activated ras oncogenes.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inhibitors of DNA topoisomerase II are widely used as chemotherapeutic agents in cancer treatment (1-3). The inhibitors include DNA-intercalating anthracyclines, anthraquinones, ellipticines, acridines, and non-DNA-intercalating epipodophyllotoxin derivatives (1-3). DNA is the major target for topoisomerase II inhibitors, and the stabilization of topoisomerase II-DNA cleavable complexes, rather than the inhibition of topoisomerase II catalytic activity, is essential for drug cytotoxicity (1-3). The cleavable complexes induced by topoisomerase II inhibitors are only potentially cytotoxic, since the complexes are readily reversible upon drug depletion (1-3). However, when the cleavable complexes persist, permanent DNA damage occurs, resulting in cell death mainly by apoptosis (1-3). Multidrug resistance (MDR), mediated by the P-glycoprotein (Pgp) efflux pump encoded by the mdr-1 gene, facilitates cellular resistance to topoisomerase II inhibitors (4,5). In addition, non-Pgp-associated MDR, such as the MDR-associated protein (MRP) or the lung resistance protein (LRP), have also been implicated in resistance to topoisomerase II inhibitors (6,7). Although much is known about the biochemical effects of topoisomerase II inhibitors, little is known about cellular factors that might influence drug cytotoxicity or tumor cell sensitivity.

The ras oncogene is the most frequently occurring gain-of-function mutation found in human cancer (8,9). While its function in cellular transformation and tumorigenesis has been well characterized (8,9), a role in the chemosensitivity of tumor cells has only recently been discovered when the oncogene was identified as a potential sensitivity factor in tumor cell lines (10) and in the treatment of acute myeloid leukemia (AML) (11) for the commonly used antineoplastic drugs, cytarabine and topoisomerase II inhibitors. Beginning with the National Cancer Institute's In Vitro Antineoplastic Drug Screen (NCI-ADS) database, we identified the anthracycline analogue, NSC 284682 (3'-hydroxydaunorubicin) (12) and the ellipticine/olivacine derivative NSC 659687 [9-hydroxy-5,6-dimethyl-1-(N-{2(dimethylamino)ethyl}carbamoyl)-6H-pyrido-(4,3-b)carbazole] (13) as the only topoisomerase II inhibitors that were more cytotoxic to human tumor cells harboring ras oncogenes than to those with wild-type ras alleles (10).

In this article, we describe experiments demonstrating that the cytotoxicity of those two topoisomerase II inhibitors is far less affected by multidrug-resistant phenotypes than that of other topoisomerase II inhibitors in clinical use. We further investigated whether, in the absence of MDR influence, the presence of activated ras oncogenes in human tumor cells was associated with enhanced sensitivity to other commonly used topoisomerase II inhibitors. We also used an immortalized human kidney epithelial (IHKE) cell line ectopically expressing a ras oncogene (IHKEras) and determined its sensitivity to a variety of topoisomerase II inhibitors, and DNA damage and apoptosis induced in this cell line by the compounds were compared with those observed in the syngeneic parental IHKE cells.


    MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell lines and compounds. The human colon carcinoma HT-29par and multidrug-resistant HT-29mdr1 cell lines were obtained from M. M. Gottesman (National Cancer Institute [NCI], Bethesda, MD) and were maintained as described (14). The multidrug-resistant sublines Ad5, Ad20, and Ad300, which were derived from the colon carcinoma cell line SW620 by stepwise exposure to doxorubicin (5, 20, and 300 ng/mL, respectively) (15), were provided by S. E. Bates (NCI). Twenty-two different tumor cell lines from NCI-ADS were used in this study for which the profile of ras mutations (10) and the levels of multidrug-resistant phenotypes analyzed by rhodamine efflux assay (16) had been determined previously (Table 1).Go The IHKE cells (17) were obtained from A. Haugen (National Institute of Occupational Health, Oslo, Norway) and were maintained in Dulbecco's modified Eagle medium supplemented with 5% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM L-glutamine, and 100 units/mL penicillin-100 µg/mL streptomycin (Life Technologies, Inc. [Gibco/BRL], Gaithersburg, MD). Hydroxyrubicin (18) and WP401 (19) were gifts from W. Priebe (The University of Texas, M. D. Anderson Cancer Center, Houston). All other drugs and compounds used were obtained through the Drug Synthesis and Chemistry Branch of the NCI.


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Table 1. The ras mutation status and the multidrug resistance (MDR) levels of NCI-ADS tumor cell lines used in this study

 
Transfections. Transfections were carried out by use of the N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) liposomal transfection reagent following the recommendations of the manufacturer (Boehringer Mannheim Corp., Indianapolis, IN). The IHKE cells were transfected either with the parental vector pDCR or with pDCR/Ha-ras[G12V] expressing the hemagglutinin (HA)-tagged human H-rasV12 oncogene (20). After the transfection, cells were selected for G418 resistance (700 µg/mL). To minimize clonal variations, we pooled together more than 100 G418-resistant clones from each transfection and maintained them in medium containing 350 µg/mL G418. Thus, the resulting cell lines are designated as IHKEDCR (for vector alone transfected) and IHKEras (for ras oncogene transfected).

In vitro growth inhibition assay and data calculation. The assay methodology and data calculation were described previously in detail (21). For all compounds, the starting concentration tested before dilution was 1 x 10-4 M, unless otherwise indicated. Each compound was tested at ten 10-fold dilutions when MDR-positive cell lines were used and at five 10-fold dilutions when NCI-ADS tumor lines were used. For all cell lines, each compound concentration was tested in duplicate wells. The sensitivity of IHKE-derived cell lines was determined by testing 10 fivefold dilutions of a compound in triplicate wells. After 48 hours of continuous exposure to a test compound, the percentage of relative growth in each treated well was determined on the basis of the growth in untreated control wells (21).

Western blot analysis. Western blot analysis was performed essentially as described before (22). The primary antibodies used are anti-pan ras (Transduction Laboratories, Lexington, KY), anti-mdr (Pgp) (Oncogene Science Inc., Cambridge, MA), anti-topoisomerase II{alpha} (TopoGEN, Columbus, OH), and anti-{alpha}-tubulin (Sigma Chemical Co., St. Louis, MO).

Quantitation of DNA damage. The cleavable complexes formed after 1 hour of exposure to a test compound were quantitated by an alkaline elution filter method by use of the bound-to-one-terminus model as described in detail previously (18,23). DNA breaks induced by irradiation with 3000 rads of x-ray were quantitated in parallel as a standard (18,23). Thus, the DNA-protein cross-links (quantifying "DNA damage") were expressed in rad-equivalents of the standard x-ray dose (18,23).

Quantitation of apoptosis. After 48 hours of continuous exposure of the cells to a test compound, total cells including floating cells were harvested, washed in phosphate-buffered saline (PBS) (pH 7.1), and fixed in 10% formalin. After the fixation, cells were washed in PBS, and approximately 3 x 105 cells were mounted onto a polylysine-coated slideglass by use of Cytospin centrifugation at 1500 rpm for 7 minutes at room temperature. The slides were stained with 4'6-diamidine-2'-phenyline dihydrochloride (DAPI) in 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 1 mM EDTA and then were washed twice in Tris-buffered saline. The DAPI-stained cells were examined by fluorescent microscopy. A total of 300-500 nuclei from several randomly chosen fields was examined, and the nuclei displaying the distinctive apoptosis-associated morphologic changes were scored. Apoptosis was expressed as a percentage of the total number of nuclei examined. After 24 hours of continuous exposure of the cells to a test compound, apoptosis-associated DNA fragmentation was quantitated by use of a filter elution assay as described in detail elsewhere (24). The amount of DNA fragments was expressed as a percentage of total DNA loaded onto a filter (24).

Determination of ras mutation status. Determination of ras mutations in NCI-ADS cell lines was described in detail elsewhere (10). Briefly, the mutational status of ras alleles was determined by both denaturing gradient gel electrophoresis and direct sequencing of exons 1 and 2 of each ras gene amplified by polymerase chain reaction from genomic DNA. The results are given in Table 1Go.

Determination of Pgp-mediated MDR levels. Determination of Pgp-mediated MDR levels in NCI-ADS cell lines by use of rhodamine efflux assay was described in detail previously (16). The SW620 cell line was used as a guideline, and the level of rhodamine efflux observed in these cells was 31 fluorescence units (16). The significance of efflux below this level was not known (16). The results are given in Table 1Go.

Statistical analysis. Multiple regression and correlation analyses were performed by use of data derived from topoisomerase II inhibitor sensitivity patterns of NCI-ADS tumor cell lines together with ras mutation status (10) and rhodamine efflux pattern (16) for each cell line (Table 1Go). Correlation coefficients were calculated by use of the pattern recognition program COMPARE (25). The COMPARE program searches for similarities in the response patterns of NCI-ADS tumor cell lines and ranks the similarity between patterns by a Pearson correlation coefficient (25). COMPARE analysis has been used successfully to identify compounds with related chemical structures and/or shared mechanisms of action (25). Tests of hypotheses were performed by use of analysis of variance, multiple regression analysis, and standard parametric and nonparametric methods. All statistical tests were two-sided.


    RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Only NSC 284682 and NSC 659687 were selectively cytotoxic against human tumor cell lines with activated ras oncogenes, when compared with those with wild-type ras alleles (10). We questioned why the large number of well-characterized topoisomerase II inhibitors tested in the NCI-ADS cell lines were not all selectively cytotoxic against the activated ras oncogene-containing tumor lines. Among NCI-ADS cell lines, the HOP-62 and HCT-15 cells, which harbor activated ras oncogenes (10), are also highly positive for multidrug-resistant phenotypes (16) (see Table 1Go). The high cytotoxicity of NSC 284682 and NSC 659687 against HOP-62 and HCT-15 cells raised the possibility that the activity of commonly used topoisomerase II inhibitors was suppressed by the multidrug-resistant phenotypes displayed by these cell lines. We asked if the presence of activated ras oncogene was associated with enhanced sensitivity to additional topoisomerase II inhibitors when the influence of MDR was eliminated. We performed multiple regression analyses and COMPARE correlation analyses (25) between rhodamine efflux pattern (quantifying Pgp-mediated MDR) (16), the ras mutation pattern (10), and each of 20 respective topoisomerase II inhibitor sensitivity patterns. The multiple regression analyses showed that the probabilities (two-sided) associated with the interaction between ras mutation status and Pgp-mediated MDR were in most cases statistically, not significant (see the "ras x MDR" column under heading "All cell lines" in Table 2, A),Go indicating that their effects are additive and do not interact in determining cellular sensitivity to a topoisomerase II inhibitor. The probabilities (two-sided) associated with the regression of sensitivity on ras mutation status were found to be statistically highly significant for most topoisomerase II inhibitors (see the "ras" column under heading "All cell lines" in Table 2Go, A). The Pgp-mediated MDR displayed differential contribution to each topoisomerase II inhibitor sensitivity pattern. Thus, the effects of the MDR on NSC 659687, idarubicin, NSC 284682, and elliptinium were statistically not significant (P>.05) and for all other inhibitors, the effects were either significant (P<.05 for nine compounds) or highly significant (P<.01 for seven compounds). With the MDR-positive HOP-62 and HCT-15 cell lines excluded from the analyses, the contributions of MDR and the interaction between ras mutations and MDR were all statistically not significant (P>.05), while the contributions by ras mutation status to most topoisomerase II inhibitor sensitivities were still statistically significant (P<.05) (see the columns under the heading "Without multidrug-resistant cell lines" in Table 2Go, A).


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Table 2, A. Probabilities associated with contributions of ras mutation status and/or rhodamine efflux pattern on topoisomerase II inhibitor sensitivity patterns of NCI-ADS human tumor cell lines*

 
Since the effects of MDR and ras mutation status on the sensitivity patterns are additive, we next performed correlation analyses on the same sets of data. As the correlation with MDR increases, the correlation with ras mutation status, in general, decreases (seethe columns under the heading "All cell lines" in Table 2Go, B). Thus, the sensitivity patterns of the topoisomerase II inhibitors NSC 659687 and NSC 284682 (10), together with hydroxyrubicin (18) and WP401 (19), showed the highest correlations with the ras mutation pattern (see the columns under the heading "All cell lines" in Table 2Go, B). Bisantrene, which is highly sensitive to Pgp-mediated MDR (26), had the lowest correlation with the ras mutation pattern (see the columns under the heading "All cell lines" in Table 2Go, B). However, when the MDR-positive cell lines were excluded from the analyses, the correlations with the ras mutation pattern improved for every topoisomerase II inhibitor (see the "Without multidrug-resistant cell lines" column in Table 2, B). A paired Wilcoxon signed rank test on the correlations with and without MDR-positive cell lines indicated that there was a global increase in the correlations for topoisomerase II inhibitors when the MDR-positive cell lines were excluded (P<.0001). We also determined that the topoisomerase II inhibitor sensitivities were not statistically significantly influenced by the relative growth rate of cell lines (data not shown). Furthermore, no significant correlations were found with the presence of activated ras oncogenes, when the MDR-positive cell lines were excluded, for other compounds whose activity is affected by MDR, e.g., a topoisomerase I inhibitor morpholinodoxorubicin (27), or the tubulin-modifying drugs paclitaxel and vinblastine (data not shown). These results indicate that the presence of activated ras oncogenes in tumor cells specifically enhances sensitivity to topoisomerase II inhibitors.


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Table 2, B. Correlations of topoisomerase II inhibitor sensitivity patterns of NCI-ADS human tumor cell lines with rhodamine efflux and ras mutation patterns*

 
The above analyses suggested that NSC 284682 and NSC 659687 were insensitive to the adverse effects of MDR, and reports show that hydroxyrubicin, which is closely related to NSC 284682, is less sensitive to MDR than is doxorubicin (18), and NSC 659687 is quite active in vitro and in vivo against tumor cells with MDR (28,29). To study this further, we tested NSC 284682 and NSC 659687 against HT-29mdr1 cells that overexpress the mdr-1 encoded Pgp (14) and compared them with other topoisomerase II inhibitors in clinical use. The HT-29mdr1 cells were significantly less sensitive than the parental cells (HT-29par) to doxorubicin and daunorubicin (Table 3).Go By contrast, both HT-29par and HT-29mdr1 cells showed a similar sensitivity to both NSC 284682 and NSC 659687 compounds (Table 3Go). Idarubicin and amsacrine (m-AMSA) are shown to be less sensitive to Pgp-mediated MDR (30,31). As expected, m-AMSA was similarly active against both HT-29par and HT-29mdr1 cells; however, the activity of idarubicin was moderately affected by MDR in the HT-29mdr1 cells (Table 3Go).


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Table 3. Relative sensitivity of HT-29 cells overexpressing P-glycoprotein to topoisomerase II inhibitors

 
Resistance to topoisomerase II inhibitors can be mediated by diverse mechanisms in addition to Pgp (4). We therefore tested the activities of NSC 284682 and NSC 659687 against tumor cells with "acquired" MDR. We employed the multidrug-resistant sublines Ad5, Ad20, and Ad300, which were derived from colon carcinoma line SW620 by stepwise exposure to doxorubicin (5, 20, and 300 ng/mL, respectively) (15). While a significant increase in resistance to doxorubicin and daunorubicin was observed in the multidrug-resistant sublines, NSC 284682, NSC 659687, and idarubicin showed similar resistance to the acquired MDR (Table 4Go). We also tested elliptinium, which is used clinically (1,3), and found that the multidrug-resistant sublines were more resistant to this drug than to the chemically related NSC 659687 (Table 4).Go The acquired multidrug-resistant cell lines displayed considerable resistance to m-AMSA (Table 4Go), implying that NSC 284682 and NSC 659687 compounds are unaffected by a broader range of MDR mechanisms.


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Table 4. Sensitivity of acquired multidrug-resistant sublines Ad5, Ad20, and Ad300 of SW620 cells to topoisomerase II inhibitors

 
We established a syngeneic system using the IHKE cells (17) ectopically expressing the H-rasV12 oncogene (IHKEras) to study the influence of the ras oncogene on sensitivity to topoisomerase II inhibitors. We determined the sensitivity of the IHKE-derived cell lines to a variety of topoisomerase II inhibitors as well as to vinblastine, which we used as a control. The IHKEras cells showed enhanced sensitivity to the topoisomerase II inhibitors at clinically achievable concentrations (Fig. 1, A-G).Go In addition, these analyses revealed that the enhanced sensitivity of IHKEras cells to etoposide and teniposide persisted over four orders of magnitude of drug concentrations (Fig 1Go, A and B). Mitoxantrone also displayed enhanced cytotoxicity against the IHKEras cells at high drug concentrations (Fig. 1Go, C), but high concentrations of NSC 284682, doxorubicin, and NSC 659687 were less cytotoxic and displayed a very narrow range of enhanced cytotoxicity to the IHKEras cells (Fig. 1Go, D, E and F, respectively). The response of IHKEras cells to different topoisomerase II inhibitors reveals a remarkable drug-dependent sensitivity pattern that parallels the degree to which these drugs intercalate into DNA (1,3,32,33). m-AMSA has been shown to be uniquely different from other topoisomerase II inhibitors (1,3), and the IHKEras cells showed the least differential sensitivity to this drug (Fig. 1Go, G). Moreover, all of the cells showed similar sensitivity to vinblastine (Fig. 1Go, H).



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Fig. 1. Sensitivity of IHKE (parental, {square}), IHKEDCR (vector alone, {blacklozenge}), and IHKEras (ras oncogene, {circ}) cells to topoisomerase II inhibitors and vinblastine (control). The topoisomerase II inhibitors tested are A) etoposide, B) teniposide, C) mitoxantrone, D) NSC 284682, E) doxorubicin, F) NSC 659687, G) amsacrine, and, as a control, H) vinblastine. The percentage relative growth (y-axis) is plotted against each drug concentration (M) tested (x-axis). For topoisomerase II inhibitors (A-G), a relative growth range reflecting the cytotoxicity of a topoisomerase II inhibitor (25% to -100%) is shown, and the range from 50% to -100% relative growth is shown for vinblastine (H). The full range of cellular response to etoposide (5.2 x 10-10 M to 1 x 10-3 M) is also shown (inset in A). The average values from three independent assays are shown with standard errors. The standard errors are not indicated when smaller than plot symbols.

 
The IHKE-derived cell lines were analyzed for Pgp and topoisomerase II{alpha} expression, since both can directly affect the sensitivity of cells to topoisomerase II inhibitors (4). We did not detect Pgp expression in the IHKE and IHKEras cells, and both displayed similar levels of topoisomerase II{alpha} expression (data not shown). The IHKE cells transfected and selected with vector alone (IHKEDCR) displayed a lower level of topoisomerase II{alpha} than either IHKE or IHKEras cells (data not shown). We also determined whether the enhanced sensitivity of IHKEras cells to topoisomerase II inhibitors might be conferred by drug-induced DNA damage. We used etoposide in this assay, since the drug showed significantly enhanced cytotoxicity against the IHKEras cells (Fig. 1Go, A). Etoposide efficiently forms cleavable complexes at high concentrations (1,3), which can be measured as "DNA damage" by the alkaline elution assay (23). Similar levels of DNA damage were induced by etoposide in IHKE and IHKEras cells, and both were approximately twofold higher than that in IHKEDCR cells (Table 5).Go This result indicates that the ras oncogene has no appreciable effect on the etoposide-induced DNA damage and is consistent with the lower level of topoisomerase II{alpha} detected in the IHKEDCR cells.


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Table 5. DNA damage induced by etoposide in IHKE-derived cell lines*

 
As an alternative mechanism for enhanced sensitivity to topoisomerase II inhibitors, we tested whether the IHKEras cells were more prone to apoptosis than the control IHKE cells. These analyses showed that apoptosis induced by etoposide was markedly enhanced in the IHKEras cells compared with both IHKE and IHKEDCR cells (Fig. 2, A).Go Moreover, the drug sensitivity pattern closely paralleled the pattern of apoptosis (Fig. 1Go, A; Fig. 2Go, A). We also used a filter elution assay to measure apoptosis-associated DNA fragmentation (24). Again, apoptosis as measured by DNA fragmentation was significantly enhanced in the etoposide-treated IHKEras cells compared with the control cells (Fig. 2Go, B). These results demonstrate that the greater sensitivity of IHKEras cells to etoposide parallels the enhanced apoptosis.



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Fig. 2. Apoptosis induced by etoposide in IHKE (parental, {square}), IHKEDCR (vector alone, {blacklozenge}), and IHKEras (ras oncogene, {circ}) cells. A) Apoptosis induced by etoposide was quantitated by staining DNA with 4'6-diamidine-2'-phenyline dihydrochloride (DAPI). Based on nuclear morphology and DNA condensation, apoptosis was scored and expressed as a percentage of the total number of nuclei examined. The assay was performed twice on duplicate samples. Data from a representative experiment are shown with standard deviations. Deviations smaller than plot symbols are not shown. B) Apoptosis-associated DNA fragmentation was measured with the use of a filter elution assay (24). The amount of fragmented DNA was expressed as a percentage of the total DNA. The DNA fragmentation induced in the IHKE cells was measured only for 0 and 40 µM etoposide. The assay was repeated several times with similar results. Data shown are from a representative experiment.

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Our results demonstrate that the presence of an activated ras oncogene specifically enhances tumor cell sensitivity to topoisomerase II inhibitors. This enhanced sensitivity was masked by MDR for most of the topoisomerase II inhibitors (Table 2GoGo), but the NSC 284682 and NSC 659687 compounds that were identified in the NCI-ADS as specifically targeting cell lines with activated ras oncogenes (10) were unaffected by MDR (Tables 2-4GoGoGo). Once we identified that MDR was a factor in the topoisomerase II inhibitor sensitivity patterns of NCI-ADS tumor cell lines, we were able to show the opposing effects between the Pgp-mediated MDR and the presence of ras oncogenes (Table 2Go). Independently, we also show that the IHKE cells harboring the ras oncogene that are apparently free of multidrug-resistant phenotypes are sensitized to many topoisomerase II inhibitors (Fig. 1Go). Using this system, we found that the sensitivity patterns of IHKEras cells to different topoisomerase II inhibitors provide a novel insight into the mechanism of drug action. Topoisomerase II inhibitors stabilize topoisomerase II enzyme-DNA cleavable complexes, resulting in DNA breaks that are essential for the cytotoxicity of the compounds (1-3). Potent DNA intercalators, like anthracyclines and ellipticines, efficiently induce the DNA breaks at low to intermediate concentrations; however, as the concentration increases, the induction of DNA breaks is suppressed (1,3,32,33). This suppression also occurs to a lesser degree with mitoxantrone (34). The suppression of DNA breaks is thought to be a direct result of interference by DNA intercalation (1,3,32-34). The DNA-intercalating drugs can even suppress the DNA breaks induced by epipodophyllotoxin derivatives and m-AMSA (32), which do not significantly intercalate into DNA (1,3). Thus, the IHKEras cells displayed enhanced sensitivity to etoposide and teniposide at very high concentrations and over a broad range of drug concentrations (Fig. 1Go, A and B). By contrast, the DNA-intercalating NSC 284682, doxorubicin, and NSC 659687 were less effective at high concentrations and showed a very narrow range of enhanced cytotoxicity against IHKEras cells (Fig. 1GoD, E, and F). We also found that the IHKEras cells were more sensitive to NSC 284682 over a broader dose range than to doxorubicin (Fig. 1Go, D and E). While we cannot exclude other explanations, it is possible that NSC 284682, like hydroxyrubicin (18), is a less potent DNA intercalator. Our results are consistent with the stabilization of cleavable complexes by topoisomerase II inhibitors being essential for the cytotoxicity of the drugs (1-3).

We tested whether enhanced DNA damage was responsible for the enhanced sensitivity of cells containing the ras oncogene to topoisomerase II inhibitors, but we found no correlation with the etoposide-induced damage (Table 5Go). However, apoptosis was markedly enhanced in the IHKEras cells at all cytotoxic concentrations tested (Table 5Go and Fig. 2Go). The ras oncogene has been shown to generate both pro- and anti-apoptotic signals, and the anti-apoptotic signals are thought to be dominant in transformed cells (35-38). It is possible that the topoisomerase II inhibitor-induced damage can either specifically shift the balance to favor apoptosis or add additional stimuli to a specific apoptotic threshold (39) lowered by a ras oncogene, perhaps by compromising a checkpoint function (40,41).

Apoptosis induced by DNA-damaging agents, including topoisomerase II inhibitors, is suggested to be p53 dependent (42-44). However, the topoisomerase II inhibitors have also been shown to efficiently induce p53 independent apoptosis (45-48). Based on the sensitivity patterns of the non-multidrug-resistant tumor cell lines of NCI-ADS harboring activated ras oncogenes, the topoisomerase II inhibitor-induced apoptosis was p53 independent [data not shown; (49)]. Moreover, the IHKE cells with mutant p53 (50) also displayed enhanced p53 independent and ras oncogene-dependent apoptosis (Fig. 2Go; data not shown).

Topoisomerase II inhibitors constitute one of the most effective groups of chemotherapeutic drugs used in cancer treatment, and most chemotherapeutic regimens include one or more topoisomerase II inhibitors. A topoisomerase II inhibitor has been routinely combined with cytarabine (51) in treating patients with AML, and it has been observed that the presence of ras oncogenes in AML cells significantly increases remission rate and prolongs overall survival of the patients in response to the treatment (11,52). Thus, the success of the combination therapy in AML treatment can be at least partially attributed to enhanced apoptosis in the tumor cells containing ras oncogenes induced by topoisomerase II inhibitors. Our results further suggest that, when ras oncogenes are present, epipodophyllotoxin derivatives, such as etoposide and teniposide, should have clinical benefit over the DNA-intercalating topoisomerase II inhibitors.

The discovery of MDR-resistant topoisomerase II inhibitors such as the compounds described in this article raises the possibility that they can be used in cancers that develop MDR during treatment. Furthermore, the identification of ras oncogenes as an enhancing factor for topoisomerase II inhibitor sensitivity could have a specific impact on the application of these drugs, since ras oncogene activation is the most frequently occurring gain-of-function mutation detected in human tumors (8,9).


    NOTES
 
Sponsored in part by the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under contract with ABL (contract N01C046000). We dedicate this article to Kenneth D. Paull, whose untimely death is a significant loss to the National Cancer Institute and the cancer research community. We thank M. M. Gottesman, S. Bates, A. Haugen, and W. Priebe for providing cell lines and compounds. We thank G. Taylor, T. Dipple, and K. Vousden for critical reading of the manuscript. We also thank A. Cline and M. Reed for the preparation of the manuscript.


    REFERENCES
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received July 24, 1998; revised November 25, 1998; accepted December 3, 1998.


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