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
(ABLBasic Research Program), M. Gray-Goodrich, A.
Vaigro-Wolff, A. Monks (Science Applications International
CorporationFrederick), 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).
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ABSTRACT
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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.
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INTRODUCTION
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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.
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MATERIALS AND METHODS
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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).
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
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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
(TopoGEN, Columbus, OH), and
anti-
-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 1
.
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 1
.
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 1
). 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.
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RESULTS
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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 1
).
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),
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 2
, 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 2
, 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*
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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 2
, 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 2
, 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 2
, 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*
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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).
By contrast, both HT-29par and
HT-29mdr1 cells showed a similar sensitivity to both NSC
284682 and NSC 659687 compounds (Table 3
). 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 3
).
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 4
). 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).
The
acquired multidrug-resistant cell lines displayed considerable
resistance to m-AMSA (Table 4
), 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
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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).
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 1
, A and B). Mitoxantrone also displayed enhanced
cytotoxicity against the IHKEras cells at high drug
concentrations (Fig. 1
, 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. 1
, 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. 1
, G). Moreover, all
of the cells showed similar sensitivity to vinblastine (Fig. 1
, H).

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Fig. 1. Sensitivity of IHKE (parental, ), IHKEDCR
(vector alone, ), and IHKEras (ras oncogene, ) 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.
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The IHKE-derived cell lines were analyzed for Pgp and topoisomerase
II
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
expression (data not
shown). The IHKE cells transfected and selected with vector alone
(IHKEDCR) displayed a lower level of topoisomerase II
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. 1
, 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).
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
detected in the IHKEDCR cells.
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).
Moreover, the drug sensitivity
pattern closely paralleled the pattern of apoptosis (Fig. 1
, A; Fig. 2
,
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. 2
, B).
These results demonstrate that the greater sensitivity of
IHKEras cells to etoposide parallels the enhanced apoptosis.
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DISCUSSION
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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 2
), 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 2-4

). 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
2
). 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. 1
). 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. 1
, 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. 1
D, 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. 1
, 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 5
). However, apoptosis was markedly enhanced in the
IHKEras cells at all cytotoxic concentrations tested (Table 5
and Fig. 2
). 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. 2
; 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
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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.
 |
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