© 1999 by Oxford University Press
Journal of the National Cancer Institute, Vol. 91, No. 3, 236-244,
February 3, 1999
© 1999 Oxford University Press
ARTICLES |
The ras Oncogene-Mediated Sensitization of Human Cells to Topoisomerase II Inhibitor-Induced Apoptosis
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 |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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.
|
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.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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).
|
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.
|
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.
|
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).
|
), 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.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.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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. 1D, 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 |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1 Pommier Y, Fesen M, Goldwasser F. Topoisomerase II inhibitors: the epipopodophyllotoxins, m-AMSA and the ellipticine derivatives. In: Chabner BA, Longo D, editors. Cancer chemotherapy and biotherapy. 2nd ed. Philadelphia (PA): Lippincott; 1996. p. 435-61.
2 Chen AY, Liu LF. DNA topoisomerases: essential enzymes and lethal targets. Annu Rev Pharmacol Toxicol 1994;34:191-218.[CrossRef][Web of Science][Medline]
3 Pommier Y. DNA topoisomerase II inhibitors. In: Teicher BA, editor. Cancer therapeutics: experimental and clinical agents. Totowa (NJ): Humana Press Inc.; 1997. p. 153-74.
4 Nitiss JL, Beck WT. Antitopoisomerase drug action and resistance. Eur J Cancer 1996;32A:958-66.cancerlit;96296548
5 Germann UA. P-glycoprotein—a mediator of multidrug resistance in tumour cells. Eur J Cancer 1996;32A:927-44.[CrossRef]cancerlit;96296546
6 Loe DW, Deeley RG, Cole SP. Biology of the multidrug resistance-associated protein, MRP. Eur J Cancer 1996;32A:945-57.cancerlit;96296547
7 Izquierdo MA, Scheffer GL, Flens MJ, Schroeijers AB, Van der Valk P, Scheper RJ. Major vault protein LRP-related multidrug resistance. Eur J Cancer 1996;32A:979-84.[CrossRef]cancerlit;96296550
8
Bos JL. ras oncogenes in human cancer: a review
[published erratum appears in Cancer Res 1990;50:1352]. Cancer Res 1989;49:4682-9.
9 Barbacid M. ras oncogenes: their role in neoplasia. Eur J Clin Invest 1990;20:225-35.[Web of Science][Medline]cancerlit;90316140
10
Koo HM, Monks A, Mikheev A, Rubinstein LV, Gray-Goodrich
M, McWilliams MJ, et al. Enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine
and topoisomerase II inhibitors in tumor cell lines harboring activated ras oncogenes. Cancer Res 1996;56:5211-6.
11
Neubauer A, Dodge RK, George SL, Davey FR, Silver RT,
Schiffer CA, et al. Prognostic importance of mutations in the ras proto-oncogenes in de novo
acute myeloid leukemia. Blood 1994;83:1603-11.
12 Fuchs EF, Horton D, Weckerle W, Winter-Mihaly E. Synthesis and antitumor activity of sugar-ring hydroxyl analogues of daunorubicin. J Med Chem 1979;22:406-11.[CrossRef][Web of Science][Medline]
13
Le Mee S, Pierre A, Markovits J, Atassi G, Jacquemin-Sablon
A, Saucier JM. S16020-2, a new highly cytotoxic antitumor olivacine derivative: DNA
interaction and DNA topoisomerase II inhibition. Mol Pharmacol 1998;53:213-20.
14
Pearson JW, Fogler WE, Volker K, Usui N, Goldenberg SK,
Gruys E, et al. Reversal of drug resistance in a human colon cancer xenograft expressing MDR1
complementary DNA by in vivo administration of MRK-16 monoclonal antibody. J Natl Cancer Inst 1991;83:1386-91.
15 Lai GM, Chen YN, Mickley LA, Fojo AT, Bates SE. P-glycoprotein expression and schedule dependence of adriamycin cytotoxicity in human colon carcinoma cell lines. Int J Cancer 1991;49:696-703.[Web of Science][Medline]cancerlit;92040371
16 Lee JS, Paull K, Alvarez M, Hose C, Monks A, Grever M, et al. Rhodamine efflux patterns predict P-glycoprotein substrates in the National Cancer Institute drug screen. Mol Pharmacol 1994;46:627-38.[Abstract]cancerlit;95058928
17
Tveito G, Hansteen IL, Dalen H, Haugen A. Immortalization of
normal human kidney epithelial cells by nickel (II). Cancer Res 1989;49: 1829-35.
18 Solary E, Ling YH, Perez-Soler R, Priebe W, Pommier Y. Hydroxyrubicin, a deaminated derivative of doxorubicin, inhibits mammalian DNA topoisomerase II and partially circumvents multidrug resistance. Int J Cancer 1994;58:85-94.[Web of Science][Medline]cancerlit;94284048
19 Priebe W, Perez-Soler R. Design and tumor targeting of anthracyclines able to overcome multidrug resistance: a double-advantage approach. Pharmacol Ther 1993;60:215-34.[CrossRef][Web of Science][Medline]cancerlit;94294476
20 White MA, Nicolette C, Minden A, Polverino A, Van Aelst L, Karin M, et al. Multiple ras functions can contribute to mammalian cell transformation. Cell 1995;80:533-41.[CrossRef][Web of Science][Medline]cancerlit;95171448
21
Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica
D, et al. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human
tumor cell lines. J Natl Cancer Inst 1991;83: 757-66.
22 Jeffers M, Rong S, Vande Woude GF. Enhanced tumorigenicity and invasion-metastasis by hepatocyte growth factor/scatter factor-met signalling in human cells concomitant with induction of the urokinase proteolysis network. Mol Cell Biol 1996;16:1115-25.[Abstract]cancerlit;96182116
23 Kohn KW, Ewig RAG, Erickson LC, Zwelling LA. Measurement of strand breaks and cross-links by alkaline elution. In: Friedberg EC, Hanawalt PC, editors. DNA repair—a laboratory manual of research procedures. New York (NY): Marcel Dekker, Inc.; 1981. p. 379-401.
24 Bertrand R, Kohn KW, Solary E, Pommier Y. Detection of apoptosis-associated DNA fragmentation using a rapid and quantitative filter elution assay. Drug Dev Res 1995;34:138-44.[CrossRef]
25 Paull KD, Hamel E, Malspeis L. Prediction of biochemical mechanism of action from the in vitro antitumor screen of the national cancer institute. In: Foye WO, editor. Cancer chemotherapeutic agents. Washington (DC): American Chemical Society; 1995. p. 9-45.
26 Zhang XP, Ritke MK, Yalowich JC, Slovak ML, Ho JP, Collins KI, et al. P-glycoprotein mediates profound resistance to bisantrene. Oncology Res 1994;6:291-301.[Web of Science][Medline]
27 Wassermann K, Markovits J, Jaxel C, Capranico G, Kohn KW, Pommier Y. Effects of morpholinyl doxorubicins, doxorubicin, and actinomycin D on mammalian DNA topoisomerases I and II. Mol Pharmacol 1990;38:38-45.[Abstract]cancerlit;90318342
28 Leonce S, Perez V, Casabianca-Pignede MR, Anstett M, Bisagni E, Pierre A, et al. In vitro cytotoxicity of S16020-2, a new olivacine derivative. Invest New Drugs 1996;14:169-80.[Web of Science][Medline]cancerlit;97070912
29 Guilbaud N, Kraus-Berthier L, Saint-Dizier D, Rouillon MH, Jan M, Burbridge M, et al. In vivo antitumor activity of S 16020-2, a new olivacine derivative. Cancer Chemother Pharmacol 1996;38:513-21.[CrossRef][Web of Science][Medline]cancerlit;96420795
30
Berman E, McBride M. Comparative cellular pharmacology of
daunorubicin and idarubicin in human multidrug-resistant leukemia cells. Blood 1992;79:3267-73.
31 Granzen B, Graves DE, Baguley BC, Danks MK, Beck WT. Structure-activity studies of amsacrine analogs in drug resistant human leukemia cell lines expressing either altered DNA topoisomerase II or P-glycoprotein. Oncol Res 1992;4:489-96.[Web of Science][Medline]cancerlit;93229824
32 Capranico G, Zunino F, Kohn KW, Pommier Y. Sequence-selective topoisomerase II inhibition by anthracycline derivatives in SV40 DNA: relationship with DNA binding affinity and cytotoxicity. Biochemistry 1990;29:562-9.[CrossRef][Medline]cancerlit;90148984
33
Tewey KM, Chen GL, Nelson EM, Liu LF. Intercalative
antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase
II. J Biol Chem 1984;259:9182-7.
34 Capolongo L, Belvedere G, D'Incalci M. DNA damage and cytotoxicity of mitoxantrone and doxorubicin in doxorubicin-sensitive and -resistant human colon carcinoma cells. Cancer Chemother Pharmacol 1990;25: 430-4.[CrossRef][Web of Science][Medline]cancerlit;90182830
35
Mayo MW, Wang CY, Cogswell PC, Rogers-Graham KS, et al.
Requirement of NF-kB activation to suppress p53-independent apoptosis induced by oncogenic
Ras. Science 1997;278:1812-5.
36 Chen G, Shu J, Stacey DW. Oncogenic transformation potentiates apoptosis, S-phase arrest and stress-kinase activation by etoposide. Oncogene 1997;15:1643-51.[CrossRef][Web of Science][Medline]cancerlit;98007653
37 Kauffmann-Zeh A, Rodriguez-Viciana P, Ulrich E, Gilbert C, Coffer P, Downward J, et al. Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB. Nature 1997;385:544-8.[CrossRef][Medline]cancerlit;97172278
38
Ohmori M, Shirasawa S, Furuse M, Okumura K, Sasazuki T.
Activated Ki-ras enhances sensitivity of ceramide-induced apoptosis without c-Jun NH2-terminal kinase/stress-activated protein kinase or extracellular signal-regulated kinase
activation in human color cancer cells. Cancer Res 1997;57:4714-7.
39 Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell 1994;78:539-42.[CrossRef][Web of Science][Medline]cancerlit;94349357
40 Vande Woude GF, Schulz N, Zhou R, et al. Cell cycle regulation, oncogenes, and antineoplastic drugs. General Motors cancer research foundation: 1990 views of cancer research. Philadelphia (PA): Lippincott; 1990. p. 128-43.
41 Murakami MS, Strobel MC, Vande Woude GF. Cell cycle regulation, oncogenes, and antineoplastic drugs. In: Howley PM, Israel MA, Liotta LA, editors. The molecular basis of cancer. Philadelphia (PA): Saunders; 1994. p. 3-17.
42 Lowe SW, Ruley HE, Jacks T, Housman DE. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993;74: 957-67.[CrossRef][Web of Science][Medline]cancerlit;94006528
43 Clarke AR, Purdie CA, Harrison DJ, Morris RG, Bird CC, Hooper ML, et al. Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 1993;362:849-52.[CrossRef][Medline]cancerlit;93241299
44
Chresta CM, Masters JR, Hickman JA. Hypersensitivity of
human testicular tumors to etoposide-induced apoptosis is associated with functional p53 and a
high Bax:Bcl-2 ratio. Cancer Res 1996;56:1834-41.
45 Malcomson RD, Oren M, Wyllie AH, Harrison DJ. p53-independent death and p53-induced protection against apoptosis in fibroblasts treated with chemotherapeutic drugs. Br J Cancer 1995;72:952-7.[Web of Science][Medline]cancerlit;96003581
46 Arita D, Kambe M, Ishioka C, Kanamaru R. Induction of p53-independent apoptosis associated with G2M arrest following DNA damage in human colon cancer cell lines. Jpn J Cancer Res 1997;88:39-43.[CrossRef][Web of Science][Medline]cancerlit;97197598
47 Nip J, Strom DK, Fee BE, Zambetti G, Cleveland JL, Hiebert SW. E2F-1 cooperates with topoisomerase II inhibition and DNA damage to selectively augment p53-independent apoptosis. Mol Cell Biol 1997;17:1049-56.[Abstract]cancerlit;97184429
48 Strasser A, Harris A, Jacks T, Cory S. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell 1994;79:329-39.[CrossRef][Web of Science][Medline]cancerlit;95042729
49
O'Connor PM, Jackman J, Bae I, Myers TG, Fan S,
Mutoh M, et al. Characterization of the p53 tumor suppressor pathway in cell lines of the
National Cancer Institute anticancer drug screen and correlations with the growth-inhibitory
potency of 123 anticancer agents. Cancer Res 1997;57:4285-300.
50
Maehle L, Metcalf RA, Ryberg D, Bennett WP, Harris CC,
Haugen A. Altered p53 gene structure and expression in human epithelial cells after exposure to
nickel. Cancer Res 1992;52:218-21.
51 Rohatiner AZ, Lister TA. The treatment of acute myelogenous leukemia. In: Henderson ES, Lister TA, editors. Leukemia. Philadelphia (PA): Saunders; 1990. p. 485-513.
52 Coghlan DW, Morley AA, Matthews JP, Bishop JF. The incidence and prognostic significance of mutations in codon 13 of the N-ras gene in acute myeloid leukemia. Leukemia 1994;8:1682-7.[Web of Science][Medline]cancerlit;95019809
53 Cragg G, Suffness M. Metabolism of plant-derived anticancer agents. Pharmac Ther 1988;37:425-61.[CrossRef][Web of Science][Medline]
Manuscript received July 24, 1998; revised November 25, 1998; accepted December 3, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Neubauer, K. Maharry, K. Mrozek, C. Thiede, G. Marcucci, P. Paschka, R. J. Mayer, R. A. Larson, E. T. Liu, and C. D. Bloomfield Patients With Acute Myeloid Leukemia and RAS Mutations Benefit Most From Postremission High-Dose Cytarabine: A Cancer and Leukemia Group B Study J. Clin. Oncol., October 1, 2008; 26(28): 4603 - 4609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Giovannetti, V. Mey, S. Nannizzi, G. Pasqualetti, M. Del Tacca, and R. Danesi Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer. Mol. Cancer Ther., June 1, 2006; 5(6): 1387 - 1395. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. F. V. Woude, G. J. Kelloff, R. W. Ruddon, H.-M. Koo, C. C. Sigman, J. C. Barrett, R. W. Day, A. P. Dicker, R. S. Kerbel, D. R. Parkinson, et al. Reanalysis of Cancer Drugs: Old Drugs, New Tricks Clin. Cancer Res., June 1, 2004; 10(11): 3897 - 3907. [Full Text] [PDF]
|
|
|
|
|
|
L. Maggiorella, V. Frascogna, M.-G. Poullain, M. Berlion, C. Lucas, S. Douc Razy, F. Eschwege, and J. Bourhis The Olivacine S16020 Enhances the Antitumor Effect of Ionizing Radiation without Increasing Radio-induced Mucositis Clin. Cancer Res., July 1, 2001; 7(7): 2091 - 2095. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q.-B. She, A. M. Bode, W.-Y. Ma, N.-Y. Chen, and Z. Dong Resveratrol-induced Activation of p53 and Apoptosis Is Mediated by Extracellular- Signal-regulated Protein Kinases and p38 Kinase Cancer Res., February 1, 2001; 61(4): 1604 - 1610. [Abstract] [Full Text]
|
|
|
|
 |
|
 D. W. Stacey, M. Hitomi, and G. Chen Influence of Cell Cycle and Oncogene Activity upon Topoisomerase IIalpha Expression and Drug Toxicity Mol. Cell. Biol., December 15, 2000; 20(24): 9127 - 9137. [Abstract] [Full Text]
|
|
|
|
|
|
H. Malonne, S. Farinelle, C. Decaestecker, L. Gordower, J. Fontaine, F. Chaminade, J.-M. Saucier, G. Atassi, and R. Kiss In Vitro and in Vivo Pharmacological Characterizations of the Antitumor Properties of Two New Olivacine Derivatives, S16020-2 and S30972-1 Clin. Cancer Res., September 1, 2000; 6(9): 3774 - 3782. [Abstract] [Full Text]
|
|
|
|
|
|
H.-M. Koo, M. J. McWilliams, W. G. Alvord, and G. F. Vande Woude Ras Oncogene-Induced Sensitization to 1-{{beta}}-D-Arabinofuranosylcytosine Cancer Res., December 1, 1999; 59(24): 6057 - 6062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-M. Koo and G. F. Vande Woude Re: The ras Oncogene-Mediated Sensitization of Human Cells to Topoisomerase II Inhibitor-Induced Apoptosis J Natl Cancer Inst, November 17, 1999; 91(22): 1969 - 1969. [Full Text] [PDF]
|
|
|
|
 |
|
 H.-M. Koo, M. VanBrocklin, M. J. McWilliams, S. H. Leppla, N. S. Duesbery, and G. F. V. Woude Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase PNAS, March 5, 2002; 99(5): 3052 - 3057. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||