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Vol. 54, Issue 5, 770-777, November 1998
Cancer Therapy and Research Center, Institute for Drug Development, San Antonio, Texas 78245 (J.M.W., W.G.C., C.N., M.C.S.H.), and Sanofi Research, Malvern, Pennsylvania 19355 (P.J.)
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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Oxaliplatin is a clinical anticancer drug with a pharmacological
profile distinct from that of cisplatin. Our studies compared site- and
region-specificity of lesions induced by oxaliplatin and cisplatin in
naked and intracellular DNA, respectively. Oxaliplatin adducts in naked
Simian virus 40 (SV40 DNA) were mapped by repetitive primer extension.
The sites of oxaliplatin adducts were nearly identical to the sites of
cisplatin adducts and were focused in G clusters and GNG motifs
probably reflecting intrastrand cross-links. Although alkaline agarose
electrophoresis of specific SV40 fragments showed that oxaliplatin
formed interstrand cross-links, the levels of this lesion type were
low. Drug-induced lesions in discrete loci of cellular DNA were
assessed by the polymerase chain reaction stop assay in human tumor
A2780 cells. Oxaliplatin at 200 µM induced ~1300,
~1500, ~800, and ~300 lesions/106 bp in the human
-globin, c-myc, and
HPRT genes and in mitochondrial DNA,
respectively. Cisplatin formed two to six times more lesions in the
same regions. For both drugs, lesion frequencies seem to parallel the
density of drug-binding motifs in the nuclear regions, whereas
mitochondrial DNA was disproportionately less affected. Despite less
potent induction of DNA lesions, oxaliplatin was more cytotoxic than
cisplatin against A2780 cells. Because our findings clearly demonstrate
that oxaliplatin forms covalent adducts with a similar sequence- and
region-specificity to that of cisplatin, other properties of
oxaliplatin adducts, factors other than DNA binding, or both determine
the unique features of the mechanism of action of oxaliplatin.
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Introduction |
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Summary
Introduction
Procedures
Results
Discussion
References
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DACH
[oxaliplatin; oxalato; (1R,2R)-DACH] (Fig.
1) is a member of the family of DACH
platinum complexes and is a third-generation platinum antitumor drug
(reviewed by Chaney, 1995
). The advantages of oxaliplatin over
cisplatin, the classic platinum drug, include a less severe clinical
toxicity and a retained activity against cisplatin-refractory tumors
(Mathe et al., 1989; Tashiro et al., 1989;
Christian, 1992; Kelland, 1993; Weiss and Christian, 1993; Kelland and
McKeage, 1994). In vitro and in vivo studies
confirmed that oxaliplatin exhibits different spectra of activity and
toxicity than cisplatin, often with a lack of cross-resistance with
cisplatin (Tashiro et al., 1989; Pendyala and Creaven, 1993;
Chaney, 1995). COMPARE analysis of the sensitivity pattern in a
panel of 60 tumor cell lines suggested that oxaliplatin and related
DACH-containing platinum analogs differ mechanistically from cisplatin
and other platinum drugs (Rixe et al., 1996).
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Studies with various DACH platinum complexes indicate that the
trans-(R,R) configuration of the DACH
moiety is critical for the properties of oxaliplatin, such as superior
cytotoxicity and drug binding to cellular DNA (Inagaki and Sawaki,
1995; Pendyala et al., 1995). The compounds with DACH
isomeric forms, trans-(S,S) and
cis-(R,S), are less potent. The
leaving group (oxalate) also may affect drug pharmacological
properties. Oxaliplatin undergoes a transformation under physiological
conditions to DACH PtCl2 (Fig. 1) and other
species that seem to be primarily responsible for the reaction with DNA
(Chaney, 1995; Luo et al., 1997).
The formation of adducts with cellular DNA generally is regarded as the
major determinant of the antitumor activity of platinum drugs. In
contrast to the extensively studied cisplatin, however, relatively
little is known about the DNA binding of oxaliplatin and related DACH
agents. Adducts that involved binding motifs such as d(GpG), d(ApG),
and (dG)2 were identified in drug-treated isolated DNA or short synthetic oligonucleotides using
DACH-Pt-SO4 and
DACH-Pt-(NO2)2 (Jennerwein
et al., 1989; Boudny et al., 1992). Also, the
different Pt-GG and Pt-AG adducts of oxaliplatin were detected recently
by immunochemistry in DNA from oxaliplatin-treated cells (Saris
et al., 1996). The sites of oxaliplatin adducts at the
nucleotide level, however, have not been characterized.
DACH-Pt adducts, once formed, should be identical to adducts by other
DACH compounds with different leaving groups, but it cannot be ruled
out that the oxalate DACH-Pt can behave differently than the nitrate
form. The precedence of carboplatin, which forms chemically identical
adducts as cisplatin but with apparently different nucleotide sequence
preferences (Blommaert et al., 1995), demonstrates that the
leaving group may affect drug-binding sites. Thus, it remains unclear
whether oxaliplatin and cisplatin affect the same or different subsets
of potential binding motifs in DNA sequences and consequently target
the same or different regions in cellular DNA.
The purpose of our study was to delineate differences and similarities between oxaliplatin and cisplatin in targeting naked and intracellular DNA. Adduct sites were mapped at the nucleotide level in naked SV40 DNA. Drug potential to form adducts in several discrete loci of cellular DNA in intact cells also was examined. The results demonstrate that oxaliplatin shares sequence- and region-specificity with cisplatin. Paradoxically, despite less potent induction of DNA lesions, oxaliplatin was more cytotoxic than cisplatin.
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Experimental Procedures |
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Procedures
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Discussion
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Drugs.
Oxaliplatin was obtained from Sanofi Research
(Malvern, PA). Cisplatin was from Sigma Chemical (St. Louis, MO). Stock
solutions of cisplatin were made in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4,
and 1.4 mM KH2PO4) and stock
solutions of oxaliplatin in water or in dimethylsulfoxide and stored at
20°.
Cell lines and cytotoxic activity.
Green monkey kidney BSC-1
cells were grown as described previously (Zsido et al.,
1991). The human ovarian carcinoma A2780 line was obtained from Dr.
Kevin J. Scanlon and grown as a monolayer culture in RPMI 1640 medium
supplemented with 10% serum. Growth-inhibitory activity was assayed
using a standard MTT assay (Arnould et al., 1990).
Exponentially growing cells in a 96-well microtiter plate were
incubated with the drug for three or four doubling times (3-4 days)
and subjected to colorimetric reaction with
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide. The
results are expressed as drug concentrations that inhibit cell growth
by 50% (the IC50 values).
Sites of drug adducts: RPE.
Localization of oxaliplatin and
cisplatin adducts was determined based on premature termination of
primer extension on a drug-modified DNA template. The use of thermally
stable Taq DNA polymerase and repetitive cycling in a PCR
machine enables linear amplification of the signal from the damaged
template (Jennerwein and Eastman, 1991; Bubley et al.,
1994).
-32P]dATP (3000 Ci/mmol, 10.0 mCi/ml; New
England Nuclear, Boston, MA) in a total volume of 50 µl. After a 1-hr
incubation at 37°, the unincorporated radiolabel was removed using
Sephadex G25 Quick Spin Columns (Boehringer-Mannheim, Indianapolis, IN)
equilibrated with TE buffer. The primer used in this study was ORI-U, a
21-mer (5'-TTTTTTCTTCATCTCCTCCTT-3') complementary to SV40 bottom
strand at positions 5010-5030.
RPE reactions for thermal cycling were set up using PCR Core Reagents
(Perkin Elmer, Norwalk, CT) and typically consisted of 1× buffer II,
1.5 mM MgCl2, 0.2 mM
concentration of each dNTP, 1 unit of Taq polymerase/20
µl, 1.25 µM concentration of end-labeled primer
(estimated assuming 100% recovery from the Sephadex G25 column), and
0.1 µg of template DNA. Thermal cycling was performed in a model 9600 Thermal Cycler (Perkin-Elmer). The following conditions were used:
95° for 30 sec followed by 30 cycles of 35° for 20 sec, 72° for
25 sec, and 94° for 15 sec. The final cycle was followed by an extra
6 min of extension at 72°. Equal volume aliquots of each reaction
(usually 2 µl) were analyzed on 8% polyacrylamide/urea sequencing
gels along with sequencing reactions, performed with the same primer as
in the RPE reactions, using Sequenase v.2 (USA Biochemicals, Cleveland,
OH) and [35S]dATP (1000 Ci/mmol, 12.5 mCi/ml;
New England Nuclear). After electrophoresis, the gels were dried and
subjected to autoradiography. Autoradiographs were scanned on a
Molecular Dynamics (Sunnyvale, CA) laser densitometer. Alternatively,
dried gels were exposed to phosphor screens and analyzed on a Molecular
Dynamics PhosphorImager. The images were processed using Image Quant
software (Molecular Dynamics). For representative oxaliplatin and
cisplatin lanes, the intensity of each drug-induced band in the
position range of 5083-5174 was quantified using Image Quant software.
Weak sites (<1.5% total signal intensity) were filtered out, and the
intensity of each remaining band was expressed as a percentage of the
sum of intensities of all the bands scored for the entire region (Fig. 2C).
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Interstrand DNA cross-links.
A singly end-radiolabeled
fragment of SV40 DNA (MAR, 508 bp from positions 3943-4451) was
synthesized by PCR as described previously (Woynarowski et
al., 1995). Another fragment, ORI (positions 5010-116), was
synthesized in a similar way except that different primers (high
performance liquid chromatography purified; Genosys, The Woodlands, TX)
were used: 32P-end-labeled ORI-U (see RPE assay)
and unlabeled ORI-L (21-mer, 5'-TCAGCAACCATAGTCCCGCCC-3'). The
following thermal cycling conditions were used: 95° for 30 sec
followed by 30 cycles of 45° for 20 sec, 72° for 25 sec, and 94°
for 15 sec. The final cycle was followed by an extra 6 min of extension
at 72°. 32P-End-radiolabeled PCR product was
purified on a Sepharose CL-6B Quick Spin column (Boehringer-Mannheim).
). Cross-link frequencies were
estimated based on the fraction of cross-linked DNA in drug-treated
samples using a Poisson distribution formula (Woynarowski et
al., 1995).
PCR stop assay.
A2780 cells were prelabeled by overnight
incubation in a medium containing 0.05-0.1 µCi/ml
[14C]thymidine (56.5 mCi/mmol, New England
Nuclear). The cells were additionally incubated in radiolabel-free
medium for 1 hr at 37° and then, with drugs, for 4 hr. After drug
treatment, cells were rinsed with PBS, scraped, and resuspended in PBS.
Cellular DNA was extracted and partially purified using the PureGene
kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's
protocol. Free drug was assumed to be removed in the course of DNA
purification. DNA samples were stored at 20° and used for the PCR
determinations in the course of 2-3 weeks. Because the stop assay does
not distinguish among different types of lesions as long as they
inhibit primer extension, no further attempt was made to prevent the
second-arm reaction of platinum monoadducts after DNA isolation.
). The concentrations of
purified DNA were expressed as "cell equivalents" based on total
14C radioactivity/106 cells
determined separately by liquid scintillation counting. PCR reactions
(20 µl) usually were carried out in triplicate at two or three
different levels of template DNA (1000-5000 and 100-1000 cell
equivalents per reaction for nuclear and mitochondrial regions, respectively). The reactions were set up using PCR Core Reagents (Perkin Elmer, Branchburg, NJ) and typically consisted of 1× buffer II, 1.5 mM MgCl2, and 0.2 mM concentration of each dNTP, except for dGTP (0.05 mM), 1 unit of Taq polymerase, and 0.04 µCi/reaction of [32P]dGTP (New England
Nuclear). The reactions were subjected to temperature cycling as
described below using a Perkin-Elmer model 9600 cycler.
The following primer systems and cycling conditions were used. For a
536-bp segment of the -globin gene (Daoud et
al., 1995), PCR was carried out using 5'-GGTTGGCCAATCTACTCCCAGG-3'
and 5'-GCTCACTCAGTGTGGCAAAG-3' as the upper and lower primer,
respectively. Initial denaturation at 95° for 30 sec was followed by
30 cycles of 95° for 20 sec, 55° for 20 sec, and 72° for 30 sec.
For a 2.7-kbp region in the HPRT locus (Oshita and Saijo,
1994; Oshita et al., 1995), PCR was carried out using
5'-TGGGATTACACGTGTGAACCAACC-3' and 5'-TGTGACACAGGCAGACTGTGGATC-3' as
the upper and lower primer, respectively, with initial denaturation at
95° for 30 sec followed by 27 cycles of 95° for 15 sec, 60° for
60 sec, and 72° for 60 sec. For a 743-bp region in the
c-myc ORI domain, 5'-GCCGTTTTAGGGTTTGTTG-3' and
5'-CAAAAAACATTCTTCTCATCC-3' were used as the upper and lower primer,
respectively. PCR was carried out with initial denaturation at 95°
for 30 sec followed by 26 cycles of 94° for 15 sec, 55° for 20 sec,
and 72° for 30 sec. For a 1100-bp region (Mito) in the mtDNA (Daoud
et al., 1995), oligonucleotides 5'-CCACATCACTCGAGACGTAA-3'
and 5'-GCGGTTGTTGATGGGTGAGT-3' were used as the upper and lower primer,
respectively, and initial denaturation at 95° for 30 sec was followed
by 18 cycles of 94° for 15 sec, 55° for 30 sec, and 72° for 25 sec. For all the systems, terminal extension was performed at 72° for
3-5 min.
The above conditions resulted in a linear response of signal intensity
in terms of both the amount of template DNA and increasing number of
cycles (data not shown). After electrophoresis in 1% agarose and
autoradiography, signal intensities were quantified in a Molecular
Dynamics densitometer. Results are normalized to the signal intensity
in control samples and averaged for replicates and the different levels
of template DNA.
Estimation of lesion frequency.
Amplification inhibition
data were converted to lesion frequency using a Poisson distribution
formula (Kalinowski et al., 1992) on the assumption that one
lesion in the target area was sufficient to eliminate the amplification
of that template. The frequency data are expressed as lesions/1 kbp:
f = [ln(Fa)]/L, where f is the number of breaks per bp,
Fa represents the fraction of
remaining amplification (relative to control) in drug-treated samples,
and L is the length of target DNA region in bp.
Analysis of drug-binding motifs. The numbers of binding motifs in each region were obtained by analyzing DNA sequences for these regions with a software tool in Oligo (NBI) program originally designed for restriction site searches. The motif input data included common motifs for platinum drugs such as GG, GNG, GC, and CG. These motifs are consistent with our data for cisplatin- and oxaliplatin-binding sites (see Results; Table 1). The number of binding motifs found in each region analyzed were normalized to 1 kbp to compensate for the differences in the lengths of these regions.
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Results |
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Summary
Introduction
Procedures
Results
Discussion
References
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Lesions in Naked DNA
Localization of oxaliplatin adducts in a selected region of
purified SV40 DNA.
To map the sites of oxaliplatin versus
cisplatin adducts, the technique of RPE was used. In an RPE assay,
lesions on the template strand result in the premature termination of
nascent chain elongation opposite an adducted site (Jennerwein and
Eastman, 1991; Bubley et al., 1994). Cisplatin has been
shown previously to induce such premature termination sites in naked
and intracellular DNA (Hemminki and Thilly, 1988; Jennerwein and
Eastman, 1991; Bubley et al., 1994).
; Hoffmann et al.,
1989) and for DACH-Pt-SO4 and
DACH-Pt-(NO2)2 (Jennerwein
et al., 1989; Boudny et al., 1992). The GAG sites might reflect an intrastrand cross-link between both guanines or
between adenine and guanine. Consistent with the induction of
intrastrand cross-links, oxaliplatin produced unwinding of supercoiled
(form I) SV40 DNA (data not shown). This unwinding effect, although
significant, was less profound than the unwinding by equimolar cisplatin.
Side-by-side comparison confirmed that oxaliplatin forms adducts at the
same sites as cisplatin. Relative intensities of these sites, however,
differed somewhat (Fig. 2C). Some sites seem to be slightly enhanced
for oxaliplatin, such as two sites at positions 5120 and 5160-61,
which contain a sequence 5'-ATGGA-3', and a doublet at 5138-5139 with
an AGGA motif (Table 1 and Fig. 2C). Some cisplatin sites (e.g.,
position 5105) seem to be absent from oxaliplatin-treated samples,
whereas others show decreased intensity (e.g., sites at 5090-5092).
Although the RPE experiments were not intended to quantify DNA adducts,
the results suggested a lower level of oxaliplatin adducts compared
with cisplatin. The longest nascent fragments (Fig. 2A, top)
are nearly eliminated by 8 µM cisplatin (Fig. 2A, lane 8) but not by oxaliplatin used at the same
concentration (Fig. 2A, lane 3). As much as 32 µM oxaliplatin (Fig. 2A, lane 4) was needed to
profoundly reduce this signal.
The RPE experiments are consistent with intrastrand cross-links as the
primary lesion, although RPE might not detect less frequent interstrand
cross-linking. Interstrand cross-links spanning two Gs in the
complementary strands, in addition to intrastrand adducts, cannot be
ruled out at some sites (such as the strong GGC site at position
5066, Table 1), although several other GC sites showed no indications
of adduction.
Interstrand cross-links in SV40 fragments.
Interstrand
cross-linking by DACH compounds other than oxaliplatin was proposed
based on the detection of (dG)2 products in digests from drug-treated DNA (Jennerwein et al., 1989;
Boudny et al., 1992). However, no data were available on
interstrand cross-links by oxaliplatin, and no direct detection of
cross-linked DNA strands was reported thus far for any DACH compound.
Given the potential biological significance of interstrand cross-links, the ability of oxaliplatin to form this lesion was directly examined. The induction of interstrand cross-links was assessed in two distinct fragments of SV40 DNA (MAR, positions 3943-4451, and ORI, positions 5010-116). These fragments, synthesized by PCR with
[32P] label on a single 5'-end, were treated
with drugs and next analyzed by alkaline agarose gel electrophoresis.
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Region-Specificity of Lesions in Intracellular DNA in A2780 cells
Despite a similar pattern of lesion sites in naked SV40 DNA,
oxaliplatin and cisplatin might differ in the distribution of drug
adducts among various regions of cellular DNA (region-specificity). Thus, the PCR stop assay was used to compare the vulnerability of
defined regions in cellular DNA to the actions of oxaliplatin and
cisplatin. Like the RPE assay, the PCR stop assay capitalizes on the
inability of Taq polymerase to bypass platinum adducts on
DNA, which results in reduced amplification of a target region. The PCR
stop assay is expected to reflect mainly intrastrand cross-links, which
represent the majority of drug adducts for cisplatin (Plooy et
al., 1985; Takahara et al., 1995; Saris et
al., 1996) and probably for oxaliplatin as well, as suggested by
the RPE experiments.
Lesions in nuclear versus mtDNA.
Drug effects on cellular DNA
were analyzed using PCR systems for three different nuclear regions (a
740-bp ORI domain of the c-myc oncogene, a 530-bp domain in
the -globin gene, and a 2.7-kbp region of the
HPRT locus) and for a system for mtDNA that was used
previously for cisplatin (Daoud et al., 1995). The selection of PCR systems was arbitrary and does not imply that any of these regions is a specific target for platinum drugs.
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-globin and mtDNA
(Daoud et al., 1995) and for HPRT systems (Oshita and Saijo,
1994) in cisplatin-sensitive cell lines, specifically human ovarian
carcinoma CaOv-3 (-globin and mtDNA) and human lung cancer PC9
(HPRT). The relative sensitivity of the HPRT system is consistent with
its much greater length (2.7 kbp) compared with other regions.
Table 2 shows the estimated frequencies
of drug-induced lesions after data conversion to
lesions/106 bp (Mbp) assuming the Poisson
formula. Compared with cisplatin, oxaliplatin induced roughly two to
four times fewer lesions in nuclear domains and nearly seven times
fewer lesions in mtDNA. Thus, we conclude that oxaliplatin is less
reactive than cisplatin with cellular DNA under these conditions. mtDNA
seems to be less vulnerable to both oxaliplatin and cisplatin than
nuclear DNA. This observation confirms the previously observed
preference of cisplatin for nuclear DNA (Daoud et al.,
1995).
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Region-specific DNA lesions versus drug-binding motifs. Having estimated oxaliplatin and cisplatin lesions in various regions, we next asked whether the relative vulnerability of these regions is related to the occurrence of known drug-binding sites. The sequences of DNA regions used in the PCR stop assay have been analyzed for the presence of binding motifs consistent with the RPE data (GG, GNG) and interstrand cross-linking (GC). Estimated lesions frequencies were plotted against the density of the drug-binding motifs (Fig. 5). For cisplatin, this analysis showed that more lesions were formed in those regions that are richer in drug-binding motifs. A similar trend was seen for oxaliplatin, although the lower frequency of oxaliplatin lesion makes this trend less pronounced than for cisplatin. For both drugs, mtDNA was clearly the exception from the trend confirming that mtDNA is a disfavored target.
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Cytotoxic Activity of Oxaliplatin and Cisplatin
Drug cytotoxic activity was evaluated using the standard MTT assay. In BSC-1 cells, the resulting drug concentrations that produced 50% growth inhibition (IC50, mean ± standard error) were 1.05 ± 0.06 and 4.8 ± 1.2 µM for oxaliplatin and cisplatin, respectively. In A2780 cells, respective IC50 values were 0.56 and 2.3 µM. These results demonstrate that oxaliplatin is approximately four times more cytotoxic than cisplatin in both cell lines.
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Discussion |
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Summary
Introduction
Procedures
Results
Discussion
References
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Our study characterized the sequence- and region-specificity of
oxaliplatin adducts with naked and intracellular DNA, respectively. The
localization of these adducts resembles that of cisplatin/DNA adducts.
Analysis at the nucleotide level by RPE technique indicated that
oxaliplatin binds mainly to guanines with a preference for guanine
clusters. Although the 1-2-bp resolution of the RPE technique does not
allow us unequivocally to localize the modified nucleotide residues,
our data are consistent with GG and GAG (or GNG) intrastrand cross-links as a prevailing lesion with naked DNA. Despite lower overall reactivity, oxaliplatin forms lesions at similar sequences as
does cisplatin. Not only does oxaliplatin share with cisplatin the
propensity for adduct formation in G clusters in purified DNA, but also
the relative intensities of adduction at the major sites, although not
identical, are generally similar. In the context of previous data on
types of DNA adducts for other DACH-Pt compounds (Jennerwein et
al., 1989; Boudny et al., 1992), oxaliplatin preference for G clusters is not particularly surprising, yet, our results provide
the first data on the sequence localization of DACH-Pt DNA adducts.
These findings clearly demonstrate that the presence of the bulky and
hydrophobic DACH moiety and oxalate leaving group does not
significantly influence the sequence specificity of oxaliplatin interaction with DNA.
Besides intrastrand cross-links, oxaliplatin forms other types of DNA
lesions. The formation of low levels of interstrand cross-links by
oxaliplatin is demonstrated by alkaline electrophoresis of
oxaliplatin-treated SV40 fragments. This result is the first direct
evidence for cross-linking of complementary strands by oxaliplatin
(although indirect evidence has been reported for other DACH compounds;
Jennerwein et al., 1989; Boudny et al., 1992).
Interstrand cross-links seem too infrequent to significantly contribute
to the RPE results. Still, this potentially lethal lesion may
contribute to the biological properties of the drug. We recently
confirmed interstrand cross-linking by oxaliplatin in intact cells or
isolated nuclei. In these systems, we also found that oxaliplatin can
induce DNA-protein cross-links (Woynarowski JM, Faivre S, Chapman WG,
unpublished observations).
It could not be ruled out that altered hydrophobic properties might direct oxaliplatin to different cellular compartments than cisplatin. Likewise, similar adduct sites at the nucleotide level did not preclude the possibility that oxaliplatin and cisplatin might target different domains of nuclear DNA. However, the distribution of oxaliplatin adducts among the various regions is similar to that for cisplatin adducts. The lesions in cellular DNA seem to follow the relative density of drug-binding motifs in the regions examined (except for the underprivileged mtDNA; see later). This trend suggests that even low sequence specificity of platinum drugs may determine the relative vulnerability of various genomic loci to drug adduction. Thus, sequence analysis may predict region-specificity for platinum drugs. This novel finding also is potentially significant for the efforts to design drugs targeted at specific domains of genomic DNA.
The significance of targeting mtDNA by platinum drugs remains
controversial. Cisplatin has been reported to prefer nuclear DNA over
mtDNA (Daoud et al., 1995). However, a preference for mtDNA
also has been postulated (Murata et al., 1990; Olivero
et al., 1995, 1997), and certain cisplatin lesions were
suggested to be inefficiently repaired in mtDNA (LeDoux et
al., 1992). Given that no data were available for oxaliplatin, it
was important to determine whether the drug can target mtDNA. Our
results indicate that oxaliplatin forms lesions in mtDNA. However,
unless drug adducts in the examined 1.1-kbp region of mtDNA are not
representative for the adducts in the entire ~16-kbp mtDNA, nuclear
DNA, not mtDNA, seems to be the main target for both drugs.
Collectively, our findings and other data emphasize the fact that
despite the obvious similarities in the pattern of DNA adduction, significant mechanistic differences exist between oxaliplatin and
cisplatin. Although the primary focus of this study was on lesion
localization with only a semiquantitative estimation of lesion
frequencies, the results suggest that oxaliplatin is less reactive
toward both naked and intracellular DNA than cisplatin. In contrast,
oxaliplatin is more cytotoxic in the A2780 cells used in our studies
and in numerous other cell lines (Tashiro et al., 1989;
Pendyala and Creaven, 1993; Chaney, 1995). The lower reactivity of
oxaliplatin than cisplatin with defined regions of cellular DNA is
consistent with the results of a recent study (Saris et al.,
1996) that used two other methods to compare cisplatin and oxaliplatin
binding to bulk DNA in A2780 cells:
32P-postlabeling and total platination levels by
atomic absorption. This paradoxical trend suggests that although DNA
adducts generally are believed to be the main effect of platinum drugs,
other effects may contribute to oxaliplatin antiproliferative
properties. Likewise, based on differing profiles of activity against
selected cell lines in the NCI screen (COMPARE program analysis), Rixe
et al. (1996) concluded that oxaliplatin and cisplatin
differ in their mechanisms of action, resistance, or both.
Oxaliplatin-induced lesions in cellular DNA could be more difficult to
repair or could lead to different biological consequences. Our separate
study, which characterized in-depth total platination, interstrand
crosslinks, and DNA-protein cross-links, confirmed that oxaliplatin
forms fewer primary lesions than equimolar cisplatin. However, at
comparable levels of DNA adducts, oxaliplatin was approximately twice
as effective as cisplatin in inducing apoptosis (Faivre and
Woynarowski, 1998). Studies are under way to further explore the
relative significance of DNA lesions and other effects to the
proapoptotic and antiproliferative actions of oxaliplatin.
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Acknowledgments |
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We thank Drs. Stephen Chaney, James Rake, and Esteban Cvetkovic for helpful suggestions and Makoto Wajima for MTT determinations.
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Footnotes |
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Received May 28, 1998; Accepted August 3, 1998
This study was supported in part by a grant from Sanofi-Research, a Division of Sanofi Pharmaceuticals, Inc., and by Cancer Therapy and Research Center Research Foundation. A preliminary account of this study was presented in part at the 88th Annual Meeting of the American Association for Cancer Research [Proc Am Assoc Cancer Res 38:311 (1097)].
Send reprint requests to: Jan M. Woynarowski, Ph.D., Cancer Therapy and Research Center, Institute for Drug Development, 14960 Omicron Drive, San Antonio, TX 78245-3217. E-mail: jmw1{at}saci.org
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Abbreviations |
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DACH, (trans-l-1,2,diaminocyclohexane)platinum(II); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PNK, polynucleotide kinase; mtDNA, mitochondrial DNA; HPRT, hypoxantine phosphoribosyltransferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RPE, repetitive primer extension; SV40, Simian virus 40; TE, Tris·HCl/EDTA; ORI, origin of replication; bp, base pair(s); kbp, kilobase pair(s).
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Summary
Introduction
Procedures
Results
Discussion
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tetrazolium derivative reduction (MTT) and tritiated thymidine uptakeon three malignant mouse cell lines using chemotherapeutic agents and investigational drugs.
Anticancer Res
10:
145-154[Medline].This article has been cited by other articles:
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  S. Ramachandran, B. R. Temple, S. G. Chaney, and N. V. Dokholyan Structural basis for the sequence-dependent effects of platinum-DNA adducts Nucleic Acids Res., May 1, 2009; 37(8): 2434 - 2448. [Abstract] [Full Text] [PDF]
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5') template strand that are
likely to be adducted. C, Normalized relative signal intensity for
oxaliplatin and cisplatin adducts (based on densitometric
quantification of the gels as that in A) versus DNA sequence (positions
5083-5174, bottom strand in the opposite orientation,
5'
) and oxaliplatin (
). The values of lesion
frequencies (for drugs at 200 µM) are from Table