Download DNA Polymerase Regulates Cisplatin

[CANCER RESEARCH 64, 8029 – 8035, November 1, 2004]
DNA Polymerase ␨ Regulates Cisplatin Cytotoxicity, Mutagenicity, and The Rate of
Development of Cisplatin Resistance
Fang Wu, Xinjian Lin, Tsuyoshi Okuda, and Stephen B. Howell
Department of Medicine and the Cancer Center, University of California San Diego, La Jolla, California
ABSTRACT
DNA polymerase ␨ participates in translesional bypass replication.
Here we show that reduced expression of the catalytic subunit hREV3
renders human fibroblasts more sensitive to the cytotoxic effect of cisplatin, reduces their sensitivity to the ability of cisplatin exposure to generate
drug resistant variants in the surviving population, and reduces the rate
of emergence of resistance to cisplatin at the population level. Reduction
of REV3 mRNA did not alter the rate of cisplatin adduct removal but did
impair both spontaneous and cisplatin-induced extrachromosomal homologous recombination and attenuated bypass replication as reflected by
reduced ability to express luciferase from a platinated plasmid. Cisplatin
induced a concentration- and time-dependent increase in hREV3 mRNA.
The results indicate that, following formation of cisplatin adducts in DNA,
REV3 mRNA levels increase, and polymerase ␨ functions to promote both
cell survival and the generation of drug-resistant variants in the surviving
population. We conclude that when cisplatin adducts are present in the
DNA, polymerase ␨ is an important contributor to cisplatin-induced
genomic instability and the subsequent emergence of resistance to this
chemotherapeutic agent.
INTRODUCTION
Cisplatin kills cells by forming intrastrand and interstrand adducts
in DNA. The major replicative DNA polymerases are unable to carry
out translesional synthesis across cisplatin adducts. However, a family
of DNA polymerases that can mediate such bypass replication has
been identified recently in mammalian cells that includes polymerase
␨, polymerase ␩, polymerase ␬, polymerase ␮, and polymerase ␫ (1).
Analysis of null mutants of REV3, the catalytic subunit of polymerase
␨, in Saccharomyces cerevisiae revealed that a large fraction of all of
the mutations induced by DNA damaging agents and the majority of
spontaneous mutations are attributable to the activity of polymerase ␨
(2). Inhibition of the expression of the REV3 subunit in cultured
human fibroblasts by expression of an antisense REV3 mRNA was
shown to reduce UV-induced mutagenesis, indicating that polymerase
␨ also plays a crucial role in mutagenesis in mammalian cell (3).
The activity of polymerase ␨ is of concern with respect to the use
of DNA-damaging chemotherapeutic agents such as cisplatin. The
most abundant lesions produced in DNA are intrastrand cross-links,
and these are believed to be important to both the cytotoxicity and the
mutagenicity of the drug. Its ability to function as a mutagen has been
documented in bacterial (4, 5) and mammalian cells (6 –11). Whereas
cisplatin produces gross chromosomal changes (12) and probably
gene amplification (13), the molecular basis for much of its mutagenicity is believed to be related to bypass replication across cisplatin
adducts by the eukaryotic DNA polymerase ␤ and/or members of the
class containing polymerases ␫, ␨, and ␩ (14 –16). Current evidence
suggests that in bacteria and yeast when nonerror-prone mechanisms
for repairing DNA damage, such as base excision repair, nucleotide
excision repair, and homologous recombination, are disabled or overwhelmed, cells make increased use of specialized low-fidelity errorprone DNA polymerases to bypass DNA lesions that block normal
replicative polymerases (17). This appears to be an important contributor to the mutagenicity of cisplatin adducts (18).
Several investigators reported previously that loss of polymerase ␨
function markedly reduces UV and benzo(a)pyrene diol epoxideinduced hypoxanthine guanine phosphoribosyl transferase mutations
(19, 20). A key question with respect to the clinical use of cisplatin is
whether cisplatin-induced mutagenesis contributes to the acquisition
of cisplatin resistance and whether suppression of cisplatin-induced
mutagenesis can reduce the rate of acquisition of cisplatin resistance,
because this remains the major cause of treatment failure. In this
study, we used the diploid human fibroblast cell line 9N58 and the 6I
subline that was engineered to express high levels of hREV3 antisense
RNA (19). We report here that reduction in polymerase ␨ function
renders cells more sensitive to the cytotoxic effect of cisplatin but also
markedly decreases its mutagenicity. Most importantly, it significantly reduces the rate at which cells acquire resistance to cisplatin.
These results strongly support the hypothesis that polymerase ␨ is, in
large part, responsible for the ability of cells to replicate their DNA
and survive in the face of a large cisplatin adduct load and for the
error-prone bypass replication that is important to the mutagenicity of
this drug and its ability to generate drug-resistant variants in the
surviving population.
MATERIALS AND METHODS
Cell Strains and Culture. Cell strain 9N58 is a clonal derivative of the
karyotypically stable, near-diploid, immortal human fibroblast line MSU-1.1
that contains a single X chromosome. 9N58 cells were transfected with a
vector expressing a hREV3 antisense, and the 6I clone, selected in the presence
of puromycin, was found to express a high level of REV3L antisense mRNA
by Northern blot analysis and to exhibit a marked reduction in sensitivity to
UV irradiation-induced mutagenesis (19). 9N58 and 6I cells were cultured as
described previously (19).
Measurement of Rate of Generation of Resistant Variants. The rate at
which highly drug-resistant variants spontaneously appeared in the population
was measured using the “maximum likelihood estimation” technique (21). The
frequencies of highly drug-resistant variants in the 9N58 and 6I population
were measured (vide infra), and 106 cells were subcultured and allowed to
expand exponentially for 4 days. The frequency of resistant variants was then
measured again, and the process repeated for a total of four iterations. Total
cell numbers were determined at each step, along with the plating efficiencies
from the previous selection and the exact number of population doublings
determined from the following equation: Population doubling ⫽ [Ln(total
Received 12/16/03; revised 3/12/04; accepted 4/12/04.
number of cells) ⫺ Ln(number of cells plated ⫻ plating efficiency)]/Ln2. The
Grant support: Grant CA78648 from the NIH and a grant IRG #70-002 from the
American Cancer Society. This work was conducted in part by the Clayton Foundation for
rate of generation of resistant variants was then obtained by plotting the
Research–California Division. X. Lin and S. B. Howell are Clayton Foundation investiobserved resistant variant frequency as a function of population doubling and
gators.
by calculating the slope by linear regression. The slope of the curve yields the
The costs of publication of this article were defrayed in part by the payment of page
rate of generation of resistant variants (resistant variants/clonogenic cell/
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
generation).
Requests for reprints: Stephen B. Howell, Department of Medicine 0058, University
Plasmid Reactivation Assay. The pRL-CMV vector (Promega, Madison,
of California San Diego, La Jolla, CA 92093. Phone: 858-822-1110; Fax: 858-822-1111;
WI) containing Renilla luciferase cDNA was platinated to 1.5 ⫾ 1.4 pg/␮g
E-mail: [email protected].
DNA, which is equivalent to 9.3 adducts per plasmid or 3.2 adducts per Luc
©2004 American Association for Cancer Research.
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CISPLATIN PHARMACODYNAMICS AND DNA POLYMERASE ␨
coding region as reported previously (22). Similar levels of platination have
been shown previously not to affect the efficiency of transfection (23). Twenty-four hours after transfection of 1 ␮g of platinated or control unplatinated
vector, luciferase activity was measured using the Promega Renilla Luciferase
Assay System (Promega).
Clonogenic Assay. Clonogenic assays were performed by seeding 1,000
cells into 60-mm plastic dishes in 5 mL of complete medium. After 24 hours,
appropriate amounts of cisplatin were added to the dishes, and the cells were
exposed for 1 hour. Colonies of at least 50 cells were visually scored after 10
to 14 days. Each experiment was performed a minimum of three times using
triplicate cultures for each drug concentration. IC50 values were determined by
log-linear interpolation ,and the relative cytotoxicity was determined using the
ratio of the slopes of the survival curves.
Measurement of Cisplatin Mutagenicity. The sensitivity of cells to the
mutagenic effects of cisplatin was measured by determining the frequency of
variants highly resistant to either 10 ␮mol/L 6TG or to 6 ␮mol/L cisplatin
itself in the surviving population 20 days after a 1-hour exposure to increasing
concentrations of cisplatin as reported previously (10, 11). Each experiment
was performed a minimum of three times ,and the data are presented as
mean ⫾ SEM. When testing for 6TG-resistant variants, the cells were grown
in HAT medium containing 0.4 ␮mol/L aminopterin, 16 ␮mol/L thymidine,
and 100 ␮mol/L hypoxanthine for a minimum of 14 days before testing to
reduce the number of pre-existing hypoxanthine guanine phosphoribosyl transferase mutants.
Measurement of Platinum in DNA. Aliquots of the DNA were digested in
70% nitric acid at 65°C for 2 hours and diluted to 5% nitric acid by adding
appropriate volume of double distilled deionized water. The picograms of
platinum per microgram of DNA in the hydrolysate was quantified by inductively coupled plasma optical emission spectroscopy or inductively coupled
plasma mass spectroscopy as described previously (24). When assessing the
time course of the loss of platinum from DNA, the cells were treated with 200
␮mol/L cisplatin for 1 hour to obtain quantifiable levels of platinum over the
entire period of the experiment. DNA was isolated at 0, 6, 12, 18, and 24 hours
after drug exposure.
Measurement of the Frequency of Extrachromosomal Homologous
Recombination. Homologous recombination was assayed by determining the
extent of recombination between 2 green fluorescent protein sequences in
plasmid DNA as described previously (25). The pBHRF vector contains an
intact “blue” variant of green fluorescent protein (enhanced blue fluorescent
protein) that includes an ⬃300 nucleotide sequence with perfect homology to
a second truncated nonfunctional copy of green fluorescent protein. In the
absence of homologous recombination within the vector only enhanced blue
fluorescent protein is expressed; however, homologous recombination between
the enhanced blue fluorescent protein and truncated green fluorescent protein
sequences creates a functional green fluorescent protein, and if this occurs the
cell expresses green fluorescent protein as well as enhanced blue fluorescent
protein, which is expressed from other plasmids in the cell that have not
undergone recombination. Cells were seeded into six-well plates overnight and
then exposed to 0 or 10 ␮mol/L cisplatin for 1 hour. The untreated or surviving
cells were then transfected with pBHRF 24 hours later with siPORT XP-1
transfection agent (Ambion Inc., Austin, TX) in the presence of serum according to the manufacturer’s instruction. Four hours after transfection, BoosterExpress reagent (Gene Therapy Systems, Inc., San Diego, CA) was added, and
the cells were then analyzed by two-color flow cytometry 48 hours after
transfection. The recombination frequency was calculated as [(enhanced blue
fluorescent protein⫹ and green fluorescent protein⫹) ⫹ (green fluorescent
protein⫹)]/[(enhanced blue fluorescent protein⫹ and green fluorescent protein⫹) ⫹ (green fluorescent protein⫹) ⫹ (enhanced blue fluorescent protein⫹)]
where enhanced blue fluorescent protein and green fluorescent protein represent the number of blue and green fluorescent cells, respectively, in the sample.
Quantitation of hREV3 mRNA by Reverse Transcription-PCR. Total
RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). First
strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers. For REV3 gene expression, forward (5⬘-TGATGTCTTCAGCTG GTATCATGA-3⬘) and reverse (5⬘-CCGCCCTTCAGGTT
CACTT-3⬘) primers were used for amplification under the following conditions: 5 minutes of predenaturation at 95°C, 30 seconds of denaturation at
94°C, 30 seconds of annealing at 59°C, 1 minute of extension at 72°C, and an
additional 10 minutes of extension at 72°C. The fluorochrome-labeled probe
that was displaced to yield the fluorescent signal during the reverse transcription-PCR reaction was as follows: 5⬘-TTACCAAGGATCCATAAAGCTA
CCAGCTCCTC-3⬘.
Relative Rate of Development Resistance to Cisplatin. The rate at which
the cell population became resistant to cisplatin during repeated cycles of
1-hour exposures to the drug was determined by measuring the IC50 for
cisplatin using a clonogenic assay after each round of selection. The cisplatin
concentration used for selection was the IC90 for the population under study.
For each round of selection, 106 cells were exposed to cisplatin for 1 hour.
When the cells had recovered to 90% confluence, an aliquot was used to
determine cell number and the slope of the cisplatin concentration–survival
curve in a clonogenic assay, and another aliquot was again exposed to cisplatin. Total cell number and plating efficiency was determined at each step; this
information, along with the exact number cells subcultured, was used to
calculate population doubling according to the equation described above. The
rate of acquisition of resistance to cisplatin was then calculated by plotting the
slope of the cisplatin concentration–survival curve as a function of population
doubling. The slope of the latter plot yields the rate of relative resistance
development.
RESULTS
Effect of Loss of Polymerase ␨ Function on the Spontaneous
Rate of Generation of Resistant Variants. These studies were performed using the polymerase ␨-proficient 9N58 human fibroblast cell
line and the 6I subline that had been molecularly engineered to
express an antisense mRNA directed at the hREV3 subunit of polymerase ␨. That the 6I cells express high levels of hREV3 antisense
mRNA and have a lower frequency of mutants induced by UV and
benzo(a)pyrene diol epoxide in their HPRT gene relative to the
parental 9N58 cells has been documented previously (19). To provide
additional confirmation that the 6I cells had diminished polymerase ␨
activity, the spontaneous rate of generation of variants resistant to
6TG was determined by serially measuring the frequency of resistant
variants in expanding populations. The results, presented in Fig. 1,
show that the hREV3 antisense-expressing 6I cells exhibited a 2.9-fold
decrease in spontaneous rate of generation of variants resistant to 6TG
Fig. 1. Effect of REV3 mRNA reduction on the spontaneous rate of generation of
6TG-resistant variants. f, parental 9N58 cells; ⽧, hREV3 antisense-expressing 6I cells.
Each curve shows the change in frequency of resistant variants with increasing numbers
of population doublings. The rate of generation of resistant variants is given by the slope
of the linear regression line. Each data point is the mean of three experiments. Vertical
bars, ⫾ SEM.
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as compared with the parental 9N58 cells (P ⬍ 0.01). This result is
consistent with the reduction in spontaneous mutation rate observed in
other studies of polymerase ␨-deficient cells (26, 27).
There is no practical way of measuring the relative activity of the
purified polymerase ␨ complex in the intact 9N58 and 6I cells, but the
overall translesional synthetic capability in cells can be assessed indirectly by determining the ability of the cell to successfully express the
Renilla luciferase from an expression vector that has been extensively
platinated by treatment with cisplatin before transfection. By comparing
luciferase expression in the uninjured 9N58 and 6I cells it is possible to
assess what fraction of host cell luciferase expression is due to specifically polymerase ␨-mediated adduct bypass as opposed to translesional
synthesis mediated by other polyermases. Fig. 2 shows that luciferase
activity was equivalent in the 9N58 and 6I cells when they were transfected with nonplatinated vector. When the platinated vector was transfected into the polymerase ␨-proficient 9N58 cells, there was little impairment in the generation of luciferase activity (1.4-fold reduction,
P ⬎ 0.05). However, when the same platinated vector was transfected
into the polymerase ␨ antisense-expressing 6I cells, the luciferase expression was markedly reduced (3.9-fold, P ⬍ 0.01). Thus, polymerase ␨
activity is important to the ability to express luciferase from a platinated
plasmid, and this activity was reduced in the 6I cells. Together with the
reduced rate of generation of spontaneous 6TG mutants these results
validate this experimental system for the study of the effect of polymerase
␨ on cisplatin pharmacodynamics.
Effect of Loss of Polymerase ␨ Function on Sensitivity to the
Cytotoxic Effect of Cisplatin. Clonogenic assays were used to determine the effect of reduced polymerase ␨ on sensitivity to the
cytotoxic effect of cisplatin. As shown in Fig. 3, the IC50 was
1.3 ⫾ 0.04 ␮mol/L (SEM) for the 6I cells and 2.5 ⫾ 0.01 ␮mol/L
(SEM) for the parental cells (P ⬍ 0.05). On the basis of the ratio of
the slopes of survival curves, the 6I cells were 1.4-fold (P ⬍ 0.05)
more sensitive to the cytotoxic effect of cisplatin than the polymerase
␨-replete 9N58 cells. Thus, hREV3 antisense-expressing cells demonstrated hypersensitivity to the cytotoxic effect of cisplatin.
Effect of Loss of Polymerase ␨ Function on the Ability of
Cisplatin to Generate Resistant Variants. Cisplatin is a mutagen in
human cells and generates mutations that result in high-level resistance to many classes of drugs (10, 11, 28). To determine the role of
Fig. 2. Efficiency of the generation of luciferase activity from an unplatinated or
platinated plasmid. A, solid bar, parental 9N58 cells; open bar, hREV3 antisense-expressing 6I cells. Cells were transfected with nonplatinated or platinated pRL-CMV vector.
Luciferase activity is expressed as relative light units. Data points represent the mean of
three independent experiments each performed with duplicate transfections; bars, ⫾ SEM.
Fig. 3. Effect of REV3 mRNA reduction on sensitivity to the cytotoxic effect of
cisplatin. Cisplatin concentration-survival curves for the hREV3 antisense-expressing 6I
cells (⽧) and its parental 9N58 cells (f). Each point represents the mean of three
experiments performed with triplicate cultures. Vertical bars, ⫾ SEM.
polymerase ␨ in cisplatin-induced mutagenesis, the 9N58 and 6I cells
were exposed to 10 ␮mol/L cisplatin for 1 hour, and then 20 days later
the number of clonogenic cells demonstrating high-level resistance to
6TG or to cisplatin itself was determined. When measured at the end
of the 1 hour of cisplatin exposure, the DNA of the 9N58 and 6I cells
contained 3.49 ⫾ 0.30 (SEM) and 3.32 ⫾ 0.36 (SEM) pg of platinum/
␮g, respectively, indicating no difference in the extent of initial
adduct formation. As shown in Fig. 4A, the frequency of 6TGresistant variants induced by exposure to 10 ␮mol/L cisplatin was
significantly lower (3.6-fold) in 6I cells than in the parental 9N58 cells
(P ⬍ 0.01). Fig. 4B shows that after exposure to cisplatin, the
polymerase ␨-proficient cells yielded 1.4-fold more colonies that were
highly resistant to cisplatin itself than the hREV3 antisense-expressing
cells (P ⬍ 0.01). When normalized to the extent of clonogenic cell kill
produced by a 1-hour exposure to 10 ␮mol/L, the frequency of 6TG
and cisplatin-resistant variants was 6.3- and 3.6-fold lower, respectively, in the 6I cells. Thus, cisplatin was able to generate variants in
the surviving population that were highly resistant to 6TG or to
cisplatin itself, and this mutagenic effect was reduced in the 6I cells
both on an absolute basis and when normalized for the extent of
clonogenic cell kill in the absence of any difference in the DNA
platinum content between the two cell types. This is consistent with
the hypothesis that polymerase ␨ plays an important role in generating
mutations when cisplatin adducts are present in DNA.
Effect of Loss of Polymerase ␨ Function on the Disappearance
of Platinum from DNA. The changes in sensitivity to the cytotoxic and
mutagenic effects of cisplatin could be explained by differences in initial
adduct levels or their persistence if loss of polymerase ␨ impaired DNA
adduct removal. The rate of disappearance of platinum from the DNA
accurately mirrors the rate of removal of the most common cisplatin
adducts (29, 30). The initial picogram of platinum per microgram of
DNA and the rate of disappearance of platinum from total cellular DNA
was measured in both cell lines after a 1-hour exposure to 200 ␮mol/L
cisplatin. The initial adduct levels were nearly identical, being 32.94 and
32.30 pg platinum/␮g DNA, respectively, for the 9N58 and 6I cells. Fig.
5 shows that there was no significant difference in the kinetics of
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CISPLATIN PHARMACODYNAMICS AND DNA POLYMERASE ␨
Fig. 4. Effect of REV3 mRNA reduction on the ability of cisplatin to generate
drug-resistant variants in the surviving population. A, number of 6TG-resistant colonies
per 106 clonogenic cells scored on day 20 after a 1-hour exposure to 10 ␮mol/L cisplatin.
B, number of cisplatin-resistant colonies. Each data point represents the mean of three
experiments. Vertical bars, ⫾ SEM.
Effect of Loss of Polymerase ␨ Function on Spontaneous and
Cisplatin-Induced Homologous Recombination. Cisplatin induces
sister chromatid exchange, and homologous recombination may play
a role in the ability of cisplatin to generate highly drug resistant
variants. To assess the effect of reduced polymerase ␨ function on
basal and cisplatin-induced rates of homologous recombination we
used the pBHRF recombination-sensitive reporter vector described by
Slebos and Taylor (25). This plasmid constitutively expresses an
intact, emission-shifted, “blue” variant of green fluorescent protein
(enhanced blue fluorescent protein) and also contains a COOHterminally truncated form of green fluorescent protein in which there
exists a 300-bp homologous sequence for recombination with identical nucleotide stretch within the intact enhanced blue fluorescent
protein sequence. In the absence of homologous recombination within
or between the enhanced blue fluorescent protein and green fluorescent protein sequences in the vector only enhanced blue fluorescent
protein is expressed in transfected cells; however, homologous recombination event can create a functional green fluorescent protein, in
which case the cell expresses both enhanced blue fluorescent protein
and green fluorescent protein, because most cells acquire multiple
copies of the vector during transfection, only some of which recombine. The polymerase ␨-proficient 9N58 and hREV3-antisense expressing 6I cells were exposed to 10 ␮mol/L cisplatin for 1 hour or
left untreated, and the pBHRF was transfected into the cells 24 hours
later. After an additional 48 hours the fraction of blue fluorescent
protein-positive cells that also expressed green fluorescent protein
was determined by flow cytometry. As shown in Fig. 6, the spontaneous homologous recombination frequency was 2.4-fold lower in the
6I cells than the parental cells (P ⬍ 0.05). Exposure to10 ␮mol/L
cisplatin for 1 hour increased the recombination frequency in both cell
types; however, the frequency was still significantly lower (2.5-fold,
P ⬍ 0.05) in the 6I than in the 9N58 cells. Thus, polymerase ␨
function appears to be important to both spontaneous and cisplatininduced homologous recombination.
Effect of Cisplatin on hREV3 mRNA Levels. Given its ability to
promote both survival and mutagenicity after a cisplatin exposure, it
was of interest to determine whether cisplatin induces polymerase ␨
function during the injury response after drug exposure. At the present
time, no antibody is available with sufficient avidity and specificity to
Fig. 5. Disappearance of platinum from DNA. The 9N58 and 6I cells were treated with
200 ␮mol/L cisplatin for 1 hour. DNA was isolated at the indicated times after treatment
and quantified by inductively coupled plasma mass spectroscopy. f, parental 9N58 cells;
⽧, hREV3 antisense-expressing 6I cells. Each data point represents the mean of three
measurements. Vertical bars, ⫾ SEM.
platinum disappearance from DNA in the two cell lines. Because nucleotide excision repair is responsible for removal of the majority of cisplatin
adducts, this suggests that the degree of impairment of polymerase ␨
function in the 6I cells did not interfere significantly with this pathway of
DNA repair.
Fig. 6. Effect of hREV3 mRNA reduction on the spontaneous and cisplatin-induced
homologous recombination frequency. Each data point represents the mean of three
experiments each performed with duplicate cultures. Vertical bars, ⫾ SEM.
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permit accurate Western blot analysis of hREV3 protein levels. Thus,
real-time PCR was used to measure changes in hREV3 mRNA level
using primers that directed amplification from the COOH-terminal
region of the coding sequence. Fig. 7A shows that the basal hREV3
mRNA level was higher in the parental 9N58 cells than in the
antisense-expressing 6I cells. Moreover, cisplatin induced a concentration-dependent increase in hREV3 mRNA level in the 9N58 cells
when measured at 24 hours after a 1-hour drug exposure. Interestingly, although smaller in magnitude, cisplatin also induced an increase in hREV3 mRNA level in the 6I antisense-expressing cells,
suggesting that the ability of the antisense to mediate hREV3 degradation may be overwhelmed by an increase in endogenous hREV3
mRNA production.
Fig. 7B shows the change in hREV3 mRNA level as a function of
time after exposure to 10 ␮mol/L cisplatin for 1 hour. The hREV3
mRNA level continued to increase up to 24 hours in both cell lines but
was generally lower in the antisense-expressing 6I cells than in the
parental 9N58 cells. These results indicate that cisplatin induced an
increase in hREV3 mRNA level that peaked at a time similar to the
time of maximum cell cycle arrest produced by cisplatin (11).
Whether this results in an increase in hREV3 protein level cannot
currently be determined.
Effect of Loss of Polymerase ␨ Function on the Rate of Development of Cisplatin Resistance. The emergence of drug resistance
in a population during cisplatin-repeated cycles of drug exposure may
be due to enrichment for pre-existing resistant clones, cisplatin-induced generation of new resistant variants, or some combination of
both. As shown above, loss of polymerase ␨ function reduced the
ability of cisplatin to generate drug-resistant variants in the surviving
Fig. 8. Effect of hREV3 mRNA reduction on the rate of development of cisplatin
resistance. f, parental 9N58 cells; ⽧, hREV3 antisense-expressing 6I cells. Each data
point represents the mean of three measurements. Vertical bars, ⫾ SEM.
population. If this ability of cisplatin is central to the emergence of
acquired cisplatin resistance in the whole population, then reduction
of polymerase ␨ activity would be expected to reduce the rate at which
resistance emerges. We measured the rate of development of resistance in the whole population of 9N58 and 6I cells starting with
500,000 cells. The cells were exposed to an IC90 concentration of
cisplatin for 1 hour, and the exposure was repeated as soon as log
phase growth resumed. After each round of drug treatment, the
sensitivity of the whole population to cisplatin was measured by
determining survival over 2 logs of cell kill as a function of cisplatin
concentration in a clonogenic assay. Fig. 8 shows that cisplatin
resistance emerges quite rapidly in both cell lines but that the rate of
development of resistance was reduced by an average of 3-fold
(P ⬍ 0.05) in the 6I cells relative to the 9N58 cells. Thus, polymerase
␨ plays a central role in the acquisition of cisplatin resistance. Because
loss of polymerase ␨ does not appear to alter the extent of adduct
formation or the time course of platinum removal from DNA, these
results are consistent with the concept that mutagenic translesional
synthesis across cisplatin adducts is responsible for generating drugresistant variants that become enriched in the population by subsequent rounds of cisplatin exposure.
DISCUSSION
Fig. 7. The effect of cisplatin on hREV3 mRNA level. A, induction of hREV3 mRNA
as a function of cisplatin concentration at 24 hours after a 1-hour exposure to cisplatin. B,
time course of change in REV3 mRNA levels after exposure to 10 ␮mol/L cisplatin for
1 hour. f, parental 9N58 cells; ⽧, hREV3 antisense-expressing 6I cells. Each data point
represents the mean of three independent reverse transcription-PCR measurements;
bars, ⫾ SEM.
The DNA adducts produced by cisplatin are important to its ability
to kill the cell. Cytotoxicity is proportional to the extent of adduct
formation, and cells with defects in the major DNA repair mechanism
that removes these adducts, nucleotide excision repair, are hypersensitive to cisplatin (31, 32). Although polymerase ␨ activity could not
be measured directly, the phenotypic differences between the 9N58
and 6I cells are consistent with reduced polymerase ␨ activity in
response to the expression of antisense hREV3 mRNA. Similar
changes in phenotype have been observed in fibroblasts from transgenic mice expressing antisense RNA to mREV3 (20). The results of
the current studies indicate that such impairment of polymerase ␨
function caused a moderate increase in sensitivity to the cytotoxic
effect of cisplatin without altering the rate at which total platinum
adducts were removed from DNA. This suggests that, as for other
types of adducts that block the progression of the replicative polymerases (15, 33), polymerase ␨ is involved in a pathway that normally
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carries out enough translesional synthesis to allow some cells to
complete DNA synthesis and survive. The observation that impaired
polymerase ␨ function also reduced the ability of cisplatin to generate
highly drug-resistant clones in the surviving population provides
strong evidence that the translesional synthesis pathway in which
polymerase ␨ functions is error-prone when it bypasses cisplatin
adducts. It appears that this pathway normally fosters the development
of resistance to cisplatin both by permitting the survival of cells that
contain mutagenic adducts in their DNA and by generating new
mutations in genes that mediate the resistant phenotype.
The actual extent to which polymerase ␨ function was disabled in
the 6I cells is not known. Knockout of both alleles in mice causes
embryonic lethality (34 –36), but knockout of both alleles in chicken
B cells only causes slowing of proliferation (37). On the basis of the
magnitude of the phenotypic effects relative to those observed in
knockout cells, it is likely that some polymerase ␨ function remains in
6I cells, a presumption that is supported by the persistence of measurable amounts of hREV3 mRNA. However, the extent of loss of
polymerase ␨ function was sufficient to yield an easily detectable
phenotype with respect to the pharmacodynamic effects of cisplatin.
What role the proteins of the polymerase ␨ complex might play in
DNA repair mechanisms other than translesional bypass is only just
becoming defined. The observation that, despite being 1.4-fold hypersensitive to the cytotoxic effect of cisplatin, the time course of
removal of platinum from DNA was the same in the polymerase
␨-proficient and antisense-expressing cells suggests that nucleotide
excision repair is not highly dependent on at least hREV3. However,
the finding that recombination-dependent expression of green fluorescent protein from the pBHRF vector was impaired in the 6I cells
suggests a role for hREV3 or the entire polymerase ␨ complex in at
least extrachromosomal homologous recombination. Caution is
needed in interpreting the pBHRF results, because the assay reflects
only extrachromosomal recombination; however, this finding is in
agreement with the results of studies in knockout yeast and chicken B
cells that also suggest that REV3 plays an important role in chromosomal homologous recombination (37, 38). Homologous recombination is important to the survival of cells after cisplatin exposure
(39 – 41), and it may be essential for repair of interstrand cross-links
(42– 44). The finding that cisplatin exposure enhances pBHRF recombination suggests that cisplatin up-regulates this putatively nonmutagenic repair mechanism, an effect expected to improve the ability of
the cell to survive the DNA damage produced by this agent. Polymerase ␨ may be particularly important to cisplatin pharmacodynamics
because its loss simultaneously impairs both translesional synthesis
and homologous recombination, each of which alone perhaps could
handle the adduct load if it were intact.
The expression of a large number of genes is altered after cisplatininduced injury (45), and hREV3 appears to be one of these. hREV3
mRNA levels increased in proportion to the extent of injury, and the
level peaked at 24 hours after a 1-hour exposure to 10 ␮mol/L
cisplatin. It is not known whether the increase in hREV3 mRNA
translates into increased polymerase ␨ activity, but it is reasonable to
expect that it does. Thus, not only are cisplatin adducts mutagenic
when bypassed by polymerase ␨, but the data are consistent with the
concept that cisplatin also increases polymerase ␨ levels and/or activity and, thus, additionally enhances its own mutagenicity.
The single most important finding of these studies is that reduction
of polymerase ␨ function can reduce the rate at which the whole
population of cells becomes resistant to cisplatin. Reduction in the rate
at which cisplatin resistance emerges is a key clinical goal. Whereas
we have shown previously (10) and confirmed in the current studies
that treatment with cisplatin produces clones in the surviving population that are highly resistant to cisplatin itself and several other
classes of drugs, whether this is really important to acquisition of
cisplatin resistance by the entire population remained unknown. The
current results establish three important points: (1) acquisition of
resistance by the entire population is not just due to enrichment for
drug-resistant clones that existed in small numbers before drug exposure; (2) the genes that mediate cisplatin resistance are susceptible to
adduction by cisplatin and to mutagenic bypass replication by polymerase ␨; and (3) the magnitude of the effect of polymerase ␨ on the rate
of resistance acquisition is quite large. Thus, these studies identify
polymerase ␨ as a target of which the pharmacological inhibition may
stabilize the genome during the cellular injury response triggered by
cisplatin and reduce the rate of emergence of resistance in patients
treated with this important chemotherapeutic agent.
ACKNOWLEDGMENTS
We gratefully acknowledge Dr. R.J.C. Slebos for kindly providing the
pBHRF vector and Dr. V.M. Maher for generously providing cell lines..
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DNA Polymerase ζ Regulates Cisplatin Cytotoxicity,
Mutagenicity, and The Rate of Development of Cisplatin
Resistance
Fang Wu, Xinjian Lin, Tsuyoshi Okuda, et al.
Cancer Res 2004;64:8029-8035.
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