Cancer Research Infection and Cancer: Biology, Therapeutics, and Prevention AACR Conference on Molecular Diagnostics - 2008
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[Cancer Research 64, 6469-6475, September 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

The Role of DNA Polymerase {eta} in Translesion Synthesis Past Platinum–DNA Adducts in Human Fibroblasts

Ekaterina Bassett1, Nicole M. King2,3, Miriam F. Bryant2, Suzanne Hector6, Lakshmi Pendyala6, Stephen G. Chaney1,3,4,5 and Marila Cordeiro-Stone2,3,4,5

1 Department of Biochemistry and Biophysics, 2 Department of Pathology and Laboratory Medicine, 3 Curriculum in Toxicology, 4 Lineberger Comprehensive Cancer Center, and 5 Center for Environmental Health and Susceptibility, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and 6 Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cisplatin, a widely used chemotherapeutic agent, has been implicated in the induction of secondary tumors in cancer patients. This drug is presumed to be mutagenic because of error-prone translesion synthesis of cisplatin adducts in DNA. Oxaliplatin is effective in cisplatin-resistant tumors, but its mutagenicity in humans has not been reported. The polymerases involved in bypass of cisplatin and oxaliplatin adducts in vivo are not known. DNA polymerase {eta} is the most efficient polymerase for bypassing platinum adducts in vitro. We evaluated the role of polymerase {eta} in translesion synthesis past platinum adducts by determining cytotoxicity and induced mutation frequencies at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in diploid human fibroblasts. Normal human fibroblasts (NHF1) were compared with xeroderma pigmentosum variant (XPV) cells (polymerase {eta}-null) after treatment with cisplatin. In addition, XPV cells complemented for polymerase {eta} expression were compared with the isogenic cells carrying the empty expression vector. Cytotoxicity and induced mutagenicity experiments were measured in parallel in UVC-irradiated fibroblasts. We found that equitoxic doses of cisplatin induced mutations in fibroblasts lacking polymerase {eta} at frequencies 2- to 2.5-fold higher than in fibroblasts with either normal or high levels of polymerase {eta}. These results indicate that polymerase {eta} is involved in error-free translesion synthesis past some cisplatin adducts. We also found that per lethal event, cisplatin was less mutagenic than UVC. Treatment with a wide range of cytotoxic doses of oxaliplatin did not induce mutations above background levels in cells either expressing or lacking polymerase {eta}, suggesting that oxaliplatin is nonmutagenic in human fibroblasts.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cis-diamminedichloroplatinum(II) (cisplatin) is a popular chemotherapeutic agent, but its clinical use is often confounded by intrinsic and acquired tumor resistance. Platinum (Pt) agents with different carrier ligands have been developed, such as (trans-R,R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin), which is effective against cisplatin-resistant tumors and is used for treatment of colon cancers in the United States. Mutagenicity of cisplatin in vivo (1) is also of concern, because secondary malignancies have been associated with cisplatin chemotherapy (2) . Whereas little has been reported about the mutagenicity of oxaliplatin in humans, platinum(II) complexes that form 1,2-diaminocyclohexane adducts in DNA (such as oxaliplatin) are less mutagenic than cisplatin in bacteria (3) .

Cisplatin and oxaliplatin primarily form intrastrand DNA cross-links at GG and AG sites (4) . Cisplatin AG and AGG adducts (the position of the adduct is italicized) are the most mutagenic Pt-DNA lesions in both prokaryotic and eukaryotic cells (5 , 6) . One of the postulated mechanisms of mutagenesis is error-prone translesion synthesis. Bypass replication of Pt-DNA adducts occurs in cultured cells, and its efficiency is increased in cisplatin-resistant cell lines (7 , 8) . Various DNA polymerases have been tested in vitro for their ability to replicate past Pt-DNA adducts. DNA polymerases {alpha}, {epsilon}, and {delta} are completely blocked by cisplatin adducts (9 , 10) , even in the presence of the accessory proteins PCNA and RPA (10) . Candidates for performing translesion synthesis past bulky adducts in vivo include members of the polymerase X (polymerase ß, polymerase µ, and polymerase {lambda}), polymerase B (polymerase {zeta}), and polymerase Y (Rev1, polymerase {eta}, polymerase {iota}, and polymerase {kappa}) families of DNA polymerases (11, 12, 13) . Among these, polymerase {iota}, polymerase {kappa}, and polymerase {lambda} are incapable of inserting even a single deoxynucleotide triphosphate opposite cisplatin-DNA adducts (14, 15, 16) , and polymerase {eta} is by far the most efficient at translesion synthesis past cisplatin- and oxaliplatin-DNA adducts in vitro (17, 18, 19) .

Polymerase {eta} is encoded by yeast RAD30 and human XPV genes and catalyzes efficient and accurate translesion synthesis past cis,syn thymine dimers formed in DNA by UV radiation (20 , 21) . Deletion of the yeast RAD30 gene leads to reduced survival and enhanced mutability (22) after exposure to UV radiation but does not affect sensitivity to cisplatin (23) . Mutations in the human polymerase {eta} gene (hRAD30A) result in the xeroderma pigmentosum variant phenotype (21 , 24 , 25) , which is characterized by a high incidence of sunlight-induced skin cancers. Human polymerase {eta} is capable of bypassing Pt-DNA adducts in vitro (18 , 26) and contributes to DNA strand growth in human cells treated with cisplatin (27) . In this study, we measured cisplatin-induced mutation frequencies at the HPRT locus in telomerase-immortalized human diploid fibroblasts from a normal donor and a xeroderma pigmentosum variant patient (polymerase {eta}-null). Loss of HPRT function by most base substitutions, frameshift mutations, or deletions confers resistance to 6-thioguanine (28) . We also measured cisplatin-induced mutation frequencies in a pair of isogenic cell lines differing only by the presence or absence of polymerase {eta} expression. In addition, the isogenic lines were used to evaluate the mutagenicity of oxaliplatin in human cells for the first time.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
The normal human fibroblast cell line NHF1-hTERT was derived from neonatal foreskin fibroblasts (29) and immortalized by ectopic expression of the catalytic subunit of human telomerase (30) . The xeroderma pigmentosum variant cell line GM02359-hTERT (XP115L0) was derived in the laboratory of Dr. Roger A. Shultz (University of Texas Southwestern Medical Center, Dallas, TX; ref. 31 ), and clone 1B (32) was compared with NHF1 in the studies reported here. An isogenic pair of polymerase {eta} (+) and polymerase {eta} (–) cells was generated by infection of xeroderma pigmentosum variant clone 1B with either a retroviral construct carrying the human cDNA for polymerase {eta} [xeroderma pigmentosum variant (+)], or the empty vector [xeroderma pigmentosum variant (–)]. The generation and characterization of these cell lines will be described in detail elsewhere. Briefly, xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) showed similar proliferation rates; xeroderma pigmentosum variant (+) cells demonstrated resistance to killing by UVC comparable with NHF1, and xeroderma pigmentosum variant (–) cells retained the same sensitivity observed with the parental xeroderma pigmentosum variant fibroblasts. Western blot analysis indicated that xeroderma pigmentosum variant (+) cells overexpress polymerase {eta} relative to normal diploid fibroblasts. Real-time reverse transcription-PCR analysis demonstrated that xeroderma pigmentosum variant (+) cells contain on average a 50-fold excess of exogenous transcripts for wild-type polymerase {eta} over the endogenous mutated mRNA.

NHF1 and xeroderma pigmentosum variant clone 1B cells were maintained in DMEM (Sigma Aldrich, St. Louis, MO) supplemented with 2x concentration of MEM nonessential amino acids (Life Technologies, Inc., Carlsbad, CA), 2 mmol/L L-glutamine (Life Technologies, Inc.), and 10% fetal calf serum (Hyclone Laboratories, Logan, UT or Sigma Aldrich). Xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells were maintained in the same medium with the addition of 200 µg/mL G418 (Life Technologies, Inc.). All of the cell cultures were kept at 37°C in humidified atmosphere of 95% air and 5% CO2. The experiments were performed in Falcon 100-mm tissue culture plates (Becton-Dickinson) with medium supplemented with 50 µg/mL gentamicin (Life Technologies, Inc.).

HAT Selection.
Cells were preselected for functional HPRT by expanding the cultures for 10 days in medium supplemented with 1x HAT (100x lyophilized HAT includes 10 mmol/L sodium hypoxanthine, 40 µmol/L aminopterin, and 1.6 mmol/L thymidine, Life Technologies, Inc.); several aliquots of selected cells were stored at –135°C. A new aliquot was thawed for every experiment to ensure consistency in the age of the cultures. At treatment time, the population doubling levels were ~78 (NHF1), 202 (xeroderma pigmentosum variant), 215 [xeroderma pigmentosum variant (+)], and 217 [xeroderma pigmentosum variant (–)] from the time of selection of telomerase-expressing cells.

Karyotyping.
Metaphase spreads from all of the HAT-selected cell lines were prepared by conventional methods. They were stained and analyzed as described previously (32) .

Preparation of Cisplatin.
Cisplatin (Sigma Aldrich) was dissolved at 6.6 mmol/L in 100 mmol/L NaCl by stirring overnight, filtered through 0.2-µm sterile Acrodisk filters (Gelman), and aliquots stored at –20°C. Immediately before each experiment, an aliquot of cisplatin solution was thawed at 50°C for 10 minutes and diluted to working concentrations in PBS (Life Technologies, Inc.), taking care to avoid light exposure.

Preparation of Oxaliplatin.
Oxaliplatin (Sanofi-Synthelabo, Malvern, PA) was dissolved at 6.6 mmol/L in deionized water by heating to 50°C and filtered through 0.2-µm sterile Acrodisk filters (Gelman) immediately before each experiment. Oxaliplatin was diluted to working concentrations in sterile deionized water.

Cytotoxicity.
Cells were plated at 750 per plate (6 plates per dose) and incubated for 12 hours before exposure to UVC or platinum drug. For UVC treatments, plates were rinsed once with Hanks’ balanced salt solution and placed uncovered under a short-wave UV lamp, emitting mostly 254 nm radiation. Fluences of UVC ranged from 0 to 8 J/m2. For cisplatin and oxaliplatin treatments, culture medium was replaced with serum-free medium containing increasing concentrations of the drug or equal volume of solvent and returned to the incubator for 1 hour. Drug-containing medium was aspirated, and fresh medium containing 10% fetal calf serum was added to the plates. Cells were fed every 3 to 4 days. After 2 weeks, cells were rinsed with PBS, fixed in methanol–acetic acid (3:1 v/v) for 10 min, and stained with 0.25% crystal violet (Sigma Aldrich) dissolved in methanol–acetic acid (3:1 v/v) for 5 min. Alternatively, the fixative was rinsed off, and cells were stained with 1:10 dilution of Giemsa solution (LabChem) in PBS for 30 min. Colonies containing >50 cells were counted.

Mutagenesis.
Cells were plated at 5 x 105 per plate (2 plates per treatment condition), incubated for 12 hours, and treated with 0 to 25 µmol/L cisplatin, 0 to 200 µmol/L oxaliplatin, or 0 to 8 J/m2 UVC, as described above. These cultures were maintained in logarithmic growth by replating the cells at 5 x 105 per plate every 3 to 4 days, until they underwent at least six population doublings. Mutant selection was done by replating cells at 4 x 104 per plate (50 to 125 plates per treatment condition for UVC; 100 plates per treatment condition for cisplatin or oxaliplatin) into medium containing 40 µmol/L 6-thioguanine. Colony-forming efficiency at the time of selection was determined by plating 750 cells per plate into medium without 6-thioguanine (6 plates for each treatment condition). Stock solutions of 6-thioguanine (Sigma Aldrich) were prepared in 1 mol/L NaOH, diluted to 4 mmol/L in 0.1 mol/L NaOH, filtered through 0.2-µm filters (Nalgene), and stored as 4 mmol/L aliquots at –20°C. Selection medium containing 40 µmol/L 6-thioguanine was prepared just before each plating or feeding. Cells were fed every 3 to 4 days, and colonies were stained and counted after 2 weeks. Spontaneous and damage-induced mutation frequencies were calculated as follows: (number of resistant colonies)/[(number of cells plated for selection) x (colony-forming efficiency at time of selection)].

Determination of Cisplatin and Oxaliplatin Adduct Levels in NHF1 and Xeroderma Pigmentosum Variant Cells.
Cells were plated at 5 x 105 per plate (40 plates) and 12 hours later exposed to 250 to 750 µmol/L cisplatin or 2 mmol/L oxaliplatin for 1 hour. Genomic DNA was isolated (Wizard Genomic DNA Purification kit; Promega, Madison, WI), resuspended in 5% HCl, hydrolyzed for 30 minutes at 95°C, and quantified spectrophotometrically from absorbance values at 260 nm relative to hydrolyzed calf thymus DNA standards. Total Pt was measured by graphite furnace atomic absorption spectrophotometry with Zeeman background correction (Perkin-Elmer 4100ZL, Norwalk, CT; ref. 33 ).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Karyotyping.
The cell lines used in this study were derived from human male fibroblasts. Expression of telomerase resulted in cell populations with unlimited proliferative capacity and normal diploid karyotypes (30 , 32) , even when reanalyzed at population doubling levels 78 (NHF1) and 202 (xeroderma pigmentosum variant). The normal 46,XY complement of chromosomes was also confirmed for the xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) HAT-selected cells. Careful inspection of Giemsa-stained chromosomes at the 500-band level also did not reveal any structural alterations.

Cytotoxicity of UVC and Cisplatin.
Fig. 1Citation shows that xeroderma pigmentosum variant cells were more sensitive to reduction of colony-forming efficiency by UVC than NHF1 cells (Fig. 1A)Citation . The increment of dose required to reduce survival from 100% to 37% (D0 value) was 2.1-fold lower in xeroderma pigmentosum variant. These findings are in close agreement with observations reported previously (34, 35, 36) . Similarly, xeroderma pigmentosum variant (–) cells (D0 = 3.2 J/m2) were more sensitive to UVC than xeroderma pigmentosum variant (+) cells (D0 = 6.5 J/m2; Fig. 1BCitation ). Together, these results indicate that polymerase {eta} expression in the complemented xeroderma pigmentosum variant (+) cells restored UVC cytotoxicity to the level observed in normal human fibroblasts.



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  		Fig. 1. Cytotoxicity of UVC in log phase human fibroblasts. NHF1 ({circ}) and XPV ({bullet}) cells (A) or XPV(+; {triangleup}) and XPV(–; {blacktriangleup}) cells (B), were treated with increasing fluences of UVC. Results represent the average of three to four independent determinations of relative colony-forming efficiency; bars, ±SE.

 
The survival curves for the same four human fibroblast cell lines treated with cisplatin are presented in Fig. 2Citation . Xeroderma pigmentosum variant cells appeared more resistant to cisplatin than NHF1 cells, displaying an IC50 value of 17 µmol/L compared with 11.5 µmol/L for NHF1 cells (Fig. 2A)Citation . By contrast, xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells demonstrated no significant difference in cytotoxic response to cisplatin treatment [IC50 values were 15 µmol/L for xeroderma pigmentosum variant (+) and 17 µmol/L for xeroderma pigmentosum variant (–); Fig. 2BCitation ].



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  		Fig. 2. Cytotoxicity of cisplatin in log phase human fibroblasts. NHF1 ({circ}) and XPV ({bullet}) cells (A) or XPV(+) ({triangleup}) and XPV(–) ({blacktriangleup}) cells (B) were treated for 1 hour with increasing cisplatin concentrations. Results represent the average of four to six independent determinations; bars, ±SE.

 
Levels of Pt Adducts in Genomic DNA of Human Fibroblasts.
The discrepancy in cisplatin sensitivity between the nonisogenic NHF1 and xeroderma pigmentosum variant cells could be explained by possible differences in accumulation of cisplatin adducts in these two cell lines (37) . Therefore, we measured the levels of total Pt using atomic absorption in DNA isolated from the four cell lines treated with increasing concentrations of cisplatin. The levels of Pt in genomic DNA of NHF1 cells were consistently higher than those in xeroderma pigmentosum variant cells at four different concentrations of cisplatin (Fig. 3A)Citation . From the ratio of the slopes of the linear regression lines in Fig. 3Citation A, we concluded that NHF1 cells accumulated ~3.6-fold more Pt than xeroderma pigmentosum variant cells. However, these regression lines did not extrapolate to zero and could not be used to estimate the number of Pt adducts present in the cells treated with cisplatin in the range of concentrations used in the cytotoxicity assays (Fig. 2)Citation . Nevertheless, these data seem to explain the apparent resistance of xeroderma pigmentosum variant cells to cisplatin compared with NHF1 and suggest that NHF1 and xeroderma pigmentosum variant cells process Pt differently. We also found that the isogenic xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells accumulated the same levels of Pt adducts (Fig. 3B)Citation , which were comparable with the platination levels observed with the xeroderma pigmentosum variant parental cell line.



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  		Fig. 3. Accumulation of cisplatin and oxaliplatin adducts in genomic DNA of normal and XPV human fibroblasts. NHF1 ({circ}) and XPV ({bullet}) fibroblasts (A) were treated with the indicated concentrations of cisplatin and genomic DNA isolated as described in Materials and Methods. Total Pt in samples from three independent experiments was determined using atomic absorption; bars, ±SE. B, accumulation of Pt adducts in NHF1 (bar 1), XPV (bar 2), XPV(+) (bar 3), and XPV(–) (bar 4) fibroblasts treated with 500 µmol/L cisplatin; accumulation of Pt-adducts in cells treated with 2 mmol/L oxaliplatin is shown for XPV(+) (bar 5) and XPV(–) (bar 6). DNA was isolated from treated cells in three independent experiments and total Pt determined using atomic absorption; bars, ±SE.

 
UVC- and Cisplatin-Induced Mutagenesis.
Mutation frequencies at the HPRT locus were measured in NHF1 and xeroderma pigmentosum variant cells to evaluate the potential role of polymerase {eta} in translesion synthesis past cisplatin adducts during nuclear DNA replication. We confirmed in parallel experiments that xeroderma pigmentosum variant fibroblasts displayed higher UVC-induced mutation frequencies than NHF1 cells. For instance, when these cell lines were exposed to the same dose of UVC (4 J/m2), which produced the same frequency of DNA photoproducts (36) , the induced mutation frequency was 4.3-fold higher in xeroderma pigmentosum variant (Table 1)Citation . These results are consistent with polymerase {eta} being the primary polymerase performing accurate translesion synthesis past cyclobutane pyrimidine dimers. Because cisplatin DNA binding was different in xeroderma pigmentosum variant and NHF1, and it was not possible to quantify Pt-DNA adducts at treatment doses, the mutation frequencies were compared in terms of cytotoxicity (Fig. 4)Citation rather than fluence (UVC), external concentration (cisplatin), or adduct levels. UVC-induced mutation frequencies were 2.4-fold higher in xeroderma pigmentosum variant than in NHF1 when survival was between 51% (NHF1) and 61% (xeroderma pigmentosum variant), and 2.9-fold higher when survival was 86% to 87% in both cell lines (Fig. 4A)Citation . These findings confirmed that xeroderma pigmentosum variant cells sustain 2- to 3-fold more mutations per lethal event than normal fibroblasts (34 , 35) . Fig. 4BCitation displays the mutagenicity results for cisplatin graphed against survival for NHF1 and xeroderma pigmentosum variant cells. Our averaged results indicated that the number of mutants per lethal event was 2.6-fold higher in xeroderma pigmentosum variant cells when survival was between 28% (NHF1) and 32% (xeroderma pigmentosum variant).


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  		Table 1 Mutation frequencies at the HPRT locus of NHF1 and XPV cell lines

 


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  		Fig. 4. Mutation frequencies in NHF1 and XPV cells treated with UVC or cisplatin. Mutagenesis results were graphed as a function of cell survival to illustrate the efficiency of mutation induction by UVC (A) and cisplatin (B), relative to the toxicity these DNA damaging agents caused in NHF1 ({circ}) or XPV ({bullet}) human fibroblasts. NHF1 and XPV cells exposed to 0 to 8 J/m2 UVC or 0 to 25 µmol/L cisplatin were expanded for a minimum of six population doublings to allow for mutation expression (i.e., loss of HPRT synthesized before the exposure to DNA damaging agents). Mutation frequencies for each cell line were determined in three or four independent experiments (Table 1)Citation ; bars, ±SE.

 
It is also apparent from the results in Fig. 4Citation that UVC photoproducts are significantly more mutagenic per lethal event than cisplatin-induced DNA damage. For example, a mutation frequency of 20 per 105 survivors in xeroderma pigmentosum variant cells was associated with ~30% survival in cisplatin-treated cells (Fig. 4)Citation ; the same mutation frequency would be induced by a UVC fluence killing <10% of the treated cells. In NHF1, an induced mutation frequency of 15 mutants per 105 survivors was associated with killing <40% of UVC-treated cells but required killing >80% of cisplatin-treated cells (Fig. 4)Citation . Taken together, the results in Fig. 4Citation indicate that cisplatin-induced DNA adducts are more toxic and less mutagenic to human fibroblasts than UVC-induced photoproducts.

To confirm the above conclusions in cells that did not differ in Pt accumulation, we measured UVC- and cisplatin-induced mutation frequencies in xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells (Fig. 5)Citation . We found that the average background mutation frequencies in xeroderma pigmentosum variant (+) fibroblasts were 3- to 5-fold higher than in xeroderma pigmentosum variant (–) fibroblasts (Table 2)Citation . We also found that overexpression of polymerase {eta} reduced mutation frequencies in UVC-treated xeroderma pigmentosum variant (+) cells close to background levels (Fig. 5A)Citation . Average UVC-induced mutation frequencies were lower in xeroderma pigmentosum variant (+) cells than in NHF1 cells (Fig. 4ACitation and Fig. 5ACitation ). Even at the UVC fluence that resulted in 34% survival, only 8 mutants per 105 clonogenic units were observed in the xeroderma pigmentosum variant (+) cells, which was similar to the frequency of mutants recovered in the untreated population (6 mutants per 105 survivors). In contrast, at 48% survival, 49 mutants per 105 colony-forming units were observed in xeroderma pigmentosum variant (–) cells, representing a 6-fold higher mutation frequency than detected in xeroderma pigmentosum variant (+) cells after comparable cytotoxic treatments. These data established that expression of the transduced polymerase {eta} in the xeroderma pigmentosum variant (+) cells protected them from UVC-induced mutagenesis.



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  		Fig. 5. Mutation frequencies in XPV(+) and XPV(–) cells treated with UVC or cisplatin. Mutagenesis as a function of cell survival illustrates the efficiency of mutation induction by UVC (A) and cisplatin (B), relative to the toxicity these DNA damaging agents caused in XPV(+) ({circ}) or XPV(–) ({bullet}) human fibroblasts. Experimental conditions were as described in the legend to Fig. 4Citation . Mutation frequencies for each cell line were determined in four independent experiments (Table 2)Citation ; bars, ±SE.

 

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  		Table 2 Mutation frequencies at the HPRT locus of XPV(+) and XPV(–) cell lines

 
Cisplatin-induced mutation frequencies in xeroderma pigmentosum variant (–) cells were lower than UVC-induced mutation frequencies after treatment conditions that caused similar levels of cytotoxicity (49 mutants per 105 colony-forming units at 48% survival for UVC and 22 mutants per 105 colony-forming units at 37% survival for cisplatin). When cisplatin-induced mutation frequencies in xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells were compared, we found that the number of mutants recovered in xeroderma pigmentosum variant (–) cells was ~2-fold higher than in xeroderma pigmentosum variant (+) cells at both concentrations of cisplatin tested (Fig. 5B)Citation . If the data were corrected for the higher background frequency in the xeroderma pigmentosum variant (+) cell line (Table 2)Citation , the number of cisplatin-induced mutations would be ~3-fold higher in xeroderma pigmentosum variant (–) than in xeroderma pigmentosum variant (+). Taken together with the NHF1 and xeroderma pigmentosum variant data (Fig. 4B)Citation , these results suggest that polymerase {eta} is likely to be involved in accurate bypass of at least one of the subtypes of cisplatin adducts in human fibroblast.

Oxaliplatin Cytotoxicity and Induced Mutagenesis in Xeroderma Pigmentosum Variant (+) and Xeroderma Pigmentosum Variant (–) Fibroblasts.
Oxaliplatin is less mutagenic in bacteria than cisplatin (3) , but the mutagenicity of oxaliplatin in human cells has not been reported previously. We determined oxaliplatin toxicity and mutagenicity at the HPRT locus in xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) fibroblasts, which were shown to accumulate similar amounts of Pt adducts in genomic DNA (Fig. 3B)Citation . Fig. 6ACitation illustrates that the toxicity of oxaliplatin was similar in xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells (IC50 values were 100 µmol/L for both cell lines), as demonstrated previously for cisplatin cytotoxicity (Fig. 2B)Citation . When oxaliplatin was evaluated for its ability to induce mutations in the HPRT locus of the xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells, there was no significant increase in mutation frequencies over the background in either cell line, even at the doses of oxaliplatin that allowed for only 10% cell survival (Fig. 6B)Citation .



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  		Fig. 6. Cytotoxicity and mutagenicity of oxaliplatin in human fibroblasts. XPV(+) ({triangleup}) and XPV(–) ({blacktriangleup}) cells were treated with increasing oxaliplatin concentrations. A, Cell survival results represent the averages of five independent determinations; bars, ±SE. Mutagenicity was plotted against survival in B to illustrate the low efficiency of mutation induction (if any) by oxaliplatin, relative to the toxicity oxaliplatin caused in XPV(+) ({triangleup}) or XPV(–) ({blacktriangleup}) fibroblasts. Cells were exposed to 0 to 200 µmol/L oxaliplatin and expanded for approximately six population doublings to allow for mutation expression. Mutation frequencies for each cell line were determined in four independent experiments using different oxaliplatin concentrations.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Among the DNA polymerases studied to date, only polymerase {eta}, polymerase µ, polymerase ß, and polymerase {zeta} have the capability for translesion synthesis past cisplatin and oxaliplatin GpG adducts in vitro (17, 18, 19 , 38) . Under comparable conditions, the extent of translesion synthesis past the cisplatin adduct by these polymerases is 25 ± 10% for polymerase {eta}, 10 ± 5% for polymerase µ, 5.5 ± 0.5% for polymerase ß, and 4.5 ± 0.5% for polymerase {zeta}. Translesion synthesis past the oxaliplatin adduct is 62.5 ± 12.5% for polymerase {eta}, 22 ± 12% for polymerase µ, 11.5 ± 0.5% for polymerase ß, and 8.0 ± 1.0% for polymerase {zeta} (38) . These findings point to polymerase {eta} as the best candidate enzyme for bypassing cisplatin and oxaliplatin adducts during nuclear DNA replication in human cells. Seeking to confirm this role for polymerase {eta}, cisplatin-induced cytotoxicity and mutagenicity at the HPRT locus were initially determined in diploid human fibroblasts containing (NHF1) or lacking (xeroderma pigmentosum variant) polymerase {eta}.

The participation of polymerase {eta} in the accurate replication of DNA containing thymine dimers is well characterized (20 , 21) . Xeroderma pigmentosum variant cells display slightly reduced colony-forming efficiency and much higher mutation frequency in response to UVC than normal cells (34 , 35) . Therefore, results of parallel experiments, in which the same NHF1 and xeroderma pigmentosum variant cell lines were damaged by UVC, provided a positive control for evaluating the cytotoxic and mutagenic effects of cisplatin. However, NHF1 and xeroderma pigmentosum variant cells are not isogenic, and it became evident that after treatments with equimolar concentrations of cisplatin, NHF1 cells accumulated 3.6-fold more Pt adducts in their genomic DNA than xeroderma pigmentosum variant cells. This phenomenon has been reported by others (37) and could be due to differences in membrane permeability, resulting in different cisplatin uptake or efflux rates (39) . Other plausible explanations would be differences in the levels of cisplatin-inactivating cytoplasmic constituents, such as metallothionein, glutathione, and glutathione S-transferases (39) . Therefore, mutation frequencies were compared on the basis of cytotoxicity rather than fluences of UVC or concentrations of cisplatin. Our findings indicated that the 2.6-fold enhancement of cisplatin-induced mutagenesis in xeroderma pigmentosum variant relative to NHF1 was similar to the 2.4 ratio of UVC-induced mutation frequencies observed in these same cells. These findings were reassessed in the isogenic xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells, which displayed similar Pt-DNA adduct levels (Fig. 3B)Citation and survival curves (Fig. 2B)Citation after cisplatin treatment. Again, the induced mutation frequencies at the HPRT locus were 2- to 3-fold higher for xeroderma pigmentosum variant (–) cells. Therefore, the two independent sets of mutagenicity data suggest that polymerase {eta} is likely to be involved in accurate translesion synthesis of cisplatin-DNA adducts in intact cells.

DNA polymerase inhibitors, such as gemcitabine (40) , appear to enhance the therapeutic efficacy of cisplatin and oxaliplatin, presumably by inhibiting DNA repair and decreasing the opportunity for translesion synthesis. Gemcitabine in combination with cisplatin (41) or oxaliplatin (42) is currently being evaluated in Phase II clinical trials. Gemcitabine was initially selected for its ability to inhibit the major replicative DNA polymerases. This compound is a nucleoside analog that is incorporated into DNA and inhibits DNA elongation at the +2 step (43) , i.e., after the next nucleotide is added to the growing strand. The error-prone polymerase {zeta} appears to have a relaxed specificity for elongation from mismatched nucleotides (44) , whereas polymerase {eta} prefers matched primers (26 , 45) ; therefore, translesion synthesis by polymerase {zeta} might be more resistant to gemcitabine than that catalyzed by polymerase {eta}. Thus, our data suggest that gemcitabine could have the unexpected effect of increasing the mutagenicity of cisplatin, if DNA replication would proceed at reduced rates and gemcitabine were to inhibit the more accurate polymerase {eta} to a greater extent than the error-prone polymerase {zeta}.

UVC induces pyrimidine dimers and (6–4) adducts in DNA with a ratio of ~3 dimers to 1 adduct (46) . Polymerase {eta} replicates past thymine dimers in DNA accurately, thus reducing the probability of UVC-induced mutations in normal cells. However, this translesion synthesis pathway is disabled in xeroderma pigmentosum variant cells, and error-prone bypass of pyrimidine dimers results in high levels of UVC-induced mutations (34 , 35 , 47) . The pathways that lead to mutation induction by cisplatin are more complex. The highly reactive mono-aquated species first covalently bind to DNA to form a monoadduct (Pt-G; ref. 39 ). These adducts are likely to be readily bypassed by a replication polymerase exhibiting an intrinsically low error frequency, such as polymerase {delta}. Therefore, monoadducts are not expected to contribute significantly to either cisplatin cytotoxicity or mutagenicity. Monoadducts convert to diadducts with a half-life of ~2.2 hours. The major diadducts are Pt-GG intrastrand cross-links (65% of all adducts), Pt-AG intrastrand cross-links (25%), Pt-GNG intrastrand cross-links (5–10%), and interstrand G-Pt-G cross-links (1% to 5%; refs. 48 , 49 ). In vitro studies with adducted templates have established that polymerase {eta} is capable of bypassing Pt-GG intrastrand diadducts, most of the time in an error-free manner (18) . On the basis of the available evidence, one would expect polymerase {eta} to be involved in accurate bypass of intrastrand cross-links in normal cells. On the other hand, cisplatin interstrand cross-links are an absolute block to replication by all of the DNA polymerases tested, and their repair occurs at slower rates than the removal of diadducts. In summary, the most persistent UVC-induced lesions, the pyrimidine dimers, contribute to both cytotoxicity and mutagenicity of UVC, whereas the most persistent cisplatin-induced lesions, the interstrand cross-links, are highly cytotoxic and should not contribute significantly to cisplatin mutagenicity. This could explain why the recovery of cisplatin-induced mutants in human fibroblasts is reduced compared with the recovery of UVC-induced mutants.

The role of polymerase {eta} in accurate bypass of cisplatin adducts was inferred from the higher mutation frequency in cisplatin-treated xeroderma pigmentosum variant (–) than in xeroderma pigmentosum variant (+) cells. Because no increase in mutation frequency above background was detected in oxaliplatin-treated cells, it was not possible to evaluate the role of polymerase {eta} in the bypass of oxaliplatin adducts in this study. However, oxaliplatin is clearly less mutagenic than cisplatin in both xeroderma pigmentosum variant (–) and xeroderma pigmentosum variant (+) human fibroblasts. Although mutagenicity has not been considered a major issue with cisplatin chemotherapy, secondary tumors have been observed 10 to 15 years later (2) , which could be a concern for the treatment of young patients. Furthermore, cisplatin-induced mutations have been implicated in the emergence of resistance to this drug (50) . Thus, if our observations are confirmed in other cell lines, it would suggest that oxaliplatin-based chemotherapy might be less likely than cisplatin-based chemotherapy to induce secondary tumors and drug resistance.


   ACKNOWLEDGMENTS
 
We are thankful to the laboratory of Dr. Roger A. Shultz (University of Texas Southwestern Medical Center) for providing xeroderma pigmentosum variant cells. These cells were made available to us through a material transfer agreement with Geron Corporation. We are also grateful to Dr. Fumio Hanaoka (Osaka University) for the cDNA encoding human DNA polymerase {eta}, Dr. Dennis A. Simpson (University of North Carolina at Chapel Hill) for help in generating the xeroderma pigmentosum variant (+) and xeroderma pigmentosum variant (–) cells, and Dr. Paul Juniewicz (Sanofi-Synthelabo) for providing oxaliplatin.


   FOOTNOTES
 
Grant support: NIH CA84480 (S. G. Chaney) and CA55065 (M. Cordeiro-Stone); Milheim Foundation for Cancer Research Grant 2003–27 (E. Bassett); University of North Carolina at Chapel Hill Center Grants P30-CA16086 and P30-ES10126. N. M. King was supported by the Curriculum in Toxicology Training Grant (ES07126).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: E. Bassett is currently at the Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop 70A-1118, Berkeley, CA 94720.

Requests for reprints: Marila Cordeiro-Stone, Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, 620A Brinkhous-Bullitt Building, CB# 7525, Chapel Hill, NC 27599-7525. Phone: 919-966-1396; Fax: 919-966-5046; E-mail: uncmcs{at}med.unc.edu

Received 4/14/04. Revised 6/ 3/04. Accepted 7/13/04.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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