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Molecular Cancer Therapeutics 6, 1858-1868, June 1, 2007. doi: 10.1158/1535-7163.MCT-07-0068
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

Mismatched nucleotides as the lesions responsible for radiosensitization with gemcitabine: a new paradigm for antimetabolite radiosensitizers

Sheryl A. Flanagan1, Blaine W. Robinson2, Christina M. Krokosky1 and Donna S. Shewach1

1 Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, Michigan and 2 Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Requests for reprints: Donna S. Shewach, Department of Pharmacology, University of Michigan Medical Center, 4713 Upjohn Center, Ann Arbor, MI 48109-0504. Phone: 734-763-5810; Fax: 734-763-3438. E-mail: dshewach{at}umich.edu

Abstract

Radiation sensitization by 2',2'-difluoro-2'-deoxycytidine (dFdCyd) has correlated with dATP depletion [dFdCDP-mediated inhibition of ribonucleotide reductase (RR)] and S-phase accumulation. We hypothesized that radiosensitization by dFdCyd is due to nucleotide misincorporations in the presence of deoxynucleotide triphosphate pool imbalances, which, if not repaired, augments cell death following irradiation. The ability of dFdCyd to produce misincorporations was measured as pSP189 plasmid mutations in hMLH1-deficient [mismatch repair (MMR) deficient] and hMLH1-expressing (MMR proficient) HCT116 cells. Only MMR-deficient cells showed a significant increase in nucleotide misincorporations (2- to 3-fold increase; P ≤ 0.01) after radiosensitizing concentrations of dFdCyd ± 5 Gy radiation, which persisted for at least 96 h. dFdCyd (10 nmol/L) did not radiosensitize MMR-proficient HCT116 or A549 cells, but following small interfering RNA–mediated suppression of hMLH1, this concentration produced excellent radiosensitization (radiation enhancement ratios = 1.6 ± 0.1 and 1.5 ± 0.1, respectively; P < 0.05) and a 2.5-fold increase in mutation frequency in A549 cells. Cytosine arabinoside (1-ß-D-arabinofuranosylcytosine), which can be incorporated into DNA but does not inhibit RR, failed to radiosensitize MMR-deficient cells or increase mutation frequency in the MMR-deficient and MMR-proficient cells. However, the RR inhibitor hydroxyurea radiosensitized MMR-deficient cells and increased nucleotide misincorporations (≥5-fold increase; P < 0.05), thus further implicating the inhibition of RR as the mechanism underlying radiosensitization by dFdCyd. These data showed that the presence and persistence of mismatched nucleotides is integral to radiosensitization by dFdCyd and suggest a role for hMLH1 deficiency in eliciting the radiosensitizing effect. [Mol Cancer Ther 2007;6(6):1858–68]

Introduction

Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is a nucleoside analogue with broad-spectrum activity against solid tumors in patients (1). In addition, it is a potent radiosensitizer at noncytotoxic concentrations in vitro and in animal models (2, 3). These results encouraged clinical studies, which showed that dFdCyd can significantly enhance tumor regression or control when administered concurrently with radiotherapy in patients (4–6).

Despite numerous clinical trials, the mechanism by which dFdCyd enhances radiation-induced cell killing has not been elucidated. Ionizing radiation (IR) produces many types of damage to DNA, and it is thought that the ineffective repair of DNA double-strand breaks (DSB) contributes most strongly to cytotoxicity (7). Antimetabolite radiosensitizers target DNA replication, and drugs such as 5-bromo-2'-deoxyuridine (BrdUrd) and 5-fluoro-2'-deoxyuridine have been shown to enhance cytotoxicity when administered with radiation by either increasing DNA DSBs or inhibiting their repair (8, 9). However, we and others have shown that dFdCyd neither enhances radiation-induced DNA DSBs nor decreases the rate or extent of their repair (10, 11). Thus, new mechanisms must be evaluated to account for radiosensitization by dFdCyd.

Previous studies have shown that radiosensitization with dFdCyd is most effective during S phase (12). In addition, functional homologous recombination repair (HRR) has been implicated (13), whereas nonhomologous end joining does not affect the ability of dFdCyd to radiosensitize cells (14). We have sought to determine whether the two major biological effects of dFdCyd, interference with deoxynucleotide triphosphate (dNTP) synthesis and incorporation into DNA, are involved in effecting radiosensitization with dFdCyd. Phosphorylation of dFdCyd to its diphosphate, dFdCDP, results in potent inhibition of ribonucleotide reductase (RR; ref. 15), with a decrease primarily in dATP in solid tumor cells (16). We have shown strong correlations between decreased dATP as cells accumulate in S phase and radiosensitization with dFdCyd (10, 16–18). However, radiosensitization does not correspond with phosphorylation of dFdCyd to its triphosphate, dFdCTP, or to its incorporation into DNA (16, 19).

Based on these data, we have hypothesized that decreased dATP pools during S phase produce errors in DNA replication that are responsible for radiosensitization with dFdCyd. Imbalances in dNTP pools can result in errors in DNA replication, such as single-base substitutions, insertions, or deletions, resulting in frameshift mutagenesis (20). The mismatch repair (MMR) system plays a role in correcting DNA mismatches during replication (21) and thus may contribute to dFdCyd radiosensitization. This idea was supported by the finding that MMR-deficient HCT116 cells, which lack hMLH1, were radiosensitized by dFdCyd at ≤IC50, whereas the isogenic MMR-proficient cell lines, HCT116+ch3 and HCT116 1-2, were not (22). Matched cell lines achieved similar S-phase accumulation, levels of dFdCMP incorporation, and an equal depletion of dATP, thus eliminating cell cycle redistribution to a more radiosensitive phase or altered drug metabolism as a possible explanation for the difference in dFdCyd-mediated radiosensitization. Overall, these results showed that MMR status is an important factor in dFdCyd-mediated radiosensitization.

Although we have postulated that dATP depletion and S-phase accumulation lead to nucleotide misincorporations in DNA, it has not been shown that dFdCyd can produce these lesions. In addition, although our results strongly correlate dATP depletion with radiosensitization, the concurrent presence of dFdCTP in these studies makes it difficult to completely exclude this metabolite or its incorporation into DNA as a factor in radiosensitization. The present study directly tests the hypothesis that the dNTP pool imbalances produced by dFdCyd can produce mutations in DNA and that these are the lesions that result in radiosensitization. For these studies, a shuttle vector assay was used using the plasmid pSP189, which encodes an amber suppressor tRNA sequence (supF gene) to allow expression of ß-galactosidase in MBM7070 Escherichia coli cells. A single mutation at nearly any site in the coding sequence for the supF gene will result in a nonfunctional gene product (23). To further evaluate the relative roles of RR inhibition by dFdCDP versus dFdCTP incorporation into DNA in nucleotide misincorporation and radiosensitization with dFdCyd, we also evaluated hydroxyurea (HU), a radiosensitizer and an inhibitor of RR that produces imbalances in dNTP pools similar to dFdCyd (24), and/or 1-ß-D-arabinofuranosylcytosine (araC), which is phosphorylated to its triphosphate and can be incorporated into DNA, similar to dFdCTP, but does not inhibit RR.

Materials and Methods

Cell Culture, Plasmid, and Drug Preparation
HCT116 colon carcinoma cells are MMR deficient due to inactivation of hMLH1. The HCT116+ch3 cell line contains an extra copy of chromosome 3 to correct hMLH1 deficiency. The HCT116 1-2 cell line contains wild-type hMLH1 cDNA and the HCT116 0-1 cell line contains the vector without the hMLH1 insert. A549 lung carcinoma cells are MMR proficient. All cells were maintained in DMEM (Invitrogen) supplemented with 10% FCS (Invitrogen) and 2 mmol/L L-glutamine (Fisher Scientific). Gemcitabine (a gift from Eli Lilly and Company), araC, and HU (Sigma Chemical Co.) were dissolved in PBS. Cell cycle distribution was determined by dual-variable flow cytometric analysis as described (25), and DNA synthesis was measured by BrdUrd incorporation (24).

Cell Survival and Radiosensitization Assay
Cells were left untreated or treated with dFdCyd, HU, or araC at various concentrations for 24 h before irradiation [60Co (AECL Theratron 80) at 1–2 Gy/min]. Following dFdCyd and/or IR, cells were assessed for clonogenic survival as described previously (16). Radiation sensitivity is expressed in terms of the mean inactivation dose (D-bar), which represents the area under the cell survival curve (26). Radiosensitization is expressed as an enhancement ratio, which is defined as the mean inactivation dose (control) / mean inactivation dose (drug).

Determination of Nucleotide Pools
Nucleotides were extracted from cells using 0.4 N perchloric acid and neutralized and ribonucleotides were removed using a boronate affinity column (16). Cellular dNTPs were separated and quantified using a strong anion exchange column (Whatman) with a high-pressure liquid chromatography system (Waters) equipped with a photodiode array detector and controlled by Millennium 2010 software. Nucleotides were eluted at 2 mL/min with a linear gradient of ammonium phosphate buffer [0.15 mol/L (pH 2.8) to 0.60 mol/L (pH 2.9 or 3.4)]. Nucleotides were identified based on their UV absorbance spectrum and quantified at either 254 or 281 nm by comparison with the absorbance of a known amount of authentic standard.

Transfection of pSP189 Plasmid
The pSP189 vector contains replication origins for both mammalian and bacterial cells, the supF suppressor tRNA marker gene, and ampicillin resistance gene (27). Cells were transfected using SuperFect transfection reagent (Qiagen). Medium was replaced after an overnight incubation with transfection complexes, and dFdCyd, HU, araC, or HU + araC were added for 24 h followed by irradiation where appropriate. pSP189 plasmid was harvested from untreated (control) or drug-treated cells immediately after the 24-h incubation with drug or 1.5 to 2 h following irradiation. To determine the plasmid mutation frequencies in the HCT116 0-1 cells over time, plasmids were extracted from cells every 24 h following a 24-h drug incubation. Plasmid DNA was isolated using a Qiagen Miniprep kit, incubated with DpnI (Invitrogen) to remove unreplicated plasmid DNA, and further purified by a phenol/chloroform extraction followed by precipitation with isopropanol/ethanol and dissolved in 0.5x TE buffer [10 mmol/L Tris-HCl, 1 mmol/L EDTA (pH 7.5)].

Transformation in MBM7070 E. coli
Transformation was accomplished via electroporation with 1 µL of TE containing plasmid DNA and 20 µL of electrocompetent MBM7070 E. coli. The transformation mixtures were plated onto agar plates containing 100 µg/mL ampicillin (Roche), 50 mg/mL isopropyl-L-thio-B-D-galactopyranoside (Invitrogen), and 20 mg/mL 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Roche). White and blue colonies were enumerated, and mutation frequencies were calculated as number of white colonies / number of (white + blue) colonies. DNA from some control and all mutant clones was isolated and sequenced at the University of Michigan DNA Sequencing Core using the 20-mer primer (5'-GGCGACACGGAAATGTTGAA).

Small Interfering RNA Transfection
Pooled synthetic small interfering RNA (siRNA) duplexes (SMARTpool siRNA) specific for hMLH1 (M-003906-01; Dharmacon) and siGLO siRNA for determination of transfection efficiency were used. A549 lung carcinoma cells and HCT116 1-2 MMR-proficient cells were incubated overnight before transfection with siRNA at ratios of 1 or 5 µg siRNA to 3 µL LipofectAMINE 2000 (Invitrogen) diluted in Opti-MEM (reduced serum medium; Invitrogen), respectively. Medium was replaced at 24 h and whole-cell lysates were prepared for immunoblotting periodically. For radiosensitization assays, cells were transfected with siRNA and expanded 48 h later. At 72 h after transfection, cells were treated with no drug (control), dFdCyd for 24 h ± IR, or IR only at 2, 5, 7.5, and 10 Gy. Cell survival was determined as described above.

Immunoblot Blot Analysis
Whole-cell lysates were prepared in radioimmunoprecipitation assay lysis buffer [0.5 mol/L Tris-HCl, 1.5 mol/L NaCl, 2.5% deoxycholic acid, 10% NP40, 10 mmol/L EDTA (pH 7.4)], with the addition of protease and phosphatase inhibitors [Complete Mini Protease Inhibitor Cocktail tablet (Roche)], 1 mmol/L sodium orthovanadate, and 1 mmol/L sodium fluoride at 24, 48, 72, 96, and 120 h after transfection with siRNA. Proteins were separated by SDS-PAGE on 10% acrylamide gels and transferred onto Immobilon-P transfer membrane (Millipore Corp.). Blots were probed with hMLH1 polyclonal rabbit IgG antibodies (Santa Cruz Biotechnology) at 1:100 and anti-rabbit IgG horseradish peroxidase–linked antibodies at 1:20,000 dilutions. Proteins were detected and visualized using an enhanced chemiluminescence detection system (Pierce).

Results

Mutation Frequency Induced by Drugs and/or IR
Previously, we have shown that dFdCyd at ≥IC10 produced radiosensitization in the HCT116 MMR-deficient cells, but in the HCT116 MMR-proficient cells, no radiosensitization was observed until dFdCyd was >IC90 (Table 1 ; ref. 22). To evaluate the role of nucleotide misincorporation in DNA and the susceptibility of cells to undergo radiosensitization by dFdCyd, plasmid mutation frequency in HCT116 MMR-deficient cells was compared with that in HCT116+ch3 MMR-proficient cells treated with dFdCyd. Our earlier studies revealed that equitoxic concentrations of dFdCyd produced differences in the degree of dATP depletion and dFdCTP accumulation in these MMR-deficient and MMR-proficient cells (22), whereas dFdCMP incorporation into DNA was similar, as expected, because DNA incorporation is primarily associated with cytotoxicity (28). Therefore, to compare the ability of dNTP pool imbalance to induce mutations, further studies used equimolar concentrations of dFdCyd (100 nmol/L dFdCyd for the HCT116 and HCT116+ch3 cells and 30 nmol/L for the HCT116 0-1 and HCT116 1-2 cells) that produced a similar degree of dATP depletion in these MMR-deficient and MMR-proficient cell lines but resulted in different cytotoxicity. These concentrations resulted in >80% reduction in dATP and S-phase arrest (>80% accumulation of cells in S phase) in all cell lines (Tables 2 and 3A ; data not shown; ref. 22) and produced radiosensitization in the MMR-deficient but not in the MMR-proficient cells (Table 1; ref. 22).


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  		Table 1. Effects of dFdCyd, HU, and araC on the sensitivity of MMR-deficient and MMR-proficient cells to IR

 

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  		Table 2. Endogenous dNTP pools in MMR-proficient and MMR-deficient HCT116 cells following incubation with dFdCyd, HU, araC, or HU + araC

 

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  		Table 3. Cell cycle analysis following drug and/or IR

 
Plasmid mutation frequencies were increased only at radiosensitizing concentrations of dFdCyd in MMR-deficient and MMR-proficient cells (Fig. 1A ). The spontaneous plasmid mutation frequencies in the untreated control group of all cell lines were similar in the MMR-proficient and MMR-deficient cell lines as reported by others (29). At the equimolar concentrations noted above, only plasmid replicated in the MMR-deficient cells (parental HCT116 and HCT116 0-1 cells) showed a significant increase compared with control mutation frequencies following dFdCyd treatment alone (2- to 3-fold increase; P < 0.05; Fig. 1B) and following the combination of dFdCyd and IR (2-fold increase in both cell lines; P < 0.05). IR alone increased the plasmid mutation frequency in the MMR-deficient cell lines, although this increase was significant only in the MMR-deficient parental HCT116 cell line (2-fold increase compared with control; P < 0.05).


Figure 1
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  		Figure 1. pSP189 plasmid mutation frequency in MMR-deficient and MMR-proficient cells following exposure to drug and/or IR. Cells were transfected with pSP189 plasmid overnight, washed and incubated with drug for 24 h, and/or treated with 5 Gy. Plasmids were harvested within 2 h after drug incubation or IR (A and B) every 24 h for 96 h (C) or 24 h after drug incubation (D). DNA was harvested from replicated plasmids and electroporated into E. coli, and mutation frequency was calculated as number of white colonies / number of (white + blue) colonies. The supF sequence of a portion of the control plasmids and all of the mutant plasmids was confirmed by DNA sequencing. Results are expressed as the fold increase relative to control untreated cells with plasmid. A, plasmid mutation frequencies for HCT116 and HCT116+ch3 cells following incubation with dFdCyd for 24 h at nonradiosensitizing concentrations (10 and 100 nmol/L dFdCyd, respectively) or radiosensitizing concentrations (100 and 300 nmol/L, respectively). Control mutation rates were similar between HCT116 and HCT116+ch3 cells, 0.33 ± 0.04 and 0.24 ± 0.02, respectively (P < 0.05). B, plasmid mutation frequencies following exposure to dFdCyd for 24 h (30 nmol/L for HCT116 0-1 and HCT116 1-2 cells; 100 nmol/L for HCT116 and HCT116+ch3 cells), 5 Gy IR, or a combination of dFdCyd (30 or 100 nmol/L) followed by 5 Gy IR. Control mutation rates were similar, 0.09 ± 0.01 and 0.05 ± 0.02 for HCT116 0-1 and HCT116 1-2 cDNA cell lines, respectively (P < 0.05), and 0.33 ± 0.04 and 0.24 ± 0.02 for the parental HCT116 and HCT116+ch3 cell lines, respectively. C, plasmid mutation frequencies in MMR-deficient HCT116 0-1 cells following a 24-h exposure to 30 nmol/L dFdCyd, 5 Gy IR, or a combination of 30 nmol/L dFdCyd followed by 5 Gy IR. Control mutation frequency at 0 h was 0.09 ± 0.01 and remained unchanged throughout the time course (P < 0.05). D, plasmid mutation frequencies in MMR-deficient HCT116 0-1 cell lines and MMR-proficient HCT116 1-2 cell lines following a 24-h exposure to HU (2 mmol/L for HCT116 0-1 and 0.7 mmol/L for HCT116 1-2) or to araC (7 µmol/L for HCT116 0-1 cells and 1.5 µmol/L for HCT116 1-2 cells) or to a combination of HU and araC. Control mutation rates were 0.09 ± 0.01 and 0.05 ± 0.02 for HCT116 0-1 and HCT116 1-2 cells, respectively (P < 0.05). A and B, columns, average of two separate experiments; bars, SD. C and D, columns, mean of at least three separate experiments; bars, SE. Asterisk, significantly greater than the corresponding control (no drug).

 
The data show that dFdCyd alone or with IR increased the mutation frequency to a similar degree in MMR-deficient cells. We then wished to determine whether the acquisition of mutations would cease following drug washout and/or the end of irradiation. If repair capability returned to normal following drug/irradiation, then subsequent replication of the mutated and wild-type plasmids in the cell population would eventually dilute out the mutation. However, if there were a prolonged defect in repair, then the cells would continue to replicate the plasmids with an increased mutation rate compared with plasmids in control (untreated) cells. To test these possibilities, plasmid mutation rates were determined for a period of 4 days following treatment with dFdCyd (IC50) and/or IR (5 Gy; Fig. 1C) in HCT116 0-1 MMR-deficient cells. Significant nucleotide misincorporation was observed at the conclusion of the incubation (0 h) and 24 h later, with a >2-fold increase in plasmid mutation rates in the dFdCyd and dFdCyd + IR groups. In cells treated with dFdCyd alone or IR alone, the mutation frequencies returned to control levels within 48 h after drug removal. However, the plasmid mutation frequency was 3- to 5-fold higher than controls for at least 96 h in cells treated with dFdCyd followed by IR. This suggests that only the cells treated with dFdCyd and IR maintained a defect in repair, resulting in increased cytotoxicity.

To evaluate the relative roles of RR inhibition by dFdCDP versus dFdCTP incorporation into DNA as the cause of the increase in mutation frequency with dFdCyd, we treated cells with equitoxic concentrations (IC50) of the RR inhibitor HU ± araC, a DNA replication inhibitor. In these studies, HU decreased dATP in both MMR-deficient and MMR-proficient cell lines by 4 h following HU addition (undetectable levels and 11% of control for MMR-deficient and MMR-proficient cells, respectively) and remained suppressed at 24 h (16% and 55% of control in MMR-deficient and MMR-proficient cells, respectively; Table 2). This rebound in dNTP production following an initial decrease in pools with HU treatment has previously been shown by us and others (24, 30). araC inhibits DNA synthesis and, when used alone, produces a buildup of dNTPs (31), an effect we observed in our studies in both MMR-deficient and MMR-proficient cells. The combination of HU and araC resulted in <40% reduction in dATP at 4 h followed by a large increase in dATP by 24 h in both cell lines (Table 2). The initial decrease observed with this combination is due to the inhibition of dNTP synthesis by HU. The increase in dATP (and all dNTPs) at 24 h is likely due to the waning of HU inhibition of dNTP synthesis combined with a buildup of dNTPs due to the inhibition of DNA synthesis, which is stronger with araC and HU compared with either drug alone (DNA synthesis following HU, araC, and HU + araC was 49%, 40%, and 1.8%, respectively, for MMR-deficient cells and 61%, 27%, and 16% for MMR-proficient cells compared with control). araCTP accumulation (data not shown) and S-phase accumulation (Table 3B) were similar between MMR-deficient and MMR-proficient cell lines following single drug treatment and the combination. Similar to the results with dFdCyd (IC50), HU (IC50) radiosensitized the MMR-deficient cells [radiation enhancement ratio (RER) = 1.5 ± 0.1] but not the MMR-proficient cells (RER = 1.1 ± 0.1; Table 1). Only plasmid replicated in the MMR-deficient cells showed a significant increase over control mutation frequencies following HU treatment alone (7-fold increase; P < 0.001) and following the combination of HU and araC (5-fold increase; P < 0.05; Fig. 1D). Treatment with araC failed to produce radiosensitization in the MMR-deficient cell line (Table 1) and did not increase the plasmid mutation frequency in MMR-deficient or MMR-proficient cells compared with control (Fig. 1D).

Type and Frequency of Mutations in pSP189
Single-base substitutions comprised >90% of plasmid mutations generated in untreated (control) MMR-deficient and MMR-proficient cells and in HCT116 0-1 MMR-deficient cells following all treatment conditions, with the exception of IR whereby a substantial number of deletions occurred and the percentage of base substitutions was 70%. Insertions and deletions accounted for the remainder of mutations observed within each group (Fig. 2 ). Plasmid mutations generated at high concentrations of dFdCyd in MMR-proficient cells (IC96 used in Fig. 1A) were similar to the plasmid mutations generated following an IC50 of dFdCyd in the MMR-deficient cells, with the majority of plasmid mutations (>90%) presented as single-base substitutions (data not shown). The most common mutations to occur following dFdCyd or dFdCyd and IR were A:T to G:C transitions (25% and 50%, respectively) or G:C to T:A (17% and 14%, respectively) and A:T to C:G (25% and 18%, respectively) transversions. The most common base substitutions in the presence of HU alone were A:T to G:C (52%) transitions and A:T to T:A (19%) transversions, whereas in the presence of both araC and HU the increase was composed of A:T to G:C (16%) transitions or G:C to C:G (20%) and A:T to C:G (15%) transversions. The majority of mutations occurred at random sites in pSP189 in response to each treatment condition.


Figure 2
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  		Figure 2. Type and frequency of mutations in the supF sequence in pSP189 plasmids replicated in HCT116 0-1 MMR-deficient cells. HCT116 0-1 MMR-deficient cells were transfected with pSP189 plasmid overnight and exposed to no drug (control) or to the IC50 for the following conditions (24 h): HU, araC, a combination of HU and araC, dFdCyd, or 5 Gy IR, and dFdCyd followed by 5 Gy IR. Mutant colonies were picked and grown in Luria-Bertani broth followed by plasmid extraction and DNA sequencing. n, total number of mutant colonies, all of which were submitted for DNA sequencing. Total number of colonies counted: control = 86,056; HU = 38,597; araC = 71,237; araC + HU = 30,236; dFdCyd = 26,376; IR = 12,260; dFdCyd + IR = 26,249.

 
Radiosensitization by dFdCyd in MMR-Proficient Cells following siRNA-Mediated Suppression of hMLH1 Expression
To directly evaluate the role of hMLH1 in preventing radiosensitization in MMR-proficient cells, we used siRNA to decrease the expression of hMLH1 in two MMR-proficient cell lines, HCT116 1-2 cells and A549 non–small cell lung carcinoma cells, which cannot be radiosensitized by dFdCyd at respective IC50 and IC10, and examined the effect on cytotoxicity and radiosensitization by dFdCyd. A549 cells are considered MMR proficient because they express the two major MMR proteins: hMLH1 and hMSH2 (32). Suppression of hMLH1 protein was greatest at 72 h after siRNA transfection in both cell lines, with protein levels depressed until at least 120 h after transfection (Fig. 3A and D ). Transfection efficiency was approximately 75% and 71% for HCT116 1-2 and A549 cells, respectively, as determined using siGLO siRNA (data not shown). In HCT116 1-2 cells, the suppression of hMLH1 decreased sensitivity to dFdCyd at concentrations ≤100 nmol/L (Fig. 3B), whereas the suppression of hMLH1 in A549 cells did not alter sensitivity to dFdCyd (Fig. 3E). Both HCT116 1-2 cells and A549 cells were treated with hMLH1 siRNA for 72 h and then incubated with 10 nmol/L dFdCyd (IC50 for HCT116 1-2 cells and IC10 for A549 cells) for 24 h followed by irradiation. dFdCyd (10 nmol/L) was previously determined to be nonradiosensitizing in HCT116 1-2 cells (RER = 1.1 ± 0.1; Table 1), but following suppression of hMLH1, these cells were radiosensitized at this concentration (RER = 1.6 ± 0.1; P < 0.05; Fig. 3C). A549 cells were not radiosensitized by dFdCyd at its IC10 but were moderately radiosensitized with dFdCyd at its IC50 (33). As illustrated in Fig. 3F, after suppressing hMLH1 with siRNA, a 24-h incubation with 10 nmol/L dFdCyd (IC10, noncytotoxic) produced excellent radiosensitization (RER = 1.5 ± 0.1; P < 0.05). Treatment of HCT116 1-2 or A549 cells with siRNA for hMLH1 alone did not increase radiation sensitivity (RER = 0.95 ± 0.1 and 1.0 ± 0.1, respectively). In addition, treatment with a nonspecific siRNA ± 10 nmol/L dFdCyd did not result in radiosensitization in HCT116 1-2 or A549 cells (RER = 1.1 ± 0.11 and 1.2 ± 0.09, respectively).


Figure 3
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  		Figure 3. Suppression of hMLH1 protein expression sensitizes MMR-proficient (A–C) HCT116 1-2 cells and (D–F) A549 cells to radiosensitization by dFdCyd. A and D, whole-cell lysates were analyzed by Western blotting for hMLH1. Expression of ß-actin as a loading control. Representative blot from a minimum of three separate experiments for each cell line. B and E, cells were left untreated (control cells) or transfected with siRNA targeted to hMLH1 mRNA or nonspecific (NS) siRNA for 72 h and then incubated with the indicated concentrations of dFdCyd for 24 h. Cell survival was assessed using a colony formation assay. Points, mean of three separate experiments for each cell line; bars, SE. C and F, cells were left untreated (control cells) or transfected with siRNA targeted to hMLH1 mRNA or nonspecific siRNA for 72 h and then incubated with or without 10 nmol/L dFdCyd for 24 h followed by irradiation. Cell survival was assessed using a colony formation assay. The surviving fraction was corrected for cell survival with dFdCyd and/or siRNA in the absence of radiation. Results are from a representative experiment of at least four separate experiments.

 
Effect of siRNA-Mediated Suppression of hMLH1 on Mutation Frequency with dFdCyd in A549 Cells
Because siRNA-mediated suppression of hMLH1 in A549 cells resulted in an increased sensitivity of these cells to dFdCyd-mediated radiosensitization, we wished to determine whether this corresponded to an increase in plasmid mutation frequency. A549 cells transfected with pSP189 and treated with 10 nmol/L dFdCyd for 24 h did not exhibit a significant increase in mutation frequency compared with untreated (no siRNA, no drug) control cells (0.09 ± 0.01% and 0.08 ± 0.01%; P > 0.05), but plasmid mutation rate increased 2.5-fold following the siRNA-induced suppression of hMLH1 in the presence of dFdCyd (0.21 ± 0.04% versus 0.08 ± 0.01%; P < 0.05). Addition of hMLH1 siRNA alone did not alter mutation frequency compared with untreated control A549 cells (0.07 ± 0.01% versus 0.08 ± 0.01%, respectively) nor did a nonspecific siRNA alone (0.08 ± 0.02%) or with 10 nmol/L dFdCyd (0.10 ± 0.02%) increase mutation frequency compared with control (no siRNA, no drug). Thus, similar to our results in the HCT 0-1 and HCT 1-2 cells, only a radiosensitizing condition with dFdCyd produced an increase in plasmid mutation frequency in A549 cells.

Discussion

Radiosensitization by antimetabolites, such as BrdUrd or 5-fluoro-2'-deoxyuridine, has traditionally been accounted for by two mechanisms: either the radiosensitizer (a) increased DNA DSBs before or with radiation or (b) inhibited the repair of DNA DSBs after irradiation. However, we and others have shown by pulsed-field gel electrophoresis that neither of these explanations can account for the radiosensitization observed with dFdCyd (10, 11). More recently, we have evaluated the effect of dFdCyd and radiation on induction of DNA DSBs using the more sensitive technique of measuring phosphorylated H2AX, which forms discrete foci in response to DNA damage, including DSBs (34, 35). These studies confirm that radiosensitization with dFdCyd is not due to an increase in DNA damage or a decrease in its repair. Our present study provides strong evidence that DNA mismatches are the lesions responsible for initiating radiosensitization by dFdCyd and further suggests that the MMR protein hMLH1 plays an integral role in this process. This is a novel paradigm that, to our knowledge, has not been shown as a mechanism for radiosensitization by other antimetabolite radiosensitizers.

The data presented here show that dFdCyd increased the mutation frequency in the HCT116 MMR-deficient and HCT116 MMR-proficient cells only at radiosensitizing concentrations (Fig. 1A). That the increase in mutation frequency is due to the imbalance of dNTP pools and not incorporation of the nucleotide analogue into DNA is supported by the finding that HU, a drug whose primary effect is the inhibition of RR, also increased the mutation frequency (Fig. 1D). Furthermore, araC, which has a hydroxyl substitution at the 2' position of the sugar compared with the difluoro substitution at that site on dFdCyd, can be incorporated into DNA but does not inhibit RR, failed to produce radiosensitization in the MMR-deficient cell line (Table 1) and did not increase plasmid mutation frequency in MMR-deficient or MMR-proficient cells (Fig. 1D).

The increased mutation frequency in HCT116 0-1 MMR-deficient cells after treatment with dFdCyd ± IR, or HU ± araC, is primarily the result of single-base substitutions, which would be predicted for cells with an imbalance in dNTP pools. Furthermore, the largest increase in base substitution events occurred at A:T sites under conditions where dFdCyd or HU was present, as expected for drugs that decrease dATP. The mutations produced with the combination of dFdCyd and IR, or HU and araC, resembled those observed in cells treated with dFdCyd or HU alone. Thus, the preponderance of single-base substitutions observed only after radiosensitizing concentrations of dFdCyd or HU strongly supports these as the lesions responsible for radiosensitization.

MMR has been implicated in cell cycle arrest following exposure to genotoxic agents (36), whereby failure to arrest in MMR-deficient cells results in the accumulation of unrepaired DNA damage. The examination of cell cycle effects in both MMR-deficient and MMR-proficient cells following treatment with dFdCyd, IR, and the combination of dFdCyd and IR shows that both cell lines undergo S-phase cell cycle arrest following dFdCyd treatment before reentering the cell cycle after washout. Furthermore, both cell lines exhibited a G2-M arrest in response to IR. Therefore, an inactive MMR system does not impart failure to arrest following dFdCyd, IR, or the combination, and therefore, a failure to arrest does not seem to underlie the mechanism of dFdCyd-mediated radiosensitization. These data are consistent with cell cycle data that we have published previously with dFdCyd and IR in other cell lines (18, 22, 25). Because both MMR-proficient and MMR-deficient cells continued to progress through the cell cycle after drug washout and/or irradiation (Table 3A; ref. 22), differences in mutation frequency cannot be attributed to inhibition of DNA synthesis. Therefore, the mutations that persisted 4 days only after dFdCyd and IR treatment likely represent both newly acquired mutations and initial mutations left unrepaired and strongly support the idea that these lesions form the basis for the decreased cell survival under radiosensitizing conditions.

Earlier reports have shown that, like dFdCyd, 5-iodo-2'-deoxyuridine and BrdUrd selectively radiosensitized MMR-deficient cells (37, 38). The authors attributed this difference to the lower incorporation of the thymidine analogues into DNA of MMR-proficient cells at equimolar concentrations, possibly through MMR-mediated recognition and excision of these lesions. In contrast, we observed higher incorporation of dFdCMP in DNA of MMR-proficient HCT116 cells at equimolar concentrations (22). The triphosphates of 5-iodo-2'-deoxyuridine and BrdUrd also inhibit pyrimidine production by RR (39), resulting in lowered dCTP and dTTP pools. If, as proposed here and previously (40), dNTP pool imbalances contribute to radiosensitization, such effects may also explain in part the observed differences in radiosensitization with 5-iodo-2'-deoxyuridine and BrdUrd in MMR-proficient and MMR-deficient cell lines.

Model studies in both MLH1-deficient and MSH2-deficient tumor cell lines have shown that MMR deficiency imparts cellular resistance to a variety of chemical agents used in cancer chemotherapy, most notably cisplatin (41, 42). Therefore, MMR status may be an important factor in resistance to cancer chemotherapy (43). Consistent with these studies, previously, we showed that the HCT116 MMR-proficient cells were more sensitive to dFdCyd compared with the isogenic HCT116 MMR-deficient cells (Table 1; ref. 22). Here, we show, in HCT116 MMR-proficient cells, that suppressing hMLH1 expression with specific siRNA decreased their sensitivity to dFdCyd. Thus, this shows directly that hMLH1 expression affects dFdCyd sensitivity.

To further support our hypothesis that hMLH1 and MMR play a role in dFdCyd-mediated radiosensitization, we returned the HCT116 1-2 MMR-proficient cell line to the hMLH1 deficiency status of the parental cell line via siRNA-mediated suppression of hMLH1 expression. Following hMLH1 suppression, we were able to radiosensitize these cells at a concentration of dFdCyd shown here and previously (22) to be unable to increase cytotoxicity by IR (Fig. 3C). Importantly, this result was not due to nonspecific effects of the siRNA, as dFdCyd was actually less cytotoxic in cells treated with the siRNA (Fig. 3B). We also decreased hMLH1 expression in MMR-proficient A549 non–small cell lung cancer cells. Although parental A549 cells were not radiosensitized by concentrations of dFdCyd <IC50, following siRNA-mediated suppression of hMLH1 protein expression, we were able to induce radiosensitization (Fig. 3F) at the noncytotoxic concentration of 10 nmol/L dFdCyd (Fig. 3E). Furthermore, radiosensitization in the A549 hMLH1-deficient cells was accompanied by an increase in plasmid mutation frequency. Data supporting a direct correlation between decreased hMLH1 expression, increased plasmid mutation frequency, and radiosensitization by dFdCyd in another cell line provide strong evidence that mismatched nucleotides are critical to dFdCyd-mediated radiosensitization.

Whether the function of hMLH1 in radiosensitization by dFdCyd is the result of its direct role in MMR or its role in other DNA repair pathways is not clear. Work by van Putten et al. (14) has shown that a functional nonhomologous end-joining pathway is not required for radiosensitization by dFdCyd, whereas Wachters et al. (13) showed that dFdCyd causes specific interference with HRR. An interaction between MMR and HRR has been shown (44), and both DNA repair pathways are affected by imbalances of dNTP pools (45). In addition, single-base changes have been shown to disrupt HRR by posing as a barrier to branch migration (46). That radiosensitization with dFdCyd does not occur through an alteration in DNA DSBs or their repair yet HRR seems to be involved may seem contradictory. However, a multitude of evidence indicates that HRR is also involved in the restarting of stalled replication forks, such as after HU treatment, and this activity is not dependent on the number of DNA DSBs (47). Taken together, the single-base mismatches showed herein to be caused by dFdCyd and persist only after irradiation likely cause disruption to HRR, which may prevent the restarting of stalled replication forks, resulting in a synergistic increase in cell death and hence radiosensitization. The selective suppression of other MMR and HRR proteins may help to further our understanding of the function of these repair pathways in radiosensitization by dFdCyd.

Most cell lines do not have a complete deficiency in MMR but rather express some level of MMR proteins, resulting in variable degrees of MMR proficiency (32), and many MMR-proficient cell lines are radiosensitized by dFdCyd (16–18, 25). Therefore, we hypothesize that, at low levels of dFdCyd, the existing MMR capability is sufficient to correct errors of replication resulting from dFdCyd-induced imbalance in dNTP pools, but at higher concentrations, the MMR capability is exceeded and mismatches will persist, resulting in radiosensitization. In the MMR-proficient cells, only a highly toxic concentration of dFdCyd (IC96, 300 nmol/L) produced radiosensitization (Table 1; ref. 22) with an accompanying increase in the mutation frequency in the pSP189 plasmid replicated in these cells (Fig. 1A), thereby supporting this hypothesis. Similarly, van Bree et al. (48) revealed little radioenhancement in MMR-proficient cells after long (24 h) incubations of low-concentration dFdCyd but greater radioenhancement using a high concentration in short duration, suggesting that MMR may be overwhelmed. Clinically, MMR protein expression in ovarian tumors before and after cisplatin treatment shows a decrease in the extent of expression, in particular, of hMLH1, following several cycles of treatment (49). The data presented here suggest that tumors with innate or acquired deficiency in hMLH1 would be most sensitive to radiosensitization with dFdCyd but that higher doses of drug could be used in hMLH1-expressing tumors to increase their sensitivity to dFdCyd and radiation.

Other drugs that affect nucleotide metabolism, such as 5-fluorouracil and the thymidine analogues (5-fluoro-2'-deoxyuridine, BrdUrd, and 5-iodo-2'-deoxyuridine), are among the most effective and widely used to sensitize tumor cells to radiation treatment. All of these antimetabolites are capable of depleting one or more of the endogenous dNTP pools, which could promote mismatches in DNA and enhance sensitivity to radiation, as shown here for dFdCyd and HU. We suggest hMLH1 status and MMR proficiency as a general mechanism governing radiosensitization by antimetabolites that produce an imbalance in dNTP pools. Because normal tissue should be MMR proficient, such agents could be used to selectively target MMR-deficient tumors clinically, which may enhance radiation sensitivity and improve the therapeutic index for these radiosensitizers.

Acknowledgments

We thank Dr. Michael Seidman for kindly providing the pSP189 plasmid vector, Dr. Françoise Praz for the generous gift of the HCT116 0-1 and 1-2 cell lines, Dr. Paul Boucher for critical reading of the manuscript, and Michael Im for his help with high-pressure liquid chromatography analysis.

Footnotes

Grant support: NIH grants CA83081 and CA76581.

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.

Received 1/30/07; revised 3/30/07; accepted 4/27/07.

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