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Clinical Cancer Research Vol. 12, 563-568, January 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

DNA Polymerase {zeta} Accounts for the Reduced Cytotoxicity and Enhanced Mutagenicity of Cisplatin in Human Colon Carcinoma Cells That Have Lost DNA Mismatch Repair

Xinjian Lin, Julie Trang, Tsuyoshi Okuda and Stephen B. Howell

Authors' Affiliation: Department of Medicine and the Cancer Center, University of California, San Diego, La Jolla, California

Requests for reprints: Stephen B. Howell, Department of Medicine 0058, University of California, San Diego, La Jolla, CA 92093. Phone: 858-822-1110; Fax: 858-822-1111; E-mail: showell{at}ucsd.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The mutagenicity of cis-diamminedichloroplatinum(II) (DDP; cisplatin) and the rate at which resistance develops with repeated exposure to DDP are dependent on mutagenic translesional replication across DDP DNA adducts, mediated in part by DNA polymerase {zeta}, and on the integrity of the DNA mismatch repair (MMR) system. The aim of this study was to determine whether disabling Pol {zeta} by suppressing expression of its hREV3 subunit in human cancer cells can reduce the mutagenicity of DDP and whether loss of MMR facilitates mutagenic Pol {zeta}-dependent translesional bypass. The HCT116+ch3 (MMR+/REV3+) and HCT116 (MMR–/REV3+) human colon carcinoma cell lines were engineered to suppress hREV3 mRNA by stable expression of a short hairpin interfering RNA targeted to hREV3. The effect of knocking down REV3 expression was to completely offset the DDP resistance mediated by loss of MMR. Knockdown of REV3 also reduced the mutagenicity of DDP and eliminated the enhanced mutagenicity of DDP observed in the MMR–/REV3+ cells. Similar results were obtained when the ability of the cells to express luciferase from a platinated plasmid was measured. We conclude that Pol {zeta} plays a central role in the mutagenic bypass of DDP adducts and that the DDP resistance, enhanced mutagenicity, and the increased capacity of MMR–/REV3+ cells to express a gene burdened by DDP adducts are all dependent on the Pol {zeta} pathway.

DNA damage is continually induced by both exogenous sources such as UV light and chemical agents and endogenous sources such as oxygen-free radicals. Whereas there are many DNA repair mechanisms (1), not all lesions can be completely repaired in all cells before DNA replication is initiated. Mutations generated during translesional synthesis are thought to contribute to malignant transformation (2). Whereas the major replicative DNA polymerase has a very limited translesional synthesis capacity, a family of DNA polymerases that can mediate such bypass replication has recently been identified in mammalian cells. This family includes Pol {zeta}, Pol {eta}, Pol {kappa}, Pol µ, and Pol {iota} (3). REV3, the catalytic subunit of Pol {zeta}, was first identified in Saccharomyces cerevisiae by isolation of mutant strains that displayed reduced frequencies of UV-induced mutagenesis (4). Analysis of REV3-null mutants revealed that a large fraction of all mutations induced by DNA-damaging agents, and the majority of spontaneous mutations, are attributable to the activity of Pol {zeta} (5). We and others have found that inhibition of the expression of the REV3 subunit in cultured human fibroblasts, by expression of an antisense REV3 mRNA, reduced UV- and cis-diamminedichloroplatinum(II) (DDP)–induced mutagenesis, indicating that Pol {zeta} also plays a crucial role in mutagenesis in mammalian cells (6, 7).

In addition to being involved in carcinogenic mutagenesis, the activity of Pol {zeta} is of concern with respect to the use of chemotherapeutic agents such as the DNA-damaging agent DDP. The most abundant lesions produced in DNA by DDP 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 (8, 9) and mammalian cells (10–14). Whereas DDP produces gross chromosomal changes (15) and probably gene amplification (16), the molecular basis for much of its mutagenicity is believed to be related to bypass replication across DDP adducts by the eukaryotic DNA polymerase ß, Pol {zeta}, and/or members of the Y family of polymerases containing Pol {iota} and Pol {eta} (17–19). There is accumulating evidence that in bacteria and yeast, when non-error-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, error-prone DNA polymerases to bypass DNA lesions that block normal replicative polymerases (20). This seems to be an important contributor to the mutagenicity of DDP adducts (21). In most attempts at replication of a DDP adducted template, the replicative DNA polymerases stop just before the 5' adducted G or after incorporation of a base opposite the 3' G of the GG adduct. Perhaps because the two G's are splayed apart by the adduct (22), further nucleotide incorporation stops. Occasionally, a polymerase is able to synthesize across the lesion and this results in infrequent misincorporation of noncomplementary bases that, if left unrepaired, generate point or frameshift mutations. Among the nine novel DNA mutases that are capable of replicating across various types of DNA damage that are now known (3), Pol {zeta} is of particular interest (23, 24). Pol {zeta} is not very good at adding the correct nucleotide opposite an adduct but it is the only mutase thus far identified that can continue to add more bases when the 3' end of the daughter strand is not very well matched with the template strand (25). Pol {zeta} seems to work in concert with one of several other mutases to accomplish bypass replication. In the case of DDP adducts, it is likely that Pol {zeta} works in concert with either Pol {iota} (25) or Pol {eta} (26, 27). Both Pol {iota} and Pol {eta} are able to insert bases opposite an adduct; Pol {iota} often inserts incorrect bases whereas Pol {eta} usually inserts correct bases (25, 27). However, neither is able to extend the daughter strand further in the absence of Pol {zeta}, the major role of which seems to be polymerization of only a small number of additional bases before handing the job of further extension back to one of the major replicative polymerases (e.g., Pol {delta}; ref. 25).

An important replication-associated correction function is provided by the postreplicative DNA mismatched repair/MMR system. MMR has been proposed to play a major role in the correction of DNA polymerase errors either by preventing error-prone bypass replication or by correcting the mismatches that are formed (28–31). Loss of MMR activity is well documented to result in increased mutation rates and instability of genome. Inactivation of MMR also augments the intrinsic mutagenicity of DDP and enhances the risk of developing cells highly resistant to either DDP itself or other drugs commonly used in combination with DDP (14). Deficiency of MMR in colon carcinoma cell lines has been reported to result in increased replicative bypass across DDP adducts (32). A very recent study has reported that MMR-deficient HCT116 cells are more sensitive to general DNA polymerase inhibitors, including aphidicolin, gemcitabine, and hydroxyurea, than MMR-proficient HCT116+ch3 cells, suggesting that MMR may be intimately involved in DNA polymerization (33). However, no direct link between MMR status, mutagenic bypass replication mediated by DNA polymerase {zeta} across DDP-DNA adducts, and the emergence of DDP resistance has been reported. In this study, we tested the hypothesis that many of the mutations induced by DDP are attributable to errors made by Pol {zeta} during synthesis across DDP adducts in DNA and that this mutagenic bypass is augmented when MMR is defective. We have molecularly engineered HCT116+ch3 (MMR+/REV3+) and HCT116 (MMR–/REV3+) colon carcinoma cells to suppress hREV3 expression by stable expression of a short hairpin RNA targeting hREV3 mRNA. We report here that loss of MMR caused increased reliance on mutagenic Pol {zeta}-mediated adduct bypass for cell survival and that loss of MMR enhanced the Pol {zeta}-dependent generation of drug-resistant variants in the surviving population.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
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Drugs. DDP was a gift from Bristol-Myers Squibb (Princeton, NJ). A stock solution of 1 mmol/L DDP in 0.9% NaCl was stored in the dark at room temperature. 6TG was purchased from Sigma Chemical Co. (St. Louis, MO) and dissolved in 0.2 N sodium hydroxide to form a 20 mmol/L stock solution and stored at –20°C.

Design and cloning of short hairpin RNAs. Short hairpin RNAs were designed using software available from the website of the laboratory of Dr. Gregory Hannon (http://www.cshl.org/public/SCIENCE/hannon.html). Two complementary {approx}72-nucleotide DNA oligonucleotides, short hairpin RNA sequences targeted to the sequence 5'-TAGTAGTCTGCAGTCACTATCCTTACTGGAAGCTTGCGGTGAGG ATAGTGACTGCGGACTATTACATTTTTTT-3' in REV3 mRNA, were annealed and cloned directly into the plasmid vector pSHAG-1 (34). Using the Gateway system (Invitrogen, Carlsbad, CA), the fragment containing U6 promoter–driven DNA oligonucleotides coding for the short hairpin RNAs was transferred to the destination vector pMSCV-PIG by clonase reaction (Invitrogen-Gateway LR Clonase Mix) according to the instructions of the manufacturer. The mammalian expression vector pMSCV-PIG was constructed by Dr. Hannon's Laboratory1 containing the cassette attR1-ccdB-attR2 for LR reaction, puromycin-resistant gene for stable selection, and enhanced green fluorescent protein (EGFP) gene as a reporter. The insert in the resultant vector, named pMSCV-PIG-shREV3, was sequence verified.

Cell culture, transfection, and selection. The hMLH1-deficient HCT116 cell line was obtained from the American Type Culture Collection (ATCC CCL 247); HCT116 contains a hemizygous mutation in hMLH1 resulting in a truncated, nonfunctional protein (35). A subline complemented with chromosome 3 (HCT116+ch3) was obtained from Drs. C.R. Boland and M. Koi, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan. The chromosome 3–complemented cells were competent in DNA mismatch repair (36). These two cell lines were maintained in Iscove's modified Dulbecco's medium (Irvine Scientific, Irvine, CA) supplemented with 2 mmol/L L-glutamine and 10% heat-inactivated fetal bovine serum. The chromosome-complemented HCT116+ch3 was maintained in medium containing 400 µg/mL geneticin (Life Technologies, Inc., Grand Island, NY). Cells were transfected with pMSCV-PIG-shREV3 using FuGENE 6 (Roche, Indianapolis, IN) following the recommendations of the manufacturer and then selected by continuous exposure to 1 µg/mL puromycin 48 hours after transfection. When drug-resistant colonies had formed, they were isolated, expanded, and screened for the extent of EGFP expression by flow cytometry using a Becton Dickinson (Mountain View, CA) FACScan equipped with an argon ion laser tuned to 488 nm to excite EGFP and a 515/545 nm bandpass filter to monitor the green fluorescence. The clone from each cell line that had the highest percent of EGFP-expressing cells in the population among all the puromycin-surviving clones for each line was subjected to fluorescence-activated cell sorting, and the 5% of the population with the brightest green fluorescence was selected and grown to mass culture in medium supplemented with 1 µg/mL puromycin for all subsequent experiments. The sublines grown from these cells were designated here as HCT116+ch3-kdREV3 and HCT116-kdREV3, respectively. They are referred to in this article by their phenotypes: HCT116+ch3-kdREV3, MMR+/REV3–, HCT116-kdREV3, MMR–/REV3–.

Quantification of hREV3 mRNA by real-time PCR. Total RNA was extracted with TRIzolR reagent (Invitrogen). First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen) and random primers. Real-time PCR was done using the Bio-Rad iCycler iQ detection system in the presence of SYBR Green I dye (Bio-Rad Laboratories, Inc., Hercules, CA). For the REV3 gene expression, the forward (5'-TGATGTCTTCAGCTGGTATCATGA-3') and reverse (5'-CCGCCCTTCAGGTTCACTT-3') primers were used for amplification with a iCycler protocol consisting of a denaturation program (95°C for 3 minutes), amplification and quantification program repeated 40 times (95°C for 10 seconds and 55°C for 45 seconds), and melting curve analysis. The data were analyzed by using the comparative Ct method, where Ct is the cycle number at which fluorescence first exceeds the threshold. The {Delta}Ct values from each cell line were obtained by subtracting the values for 18S Ct from the sample Ct. A 1-unit difference of Ct value represents a 2-fold difference in the level of mRNA.

Clonogenic assay. Clonogenic assays were done by seeding 1,000 cells into 60-mm plastic dishes in 5 mL of complete media. After 24 hours, appropriate amounts of DDP were added to the dishes and the cells were exposed for 1 hour. Thereafter, the cells were washed and fresh drug-free medium was added. Colonies of at least 50 cells were visually scored after 10 to 14 days. Each experiment was done at least thrice using triplicate cultures for each drug concentration. IC50 values were determined by log-linear interpolation.

Measurement of DDP mutagenicity. The sensitivity of cells to the mutagenic effects of DDP was measured by determining the frequency of variants highly resistant to 6TG in the surviving population 21 days after a 1-hour exposure to 10 µmol/L DDP as previously reported (14, 37). Aliquots of the surviving population were seeded into each of ten 100-mm tissue culture dishes at 100,000 cells per dish in the presence of 10 µmol/L 6TG. At the same time, 1,000 cells were seeded into each of three 60-mm dishes in drug-free medium for determination of cloning efficiency. After 14 days, colonies were counted after staining with 0.1% crystal violet. The frequency of highly drug-resistant variants was calculated as follows: variant frequency = a / (b x 106), where a is the number of colonies present in the 10 drug-treated dishes and b is the cloning efficiency. Each experiment was done at least thrice. Before testing for 6TG-resistant variants, the cells were grown in hypoxanthine-aminopterin-thymidine medium containing 0.4 µmol/L aminopterin, 16 µmol/L thymidine, and 100 µmol/L hypoxanthine for a minimum of 14 days before testing to remove preexisting hypoxanthine-guanine phosphoribosyltransferase mutants.

Plasmid reactivation assay. The pRL-CMV mammalian expression vector (Promega, Madison, WI) containing the 935-bp Renilla luciferase cDNA produces high-level Renilla luciferase expression in transfected mammalian cells. Thirty micrograms of plasmid DNA were dissolved in buffer containing 10 mmol/L Tris and 1 mmol/L EDTA (pH 7.4) and incubated with 5 µmol/L DDP at 37°C for 3 hours. The platinated DNA was then purified by ethanol precipitation and unbound free drug was removed. This procedure resulted in plasmid DNA that was >90% supercoiled as verified by gel electrophoresis. The platination procedure yielded 1.5 ± 1.4 pg/µg DNA, which is equivalent to 9.3 adducts per plasmid or 3.2 adducts per Luc-coding region and promoter as previously reported (38). Similar levels of platination have previously been shown not to affect the efficiency of transfection (39). One microgram of randomly platinated or unplatinated pRL-CMV vector was transfected for 6 hours into the cells as described above. Subsequently, DNA was washed off and fresh medium was added. Twenty-four hours after transfection, duplicate samples were washed with ice-cold PBS and then lysed in a Renilla Luciferase Assay Lysis Buffer (Promega). Cell lysates were stored at –70°C until assayed. Luciferase activity was measured in cell lysates with a Monolight 2010 Luminometer (Analytical Luminesence Laboratory, San Diego, CA) using Renilla Luciferase Assay System (Promega).


   Results
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 Materials and Methods
 Results
Discussion
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Generation and characterization of hREV3 knockdown cells. MMR-proficient and MMR-deficient HCT116 sublines were transfected with the pMSCV-PIG-shREV3 vector which expresses three major components: a puromycin-resistant gene to permit facile selection of transfected clones; an EGFP to permit further selection by cell sorting; and a sequence from which a short hairpin RNA is transcribed that is subsequently processed into an interfering RNA targeted to REV3. Individual puromycin-resistant clones were expanded and the resulting populations were checked for EGFP expression. The clones with the highest percent of EGFP-expressing cells in the population were further subjected to cell sorting to isolate the 5% of the population with the brightest green fluorescence. The resulting MMR+/REV3– and MMR–/REV3– cell lines were used for subsequent experiments. The REV3 mRNA level was measured by quantitaive real-time PCR in the parental cell lines and REV3 knockdown sublines. Interestingly, the steady-state level of hREV3 mRNA in MMR–/REV3+ cells was already 2.5-fold higher (P < 0.01) than in MMR+/REV3+ cells. Because no sufficiently specific antibody to REV3 has yet been developed, it was not possible to determine whether the higher level of hREV3 mRNA was accompanied by an increased level of REV3 protein, but the higher level of hREV3 mRNA suggests that proficiency of the MMR system directly influences the abundance of hREV3. As shown in Fig. 1, the steady-state endogenous hREV3 mRNA level in MMR+/REV3– and MMR–/REV3– cells was found to be reduced to 30 ± 0.5% and 10 ± 0.2% (P < 0.01 for both), respectively, of that in MMR+/REV3+ cells.


Figure 1
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  		Fig. 1. Relative hREV3 mRNA levels in MMR+/REV3+, MMR+/REV3–, MMR–/REV3+, and MMR–/REV3– cells. The expression of hREV3 mRNA was assessed by real-time PCR using the Bio-Rad iCycler iQ detection system in the presence of SYBR Green I dye. The relative expression level of hREV3 was determined by normalizing the {Delta}Ct value to the MMR+/REV3+ cell value. The level of hREV3 mRNA in the MMR+/REV3+ cells was arbitrarily set to 1.

 
Effect of the knockdown of REV3 and the loss of MMR on sensitivity to the cytotoxic effect of DDP. Clonogenic assays were used to determine the effect of loss of MMR, REV3, or both on sensitivity to the cytotoxic effect of DDP. Figure 2 shows the survival of the four cell lines as a function of drug concentration following a 1-hour exposure to DDP. As shown in Table 1, the mean ± SE IC50 values were 12.1 ± 0.8, 7.7 ± 0.4, 23.0 ± 0.5, and 7.7 ± 1.2 µmol/L for the MMR+/REV3+, MMR+/REV3–, MMR–/REV3+, and MMR–/REV3– cells, respectively. Suppression of REV3 expression in the MMR+/REV3– cells increased sensitivity to DDP by only 1.6-fold. This result is consistent with the greater reduction in steady-state REV3 mRNA from baseline in the MMR-deficient than in the MMR-proficient cells. As previously reported (40), and confirmed by the current results, loss of MMR in this model system resulted in 2-fold resistance to DDP. The effect of knocking down REV3 expression was to completely offset the resistance mediated by loss of MMR and reduce the IC50 value to the same level as that observed when REV3 expression was knocked down in the MMR-proficient cells. It is noteworthy that there was a strong correlation between the relative REV3 mRNA level and the IC50 values (r2 = 0.990, P < 0.01), suggesting that even small changes in REV3 mRNA have a biological effect.


Figure 2
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  		Fig. 2. DDP concentration-survival curves for the MMR+/REV3+ ({blacktriangleup}), MMR+/REV3– ({bigtriangleup}), MMR–/REV3+ ({blacksquare}), and MMR–/REV3– ({square}) cells. Points, mean of three experiments each done with triplicate cultures; bars, SE.

 

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  		Table 1. Effect of the loss of MMR, REV3, or both on DDP cytotoxicity, mutagenicity, and platinated plasmid reactivation

 
Effect of the knockdown of REV3 and the loss of MMR on the ability of DDP to generate resistant variants. DDP is a mutagen in human cells (10, 12–14) and generates mutations that result in high-level resistance to many classes of drugs (14). To determine the role of hREV3 in DDP-induced mutagenesis in the context of adequate or inadequate MMR function, the four cell lines were exposed to 10 µmol/L DDP for 1 hour and then, 14 days later, the fraction of clonogenic cells showing high-level resistance to 6TG was determined. As shown in Table 1 and Fig. 3, DDP produced ~ 2.5-fold more 6TG-resistant clones per 106 clonogenic cells in the surviving MMR-deficient cells than in the MMR-proficient cells. In MMR+/REV3+ cells, knockdown of hREV3 had only a modest effect on the ability of DDP to generate variants resistant to 6TG; it reduced the frequency of such clones by 37.5 ± 3.1% (mean ± SE). In contrast, knockdown of hREV3 in the MMR-deficient cells had a more profound effect on DDP mutagenicity and decreased the generation of resistant variants by 58 ± 0.5% (mean ± SE). Thus, reduction of REV3 mRNA produced parallel effects on sensitivity to both the cytotoxic and mutagenic effects of DDP that were proportional to the magnitude of the relative degree of REV3 knockdown. The greater effect of REV3 knockdown in the MMR-deficient cells might be attributed to the lower level of REV3 mRNA in MMR–/REV3– than in MMR+/REV3– cells; however, the fact that the absolute frequency of 6TG-resistant cells was higher in the MMR–/REV3– than in MMR+/REV3– cells indicates that loss of MMR may allow other non-REV3-dependent mutagenic pathways to become involved in processing adducts. This concept is supported by the fact that the correlation between REV3 mRNA level and 6TG mutant frequency was not strong (r2 = 0.779, P < 0.05). Nevertheless, these results indicate that REV3 abundance is important both when MMR is fully functional and when it is disabled, suggesting that the highly error-prone Pol {zeta} pathway is as important, or perhaps even more important, to the mutagenic bypass of DDP adducts in the MMR-deficient as in the MMR-proficient cells.


Figure 3
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  		Fig. 3. Effect of the knockdown of hREV3, loss of MMR, or both on the ability of DDP to generate drug-resistant variants in the surviving population. The number of 6TG-resistant colonies per 106 clonogenic cells was scored on day 14 after a 1-hour exposure to 10 µmol/L DDP. Columns, mean of three experiments; bars, SE.

 
Effect of REV3 and MMR on plasmid reactivation. There is no practical way of measuring the relative activity of the purified Pol {zeta} complex in the intact cells, but the overall capacity to synthesize a functional protein from a gene encumbered by DDP adducts can be assessed 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 DDP before transfection. Thus, the effect of loss of REV3 and/or MMR on the function of a gene inactivated by DDP adducts can be examined by comparing the ability of the MMR+/REV3+, MMR+/REV3–, MMR–/REV3+, and MMR–/REV3– cells to express luciferase following transfection of an unplatinated or platinated form of the same luciferase expression vector. All four cell lines expressed equivalent levels of luciferase activity following transfection with nonplatinated vector. The data presented in Table 1 show that when the platinated vector was transfected into the MMR–/REV3+ subline, there was little impairment [1.3 ± 0.03 (SE)-fold reduction] of the generation of luciferase activity, indicating substantial capacity to synthesize the protein despite the initial presence of adducts in the coding sequence. However, when the same platinated vector was transfected into the MMR+/REV3+ subline, the luciferase activity was significantly reduced by 2.6 ± 0.05 (SE)-fold. This result is consistent with our prior finding that the MMR-proficient cells are less capable of generating luciferase activity from the platinated vector than the MMR-deficient cells (38). Notably, when REV3 expression was knocked down, luciferase expression was impaired to the same degree in MMR-proficient and MMR-deficient cells. Thus, both the basal capacity to express luciferase from a platinated plasmid observed in the MMR-proficient cells and the enhanced capacity observed in the MMR-deficient cells are dependent on Pol {zeta} activity. Knockdown of REV3 mRNA completely eliminated the enhanced capacity in MMR-deficient cells. As noted for sensitivity to the cytotoxic effect of DDP, there was a good correlation between the relative REV3 mRNA levels in the four cell variants and the extent of platinated plasmid reactivation (r2 = 0.987, P < 0.01), as well as between the IC50 values and plasmid reactivation (r2 = 0.999, P < 0.01), suggesting that these three variables are reflecting the activity of the same process.


   Discussion
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References
 
A single exposure of colon cancer cells to DDP results in the appearance of a large number of highly drug-resistant clones in the surviving population (14, 30, 37). This phenomenon is observed when testing for resistance to DDP itself and to a wide variety of other classes of drugs, including 6TG, etoposide, topotecan, paclitaxel, and gemcitabine (14). The number of resistant clones is directly related to the concentration of DDP used to mutagenize the cells (14, 37). As measured by this type of assay, loss of MMR renders colon carcinoma cells more resistant to the cytotoxic effect of DDP but more sensitive to DDP-induced mutagenicity (14), consistent with the concept that when MMR is lost, the cell relies more heavily on bypass replication to complete duplication of a genome loaded with DDP adducts. We now have substantial evidence that Pol {zeta} plays a key role in this mutagenic bypass of DDP adducts. Suppression of REV3 mRNA levels using an antisense mRNA in human fibroblasts both enhanced sensitivity to the cytotoxic effect of DDP and reduced its mutagenicity (7). This has been interpreted as indicating that Pol {zeta} helps preserve viability by allowing completion of DNA synthesis but at the price of introducing additional mutations in the genome, some of which result in drug resistance. The higher REV3 mRNA levels in the MMR-deficient cells raise the question of whether the DDP resistance that accompanies loss of MMR function is really due to secondarily enhanced Pol {zeta} activity and increased bypass rather than to tolerance of DDP-induced damage caused by the failure of the MMR system to recognize or process the DNA adducts.

The results of the current study provide further evidence for a central role of Pol {zeta} in mediating DDP-induced mutagenicity. In the MMR-proficient cells, suppression of REV3 expression clearly reduced the ability of DDP to generate highly 6TG-resistant clones in the surviving population and impaired the ability of the cell to express luciferase from a platinated plasmid. The magnitude of the reduction of REV3 mRNA was somewhat greater in MMR-deficient than in MMR-proficient cells; thus, one cannot determine from these data whether MMR-deficient cells are even more dependent on Pol {zeta} than MMR-proficient cells. However, we can firmly conclude that REV3, and by extension Pol {zeta}, is important to DDP-induced cytotoxicity and mutagenicity in both MMR-proficient and MMR-deficient fully malignant colon carcinoma cells.

The MMR–/REV3+ cells were found to be 3.7 ± 1.3-fold more sensitive to the mutagenic effect of DDP than the MMR+/REV3+ cells over a wide range of DDP concentrations when mutagenicity was scored on the basis of the generation of 6TG-resistant variants (30). We have previously noted that loss of MMR is associated with enhanced capacity to express a protein from an adducted gene (38). The current results confirm this observation. One interpretation is that MMR normally suppresses bypass replication and that, when MMR is lost, Pol {zeta} is freed to act on a larger proportion of adducts remaining in the genome. Although this may be true, the current study showed that the steady-state level of REV3 mRNA was also markedly increased in MMR-deficient cells. This suggests the alternative explanation that there is simply more Pol {zeta} available in these cells. The lack of a sufficiently specific antibody to REV3 protein precludes direct determination of whether REV3 protein is increased in proportion to its mRNA. However, irrespective of how the level of REV3 protein is altered, the higher level of REV3 mRNA in the MMR-deficient cells indicates a regulatory interconnection between MMR and the Pol {zeta}-mediated bypass mechanism.

In previous studies, we have shown that suppression of REV3 mRNA expression is associated with impaired homologous recombination between two genes on a transfected plasmid, suggesting that REV3 plays an important role in this DNA repair mechanism as well as in adduct bypass (7). Although loss of MMR generally increases homologous recombinational repair, the presence of a DDP adduct in one strand has been reported to reduce recombination (41), providing evidence that homologous recombination is an unlikely source of most of the DDP-induced mutations.

When MMR is fully active and the Pol {zeta}-dependent mechanism bypasses an adduct, the MMR protein complex would be expected to recognize and bind to the adduct again and initiate another round of Pol {zeta}-dependent bypass replication. This would be expected to give the Pol {zeta} pathway many chances to insert an incorrect base and thus amplify the mutagenicity of a single adduct. However, loss of MMR renders DDP more, not less, mutagenic when REV3 is available. Because DDP-induced mutagenesis is still highly dependent on REV3 in MMR-deficient cells, potential futile cycling of MMR seems to be less important than the ability of MMR to suppress Pol {zeta}-dependent bypass. The MMR protein complex recognizes and binds to DDP adducts in DNA (42–44). Thus, this suppression may be the result of physical blockade by the MMR proteins of the access of Pol {zeta} to adducts.

The central role played by Pol {zeta} in regulating the mutagenicity of DDP has substantial clinical importance. We have recently shown that suppression of the expression of either REV3 or REV1, a separate polymerase that supports the activity of Pol {zeta}, reduces the rate at which DDP resistance evolves when a population of cells is repeatedly exposed to DDP on a schedule that mimics the way DDP is used in the clinic (7, 45). Thus, pharmaceutical inhibition of Pol {zeta} would be expected to both render tumors more sensitive to DDP and reduce the rate at which resistance evolves during a course of treatment. The results reported here indicate that Pol {zeta} is an important target for reducing the rate of development of resistance and that it is an even more important target in tumors in which MMR has been lost.

In summary, we conclude that Pol {zeta} plays a central role in the mutagenic bypass of DDP adducts and that the DDP resistance, enhanced mutagenicity, and the increased capacity of MMR–/REV3+ cells to express a gene burdened by DDP adducts are all dependent on the Pol {zeta} pathway. The good correlations between REV3 mRNA level and both DDP IC50 and plasmid reactivation and between the latter two variables provide evidence that each quantitatively reflects the activity of a highly REV3-dependent process.


   Footnotes
 
Grant support: NIH grant CA78648 and in part by a grant from the Foundation for Research.

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: Drs. X. Lin and S.B. Howell are Foundation for Research investigators.

1 Unpublished data. Back

Received 6/24/05; revised 11/ 3/05; accepted 11/ 8/05.


   References
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 Abstract
 Materials and Methods
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 Discussion
 References
 

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