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Carcinogenesis Advance Access originally published online on May 23, 2007
Carcinogenesis 2007 28(10):2089-2095; doi:10.1093/carcin/bgm125
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Adenomatous polyposis coli-mediated hypersensitivity of mouse embryonic fibroblast cell lines to methylmethane sulfonate treatment: implication of base excision repair pathways

Chanakya N. Kundu, Ramesh Balusu, Aruna S. Jaiswal and Satya Narayan*

Department of Anatomy and Cell Biology and UF Shands Cancer Center, University of Florida, Gainesville, FL 32610, USA

* To whom correspondence should be addressed. Tel: +1 352 273 8163; Fax: +1 352 273 8285; Email: snarayan{at}ufl.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
The role of adenomatous polyposis coli (APC) has been implicated in various cellular functions including cell migration, cell–cell adhesion, cell cycle control, chromosomal segregation and apoptosis. Recently, we discovered a novel role of APC in DNA base excision repair (BER) and showed that APC interacts with DNA polymerase ß (Pol-ß) and flap endonuclease 1 and interferes long-patch base excision repair (LP-BER) by blocking strand displacement synthesis. Many times, the chemotherapeutic drugs induce DNA alkylation damage, which is primarily repaired by the BER pathway. Thus, the efficacy of such drugs can be increased by APC resulting in the blockage of LP-BER. In the present study, we tested this hypothesis by using isogenic wild-type and Pol-ß-knockout mouse embryonic fibroblast (MEF) cell lines in which the Apc gene was knocked down by the small interfering RNA technique and treated with methylmethane sulfonate (MMS). The MEF-Apc(WT)/Polß–/– cells were hypersensitive to MMS treatment compared with the MEF-Apc(WT)/Polß+/+ cells. However, once the Apc gene was knocked down, these cells became more resistant to MMS treatment, suggesting that the MMS-induced hypersensitivity was associated with Apc. We then determined whether the hypersensitivity of MEF-Apc(WT)/Polß–/– and MEF-Apc(WT)/Polß+/+ cell lines were due to decreased Pol-ß-independent and Pol-ß-dependent LP-BER pathways, respectively. The results of in vivo and in vitro LP-BER assays supported our findings. Furthermore, Apc-mediated hypersensitivity to MMS treatment was correlated with increased apoptosis of MEF-Apc(WT)/Polß–/– and MEF-Apc(WT)/Polß+/+ as compared with MEF-Apc(KD)/Polß–/– and MEF-Apc(KD)/Polß+/+ cells. These results suggest that an increased level of Apc can increase the efficacy of DNA-alkylating drugs used as a curative therapy.

Abbreviations: AP, apurinic/apyrimidinic; Apc, adenomatous polyposis coli; APE, apurinic/apyrimidinic endonuclease; BER, base excision repair; dRP, deoxyribose phosphate; Fen-1, flap endonuclease 1; LP-BER, long-patch base excision repair; MEF, mouse embryonic fibroblast; MMS, methylmethane sulfonate; Pol-ß, DNA polymerase ß; SP-BER, short-patch base excision repair


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
DNA-alkylating agents are widely distributed environmental mutagens and carcinogens (1). On the other hand, several DNA-alkylating drugs are used in cancer chemotherapy (2,3). Insight into the molecular mechanisms of cellular defense against DNA-alkylating agents could impact important public health issues, such as risk evaluation, dose-risk considerations, adaptive phenomena and increase in the efficacy of cancer chemotherapy. DNA-alkylating agents induce a variety of DNA lesions, which may have individual contributions to cytotoxicity and various genotoxic end points, including chromosomal aberrations that are not yet fully understood (4). Data obtained from agent comparison, molecular dosimetry and genetically engineered cell lines indicate that both O- and N-alkylation products of DNA bases are genotoxic and that the extent of toxicity depends on the agent’s pharmacokinetic properties as well as the repair capacity of the cell (5,6). N-alkylation lesions, which represent the majority of alkylation-induced DNA damage (7), are removed from DNA by the base excision repair (BER) pathway. The BER system of both prokaryotes and eukaryotes is directed toward the repair of modified bases and strand breaks (8). It is mediated through two pathways that are differentiated by the size of the repair gap and the enzymes involved (9,10). One pathway is designated as ‘single nucleotide or short-patch base excision repair (SP-BER)’. The other pathway is known as ‘multiple nucleotide (2–11) or long-patch base excision repair (LP-BER)’. In both pathways, repair is initiated by the removal of the damaged base by a DNA glycosylase, leaving an abasic or apurinic/apyrimidinic (AP) site. Based upon the type of nucleotide damage, there are several mammalian DNA glycosylases including 3-methyladenine-DNA glycosylase (AAG), 8-oxoguanine-DNA glycosylase (OGG), endonuclease II-like (Nth) DNA glycosylase and the endonuclease VIII-like (NEIL) DNA glycosylase (11). These glycosylases can be either monofunctional, which remove the damaged base and create an AP site, or bifunctional which additionally catalyze strand incision at the AP site after damaged base removal. In the case of monofunctional DNA glycosylase activity, the generated AP site is recognized by apurinic/apyrimidinic endonuclease (APE) which creates an incision at the 5' end of the AP site and generates a 3'-OH and 5'-deoxyribose phosphate (dRP) termini in the gap. Then DNA polymerase ß (Pol-ß), which also possesses the 5'-dRP lyase activity, synthesizes the correct nucleotide and removes the 5'-dRP moiety leaving the 5'-phosphate for DNA ligase I or III to seal the gap (9). This is called the SP-BER. However, this becomes complicated once the abasic site becomes oxidized or reduced. In this case, the 5'-dRP lyase activity of Pol-ß is interrupted and the repair of DNA is accomplished through LP-BER. Under these circumstances, the Pol-ß-dependent strand displacement synthesis generates a longer repair patch and a single-nucleotide DNA flap with a modified sugar at its 5' end. The DNA flap is cleaved by flap endonuclease 1 (Fen-1) and the gap is sealed by DNA ligase I or III (9). In the absence of Pol-ß, the strand displacement synthesis can be accomplished by an alternative pathway involving Pol-{delta}/{epsilon} (12,13). However, the Fen-1 activity is common in both Pol-ß- or Pol-{delta}/{epsilon}-dependent LP-BER pathways and thus can play an important role in the DNA alkylation damage-induced cytotoxicity of cells.

Germ line knockouts, resulting in a deficiency in any one of the BER proteins, such as Pol-ß, APE, Fen-1 or DNA ligase I are all embryonically lethal (14,15). This suggests that a correct performance of BER is highly important for development. Although Pol-ß-deficient null mice are not viable (15), the corresponding embryonic cells survive in culture (16), indicating that Pol-ß is not essential for cell viability. However, Pol-ß is required in response to genotoxic stress, as indicated by the fact that pol-ß-deficient cells are hypersensitive to the cytotoxic effect of methylating agents (16). It is well known that Pol-ß-knockout cells are dramatically more sensitive than wild-type cells with regard to chromosomal breakage induced by various mono- and bifunctional DNA-alkylating agents, displaying a higher frequency of apoptosis and necrosis (16).

Mutations in the adenomatous polyposis coli (APC) gene are the earliest events in the development of colorectal carcinogenesis (17). The APC gene contains 8535 nucleotides which encodes 2843 amino acids or a 312 kDa protein. Most of the somatic mutations are clustered between codons 1284 and 1580, also called the mutation cluster region (17,18). APC plays a diversified role in a broad spectrum of functions ranging from cell adhesion to cell migration, Wnt/ß-catenin signaling (18), cell cycle control (18,19), apoptosis regulation (19,20) and chromosomal segregation (21). Our recent findings implicate another role of APC in the regulation of BER (22–25), which we are currently pursuing in our laboratory to establish its significance in biological functions.

We have previously shown that APC gene expression is induced in cancer and normal breast and colon epithelial cell lines upon exposure to the DNA-alkylating agents, N-methyl-N'-nitro-N-nitrosoguanine, methylmethane sulfonate (MMS) and dimethylhydrazine, as well as the cigarette smoke carcinogen, dimethylbenzanthracine (25–28). Recently, we have shown that APC interacts with Pol-ß and Fen-1 and blocks LP-BER by blocking strand displacement synthesis (22,25). We described that the altered level of APC in different breast cancer cell lines was associated with the altered LP-BER. Our studies suggested a role of increased level of APC in compromised LP-BER in benzo[a]pyrene and cigarette smoke condensate-induced transformation of pre-malignant breast epithelial cells (24). In the present communication, we report that MMS induces significant DNA damage that is repaired by the BER pathway. We present evidence that the increased level of Apc protein causes hypersensitivity of MEF cells to MMS due to decreased LP-BER by both Pol-ß-dependent and Pol-ß-independent pathways.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell lines and treatment
Isogenic mouse embryonic fibroblast (MEF) cell lines containing either the wild-type (MEF-Polß+/+) or Pol-ß gene knockout (MEF-Polß–/–) were grown in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin and 80 µg/ml of hygromycin B at 37°C under a humidified atmosphere of 5% CO2 (29). These cell lines were developed from primary cultures of wild-type or Pol-ß-deficient embryonic fibroblasts transfected with the DNA plasmid construct ptsA58H, which contains a coding sequence for both a hygromycin-resistant gene and immortalized with the DNA plasmid construct tsA58, which expresses a temperature-sensitive SV40 Tag (29). After 70% confluence, cells were treated with MMS (Sigma–Aldrich Chemicals Co., St Louis, MO), as indicated in figure legends.

Silencing of Apc in MEF cell lines
The wild-type Apc gene expression was silenced in MEF-Polß+/+ and MEF-Polß–/– in cell lines by the procedure described earlier (25). Briefly, for Apc gene silencing, the small interfering RNA sense and anti-sense oligonucleotides 5'-GATCCGCAACAGAAGCAGAGAGGTTTCAAGAGAACCTCTCTGCTTCTGTTGCTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAAGCAACAGAAGCAGAGAGGTTCTCTTGAAACCTCTCTGCTTCTGTTGCG-3', respectively, were annealed and subcloned into the pSilencer 2.1 vector system to generate pSiRNA-Apc plasmid (Ambion, Austin, TX). A 19-nucleotide sequence of the pSiRNA-Apc plasmid was scrambled to generate the pSiRNA-Apcmut plasmid. The sense and anti-sense nucleotide sequences for the pSiRNA-Apcmut plasmid were 5'-GATCCGGACAGTAAGGAAGAACGCTTCAAGAGAGCGTTCTTCCTTACTGTCCTTTTTTGGAAA-3' and 5'-AGCTTTTCCAAAAAAGGACAGTAAGGAAGAACGCTCTCTTGAAGCGTTCTTCCTTACTGTCCG-3', respectively. Specificity of the mutant 19-nucleotide sequence was confirmed by a BLAST search against the human genome sequence. Isogenic MEF-Polß+/+ and MEF-Polß–/– cell lines were grown in 60 mm tissue culture dishes to 60% confluence and then transfected with 4 µg of pSiRNA-Apc or pSiRNA-Apcmut plasmids with 9 µl of Lipofectamine transfection reagent (Invitrogen Life Technologies, Carlsbad, CA). The Apc protein level was determined by western blot analysis as described by Narayan et al. (28). MEF cell lines transfected with pSiRNA-Apcmut plasmid were named as MEF-Apc(WT)/Polß+/+ and MEF-Apc(WT)/Polß–/–. On the other hand, MEF cell lines transfected with pSiRNA-Apc plasmid, which knocks down the Apc gene expression, were named as MEF-Apc(KD)/Polß+/+ and MEF-Apc(KD)/Polß–/–.

MTT assay
The growth of MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines after MMS treatment was measured using a MTT [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] cell proliferation assay kit from American Type Culture Collection (Manassas, VA). Briefly, 500 cells were plated in triplicate in 96-well flat-bottom tissue culture plates and treated with MMS as indicated in figure legends. Then, 10 µl of MTT reagent was added to each well and incubated at 37°C for 4 h to allow the formation of purple color crystals of formazan. A 100 µl of detergent solution was added to each well and the reaction mixture was incubated in the dark for 2–4 h at room temperature. The color density was then measured spectrophotometrically at 570 nm using the microplate reader (Vmax Kinetic Microplate Reader from Molecular Device, Sunnyvale, CA).

Western blot analysis
The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines were treated with 500 µM of MMS for 24 h and nuclear extracts were prepared as described by Shapiro et al. (30). The protein levels of Apc, Pol-ß, Pol-{delta}, APE, Fen-1, Proliferating cell nuclear antigen (PCNA) and {alpha}-tubulin were determined by western blot analysis. The anti-Apc and -{alpha}-tubulin antibodies were obtained from EMD Biosciences (La Jolla, CA) and (Sigma-Aldrich Chem. Co., St. Louis, MO) respectively. The anti-Pol-{delta}, -APE and -Fen-1 antibodies were purchased from Novus Biologicals (Littleton, CO). The anti-PCNA antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the anti-Pol-ß polyclonal antibody was provided by Drs Rajendra Prasad and Samuel H.Wilson from National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Preparation of the substrate for LP-BER
The C-residues of a plasmid DNA of p21(Waf1/Cip1) promoter (pGL3-p21) were deaminated by 3 M of sodium bisulfite in the presence of 50 mM of hydroquinone (31). The deamination of C-residues produce U-residues (U-p21P). The resulting U-p21P DNA was further treated with uracil-DNA glycosylase (UDG) (New England Biolabs, Ipswich, MA) and then reduced with 0.1 M sodium borohydride to generate reduced AP sites (R-p21P), which becomes a substrate for LP-BER. The DNA was gel purified and used for BER study (22,24,32).

In vivo LP-BER
The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines were grown to 60–70% confluence in 60 mm tissue culture dishes and transfected with 2 µg/ml of R-p21P plasmid and 0.5 µg/ml of pCMV-ß-galactoside plasmid using 6 µl/ml of Lipofectamine reagent (Invitrogen Life Technologies, Carlsbad, CA). The pCMV-ß-galactoside served as an internal control to correct the differences in the transfection efficiency. After transfection, cells were acclimated for 5 h. One set of cells was harvested at this time point for promoter activity, which was considered a zero time point. The medium of the remaining dishes was aspirated and replaced with complete medium supplemented with 10% of fetal bovine serum. Cells were harvested at different time intervals as shown in figure legends. The luciferase gene reporter activity of the cellular lysate was determined by using a Moonlight 3010 Illuminometer (Promega, San Diego, CA). The reporter activity was interpreted as the extent of DNA repair in these cells.

In vitro LP-BER
To determine the ability of these substrates to undergo Pol-ß and Apc-dependent or -independent DNA repair, the BER reaction was assembled at 25°C with the nuclear extracts of MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines with R-p21P substrate. The BER-proficient nuclear extracts were prepared by the procedure of Shapiro et al. (30). The reaction mixture in a 25 µl final volume contained the following: 15 µg of nuclear extract, 50 mM of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.9, 2 mM of dithiothreitol, 10 mM of MgCl2, 0.5 mM of nicotinamide adenine dinucleotide, 5 mM of diTris-phosphocreatine, 6 U phosphocreatine kinase, 20 µM of ATP and 20 µM each of deoxynucleoside triphosphates and 100 ng of R-p21P. The BER was initiated by the addition of 5–10 µCi of [{alpha}-32P]dCTP. The BER reaction was stopped by adding 0.4% (w/v) of sodium dodecyl sulfate (final concentration) and 2 µg of proteinase K. The incubation was continued for an additional 30 min at 37°C. The DNA was recovered by chloroform–phenol extraction and ethanol precipitation and analyzed on a 1% agarose gel. After electrophoresis, the gel was dried and the amount of radioactivity in the repaired DNA was quantified with InstantImager (Packard Instrument Co., Meriden, CT).

FACS analysis
A detergent and proteolytic enzyme-based technique was used for nuclear isolation and DNA content analysis of cells in different phases of the cell cycle. The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines were treated with 500 µM of MMS for 24 h. Cells were harvested and processed for propidium iodide staining of the nuclei as described earlier (33). The cellular DNA content was analyzed by Becton-Dickinson FACScan flow cytometer. The ranges for G0/G1, S, G2/M and subG1 phase cells were established based upon their corresponding DNA content of histograms (32,34). At least 10 000 cells per sample were considered in the gated regions used for calculations.


   Results
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 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Apc increases the sensitivity of MEF-Polß–/– cells to MMS-induced DNA damage
In the past, several lines of evidence supported that MEF-Polß–/– cells are more sensitive to MMS treatment than MEF-Polß+/+ cells (4,16,35). These results implicated that the decreased BER sensitized MEF-Polß–/– cells to MMS-induced DNA damage, which can be repaired by BER. Since APC interacts with Pol-ß and Fen-1 and blocks Pol-ß-mediated LP-BER (22,25), we expected that the knockdown of Apc would decrease the sensitivity of MEF-Polß+/+ cells to MMS treatment due to increased DNA repair. The Apc expression was transiently knocked down in MEF-Polß+/+ and MEF-Polß–/– cell lines by using small interfering RNA technique as described in our earlier studies (22,24,25). More than 95% of Apc gene expression was knocked down in MEF-Polß+/+ and MEF-Polß–/– cell lines (Figure 1A, compare lane 2 with 3 and 4 with 5). The APC protein level in HCT-116 was used as a reference (Figure 1A, lane 1). This experimental approach for the knocking down of Apc in MEF cell lines was used in the entire study.


Figure 1
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  		Fig. 1. Wild-type Apc increases MMS-induced sensitivity of MEF cells in a dose-dependent manner. Panel (A) depicts the Apc-knockdown in MEF cell lines. The MEF-Polß–/–and MEF-Polß+/+cell lines were grown to 60–70% confluence in 60 mm tissue culture dishes and transiently transfected with pSiRNA-Apc and pSiRNA-Apcmut plasmids for 36 h. A decreased level of Apc in pSiRNA-Apc (lane 3 and 5), but not in pSiRNA-Apc mut-transfected cells (lane 2 and 4) is shown. The nuclear extract from HCT-116 cells, which carries wild-type Apc and Pol-ß genes, was used as a control (lane 1). The protein levels of {alpha}-tubulin served as a loading control. Panle (B) shows the growth of MEF cells after MMS treatment. The MEF cell lines transiently transfected with pSiRNA-Apc and pSiRNA-Apc mut plasmids, designated as MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/–and MEF-Apc(KD)/Polß–/–, were grown in a 96-well plate. They were treated with different concentrations of MMS for 24 h. The growth of the cells was measured by MTT assay. Data are the mean ±SE of three different experiments.

 
To test our hypothesis that wild-type Apc increases sensitivity to MMS treatment, we treated these cell lines with different concentrations of MMS for 24 h (Figure 1B) or with 500 µM of MMS for different time periods (Figure 2) and determined the growth of these cell lines by MTT assay. The results showed that the MEF-Apc(WT)/Polß–/– cells were more sensitive to MMS treatment than the MEF-Apc(WT)/Polß+/+ cells in a concentration- (Figure 1B) and time-dependent manner (Figure 2). This is consistent with the observations made by other investigators (4,16,35). Once the Apc was knocked down, the sensitivity of MEF-Apc(KD)/Polß+/+ cells decreased as compared with MEF-Apc(WT)/Polß+/+ cells (Figures 1B and 2). The decreased sensitivity of MEF-Apc(KD)/Polß+/+ cells could be due to increased LP-BER in the absence of Apc. Interestingly, once the Apc was knocked down, the sensitivity of MEF-Apc(KD)/Polß–/– cells to MMS treatment also decreased as compared with MEF-Apc(WT)/Polß–/– cells (Figures 1B and 2). These results suggested that the knockdown of Apc in MEF-Polß+/+ cells decreased sensitivity to MMS treatment perhaps due to Pol-ß-dependent increased LP-BER. However, the decreased sensitivity to MMS treatment in MEF-Apc(KD)/Polß–/– cells as compared with MEF-Apc(WT)/Polß–/– cells could be due to the involvement of Apc in the blockage of Pol-ß-independent LP-BER.


Figure 2
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  		Fig. 2. Apc increases MMS-induced sensitivity of MEF cells in a time-dependent manner. The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/–and MEF-Apc(KD)/Polß–/–cell lines were treated with 500 µM of MMS for different time intervals. After treatment, the growth of the cells was measured by MTT assay. Data are the mean ±SE of three different experiments.

 
Apc, Pol-ß and Fen-1 protein levels are increased in MEF cell lines after MMS treatment
To examine whether the increased sensitivity of MEF cell lines were due to increased levels of Apc or due to the alteration of BER proteins, we performed a western blot analysis of the nuclear extracts of MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines. These cell lines were either treated or untreated with 500 µM of MMS for 24 h. The Apc protein levels were significantly increased in MEF-Apc(WT)/Polß+/+ and MEF-Apc(WT)/Polß–/– cell lines after MMS treatment (Figure 3, compare lane 1 with 2 and 5 with 6). The Pol-ß protein levels were significantly increased only in MEF-Apc(WT)/Polß+/+ cells but unaffected in MEF-Apc(KD)/Polß+/+ cells (Figure 3, compare lane 1 with 2 and 3 with 4). The increased protein level of Pol-ß after MMS treatment is consistent with previous findings (36). The Pol-{delta} protein levels were also increased in MEF-Apc(WT)/Polß+/+ and MEF-Apc(WT)/Polß–/– cells after MMS treatment (Figure 3, compare lane 1 with 2 and 5 with 6). We observed two protein bands of Pol-{delta}, which were recognized by the antibody we used in these experiments. The upper protein band seems to be the correct Pol-{delta} protein band. However, at this stage, we could not verify whether the extra protein band is non-specific or a splicing product of the same gene. Pol-{delta} is an alternative polymerase that replaces Pol-ß for LP-BER (9,13,37). Since Pol-ß-mediated LP-BER activity can be blocked by Apc–Pol-ß interaction, the increased level of Pol-{delta} in MEF-Apc(WT)/Polß+/+ cells after MMS treatment can substitute Pol-ß for LP-BER. The Fen-1 protein levels were also increased in all the cell lines, irrespective of Apc expression, after MMS treatment (Figure 3, compare lane 1 with 2, 3 with 4, 5 with 6 and 7 with 8 for MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines). The APE and PCNA protein levels were unaffected in all the MEF cell lines in response to MMS treatment. From these results, it appears that the increased protein levels of Apc, Pol-ß, Pol-{delta} and Fen-1 may play an important role in MMS-induced sensitivity of MEF cells.


Figure 3
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  		Fig. 3. Apc and BER protein levels of MEF cells after MMS treatment. The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/–cell lines were treated with 500 µM of MMS for 24 h. Nuclear extracts were prepared and processed for western blot analysis for determining the protein levels of APC, APE, Pol-ß, Pol-{delta} and PCNA. The protein levels of {alpha}-tubulin served as a loading control. Data are representative of three different experiments.

 
Increased level of Apc decreased BER in vivo and in vitro
Recently, we have shown that APC interacts with Pol-ß and Fen-1 and blocks strand displacement synthesis of LP-BER in vitro (22,25). We also examined the role of APC in cigarette smoke condensate-induced blockage of LP-BER in vivo (24). For in vivo studies, we used a plasmid-based LP-BER assay system as described in our previous studies (22,24). We randomly modified multiple cytosine (C) residues of p21(Waf1/Cip1)-luciferase promoter DNA into a reduced abasic p21P (R-p21P) DNA by chemical modifications as described in Materials and methods (32) (Figure 4A). The principle behind this assay is that the R-p21P plasmid when transfected into cells should show poor promoter activity due to alterations in the C-residues as compared with the unmodified p21P plasmid. However, the promoter activity can be restored if the R-p21P DNA is allowed to go through DNA repair processes in the cell.


Figure 4
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  		Fig. 4. Increased levels of Apc decreased LP-BER in vivo. Panel (A) describes the protocol for the modification of p21P plasmid into R-p21P as a LP-BER substrate. Panel (B) shows a Luciferase gene reporter assay of R-p21P plasmid transfected into MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/–and MEF-Apc(KD)/Polß–/–cell lines. Data are the mean ±SE of three different experiments.

 
The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines were transfected with the R-p21P plasmid. After transfection, cells were acclimated for 5 h. The medium containing DNA–lipid complex was removed, a fresh medium was added and the incubation was continued for additional 30 h. At 5 h time point, although a significant repair was observed, the difference in the R-p21P promoter activity between MEF-Apc(WT)/Polß+/+ versus MEF-Apc(KD)/Polß+/+ and MEF-Apc(WT)/Polß–/– versus MEF-Apc(KD)/Polß–/– was not significant (Figure 4B). These results indicate that the 5 h time period after R-p21P plasmid transfection was not sufficient to see the effect of Apc on BER in these cells, especially when the cells were going through the recovery of serum starvation during transfection. However, once cells were acclimated in serum, the R-p21P promoter activity at the 30 h time point as compared with the 5 h time point was 7.9-fold higher in MEF-Apc(KD)/Polß+/+ cells than in MEF-Apc(WT)/Polß+/+ cells (Figure 4B). These results suggested that the absence of Apc increases LP-BER through a Pol-ß-dependent pathway. The R-p21P promoter activity was also 2.3-fold higher at the 30 h time point as compared with the 5 h time point in MEF-Apc(KD)/Polß–/– cells than in MEF-Apc(WT)/Polß–/– cells (Figure 4B). In Polß–/– cell lines, the decreased R-p21P promoter activity in the presence of Apc indicates that the Apc also influences the repair of R-p21P promoter DNA through a Pol-ß-independent pathway, perhaps through the Pol-{delta}/{epsilon}-dependent pathway.

The effect of Apc on in vivo LP-BER in MEF cells on Pol-ß-dependent and -independent pathways was further recapitulated in an in vitro LP-BER assay system. Since previous studies have shown that LP-BER is more efficient on covalently closed circular DNA than on the linear DNA substrates (38), we applied this strategy in our in vitro studies. The covalently closed circular DNA (R-p21P), which was used in the in vivo LP-BER assays, was also used in these experiments. The in vitro assay system is described in our earlier study (32). MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines were treated with 500 µM of MMS for 24 h. The nuclear extracts were prepared and the protein levels of Apc, APE, Pol-ß, Pol-{delta}, Fen-1 and PCNA were determined as shown in Figure 3. These nuclear extracts were used for in vitro LP-BER assays. Since MMS treatment increases Apc level (Figure 3), it was expected that the increased level of Apc should block Pol-ß-mediated in vitro LP-BER. The results indeed showed a decreased level of LP-BER with the nuclear extract prepared from MMS-treated MEF-Apc(WT)/Polß+/+ cells (Figure 5A and B, compare lane 1 with 2 for the repair of Form I and II DNA, respectively) as compared with MEF-Apc(KD)/Polß+/+ cells (Figure 5A and B, compare lane 3 with 4 for the repair of Form I and II DNA, respectively). Repair of the R-p21P plasmid in MEF-Apc(WT)/Polß–/– or MEF-Apc(KD)/Polß–/– cell lines was very poor (Figure 5A and B, compare lane 5 with 6 and 7 with 8 for the repair of Form I and II DNA, respectively); therefore, the effect of Apc on Pol-ß-independent LP-BER could not be assessed clearly from these experiments. These results suggest that the Pol-ß-dependent LP-BER pathway is the predominant pathway in MEF cell lines, which is blocked by the MMS-induced level of Apc.


Figure 5
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  		Fig. 5. Increased level of Apc decreased LP-BER in vitro. Panel (A) shows a typical result of an in vitro LP-BER. The MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/–and MEF-Apc(KD)/Polß–/–cell lines were treated with 500 µM of MMS for 24 h. After treatment, cells were processed for nuclear extract preparation. The LP-BER activity of nuclear extracts was determined with the repair of Form I and II of R-p21P plasmids as described in Materials and methods. Photograph of the autoradiogram is representative of three independent experiments. Panel (B) shows a quantitative analysis of the data for the repair of Form I and II of R-p21P plasmids. Data are the mean ±SE of three different experiments.

 
Apc-mediated blockage of LP-BER induces apoptosis in MEF cells
Finally, we examined whether the increased level of Apc and the decreased LP-BER activity are associated with increased MMS sensitivity of the MEF cells. Also, we determined whether the Pol-ß status can distinguish the role of Apc in this process. For these experiments, we treated MEF-Apc(WT)/Polß+/+, MEF-Apc(KD)/Polß+/+, MEF-Apc(WT)/Polß–/– and MEF-Apc(KD)/Polß–/– cell lines with 500 µM of MMS for 24 h and performed the FACS analysis to determine the cell cycle profile and apoptosis of control versus MMS-treated cells. The results showed an increased S-phase arrest of all the MEF cell lines after MMS treatment, which was independent of the status of Apc or Pol-ß level (Table I). These results suggested a general cell cycle arrest at the S-phase before DNA replication in response to alkylation damage. We observed an increased subG1 or apoptotic population of MEF-Apc(WT)/Polß–/– cells than the MEF-Apc(WT)/Polß+/+ cells after MMS treatment (Table I). Interestingly, once the Apc expression was knocked down, the population of apoptotic cells was reduced in both MEF-Apc(KD)/Polß+/+ and MEF-Apc(KD)/Polß–/– cell lines. These are in agreement with the growth assay results (Figures 1 and 2). These results suggest that after MMS treatment the increased apoptosis in MEF-Apc(WT)/Polß–/– and MEF-Apc(WT)/Polß+/+ cell lines compared with MEF-Apc(KD)/Polß+/+ and MEF-Apc(KD)/Polß–/– cell lines, respectively, is due to Apc-mediated decrease in LP-BER.


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  		Table I. Effect of MMS on cell cycle regulation and apoptosis of MEF cell lines

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
In earlier studies, we have shown that the APC protein level increases in human normal breast epithelial cells after treatment with cigarette smoke condensate, and this suggested a role of APC in compromised LP-BER activity and transformation of pre-malignant human breast epithelial cells (24). The APC-mediated blockage of LP-BER was shown due to the blockage of Pol-ß-mediated strand displacement synthesis and the blockage of Fen-1 activity (22,25). In previous studies, it has been reported that LP-BER can be processed by both Pol-ß-dependent and -independent pathways (9). The Pol-ß-independent pathway is accomplished by the Pol-{delta}/{epsilon} pathway (13,37). Due to the availability of the MEF-Polß–/– cell line, it became possible to determine the contribution of these two pathways in DNA damage-induced sensitivity. The major cause of the hypersensitivity of MMS treatment is suggested due to a failure to repair the BER intermediate 5'-dRP (16,39,40). In recent studies, the role of Pol-{lambda} and Pol-{iota}, which also encode a 5'-dRP lyase activity, has been suggested to participate in the removal of toxic lesions of 5'-dRP intermediates produced by DNA alkylation damage (41–43). Now, the contribution of other cellular components in the repair of alkylation-induced DNA damage is beginning to emerge. Our finding of the involvement of APC in BER pathway is one of the latest examples (22,24,25). It is well established that MEF-Polß–/– cells are hypersensitive as compared with MEF-Polß+/+ cells to the treatment of MMS, methylnitrosourea (35) and temozolomide (44).

Based upon our previous findings that APC blocks LP-BER, we initiated the present study to determine whether the MMS-induced Apc level can distinguish the sensitivity of MEF cells based upon the Apc level as well as the Pol-ß-dependent and -independent LP-BER pathways. We approached our study by knocking down the Apc gene in MEF-Polß+/+ and MEF-Polß–/– cell lines. The MEF-Apc(WT)/Polß–/– cells were more hypersensitive than the MEF-Apc(WT)/Polß+/+ cells to MMS treatment, which is already reported by previous investigators (4,16,35). In order to understand whether Apc plays a role in MMS-induced hypersensitivity of these cell lines, we compared the growth of MEF-Apc(WT)/Polß+/+ and MEF-Apc(WT)/Polß–/– cell lines with MEF-Apc(KD)/Polß+/+ and MEF-Apc(KD)/Polß–/– cell lines. One should expect that the knocking down of Apc may increase LP-BER capacity of these cells that may result in more resistance to MMS treatment. In fact, our results supported this hypothesis. Although the increased resistance of MEF-Apc(KD)/Polß+/+ cells to MMS treatment was expected, we also found a similar resistance of MEF-Apc(KD)/Polß–/– cells to MMS treatment. In MEF-Apc(KD)/Polß+/+ cells, the resistance to MMS treatment can be attributed to improved LP-BER, which was supported by in vitro LP-BER assays. However, in MEF-Apc(KD)/Polß–/– cells, the increased resistance can be due to the involvement of a Pol-ß-independent LP-BER pathway, especially the Pol-{delta}/{epsilon} pathway. However, experimentally it needs to be proven that there is a direct role of Pol-{delta}/{epsilon}-dependent pathway in APC-mediated resistance to MMS treatment.

The involvement of APC in Pol-{delta}/{epsilon}-dependent LP-BER is possible due to the role of Fen-1 in this pathway (9,12,13). In earlier studies, we have shown that Apc interacts and blocks the 5'-flap endonuclease as well as the 5'–3' exonuclease activities, which are essential for the completion of LP-BER (22). Therefore, if the Fen-1 activity is critical in Pol-{delta}/{epsilon}-dependent LP-BER pathway, then in the MEF-Apc(KD)/Polß–/– cells, the effect of APC on the Fen-1 activity will be relieved, and the LP-BER will be accomplished through the Pol-{delta}/{epsilon}-dependent pathway. If this is the case, then the improved LP-BER will cause resistance to MMS treatment in MEF-Apc(KD)/Polß–/– cells, which is consistent with our results. Although we did not find much effect of Apc-knockdown on Pol-ß-independent in vitro LP-BER, it was clearly seen with the in vivo LP-BER. Since the Pol-ß-independent LP-BER in MEF cells is very poor, our in vitro assay system may have not been sensitive enough to determine the effect of Apc on LP-BER. However, the effect of Apc on LP-BER was observed with the plasmid-based in vivo assay. This suggests that our in vivo LP-BER assay is more sensitive and can be a useful tool in determining the effect of alkylating drugs on LP-BER. This assay system can also be useful for BER pathway in general by the use of U-p21P DNA, which can be repaired by both SP-BER and LP-BER pathways. However, whether Apc plays a role in SP-BER is currently not clear.

In many cases, the decreased DNA repair is associated with cell cycle arrest leading to apoptosis (44–46). In our studies, we also found that the MMS-induced level of Apc, which blocked LP-BER in Pol-ß-dependent and -independent pathways, was associated with cell cycle arrest and apoptosis. The MEF cells were more sensitive to apoptosis after MMS treatment in the presence of Apc, and the sensitivity was higher in Polß–/– versus Polß+/+ cells. The knockdown of Apc decreased the amount of apoptotic cells in both Polß+/+ and Polß–/– cells. From these results, we conclude that Apc blocks the repair of alkylation damage induced by MMS and leads the target cell to apoptosis. These findings can be clinically significant. In future studies, it could be possible to increase the efficacy of DNA-alkylating drugs in cancer cells by increasing Apc levels and decreasing BER activity.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Cancer Institute–National Institutes of Health (CA-097031 and CA-100247) and Flight Attendant Medical Research Institute 24027, Miami, FL, to S.N.


   Acknowledgments
 
We would like to thank Drs Rajendra Prasad and Samuel H.Wilson from National Institute of Environmental Health Sciences, Research Triangle Park, NC, for providing the MEF-Polß+/+ and MEF-Polß–/– cell lines. We are also thankful to Ms Mary Wall and Ms Melissa Armas for the proofreading of the manuscript.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received January 26, 2007; revised May 8, 2007; accepted May 10, 2007.


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