Cells with pathogenic biallelic mutations in the human MUTYH gene are defective in DNA damage binding and repair

Antony R. Parker 1, 5, *, Oliver M. Sieber 4, Chanjuan Shi 1, Li Hua 1, Masashi Takao 3, Ian P. Tomlinson 4 and James R. Eshleman 1, 2

1 Department of Pathology and 2 Department of Oncology Johns Hopkins University, Baltimore, MD 21205, USA, 3 Department of Molecular Genetics, Tohoko University, Sendai, Japan and 4 Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, London, UK

* To whom correspondence should be addressed. Tel: +44 121 436 1016; Fax: +44 121 430 7061; Email: parkerco21229{at}yahoo.com


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Inherited biallelic mutations in the human MUTYH gene are responsible for the recessive syndrome—adenomatous colorectal polyposis (MUTYH associated polyposis, MAP)—which significantly increases the risk of colorectal cancer (CRC). Defective MUTYH activity causes G:C to T:A transversions in tumour APC and other genes thereby altering genomic integrity. We report that of the four established cell lines, derived from patients with the MAP phenotype and containing biallelic MUTYH mutations, three contain altered expressions of MUTYH protein (MUTYH Y165C–/–, MUTYH 1103delC/G382D and MUTYH Y165C/G382D but not MUTYH G382D–/–), but that all four cell lines have wild type levels of MUTYH mRNA. Mutant MUTYH proteins in these four cell lines possess significantly lowered binding and cleavage activities with heteroduplex oligonucleotides containingA·8-oxoG and 8-oxoA·G mispairs. Transfection of mitochondrial or nuclear MUTYH cDNAs partially correct altered MUTYH expression and activity in these defective cell lines. Finally, we surprisingly find that defective MUTYH may not alter cell survival after hydrogen peroxide and menadione treatments. The Y165C and 1103delC mutations significantly reduce MUTYH protein stability and thus repair activity, whereas the G382D mutation produces dysfunctional protein only suggesting different functional molecular mechanisms by which the MAP phenotype may contribute to the development of CRC.

Abbreviations: 8-oxoG, 8-hydroxyguanosine; APC; adenomatous polyposis coli; CRC, colorectal cancer; MAP, MUTYH associated polyposis; MMR, mismatch repair; MUTYH, MutY homolog


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cellular DNA damage can arise from a variety of sources such as ionizing radiation, exogenous chemicals and normal cellular metabolism and can create, among others, strand breaks, base alterations, and apuriminic/pyrimidinic sites. These deleterious modifications of genomic DNA can increase the cell's mutation rate and ability to undergo programmed cell death and thus have been implicated in aging and diseases such as cancer and neurodegeneration (14).

Among the DNA lesions produced from DNA oxidation, arguably the most mutagenic is 8-hydroxyguanosine (8-oxoG). The carcinogenic potential of 8-oxoG arises from its ability to readily mispair with adenine, which upon subsequent cycles of replication can produce G:C to T:A and A:T to C:G transversions (5,6). In mammalian cells, the human MutY homolog (MUTYH) is responsible for the removal of adenine bases mispaired with 8-oxoG so that a cytosine can be inserted and the 8-oxoG excised and repaired; therefore, cells defective in MUTYH should exhibit a mutator phenotype (710).

Inherited biallelic mutations in the human MUTYH gene have been implicated as a cause of adenomatous colorectal polyposis (MUTYH associated polyposis, MAP) resulting in an increased risk of colorectal cancer (CRC) (1117). Furthermore, the MUTYH associated pathway of carcinogenesis appears to be distinct from both the chromosomal instability and microsatellite instability associated pathways (16). While investigating the cause of multiple colorectal adenomas and carcinoma in British family members, Al-Tassan and colleagues discovered unique G:C to T:A transversions in the adenomatous polyposis coli (APC) genes of their tumours, and these mutations were not present in their germline DNA (11). They sequenced the OGG1, MTH1 and MUTYH cDNAs and discovered two mutations that produced two non-conservative amino acids variants in MUTYH, Y165C and G382D. All three affected individuals were compound heterozygotes for both variants. Jones and colleagues also identified biallelic MUTYH mutations, including Y165C and G382D, in 7/21 of unrelated patients affected with multiple colorectal adenomas. Analysis of the APC cDNA in these adenomas again revealed an excess of G:C to T:A transversions (12). Furthermore, Sampson and colleagues discovered that 25/111 unrelated patients presenting with multiple colorectal adenomas also possessed biallelic mutations of the MUTYH gene, confirming the mutational basis for MAP (18). Recently, Sieber and co-workers identified biallelic MUTYH mutations in 6/152 patients presenting with multiple colorectal carcinomas (13). Among the six biallelic MUTYH mutations discovered were homozygotes with Y165C or G382D, and the compound heterozygotes Y165C/G382D and 1103delC/G382D.

Site directed Escherichia coli MutY mutant proteins (11,19), mimicking the mutations in CRC, have suggested that the Y165C mutant (analogous to Y82C in E.coli) is inactive and unable to complement the mutator phenotype in MutY defective E.coli cells. In contrast, the G382D mutation (G253D in E.coli) appears to retain most of its in vitro activity, but also cannot functionally complement the E.coli MutY mutator phenotype. The use of site directed human MUTYH mutant proteins, containing MUTYH mutations responsible for MAP, has also suggested that the mutations found in CRC inactivate MUTYH (20). This has been recently supported by the lack of complementation of the elevated mutation rate in MUTYH–/– mouse embryonic stem cells (10) with a murine G382D mutant. However, these studies do not address the contribution of MUTYH protein expression in the molecular pathogenesis of defective MUTYH and at present there are no reports of the relative expression and activity of the mutant MUTYH proteins in cells derived from patients exhibiting the MAP phenotype.

In this report we examined human lymphoblastoid cell lines, established from patients affected with multiple colorectal adenomas and which contain biallelic mutations in the MUTYH gene, to determine how these mutations cause dysfunctional protein activity. Three of the four lymphoblastoid cell lines contained altered levels of the human MUTYH protein but all four cell lines had wild type levels of MUTYH mRNA. Cell extracts and MUTYH proteins immunoprecipitated from all four MUTYH defective cell lines had significantly lower in vitro binding and cleavage activities with A·8-oxoG and 8-oxoA·G mispairs. Further we show that these defective activities can be partially corrected by exogenous complementation with transient transfected mitochondrial or nuclear MUTYH cDNAs. Surprisingly, defective MUTYH may not consistently alter the killing curves after treatment with either hydrogen peroxide or menadione, both of which cause oxidative DNA damage. These findings suggest a direct mechanism for the pathogenesis of the biallelic MUTYH mutations, in the majority of cases through a contribution of defective expression and activities, which cause the MAP mutator phenotype and possibly the development of CRC.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
Establishment of MUTYH lymphoblastoid cell lines
Briefly, 25 ml of patients' peripheral blood were collected into a tube containing 25 ml of RPMI-1640/0.6% tri-sodium citrate/0.04 mM mercaptoethanol, and maintained at room temperature for 24–72 h during shipment to the laboratory. After adding 600 µl of 1 M CaCl2, the blood was defibrinated in a conical glass flask containing sterile glass beads by shaking at 250 r.p.m. for 15 min on a Gyrotory shaker (New Brunswick Scientific, UK). Lymphocytes were isolated from the defibrinated blood using LymphoprepTM solution according to the manufacturer's instructions (Axis-Shield Diagnostics, UK). The isolated cells were counted and resuspended in 1 ml of cyropreservation solution consisting of 90% FCS/10% DMSO. The samples were frozen at –80°C before storage in liquid nitrogen.

For infection with Epstein–Barr virus (EBV), frozen lymphocyte suspensions were rapidly thawed at 37°C and washed in 10 ml RPMI-1640/20% FCS. The cell pellets (~2 x 106 cells) were incubated for 1.5 h with 200 µl of EBV stock prepared from the culture supernatants of transformed B95-8 marmoset leukocytes constitutively releasing EBV. The samples were washed with 10 ml RPMI-1640/20% FCS and resuspended in 4 ml RPMI-1640/20% FCS containing 1 µg/ml Cyclosporin A (Sigma, St Louis, MO). The suspensions were added to feeder layers of human fetal foreskin fibroblasts and incubated at 37°C in a humidified 5% CO2 atmosphere. The transformation, seen as large floating clumps of cells, was followed by microscopical examination over a period of 4 weeks. Cells were propagated and maintained in RPMI-1640/20% FCS.

Written informed consent was obtained from all individuals. DNA sequencing confirmed the presence of each mutation in the derived cell lines as well as wild-type MUTYH sequence in two control cell lines (data not shown). MUTYH Y165C–/– is homozygous for an A to G transition at position 494 in both MUTYH alleles, and MUTYH G382D–/– is homozygous for a G to A transition at position 1145 in both MUTYH alleles. MUTYH 1103delC/G382D is compound heterozygous with a G to A transition at position 1145 in one allele and a deleted cytosine nucleotide at position 1103 in the other. MUTYH Y165C/G382D is a compound heterozygote containing the above MUTYH mutations. Cells were maintained as described previously (21).

Preparation of whole cell extracts, nuclear extracts and mitochondrial extracts
Whole cell extracts (WCEs), nuclear extracts and mitochondrial extracts were prepared as reported previously (22,23).

Immunoprecipitation of MUTYH and western blotting
Immunoprecipitations of MUTYH were carried out essentially as published previously (22). Briefly, extracts were diluted 2-fold in phosphate-buffered saline containing Sigma protease inhibitor mixture, 5 µg/ml leupeptin, pepstatin and chymostatin, and then precleared by adding 20 µl of protein A/G-agarose (Invitrogen, Carlsbad, CA) and gently rocking for 1 h at 4°C. After centrifugation at 1000x g the supernatant was incubated with 1 µg of anti-human MUTYH antibody (Alpha Diagnostics, San Antonio, TX) overnight at 4°C. Protein A/G-agarose (20 µl) was added and incubated for 4–12 h at 4°C. After centrifugation at 1000x g the supernatant was stored and the pellet was washed three times with 800 µl of phosphate-buffered saline. A control was run concurrently without the anti-human MUTYH antibody. The pellets were reconstituted in 3x SDS loading buffer and then resolved on 12% SDS polyacrylamide gels and transferred to a nitrocellulose membrane. Western blotting analyses were performed as previously described (22) using the anti-human MUTYH antibody and an anti-human Actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at dilutions of 1:100 and 1:200, respectively. Anti-PCNA and anti-HSP60 antibodies were purchased from Santa Cruz Biotechnology and were both used at dilutions of 1:1000.

Real-time quantitative PCR analysis
Total RNA was isolated using the Qiagen RNeasy Kit (Qiagen). Reverse transcription was performed using reverse transcriptase (40 U, Roche Diagnostics, Indianapolis, IN), 1 µg total RNA and 3 µg random hexamer primers (Invitrogen) according to the manufacturer's instructions, followed by spin column purification (Princeton Apparatus, Aldephia, NJ). Quantitative PCR (Q-PCR) was performed on a Cepheid Smartcycler using a MUTYH assay in demand kit (4331182, Applied Biosystems, Foster City, CA) with a minor groove binding probe directed at the junction of exons 1 and 2, PlatinumQ-PCR Supermix-UDG (Invitrogen) and 2 µl of the reverse transcriptase product. A standard curve was produced using a MUTYH plasmid containing the MUTYH cDNA cloned into a pCR2.1 vector (Invitrogen). PCR conditions were 50°C for 120 s and 95°C for 120 s, followed by 50 cycles of 95°C for 15 s and 60°C for 60 s.

Transient transfection of human MUTYH cDNA
Nuclear and mitochondrial MUTYH cDNA vectors were constructed as described previously (9,24). Cells were seeded into six well plates (~0.3 x 106 cells per well) and then incubated for 24 h at 37°C. The nMUTYH cDNA, mMUTYH cDNA or vector alone was added to 8 µl of lipofectamine 2000 (Invitrogen) and incubated for 20 min at room temperature. The DNA/liposome complex was added to 1.5 ml of OPTI-MEM serum-free medium (Life Technologies, Carlsbad, CA), kept for 5 min and then overlaid onto the cells rinsed with serum-free medium. After incubation for 6 h at 37°C, the media was replaced with regular MEM media and the cells were further incubated for a total of 48 h at 37°C before being harvested.

UV A·8-oxoG and 8-oxoA·G binding assay
Protein samples were incubated with 5 fmol 32P 3'-end labelled or unlabelled 20mer duplex DNA in a 20 µl reaction mixture containing the same buffer used in the glycosylase assay except that 20 ng of poly(dI–dC), 20 mM NaCl and 25 mM EDTA were added. Following a 0–60 min incubation at 37°C, the samples were exposed to ultraviolet (UV) light (254 nm; UVC 500 UV Crosslinker; Pharmacia Biotech, San Francisco, CA) and at time intervals aliquots were taken and stored on dry ice. All samples were diluted with SDS loading buffer and resolved by SDS–PAGE. Fold increase was calculated relative to time 0.

A·8-oxoG and 8-oxoA·G glycosylase assay
A·8-oxoG glycosylase activity was performed as described previously (22) except that the reaction buffer contained 10 mM Tris–HCl, pH 7.6, 0.5 mM dithiothreitol, 5 µM ZnCl2, 30 mM NaCl and 5 mM MgCl2. The sequences for 8-oxoG sense and antisense oligonucleotides were the same as published previously (25), and the sequence for the 8-oxoA sense oligonucleotide was 5'-CCGAGGAATT8-oxoAGCCTTCTG-3', where 8-oxoA is 8-oxoadenine and for the 8-oxoA antisense oligonucleotide it was 5'-GCAGAAGGCGAATTCCTCG-3', where G is mispaired with 8-oxoA (Midland Certified Reagent Company, Midland, TX). After hybridization of the two corresponding sense and antisense oligonucleotides, the substrate was 3' radiolabelled as previously described (22). Briefly, to the immunoprecipitated MUTYH samples the reaction buffer containing the radiolabelled oligonucleotide (5 fmol) was added. Approximately equal amounts of immunoprecipitated MUTYH, normalized to the amount of MUTYH immunoprecipitated from MUTYH Y165C–/–, were used in the reactions. Cleavage of the 3' radiolabelled 20 bp mispaired oligonucleotides, at the adenine or 8-oxoA nucleotides, produces 10 bp products. The 20 bp uncut oligonucleotide and 10 bp products were separated by denaturing gel electrophoresis and detected by autoradiography. The amount of radiolabelled 10 bp product from cleavage is calculated as a percentage of the total amount of radioactivity (the sum of the uncut 20 bp oligonucleotide and the 10 bp product). A control was run concurrently in the absence of immunoprecipitated MUTYH. Equal lane loading was confirmed with SDS–PAGE and coomassie blue staining.

Cell viability with H2O2 and menadione
For the cell viability assay, exponentially growing cells were trypsinized and washed twice in serum-free minimal essential medium (SF-MEM). Washed cells were resuspended in SF-MEM and treated with 0–30 µM H2O2 (Sigma) or 0–6 µM menadione (Sigma) for 60 min at 37°C. After treatment, the cells were washed once with MEM2+ media (21), resuspended in fresh growth medium and seeded (~500 000 cells/well in six well plates). The cells were grown for 5 days, subcultured and counted using trypan blue exclusion by using a haemocytometer. Experiments were performed three times, each in triplicate.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Human MUTYH protein levels, but not MUTYH mRNA levels, are decreased in MUTYH defective lymphoblastoid cell lines
Mutations in both alleles of human MUTYH correlate with an increase in G:C to T:A transversions in the somatic APC gene in the tumors of MAP patients suggesting that MUTYH dependent repair is defective (11,13). Whether this defect is due to the absence of protein because of instability or due to present but dysfunctional protein is unclear. Western blot analyses for mutated MUTYH proteins in the WCE of the MUTYH Y165C–/–, 1103delC/G382D and Y165C/G382D cell lines showed that the levels of MUTYH protein were significantly reduced (Figure 1A and B). There was barely detectable MUTYH protein in MUTYH Y165C–/– (~5–10% of the wild type levels), and the MUTYH 1103delC/G382D and Y165C/G382D cell lines possessed only ~40–50% MUTYH protein (Figure 1B) relative to the wild-type controls. Interestingly, no faster migrating protein band was observed in MUTYH 1103delC/G382D, which one may have predicted from the frameshift created with the deletion at the nucleotide 1103. MUTYH G382D–/– possessed levels of MUTYH protein comparable with the control cell lines.



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Fig. 1. Mutated MUTYH expression levels are altered in some of the MUTYH defective lymphoblastoid cell lines but the MUTYH mRNA levels are normal. (A) Western blot analysis of WCE (100 µg) of two control and four MUTYH defective cell lines. WCE were resolved on 12% SDS–PAGE and transferred overnight onto a nitrocellulose membrane. The blots were probed for MUTYH and actin proteins as described. WT represents wild-type control cell lines. (B) Quantitation of western blot data using a Storm 840 Phosphoimager. (C) Validation of MUTYH Q-PCR assay and analysis of MUTYH mRNA from the four MUTYH defective cell lines. (D) Q-PCR assay of MUTYH mRNA from two control and four MUTYH defective cell lines. The diamonds represent the serial dilution of the plasmids. (E) Correction of defective MUTYH expression in Y165C –/– with exogenous mitochondrial and nuclear MUTYH protein. Nuclear extracts (70 µg) from Y165C–/– and Y165C–/– transfected with nuclear MUTYH cDNA and mitochondrial extracts (70 µg) from Y165C–/– and Y165C–/– transfected with mitochondrial MUTYH cDNA were resolved on 12% SDS–PAGE and transferred overnight. The blots were probed for MUTYH as described. Equal loading was monitored by western blot analyses of duplicate gels for PCNA (nuclear) and HSP60 (mitochondrial).

 
We then tested whether messenger RNA instability was the cause of the low MUTYH protein in some of the cell lines. We first validated a real-time Q-PCR assay using cloned MUTYH cDNA that was serially diluted (Figure 1C and D). We determined that in contrast to the decreased protein levels in three of the four MUTYH defective cell lines, the MUTYH mRNA levels were similar in all cell lines when compared with the levels in the MUTYH proficient control cell lines (Figure 1C and D).

If the defective MUTYH protein levels are the direct result of insufficient protein levels or defective protein function they should be complemented by the expression of exogenous MUTYH protein produced from a transfected cDNA expression vector. Alternatively, if the mutation causes a dominant negative protein, then exogenous supplementation with wild-type cDNAs should not restore function. Figure 1E shows that the MUTYH protein levels were complemented in part by transient transfection of nuclear and mitochondrial MUTYH cDNAs into the MUTYH Y165C–/– cell line. Functional analysis of the cDNA complemented cell lines are presented below.

Binding activities of A·8-oxoG and 8-oxoA·G mispairs are defective in MUTYH defective cell lines
Since the MUTYH protein levels were significantly lower in at least three of the four MUTYH polyposis cell lines, we asked whether the binding of mutated MUTYH proteins to mutagenic mispairs would be affected in these cell lines. We have previously found that MUTYH may cleave 8-oxoA from a QJ;8-oxoA:G mispair (Parker and Eshelman et al., submitted) and Jensen et al. (26) have reported that8-oxoA:C, but not 8-oxoA:G, is cleaved by OGG1. However, they did not identify what was responsible for cleavage of the 8-oxoA:G mispair. Since both bacterial MutY and human MUTYH cleave an adenine derivative, 2-hydroxyadenine (7,27), we hypothesized that MUTYH may play a role in the repair of the adenine derivative 8-oxoA from the 8-oxoA:G mispair and that cells from patients with the MAP phenotype may have impaired binding and cleavage of this mispair. Figure 2A shows that radioactively labelled oligonucleotides containing A·8-oxoG, a known MUTYH substrate, and8-oxoA·G mispairs are efficiently bound and UV cross-linked to a single protein, as determined by SDS–PAGE, in extracts of a MUTYH control cell line. It has been suggested that MUTYH is the major protein that binds these substrates [Parker and Eshleman et al., submitted; (28)] under these conditions. This was confirmed by cross-linking the same extracts with labelled substrates and immunoprecipitating MUTYH from the cell extracts. Figure 2B shows that these higher molecular mass radiolabelled bands can be detected after immunoprecipitation with the specific anti-MUTYH antibody but only after cross-linking with the substrates. Furthermore, western blot analysis with the same MUTYH antibody also identified shifted MUTYH protein bands using unlabelled substrates only after cross-linking (Figure 2C), whose size correlates with the cross-linked radiolabelled band (Figure 2B). Having established that this assay is relatively specific for MUTYH binding we measured the rate of binding of these substrates in the control cell lines and the four MUTYH defective cell lines. Using equal amounts of cell extract all four cell lines show lower rates of binding to both substrates when compared with the control cell lines, as shown in Figure 2D and E. MUTYH Y165C–/– consistently exhibited the lowest level of binding to both substrates, whereas MUTYH 1103delC/G382D and Y165C/G382D possessed ~40–50% binding. MUTYH G382D–/– cell extracts possessed ~70% binding of the controls cell lines. Since equal amounts of cell extracts were used, these results are consistent with and confirm the western blot data (Figure 1A and B).



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Fig. 2. MUTYH binding activities are defective in WCE that possess defective MUTYH. (A) UV cross-linking of radiolabelled A·8-oxoG and 8-oxoA·G mispaired oligonucleotides (60 min incubation) in WCE of a control cell line. Approximately 50 µg of WCE was loaded onto the gels for resolution. The gel was dried and exposed to X-ray film. (B) Immunoprecipitation of cross-linked radiolabelled A·8-oxoG and 8-oxoA·G mispaired oligonucleotides with anti-MUTYH antibody. (C) Western blot analysis of shifted MUTYH protein after cross-linking of unlabelled oligonucleotide mispairs in WCE (50 µg) of the control cell line. (D and E) Rates of binding of the cross-linked radiolabelled A·8-oxoG and 8-oxoA·G mispaired oligonucleotides in WCE (30 µg) of two control lines and the four MUTYH defective cell lines over 60 min incubation.

 
Functional repair activities of A·8-oxoG and 8-oxoA·G mispairs are also defective in MUTYH defective cell lines
Several of the MUTYH defective cell lines showed marked reduction in protein levels and corresponding ability to bind oligonucleotide heteroduplexes containing A·8-oxoG and8-oxoA·G mispairs. For these, we wanted to know whether the small levels of MUTYH protein that are present retained the function or not. For the G382D mutant, we hypothesized that it must be dysfunctional since its protein steady-state levels are relatively normal. We therefore examined the functional activities of these mutated MUTYH proteins to cleave the adenine and 8-oxoadenine from these substrates (Figure 3A and B). To obtain the most accurate estimation of each MUTYH protein's function directly, we first immunoprecipitated the cell extracts with a MUTYH specific antibody, under identical conditions, and assayed the activities of the immunoprecipitated proteins. Since MUTYH from MUTYH Y165C–/– cell line was present at the lowest steady-state level (Figure 1A and B), the levels of immunoprecipitated MUTYH proteins from all cell lines were diluted to approximately the same level as MUTYH present in the precipitates from MUTYH Y165C–/–. None of the MUTYH precipitates from the four defective cell lines possessed any significant cleavage activities of the A·8-oxoG mispair compared with the MUTYH precipitated from control cell lines (Figure 3A, compare lanes 3–6 with lanes 1, 2 and 7). Mutated MUTYH from the MUTYH Y165C–/– cell line also possessed low levels of cleavage of the oligonucleotide substrate containing the 8-oxoA·G mispair (Figure 3B, lane 3), whereas the MUTYH proteins from the other three defective cell lines possessed cleavage activity approximately similar to that observed in MUTYH pelleted from the control cell lines. To examine these activities further, we compared the rates of cleavage of both mispaired substrates. As expected, the rates of cleavage of the A·8-oxoG mispair by the four MUTYH pellets from defective cell lines were barely detectable, even after 60 min incubation, when compared with MUTYH from the two control cell lines (Figure 3C). However, although MUTYH precipitated from MUTYH G382D–/–, MUTYH 1103delC/G382D and MUTYH Y165C/G382D cell lines all possessed somewhat similar amounts of cleavage of the 8-oxoA·G mispair after 1 h of incubation (Figure 3B), a time course of cleavage suggested that all three cell lines actually possessed somewhat reduced rates of cleavage of this mispair (Figure 3D). MUTYH pelleted from Y165C–/– cells again had barely detectable activity with the 8-oxoA·G mispair.



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Fig. 3. Immunoprecipitated mutated MUTYH protein has defective cleavage activities. MUTYH proteins were immunoprecipitated from the four defective cell lines and two wild-type cell lines. The amount of MUTYH used in the assay was normalized to the amount of MUTYH pelleted from MUTYH Y165C–/–. Cleavage of radiolabelled A·8-oxoG (A) mispaired oligonucleotide after 60 min incubation with immunoprecipitated MUTYH from control cell lines (lanes 1 and 2), Y165C–/– cells (lane 3), G382D–/– cells (lane 4), 1103delC/G382D cells (lane 5), Y165C/G382D cells (lane 6) and the no protein control (lane 7). S represents uncleaved substrate and P represents cleaved product. Lanes 5–7 are from a non-concurrent experiment. (B) Cleavage of radiolabelled 8-oxoA·G mispaired oligonucleotide after 60 min incubation with immunoprecipitated MUTYH from control cell lines (lanes 1 and 2), Y165C–/– cells (lane 3), G382D–/– cells (lane 6), Y165C/G382D cells (lane 5), 1103delC/G382D cells (lane 4) and the no protein control (lane 7). Rates of cleavage of radiolabelled A·8-oxoG (C) and 8-oxoA·G (D) mispaired oligonucleotides by immunoprecipitated MUTYH proteins as in (A and B). (E and F) Correction of defective MUTYH glycosylase activity in MUTYH Y165C–/– cell line with transiently expressed exogenous mitochondrial and nuclear MUTYH wild-type protein. Total MUTYH was immunoprecipitated from MUTYH Y165C–/– and MUTYH Y165C–/– transfected with either nuclear or mitochondrial MUTYH cDNA, and incubated for 60 min at 37°C with radiolabelled A·8-oxoG (E) or 8-oxoA·G (F) mispaired oligonucleotides. (G and H) Correction of defective MUTYH glycosylase activity in MUTYH G3820–/– cell line with transiently expressed exogenous mitochondrial and nuclear MUTYH wild-type protein. Total MUTYH was immunoprecipitated from MUTYH G382D–/– and MUTYH G382D–/– transfected with either nuclear or mitochondrial MUTYH cDNA, and incubated for 60 min at 37°C with radiolabelled A·8-oxoG (G) or 8-oxoA·G (H) mispaired oligonucleotides.

 
Expression of exogenous human nMUTYH and mMUTYH cDNAs complement the glycosylase activities in MUTYH immunoprecipitations from the four defective cell lines
If the observed defective repair activities, in the MUTYH precipitations, are directly due to dysfunctional MUTYH protein produced from the known MUTYH mutations in these cell lines, then the activity should be restored by the expression of exogenous wild-type MUTYH protein. We therefore performed transient transfection of these cells with cDNA expression vectors containing either the mitochondrial or nuclear MUTYH cDNAs. Figure 3E–H shows that immunoprecipitation of MUTYH, from genetically complemented cells expressing either recombinant nMUTYH and mMUTYH, could complement the defective adenine and 8-oxoadenine cleavage activities observed in MUTYH pellets from untransfected MUTYH Y165C–/– (Figure 3E and F) and MUTYH G382D–/– (Figure 3G and H). Exogenous expression of MUTYH also corrected activity for the cell lines bearing 1103delC/G382D and Y165C/G382D mutations (data not shown).

Defective MUTYH may not significantly affect the killing curves after treatment with H2O2 and menadione
MUTYH is essential for protecting the genome from the deleterious effects of oxidative DNA damage and thus in the absence of MUTYH one may expect increased sensitivity upon treatment with free radical producing chemicals. In fact, the idea of capitalizing on the DNA repair defect as a therapeutic strategy, by treating cancer cells with mutagens that create lesions that the cells are incapable of repairing, was previously established by treating mismatch repair (MMR) defective cells with frameshift mutagens (29). However, similar to previous observations (10), treatment of MUTYH defective cells with either H2O2 or menadione, chemicals that produce oxidative DNA damage, had no significant consistent effect on the killing curves when compared with the control cell lines (Figure 4).



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Fig. 4. MUTYH defective cell lines may not have altered killing curves when treated with chemicals that produce oxidative DNA damage. Four MUTYH defective and two control cell lines were incubated with 0–30 µM H2O2 (A) or 0–6 µM menadione (B) and incubated for 60 min at 37°C. Cells were washed and seeded in regular media. After incubation for a further 5 days at 37°C, cells were trypsinized, washed and counted using trypan blue exclusion.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Defective MUTYH has been proposed to be a causative agent for the development of multiple colorectal adenomas, the so called MAP syndrome. In an attempt to understand the pathogenesis of defective MUTYH, most studies have used site directed mutants to analyse the biochemical implications of the MUTYH mutations identified in patients (11,19,20). However these studies do not fully explain the mechanisms of colorectal pathogenesis and do not address issues such as the levels of expression of mutant MUTYH proteins in patients' cells that arise from biallelic mutations in the human MUTYH genes. In this report we have examined four lymphoblastoid cell lines, with well-defined biallelic MUTYH mutations, established directly from affected MAP patients.

Previous reports have suggested that the Y165C mutation may reduce the ability of MUTYH to recognize the 8-oxoG and guanine component of adenine mispairs (11). Y165 is part of one the two helix–hairpin–helix motifs, which is thought to be crucial for recognition and binding of the mispaired substrates and is highly evolutionarily conserved among MUTYH homologs (30). Fromme et al. (31) have recently showed that Y88 in Bacillus stearothermophilus MutY (homologous to Y82 in E.coli) actually residues between the 8-oxoG residue and nucleotides 5' to 8-oxoG and forms a hydrogen bond with a serine that forms a hydrogen bond with 8-oxoG. It has been suggested that Y165 maybe required for the ‘flipping out’ of8-oxoG molecules or stabilizing the gap left after ‘flipping out’. It is also possible that this residue is involved in a conformational change that occurs upon substrate binding. Chmiel et al. (19) showed that this mutation in bacterial MutY enhances the turnover rate and thus may contribute to the pathogenesis of this mutant by early inappropriate release of the pro-mutagenic mispairs AP¥G or AP¥8-oxoG. In this report, we demonstrate that Y165C containing MUTYH protein is dysfunctional for two separate reasons. First, cells expressing biallelic Y165C MUTYH mutations contain low levels of the MUTYH protein (~5% of normal cells), which has not been observed when studying Y165C mutants expressed from exogenous vector DNA (11,19,20) (Figure 1A and B), and second the protein instability is not due to mRNA instability. Using immunoprecipitated MUTYH from Y165C–/– cells, at similar levels to that precipitated from wild type cells, the rates of MUTYH catalysed binding and cleavage of the A·8-oxoG and 8-oxoA·G mispairs were significantly reduced by the biallelic Y165C mutation, with the levels just above baseline. Thus the Y165C containing MUTYH protein is both unstable in the cell and is also dysfunctional when tested at levels comparable with those present in normal cells.

The G382D–/– biallelic mutation produces only G382D mutation bearing protein. The pathogenesis of this mutation is thought with involve altered recognition of 8-oxoG, which is evident by the significantly lowered glycosylase activity of the bacterial mutant protein (11) and the lack of activity in human MUTYH (20), and our results support the same conclusion. Cells expressing biallelic G382D MUTYH mutations contain mRNA and protein at levels comparable with other cells, and so, unlike Y165C (Figure 1A and B) this mutation does not grossly affect protein stability. When binding activity was examined (Figure 2D and E), this MUTYH mutation showed ~50% binding activity relative to wild-type protein. While this binding is clearly somewhat reduced, the major effect is demonstrated functionally where these proteins exhibit defective repair of an A·8-oxoG mispair and lower rates of repair of 8-oxoA·G (Figure 3A–H). Cell extracts from MUTYH defective mouse cells stably transfected with a G365D mutant also possess no cleavage activity of adenine mispaired with 8-oxoguanine, which is correlating with our data (10). However, E.coli expressed G365D MUTYH protein exhibits in vitro cleavage activity in levels very similar to the wild-type MUTYH for the A:8-oxoG mispair (3234) and has a more pronounced defect in the cleavage of the adenine derivative2-hydroxyadenine from a 2-OH-A:G mispair (33). To explain this apparent difference between in vitro activity and in vivo activity, it has been suggested that this mutation may affect the phosphorylation status of MUTYH (10) and this is correlated with previous results from our laboratory showing that MUTYH cleavage of A·8-oxoG is in fact influenced by phosphorylation, which is defective in some CRC cell lines with wild-type MUTYH genes (10,22).

Both 1103delC/G382D and Y165C/G382D cell lines possess ~50% of MUTYH steady-state protein levels, which is most easily explained by protein instability of the frameshift mutant 1103delC and the instability of the Y165C mutant (relatively, see above) with residual MUTYH levels due to expression of the G382D mutant allele. The observed binding and cleavage activities from these cells also are most probably due to the presence of G382D mutant MUTYH protein (Figure 2D and E, Figure 3C and D).

Two interesting observations arise from this work. First, none of the four MUTYH defective cell lines showed altered survival after challenging them with the oxidative damaging agents, H2O2 and menadione. Hirano et al. (10) also reported the same phenotype for MUTYH–/– murine cells. The explanation of this result is not clear at this time since one would certainly intuitively expect that cells that are defective in repair of a certain lesion should be sensitive to chemicals known to produce these lesions (29), in contrast to the results with N-methyl-N'-nitro-N-nitrosoguanidine and MMR defective cell sensitivity. The most likely explanation of these data may be the redundancy of this repair system such that the mutations are repaired by the other two members, MTH1 and OGG1.

Second, is the finding that MUTYH defective cells are impaired in their cleavage of 8-oxoA from an 8-oxoA·G mispair. It is interesting that the spontaneous mutation spectrum in MutY defective E.coli reveals a huge increase in G–T transversions but no detectable increase in A to G transitions or A to C transversions, which would be reminiscent of8-oxoA incorporation and lack of repair. This leaves the question to whether MUTYH catalysed repair of 8-oxoA is biologically significant, but this may also depend on the relative frequency of 8-oxoA:G mispair formation and the relative removal of 8-oxodATP from the system.

In conclusion, our findings demonstrate different mechanisms of pathogenesis caused by different MUTYH mutations in patients with MUTYH polyposis. The Y165C mutation subverts function in two ways, protein stability and MUTYH function. Incorporating the fact that the G382D homozygous cells contain wild-type levels of MUTYH protein, the 1103delC mutation also causes MUTYH protein instability. The major effect of the G382D mutation, in contrast, is not protein instability, but rather is its manifestation as a dysfunctional protein. Additional work will be needed to characterize the mutations present in different ethnic backgrounds to aid in identifying the biallelic individuals in mutation bearing families so that more aggressive screening can be applied and colon cancer prevented.


    Notes
 
5Present address: The Binding Site, ELISA Department, PO Box 11712, Birmingham, B14 4ZB, UK Back


    Acknowledgments
 
This work was supported by grants R01CA81439-01 and K08CA66628-01A1 (JRE). The authors would like to thank Drs Sanford Markowitz, Jim Willson, Bert Vogelstein and Victor E. Velculescu for the helpful discussions.Conflict of Interest Statement: None declared.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 24, 2005; revised June 16, 2005; accepted June 18, 2005.





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