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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: 8-oxoG, 8-hydroxyguanosine; APC; adenomatous polyposis coli; CRC, colorectal cancer; MAP, MUTYH associated polyposis; MMR, mismatch repair; MUTYH, MutY homolog
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For infection with EpsteinBarr 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 412 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(dIdC), 20 mM NaCl and 25 mM EDTA were added. Following a 060 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 SDSPAGE. 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 TrisHCl, 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 SDSPAGE 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 030 µM H2O2 (Sigma) or 06 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 SDSPAGE, 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 4050% 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).
|
|
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).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 helixhairpinhelix 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 3AH). 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 GT 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 |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|