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[Cancer Research 62, 7230-7233, December 15, 2002]
© 2002 American Association for Cancer Research

Carcinogenesis

8-Hydroxyguanosine Repair Is Defective in Some Microsatellite Stable Colorectal Cancer Cells1

Antony R. Parker, Robert N. O’Meally, Dwight H. Oliver2, Li Hua, William G. Nelson, Theodore L. DeWeese and James R. Eshleman3

Departments of Pathology [A. R. P., D. H. O., L. H., J. R. E.], Oncology [W. G. N., T. L. D., J. R. E.], Urology [R. N. O., W. G. N., T. L. D.], and Pharmacology and Medicine [W. G. N.], Johns Hopkins University, Baltimore, Maryland 21205


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mutator phenotypes are involved in the carcinogenesis of some cancers, e.g., defects in mismatch repair produce a mutator phenotype that drives carcinogenesis and causes microsatellite instability in hereditary nonpolyposis colon cancers and some sporadic colorectal cancers (CRC). Less understood, however, is the potential role of mutator phenotypes in microsatellite stable (MSS) CRC carcinogenesis. A novel transversion mutator phenotype was reported recently in an MSS CRC cell line. We hypothesized that 8-hydroxyguanosine could be involved and found elevations in 5 of 15 (33%) MSS CRC cell lines analyzed. Repair of an adenine{bullet}8-hydroxyguanosine mispair was functionally defective in the same five cell lines. The human MutY homologue transcript and MutY homologue protein levels were also decreased. These findings may reflect a MSS mutator phenotype contributing to the development of CRC.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The malignant phenotype, in many cancers, requires altered expression of multiple gene products (1) . This, coupled with the low mutation rate in normal cells, led Loeb to hypothesize that during multistep carcinogenesis, premalignant cells must acquire a "mutator phenotype" (2) . By increasing the mutation rate, mutator phenotypes produce sufficient numbers of alterations in oncogenes and tumor suppressor genes, which if selected for during clonal evolution and expansion, are then seen in the resulting tumor. Consistent with this hypothesis, mutations in the genes encoding components of the DNA MMR4 system cause mutator phenotypes that profoundly increase the rates of both base substitutions and frameshift mutations (3) . As a result, important cellular genes with long mononucleotide repeats are especially susceptible to inactivation, e.g., transforming growth factor ß receptor type-II (4) . This high mutation rate also causes microsatellite instability (replication error phenotype; Refs. 5 and 6 ). Whether another mutator phenotype is also involved in MSS CRC carcinogenesis remains unanswered.

A novel transversion mutator phenotype, in an MSS- and MMR-proficient CRC cell line, has been reported recently (7) . The spectrum of mutations, which arises in these cells, does not include frameshift mutations, as seen in MMR-defective cells, but instead predominates in transversions. Among the DNA lesions that readily result in transversions is 8-oxo-G (8) , which arise from the attack on genomic DNA by reactive oxygen species and are generated at a frequency of ~104 8-oxo-G residues daily per cell (9) . The carcinogenic potential of 8-oxo-G arises from its ability to readily mispair with adenine producing G:C to T:A and A:T to C:G transversions (9) . In Escherichia coli, MutY and MutT have been shown to directly prevent the formation of, or repair of, an A{bullet}8-oxo-G mispair in the genome (9 , 10) . MutY catalyzes the removal of adenine bases mispaired with 8-oxo-G, and MutT is an 8-oxo-GTPase, which converts the triphosphate 8-oxo-G form to an inactive monophosphate form. Human homologues of both MutY and MutT have been cloned, MYH and MTH1, respectively (11 , 12) .

Because elevations of 8-oxo-G might explain a mutator phenotype with a spectrum dominant in transversions, we hypothesized that an increase in genomic 8-oxo-G may be responsible for the novel transversion mutator phenotype. We report that 5 of 15 MSS CRC cell lines possess elevations of 8-oxo-G in their genomic DNA and decreased A{bullet}8-oxo-G repair activity, in addition to decreased levels of MYH mRNA and MYH protein.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Preparation of WCE, and Western Blotting.
The 15 human colorectal MSS cell lines used in this study were grown as described (13) . Those bearing a VACO prefix were provided generously by Dr. James Willson (Ireland Cancer Center and Case Western Reserve University), whereas the others were obtained from the American Type Culture Collection (Manassas, VA). Cells were harvested as described previously (14) . Western blotting analyses were performed as described previously (15) using an antihuman MYH antibody (Alpha Diagnostics, San Antonio, TX) and an antihuman actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at dilutions of 1:100 and 1:200, respectively.

LC/MS/MS Measurement of 8-Oxo-G.
Genomic DNA was extracted from cultured cells using the Genomic DNA Extraction Kit (Qiagen, Valencia, CA). Working standard solutions of 8-oxo-G and dG (Sigma, St. Louis, MO) were prepared daily from stock solutions frozen at -80°C. The concentration of each standard was checked by UV absorbance before each sample run. For each sample, at least three independent measurements of dG and 8-oxo-G, each with independent calibrations, were performed. All analyses were performed in a blinded fashion. DNA was hydrolyzed to individual nucleosides as described previously (16) , with the exception that ammonium acetate was substituted for sodium acetate to reduce adduct formation. A PE Biosystems high-performance liquid chromatography equipped with two series 200 micropumps, one series 200 autosampler (Perkin-Elmer, Norwalk, CT), and a Phenomenex Aqua high-performance liquid chromatography column (150 x 2 mm, 5 microns; Phenomenex, Torrance, CA) was used to separate hydrolyzed DNA bases. This system was coupled to a series 200 UV/visible detector, as well as a PE Biosystems API 3000 triple quadruple mass spectrometer with a turboionspray source, controlled by Analyst software (Version 1.2; PE Biosystems). Sample analysis was performed as follows: (a) the mobile phase consisted of eluent A, 2 mM ammonium acetate, adjusted to pH 3.0 with formic acid and eluent B, 90% acetonitrile with 10% miliQ water; (b) separation was accomplished via isocratic followed by gradient elution at a flow rate of 200 µl/min; (c) electrospray ionization was performed in the positive ion mode for 8-oxo-G by monitoring the optimal parent-daughter ion transition of 284 to 168; and (d) dG was quantified by monitoring at 252 nm with the UV/visible detector. This method allows for simultaneous measurement of both analytes. The data are expressed as amount of 8-oxo-G per 106 dG.

A{bullet}8-Oxo-G Glycosylase Assay.
A{bullet}8-oxo-G glycosylase activity was performed as described previously (15) , except the reaction buffer contained 10 mM Tris-HCl (pH 7.6), 0.5 mM DTT, 5 µM ZnCl2, 30 mM NaCl, and 5 mM MgCl2. Equal lane loading was confirmed with SDS-PAGE.

Reverse Transcriptase-PCR Analysis.
Total RNA was isolated using the Qiagen RNeasy Kit (Qiagen). First-strand cDNA was synthesized in 50 mM Tris-HCl, 8 mM MgCl2, 30 mM KCl, 1 mM DTT (pH 8.5) with 3 µg of total RNA using avian myeloblastosis virus reverse transcriptase (Roche Diagnostics, Indianapolis, IN), 1 µg of oligodeoxythymidylic acid12–18 primer, 1 mM deoxynucleotide triphosphates, and 40 units of RNase inhibitor at 70°C for 2 min, 42°C for 60 min, and 94°C for 5 min. For PCR amplification, the reactions contained 200 µM deoxynucleotide triphosphates, 2 mM MgSO4, 0.2 µM each primer (Research Genetics, Huntsville, AL), 5 µl of cDNA, and 2.5 units of Hi-Fidelity Taq Polymerase (Invitrogen, Carlsbad, CA). PCR amplification was performed for 94°C for 1 min; 35 cycles of 94°C for 1 min, 52°C for 1 min, and 68°C for 3 min; followed by 69°C for 10 min. We designed PCR primers to detect all reported MYH (F: 5'-AGAAGATGAGATGGACCTGGACAGG-3', R: 5'-TAGCTCCATGGCTGCTTGTGGT-3') and MTH1 mRNA isoforms (17, 18, 19) . Internal controls were GAPDH (5'-CGGAGTCAACGGATTTGGTCGTAT-3' and 5'-AGCCTTCTCCATGGTGGTGAAGAC-3') and ß-2 microglobulin (5'-ACTCACGTCATCCAGCAGAGAATG-3' and 5'-CATCAAACATGGAGACAGCACTCA-3') mRNAs.

MYH cDNA Sequencing.
Full-length MYH cDNA was prepared as described above and with three more additional sets of primers: MYH exons 1–3 (-146 to 398, F: 5'-TCTGAAGCTTGAGGAGCCTCTAGAACT-3', R: 5'-TTGATCACAGTGGCAACCTGGGT-3'), MYH exons 3–9 as above, MYH exons 8–12 (724–1211, F: 5'-ACCCTTGTTTCCCAGCAGCT-3', R:5'-TTGCGCTGAAGCTGCTCTGA-3'), and MYH exons 12–16 (1114–1650, F: 5'-CAAATTCTGCTGGTGCAGAGG-3', R: 5'-ACTAACAACAGGATTCTCAGGGAA-3'). MYH cDNA samples from the cell lines VACO8, VACO411, VACO425, VACO429, and VACO489 were sequenced using the above primers for forward and reverse BigDye 3.0 cycle sequencing on an ABI377 sequencer (Applied Biosystems, Forest City, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Genomic 8-Oxo-G Levels Are Increased in MSS CRC Cells.
We hypothesized that increased 8-oxo-G in genomic DNA might be, in part, responsible for the novel transversions in an MSS CRC cell line (7) . We measured the level of 8-oxo-G in the genomic DNA of VACO411 using LC/MS/MS (Fig. 1A)Citation and found a 14-fold increase when compared with the nonmutator control cell line SW480 (13) . Because elevated 8-oxo-G levels might contribute to MSS CRC carcinogenesis more generally, we measured 8-oxo-G levels in 14 additional MSS CRC cell lines. We found four additional MSS lines (5 of 15 total cell lines, 33%), VACO8, VACO411, VACO425, VACO429, and VACO 489, contained ~10–20 times more genomic 8-oxo-G than the control line SW480 (P = 0.0001).



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  		Fig. 1. MSS CRC cells contain increased 8-oxo-G and defective repair of A{bullet}8-oxo-G mispairs. In A, the 8-oxo-G content was measured using LC/MS/MS. The results represent the mean 8-oxo-G content from three to four independent preparations of genomic DNA per cell line. Bars, 2 SE. The results were analyzed statistically using an unpaired t test. B, in vitro repair activity with the A{bullet}8-oxo-G mispair by MSS CRC WCE (9 µg) after incubation for 4 h at 37°C. M signifies the free A{bullet}8-oxo-G mispair, and P is the cleaved product. Lane 10 is from a nonconcurrent experiment. C, rate of repair activity with the A{bullet}8-oxo-G mispair by MSS CRC WCE (9 µg) after incubation for 0–4 h at 37°C. Symbols: {blacksquare}, SW480; {blacktriangledown}, VACO429; {blacktriangleup}, VACO489; {diamondsuit}, VACO425; {bullet}, VACO8; {square}, VACO411; {triangleup}, VACO9M; {triangledown}, VACO400; {diamond}, SW837. Bars, SE from three experiments. D, repair activity with the A{bullet}8-oxo-G mispair by various amounts of MSS CRC WCE (0–9 µg) after incubation for 4 h at 37°C. Symbols as above.

 
Repair Activity of A{bullet}8-Oxo-G Is Defective in MSS CRC Cells.
The increase in genomic 8-oxo-G levels may reflect decreased repair of A{bullet}8-oxo-G, so we examined the repair activity using cell extracts of the above cell lines and an oligonucleotide containing this mispair (Fig. 1B)Citation . The repair activity in the five MSS CRC cell lines, which exhibited elevated 8-oxo-G levels, was generally lower than the control SW480 cell line. Three of these five cells lines, VACO425, VACO8, and VACO411 (Lanes 8–10), showed marked reductions in repair (all ~0.0044 fmol/µg/4 h) when compared with SW480 (0.068 fmol/µg/4 h), whereas the two other lines, VACO429 and VACO489 (Lanes 4 and 5), exhibited only slightly slower repair activities of 0.044 and 0.042 fmol/µg/4 h. Interestingly, the cell lines with normal 8-oxo-G levels, VACO9M, VACO400, and VACO837 had repair activities roughly comparable with SW480 (Lanes 2, 3, 6, and 7). Because VACO429 and VACO489 still repaired the mispair to some extent, we examined the rate of A{bullet}8-oxo-G repair. Using a constant concentration of WCE, the rate of repair activity for VACO429 and VACO489 was approximately half that of the rate exhibited by the control lines (Fig. 1C)Citation . As expected, VACO8, VACO411, and VACO425 again showed only minimal repair activity. A similar result was obtained when repair was measured by varying WCE concentrations over a fixed time (Fig. 1D)Citation .

MYH mRNA and MYH Protein Levels, but not that of MTH1, Are Decreased in MSS CRC Cells.
Because the data indicate ineffective repair of A{bullet}8-oxo-G, we hypothesized that the human MYH or MTH1 might be defective in these cell lines, thereby accounting for both the increased levels of 8-oxo-G and the lack of A{bullet}8-oxo-G repair. Measurements of MYH mRNA levels were altered in three (VACO489, VACO425, and VACO411; Fig. 2ACitation ) of the five previous suspect cell lines with elevated 8-oxo-G. In contrast to MYH mRNA alterations, the expression of MTH1 mRNA did not appear to be substantially different in any of the five MSS CRC lines with elevated genomic 8-oxo-G (data not shown). Corresponding to the decrease in MYH mRNA, Western blot analysis for MYH protein in the WCE was also decreased significantly (P = 0.0001) in the five identified cell lines (Fig. 2B)Citation . VACO411, VACO429, and VACO489 retained slight expression of MYH protein (10–20% of control lines), whereas VACO8 and VACO425 had no detectable expression of the protein when compared with the control lines (SW480, SW837, VACO400, and VACO9M) containing basal levels of 8-oxo-G.



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  		Fig. 2. MYH mRNA and protein levels are decreased in the MSS CRC cell lines with elevated genomic 8-oxo-G. A, MYH mRNA levels in six MSS CRC cell lines were assessed by reverse transcriptase-PCR and quantified. GAPDH and ß-2 microglobulin were used as internal controls. SE of three experiments. B, Western blot analyses of the five MSS CRC cell lines, with elevated 8-oxo-G, and four control lines were performed as described previously. The membranes were probed with anti-MYH or anti-actin antibodies. The Western blots were quantified; bars, SE of three individual experiments. The results were statistically analyzed using an unpaired t test.

 
MYH cDNA Is Not Mutated in the Defective CRC Cell Lines.
The decreases in MYH mRNA and MYH protein levels, as well as A{bullet}8-oxo-G repair activities in five MSS CRC cell lines, suggested that mutations may exist in MYH cDNA. Complete sequencing of full-length MYH cDNA from the cell lines VACO8, VACO411, VACO425, VACO429, and VACO489 showed that no mutations were present in their respective cDNAs when compared with the published primary transcript sequence (data not shown; Ref. 11 ). Interestingly, in addition to the major primary transcript, in VACO429, the MYH{alpha}4 isoform (18) was also present, whereas in the other four cell lines, the MYH{alpha}2 isoform was also present.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data suggest that the repair of 8-oxo-G is defective in 5 of 15 (33%) MSS CRC cell lines tested and that decreased MYH expression and activity are correlated with it. We hypothesize that defective postreplication repair of 8-oxo-G may be part of the mechanism for the transversion mutator phenotype observed in VACO411 and for some portion of MSS CRC carcinogenesis. A comparison of the results with age and sex of the individuals, whose tumors were used to generate the cell lines, revealed no obvious bias toward a younger age group or particular gender.

Two of the five cell lines (VACO411 and VACO425) show consistent elevations in 8-oxo-G, decreased A{bullet}8-oxo-G binding, and repair activity, in addition to decreased mRNA and protein levels of MYH (Table 1)Citation . The remaining three cell lines are more complex. In VACO429 and VACO8, the mRNA levels appear approximately normal, but MYH protein, binding, and repair activity are absent or moderately decreased. However, levels of 8-oxo-G in these two cell lines are as elevated as in the other three. These data may reflect dysfunctional protein, increased MYH protein turnover, or reduced cofactor levels (e.g., APE1). In VACO489, mRNA and protein levels are markedly decreased, whereas binding and repair activity are only modestly decreased. This may reflect relative sensitivities of the two assays, such that moderate decreases in protein levels produce only modest decreases in functional activity.


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  		Table 1 Summary of results

 
Mutations in MYH have been identified previously in lung cancer, further suggesting a role for defective MYH as a cause of a mutator phenotype. Shinmura et al. (20) showed that a missense mutation, R170P (G to C), was present in type-1 MYH in the lung cancer cell line NCI-H157, although no functional data were presented. Al-Tassan et al. (21) described a single family with an increased incidence of CRC correlating with MYH gene mutations. Because APC gene sequencing revealed transversions instead of truncating mutations, they sequenced the MYH gene and found that affected kindred members had inherited two defective MYH alleles. In our study, however, sequencing of the full-length MYH cDNA from the five suspect MSS CRC cell lines did not reveal any mutations. Interestingly, MYH activity has been shown previously to be affected by phosphorylation (22) , and we are at present examining promoter methylation and protein phosphorylation status in these cell lines.

Although MYH appears defective, many other proteins have also been implicated in the repair of 8-oxo-G. Human Mut S homologues 2 and 6 heterodimer binds to 8-oxo-G-containing oligonucleotides, stimulating its ATPase and ADP->ATP exchange activities, and MSH2 -/- cells possess elevated 8-oxo-G levels (23 , 24) . Recently, the MSH6 component of Mut S homologues 2 and 6 heterodimer has been shown to interact with, and stimulate the glycosylase activity of, MYH (25) . The Cockayne syndrome B gene product is required for general genome repair of 8-oxo-G (26) , and both BRCA1 and BRCA2 participate in transcription-coupled repair of 8-oxo-G (27) . 8-Hydroxyguanine glycosylase removes 8-oxo-G, in nontranscribed DNA, from opposite cytosine (9) , whereas endonuclease III homologue repairs 8-oxo-G mispaired with guanine (9) . So, with these overlapping substrate specificities, it may be of significance to determine whether any of these gene products are also defective in MSS CRC.

Increases in the levels of oxidative stress and genomic 8-oxo-G have been detected in patients with several forms of cancer. These results demonstrate, for the first time, a possible link between the mutator phenotype and elevations of 8-oxo-G in colon cancer carcinogenesis. We postulate that defective 8-oxo-G repair could be an early event in the development of carcinogenesis and the mutator phenotype in MSS CRC cells, leaving the cell susceptible to oxidative stress. It is interesting that MYH defects might be responsible.


   ACKNOWLEDGMENTS
 
We thank Dr. James Willson (Case Western Reserve University) for generously providing the human colon cancer cell lines. We also thank Drs. Christophe Rosty and Michael Goggins for the DNA primers for the GAPDH gene.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by National Cancer Institute Grants R01CA81439 and K08CA66628 (J. R. E.). Back

2 Present address: University of Texas, Department of Pathology, MSB, Rm 2.292, 6431 Fannin, Houston, TX 77025. Back

3 To whom requests for reprints should be addressed, at The Johns Hopkins University, School of Medicine, Department of Pathology, 720 Rutland Avenue, 632 Ross Building, Baltimore, MD 21205. Phone: (410) 955-3511; Fax: (410) 614-0671; E-mail: jeshlema{at}jhmi.edu Back

4 The abbreviations used are: MMR, mismatch repair; 8-oxo-G, 8-hydroxyguanosine; A{bullet}8-oxo-G, adenine{bullet}8-hydroxyguanosine; CRC, colorectal cancer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LC/MS/MS, liquid chromatography and tandem mass spectroscopy; MSS, microsatellite stable; dG, guanosine; MTH, MutT homologue; MYH, MutY homologue; WCE, whole cell extract. Back

Received 5/31/02. Accepted 10/28/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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X. Ouyang, T. L. DeWeese, W. G. Nelson, and C. Abate-Shen
Loss-of-Function of Nkx3.1 Promotes Increased Oxidative Damage in Prostate Carcinogenesis
Cancer Res., August 1, 2005; 65(15): 6773 - 6779.
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A. R. Parker, R. N. O'Meally, F. Sahin, G. H. Su, F. K. Racke, W. G. Nelson, T. L. DeWeese, and J. R. Eshleman
Defective Human MutY Phosphorylation Exists in Colorectal Cancer Cell Lines with Wild-type MutY Alleles
J. Biol. Chem., November 28, 2003; 278(48): 47937 - 47945.
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