Skip Navigation


Carcinogenesis Advance Access originally published online on March 14, 2007
Carcinogenesis 2007 28(7):1499-1503; doi:10.1093/carcin/bgm056
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/7/1499    most recent
bgm056v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Li, L.
Right arrow Articles by Pereira, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, L.
Right arrow Articles by Pereira, M. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published by Oxford University Press 2007.

Modulation by budesonide of a CpG endonuclease in mouse lung tumors

Long Li1,4, Lianhui Tao2, Ronald A. Lubet3, Vernon E. Steele3 and Michael A. Pereira1,2,*

1 Department of Biochemistry and Cancer Biology, Medical University of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA
2 Division of Hematology and Oncology, Department of Internal Medicine, College of Medicine, Ohio State University, 1148 CHRI, 300 West 10th Avenue, Columbus, OH 43210-1240, USA
3 Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892, USA
4 Present address: Division of Biology, California Institute of Technology, Mail Code 156-29, 1200 East California Boulevard, Pasadena, CA 91125, USA

* To whom correspondence should be addressed. Tel:+1 614 293 6864; +1 614 293 4072; Email: michael.pereira{at}osumc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
CpG endonuclease activity was identified in nuclear extracts obtained from mouse lung tumors. Enzyme activity was determined using a 333 bp polymerase chain reaction product of the estrogen receptor-{alpha} gene that contained either radiolabeled cytosine or tritium-labeled methyl groups at CpG sites. Activity was measured as the release of radioactivity from the substrate. The product of the nuclease activity was identified by high pressure liquid chromatography (HPLC) as either 5-methyl-2'-deoxycytidine when the CpG sites in the substrate were methylated or 2'-deoxycytidine when the CpG sites were not methylated. The CpG endonuclease activity was dependent on nuclear protein and temperature, had a proclivity for double-stranded over single-stranded DNA and was inhibited by ethylenediaminetetraacetic acid or 2-mercaptoethanol. Strain A/J mouse lung tumors induced by vinyl carbamate had a greater level of CpG endonuclease activity than non-involved lung tissue. Budesonide, a potent chemopreventive agent in mouse lung, not only prevented an increase in CpG endonuclease activity in lung tumors but, when administered to mice with established tumors, also decreased the level of endonuclease activity in the tumors. The effect of budesonide on CpG endonuclease activity in lung tumors was inversely related to its published effect on DNA methylation in mouse lung tumors, i.e. the drug decreased CpG endonuclease activity and increased the methylation of DNA. The increased CpG endonuclease activity in mouse lung tumors and its inhibition by budesonide would suggest this endonuclease as a potential molecular target for chemoprevention.

Abbreviations: 5-Me-C, 5-methyl-cytosine; HPLC, high pressure liquid chromatography; PCR, polymerase chain reaction


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Lung cancer is the most common form of cancer diagnosed and the leading cause of cancer-related death worldwide. The development of agents that prevent lung cancer is needed to decrease the high incidence of and mortality from lung cancer. Surrogate end-point biomarkers are being developed for use in determining the potential efficacy of chemopreventive agents without having to rely on an invasive tumor end-point. One early molecular alteration found in most tumors, including lung cancer in humans and laboratory animals, is a decrease in DNA methylation, i.e. DNA hypomethylation (1). In normal tissue, ~5% of the cytosine in DNA is methylated as 5-methyl-cytosine (5-Me-C) in CpG sites dispersed throughout the DNA, including repetitive sequences and the bodies of genes (2). It is this dispersed global methylation that is decreased in tumors (3).

Budesonide, an anti-inflammatory glucocorticoid, has been shown to be very effective in preventing chemically induced mouse lung tumors when administered either by inhalation or in the diet (4–7). Budesonide has also been shown to be very effective in preventing DNA hypomethylation in mouse lung tumors (7–9). When administered to mice with lung tumors, budesonide very rapidly, within days, increased the methylation of tumor DNA resulting in DNA that was no longer hypomethylated (10). DNA hypomethylation in tumors could result either from a decrease in the methylation of DNA or from an increase in the demethylation of DNA. DNA methylation is catalyzed by DNA methyltransferases, including DNA methyltransferase 1, 3a and 3b, but it is unlikely that DNA hypomethylation results from a decrease in the methylation of DNA since many cancers, including lung tumors, have been shown to have increased activity of DNA methyltransferases (1,11). DNA hypomethylation could result from the demethylation of methylated DNA; however, the enzymes responsible for DNA demethylation have not been identified (12,13). The enzyme responsible for DNA hypomethylation could possibly be a 5-Me-C DNA demethylase that removes only the methyl group, a glycosylase that removes 5-Me-C or a nuclease that results in the release of 5-methyl-2'-deoxycytidine monophosphate or 5-methyl-2'-deoxycytosine from DNA.

In this manuscript, we describe a CpG endonuclease activity in a nuclear protein extract that excises both methylated and unmethylated deoxycytosine specifically from CpG sites in DNA. We also report that the CpG endonuclease activity is increased in mouse lung tumors and that budesonide prevents or reduces its activity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Experimental design and animal treatment
Lung tissue and tumors used in this study were from previously published experiments (7,10). Lung tumors were induced in female A/J mice by intra-peritoneal injection of 16 mg/kg vinyl carbamate administered once a week for two consecutive weeks. In one experiment, 2.4 mg/kg budesonide was administered in the diet by three treatment regimens, i.e. 4–20, 4–35 or 20–35 weeks after the second dose of vinyl carbamate with all the mice being killed at week 35 (7). Budesonide administered from weeks 4 to 35 but not from weeks 4 to 20 or 20 to 35 decreased lung tumor multiplicity at week 35. However, all the three treatment regimens did decrease the size of the tumors and the progression of adenomas to carcinomas. Budesonide administered from weeks 4 to 35 and 20 to 35 increased DNA methylation in the tumors, reversing the hypomethylation in them. In the other experiment, the mice were administered 2.0 mg/kg budesonide in their diet for 0, 2, 7 or 21 days prior to killing that was performed 27 weeks after the first dose of vinyl carbamate (10). Other mice received budesonide for 14 days followed by a 7-day holding period prior to killing. In this experiment, tumor multiplicity was not altered, while two days of treatment with budesonide was sufficient to decrease the size of the lung tumors and to increase DNA methylation in them, with both effects requiring continued treatment. In both experiments, the left lobe of the lungs was frozen in liquid nitrogen and stored at –70°C.

Nuclear protein extraction
Lung tissue and tumors were homogenized and incubated for 15 min on ice with 10 mM Tris–HCl (pH 8.0), 3 mM MgCl2, 50 mM KCl, 0.5% Nonidet P-40, 0.01 µM aprotinin, 1 µM leupeptin and 0.4 mM Add: 4-(2-aminoethyl)benzenesulfonyl fluoride. The homogenate was then centrifuged at 1000g for 15 min at 4°C. The pellet was re-suspended in 0.8 M KCl, 50 mM Tris–HCl (pH 7.8), 0.01 µM aprotinin, 1 µM leupeptin and 0.4 mM AEBSF and incubated on ice for 10 min. The nuclei were then lyzed by diluting the incubations to 0.3 M KCl with 10 mM Tris–HCl and incubating on ice for 30 min. The nuclear protein extract was obtained by centrifugation at 12 000g for 30 min at 4°C. The supernatant was adjusted to 10% glycerol using 50% glycerol containing 50 mM MgCl2 and 10 mM Tris–HCl (pH 7.4). Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad, Richmond, CA). The nuclear protein was stored at –70°C.

CpG nuclease activity assay
A DNA substrate containing 29 CpG sites in a CpG island of the rat estrogen receptor-{alpha} gene was synthesized by polymerase chain reaction (PCR) amplification using an upstream primer: 5'-TGACCCTTCACACCAAAGCCTC-3' and a downstream primer: 5'-ATCAGCGGACTGGGCGACAC-3' (GenBank accession number X98236, nt 4515–4848). Some PCRs included dCTP labeled with tritium at carbon-5 (37 MBq/ml, GE Healthcare, Piscataway, NJ) to obtain the DNA substrate that was used to determine whether the nuclease will cleave unmethylated CpG sites. An aliquot of all PCR products was electrophoresed in a 1.5% agarose gel containing ethidium bromide to confirm a single band of 333 bp. The PCR product was methylated using SssI CpG methylase (New England Biolabs, Ipswich, MA) and S-adenosyl-L-methionine with a tritium-labeled methyl group (37 MBq/ml, GE Healthcare). The reaction was stopped by incubation at 65°C for 20 min and the PCR product was recovered using a Microcon YM-100 filter membrane and stored at –20°C.

Nuclease activity was determined in an incubation mixture containing 5 µg of nuclear protein, PCR product (42 000 d.p.m.), 10 mM MgCl2 and 10 mM Tris–HCl (pH 7.4) at 37°C for 90 min. The reaction was stopped by placing in ice and then either extracted twice with eight volumes of water-saturated 1-butanol or filtered through a Microcon YM-10 membrane to separate the products released by the nuclease activity from the butanol-insoluble and higher molecular weight PCR product. Radioactivity was measured by liquid scintillation counting using a LS 6500 Scintillation Counter.

Identification of the products of nuclease activity by HPLC analysis
The products released from the DNA substrate by the nuclease activity were analyzed using a Waters Model 510 HPLC system equipped with a Whatman Partisphere C18 column (4.6 x 250 mm, 5 µm particle), a Model 481 Lambda Max UV/visible LC spectrophotometer and Laura Lite modulate software. The column was eluted at room temperature with a 20 min isocratic mobile phase consisting of 100 mM ammonium acetate (pH 4.25) containing 0.5% acetonitrile with a flow rate of 1.2 ml/min. Fifteen fractions were collected at 1 min intervals. Reference standards of cytosine, 2'-deoxycytidine, 5-methyl-cytosine, 5-methyl-2'-deoxycytidine, 5-methyl-2'-deoxycytidine monophosphate, thymidine and thymine were used to identify the products that were released by the nuclease from the PCR product and were present in the eluate from the HPLC. The radioactivity of each fraction was measured by liquid scintillation using a LS 6500 Scintillation Counter.

Ion exchange chromatography
The nuclease extract from mouse lung was loaded onto an Alltech Macrosphere WAX column (4.6 x 250 mm, 7 µm particles). The column was eluted at 4°C for 3 min with an isocratic mobile phase consisting of Solution A (20 mM Tris–HCl, pH 7.4), for 12.5 min with linear gradient mobile phase changing from 100% Solution A to 100% Solution B (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) and then for 9.5 min with Solution B. The flow rate was 1.0 ml/min. Twenty-five fractions were collected at 1 min intervals. From each fraction, a 0.5 ml aliquot was used to determine the CpG nuclease activity.

Statistical analysis
Results are expressed as means ± SEs and were analyzed for statistical significance by either the t-test or by a one-way analysis of variance followed by the Bonferroni t-test, P-value < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Product and specificity of the CpG nuclease
The products produced by the CpG nuclease activity were identified by HPLC (Figure 1). Figure 1A depicts the HPLC elution profile of standards for the various nucleosides and nucleotides of cytosine and thymine. The PCR product was radiolabeled using SssI CpG methylase and tritium-labelled s-(5'-adenosyl)-L-methionine. The SssI CpG methylase only methylates cytosines at CpG sites so that only these cytosines are radiolabeled with the 3H-methyl group. After incubation of this PCR product with the nuclear extract from either lung tissue or tumors, the major peak of released radioactivity had a retention time 11.8 min (Figure 1B). This retention time was the same as 5-methyl-2'-deoxycytidine (Figure 1A), indicating that the nuclear extract caused the release of the methylated nucleotide of cytosine. An unmethylated PCR product was synthesized using tritium-labelled 2'-deoxycytidine 5'-triphosphate, so that all cytosines both at and outside of CpG sites were radiolabeled. When this PCR product was incubated with the nuclear extract, the released radioactivity had a retention time of 6.8 min, similar to that of 2'-deoxycytidine (Figure 1C). Thus, the nuclease activity in the extract could release the unmethylated nucleotide of cytosine. However, when the PCR product containing tritium-labeled cytosine was methylated using the SssI CpG methylase and unlabeled SAM, the peak of released radioactivity was eluted at 11.8 min, i.e. 5-methyl-2'-deoxycytidine. Consequently, when the PCR product is methylated, 2'-deoxycytidine was no longer released by the nuclear extract, but rather 5-methyl-2'-deoxycytidine was released. The 333 bp PCR product contains a 3-fold greater number of cytosines outside of CpG sites (178 cytosines) than inside the sites (58 cytosines). Should the nuclease not have a preference for CpG sites, then it should had released 3-fold more 2-deoxycytidine than 5-methyl-2'-deoxycytidine from the methylated 3H-cytosine-labeled PCR product. Thus, the release of only 5-methyl-2'-deoxycytidine from the methylated PCR product supports the supposition that the nuclease activity is specific for CpG sites, while demonstrating that the CpG sites do not have to be methylated in order to be a substrate of the nuclease.


Figure 1
View larger version (15K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Substance released from the PCR product by the CpG nuclease. After incubation with the nuclear extracts, the released radioactivity was separated from the PCR product by filtration through an YM-10 filter membrane and loaded onto a C-18 Reverse Phase HPLC column. The column was eluted with an isocratic mobile phase consisting of 100 mM ammonium acetate (pH 4.25) containing 0.5% acetonitrile and a flow rate of 1.2 ml/min. (A) Elution profile of nucleoside and nucleotides of cytosine, 5-Me-C and thymine. (B) Elution profiles of radioactivity released from 3H-Me-CpG containing PCR product as the result of incubation with nuclear protein extract from normal lung (solid line) and lung tumor (dashed line). (C) Elution profiles of radioactivity released from 3H-cytosine containing PCR product as the result of incubation with nuclear protein extract from lung tumor. Methylated PCR product (dashed line) and unmethylated PCR product (solid line).

 
The integrity of the PCR product after incubation with the nuclear protein extract was checked by electrophoresis in a 2% agarose gel containing 1% sodium dodecyl sulfate (Figure 2). After a 3 h incubation of the methylated PCR product with the nuclear extract, there was no indication that the product was being overtly degraded, even though 5-methyl-2'-deoxycytidine had been released from ~40% of the Me-CpG sites. Thus, it appears that 5-methyl-2'-deoxycytidine is cleaved from the PCR product without significant fragmentation of the product.


Figure 2
View larger version (75K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Integrity of the DNA substrate after CpG nuclease activity. 3H-cytosine containing PCR product (42 000 d.p.m.) was incubated with 5 µg of nuclear extracts from mouse lung for 10 (lane 3 and 4), 90 (lane 5 and 6) and 180 (lane 7 and 8) min. Lanes 1 and 2 contain PCR product (42 000 d.p.m.) that was not incubated with the nuclear extract. The samples were electrophoresed using a 2% agarose gel with 1% sodium dodecyl sulfate and the gel was visualized with 254 nm UV light.

 
Time and concentration dependence of CpG nuclease activity
The reaction time and nuclear protein concentration dependency of the CpG nuclease activity were determined (Figure 3). Nuclease activity increased linearly over the 6 h of incubation and was dependent on protein concentration. After 6 h of incubation, 1 µg of nuclear protein released 2378 ± 14 d.p.m. from the PCR product, whereas 5 µg of nuclear protein released 11 630 ± 13 d.p.m. The increase in enzyme activity, resulting from a 5-fold increase in protein, demonstrates a direct relationship between activity and protein concentration.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Effect of protein concentration and time on CpG nuclease activity. 3H-cytosine containing PCR product (42 000 d.p.m.) was incubated with 1 or 5 µg of nuclear protein from mouse lung for up to 6 h. The amount of released radioactivity was determined by filtration through an YM-10 membrane. Results are means ± SEs for duplicate incubations. The dashed line is the linear regression and the solid line connects the data points.

 
Properties of CpG nuclease
The molecular weight of the CpG nuclease was estimated by filtration of the nuclear extracts through YM-100 membranes. Nuclease activity was determined using PCR product containing 42 000 d.p.m. and 90 min of incubations. The activity of the nuclear extract applied to the membrane released 4416 ± 114 d.p.m. from the PCR product. The filtrate obtained by passing the nuclear extract through an YM-100 membrane released only 170 ± 18 d.p.m. In contrast, the nuclease activity washed from the YM-100 membrane released 10 640 ± 143 d.p.m. Consequently, the molecular weight of the CpG nuclease would appear to be >100 000 Da. However, we cannot rule out that it is of smaller molecular weight and is part of a higher molecular weight multiprotein complex. Furthermore, nuclease activity in the extract appeared to increase after separation from material of >100 000 Da.

The nuclear extract from mouse lung was subjected to WAX ion exchange chromatography using a gradient from 20 mM Tris to 20 mM Tris + 500 mM Nacl (Figure 4). The nuclease activity was eluted at fraction 20, i.e. after the salt concentration had increased to 500 mM. Hence, the activity was stable to high salt concentration and could be fractionated by ion-exchanged chromatography.


Figure 4
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. WAX ion exchange chromatography of the nuclease activity. The nuclease extract from mouse lung was loaded onto a WAX column (4.6 x 250 mm, 7 µm particles) at 4°C and eluted for 3 min with an isocratic mobile phase consisting of Solution A (20 mM Tris–HCl, pH 7.4), for 12.5 min with gradient mobile phase changing from 100% Solution A to 100% Solution B (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) and then for 9.5 min with Solution B. The flow rate was 1.0 ml/min. One-minute fractions were collected. A major peak of nuclease activity was found around fraction 20.

 
The CpG nuclease had a predilection for double-stranded over single-stranded DNA. Incubations of double-stranded PCR product (42 000 d.p.m.) with nuclear extracts resulted in the release of 1739 ± 87 d.p.m., whereas incubations containing 42 000 d.p.m. of single-stranded product resulted in the release of 872 ± 35 d.p.m., a 50% reduction in nuclease activity. The effect of ethylenediaminetetraacetic acid on CpG nuclease activity was determined. In the presence of 4 mM of Mg2+, concentrations of 0, 4, 8, 16 and 32 mM ethylenediaminetetraacetic acid resulted in the release from the PCR product of 1739 ± 87, 20 ± 49, 227 ± 62, 150 ± 87 and 7.74 ± 4.47 d.p.m., respectively, suggesting that magnesium is a required co-factor for CpG nuclease activity. The effect of 2-mercaptoethanol on CpG nuclease activity was also determined. There was a dose-dependent reduction of CpG nuclease activity with increasing concentrations of 2-mercaptoethanol. Concentrations of 0, 0.1, 0.3 and 1.0 g/l of 2-mercaptoethanol resulted in the release of 1739 ± 87, 1077 ± 39, 603 ± 25 and 126 ± 31 d.p.m., respectively.

Effect of budesonide on CpG nuclease activity in mouse lung tumors
The effect of budesonide on CpG nuclease activity in mouse lung tumors was determined using tumors from two of our previously published studies (3,10). In the first study, tumors were induced by vinyl carbamate followed by budesonide administered from weeks 4 to 20, 4 to 35 or 20 to 35, with the mice being killed at 35 weeks. Nuclease activity in the nuclear extract obtained from lung tumors of mice not administered budesonide was significantly increased relative to non-involved lung tissue (Figure 5). When administered from weeks 4 to 35 and 20 to 35, budesonide prevented the increase in nuclease activity, so that it was no longer different from the activity in non-involved tissue. However, the increase in nuclease activity was not prevented in tumors when treatment with budesonide (weeks 4–20) was followed by a holding period of 15 weeks, suggesting that continued treatment with the drug was necessary to maintain the prevention in activity.


Figure 5
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Effect of budesonide of CpG nuclease activity in mouse lung tumor. CpG nuclease activity was determined in nuclear extracts obtained from mice administered budesonide from 4 to 20, 4 to 35 and 20 to 35 weeks, as well as from non-involved lung from mice not administered the drug. All the mice were killed at week 35. The amount of released radioactivity was determined by filtration through an YM-10 membrane. Results are mean ± SE of duplicate determinations for eight tumors, each from a different mouse. Bars with different letters were statistically different as determined by an analysis of variance followed by the Bonferroni t-test, P-value < 0.05.

 
In the second study, lung tumors were also induced by vinyl carbamate, however, in this study budesonide was not administered until after tumors had occurred. Thus, budesonide was administered for 2, 7 or 21 days prior to killing at week 27 after the first dose of vinyl carbamate. Within 2 days of treatment, budesonide significantly decreased the nuclease activity in lung tumors that remained decreased >21 days of treatment (Figure 6). Hence, budesonide decreased nuclease activity in established lung tumors and similar to its prevention of nuclease activity in tumors continued treatment with the drug appeared to be necessary to maintain the decrease in activity.


Figure 6
View larger version (10K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Effect of short-term treatment with budesonide on CpG nuclease activity in mouse lung tumors. CpG nuclease activity was determined in nuclear extracts obtained from mice with established vinyl carbamate-induced mouse lung tumors and then treated with budesonide for 2, 7 and 21 days or for 14 days followed by a 7-day holding period. The amount of released radioactivity was determined by filtration through an YM-10 membrane. Results are mean ± SE with each treatment group containing tumors from eight different mice. The asterisk indicates a significant difference from the control incubation (P-value < 0.001).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
A CpG-specific endonuclease activity was identified that excises deoxycytosine from CpG sites in DNA, irrespective of the methylation status of the cytosine, i.e. both methylated and unmethylated deoxycytidine were released. However, deoxycytidine not at CpG sites were apparently not excised. The nuclease appears to be a high molecular weight >100 000 Da, although we have not ruled out that it is of smaller molecular weight but part of a larger protein complex. The nuclease has a proclivity for double-stranded over single-stranded DNA. The CpG endonuclease requires divalent ions, being inhibited by ethylenediaminetetraacetic acid. It is also inhibited by thiol-modifying reagents, suggesting that disulfide bonds are involved.

The specificity of the nuclease for CpG sites would suggest an association with DNA methylation, since methylation occurs primarily at CpG sites. A recombinant human methylated DNA-binding protein, MBD4, has specificity for CpG sites and was found to have G/T mismatch as well as 5-Me-C DNA glycosylase activities (14,15). One interesting property of MBD4's 5-Me-C DNA glycosylase activity is that it demethylates hemimethylated DNA substrate. The CpG/CpG hemimethylated site is similar to TpG/CpG mismatch due to the fact that 5-Me-C and thymine only differ at the C-4 position. It is possible that function of MBD4 in living cells is G/T mismatch glycosylase and the DNA demethylation activity was an artifact of the high level of protein expression of recombinant MBD4.

The lung tumors reported here to contain increased CpG nuclease activity were previously reported by us to contain hypomethylated DNA (3,10). The inverse relationship of CpG endonuclease activity and DNA methylation was further demonstrated by the effect of budesonide; when CpG nuclease activity was decreased by budesonide in lung tumors, there was a concurrent increase in DNA methylation. In one of the studies reported here, lung tumors had a 38.1% increase in nuclease activity that after budesonide treatment of 4–35 and 20–35 weeks was no longer significantly different from non-involved lung. We have reported previously that lung tumors from this study had a 40.3% reduction in global DNA methylation that after treatment with budesonide for 4–35 or 20–35 weeks was no longer different from non-involved lung, i.e. 98.4 and 92.8% of lung tissue (3). Further, when treatment with budesonide ceased at 20 weeks (15 weeks prior to killing of the mice), neither nuclease activity nor DNA methylation were back to their level in non-involved lung, i.e. nuclease activity was increased by 62.5% and DNA methylation was decreased by 46.2%, relative to lung tissue. In the other study reported here, budesonide treatment for 2, 7 and 21 days decreased nuclease activity in lung tumors by 47.7, 69.9 and 47.4%, respectively. Using tumors from this same study, we have reported previously that DNA methylation in the tumors was increased 46.0, 74.5 and 61.1% by 2, 7 and 21 days of budesonide treatment. Thus, in both studies, budesonide decreased nuclease activity whereas increasing DNA methylation. The inverse relationship between the ability of budesonide to decrease CpG nuclease activity and to increase DNA methylation in lung tumors would suggest a cause and effect relationship in which the nuclease excises 5-methyl-2'-deoxycytidine from DNA to be replaced by unmethylated 2-deoxycytidine. However, even if a cause and effect relationship does not exist, the fact that both CpG nuclease activity and DNA methylation are altered within two days of treatment with budesonide would suggest that at least a common upstream event exists between them.

DNA hypomethylation is usually an early molecular alteration in carcinogenesis (1,2). Our results suggesting an association between DNA hypomethylation and increased CpG nuclease activity in lung tumors would further suggest that the increase in nuclease activity is also an early event. Since many tumor promoters and non-genotoxic carcinogens have been shown to cause DNA hypomethylation (11,16,17), it is possible that an increased CpG nuclease activity may also be involved in the tumor enhancing activity of these agents. In mice, prevention by methionine of liver tumors induced by the non-genotoxic carcinogen, dichloroacetic acid, was associated with its ability to prevent DNA hypomethylation (18). Drugs that prevent mouse lung cancer including R115777 (a farnesyl transferase inhibitor) and Targretin (19,20), as well as aspirin, calcium chloride, celecoxib, {alpha}-diflouromethylornithine, piroxicam and sulindac that prevent colon cancer in rats (21,22) and have been shown to prevent and reverse DNA hypomethylation in tumors. These chemopreventive agents, similar to budesonide, may also decrease CpG nuclease activity in lung and colon tumors. The speculation that increased CpG nuclease activity is involved in the promotion of cancer and that decreased activity is involved in the prevention of cancer warrants further investigation.


   Acknowledgments
 
This study is supported in part by grants R01 CA-096129 and R03 CA-117520 and contract N01-CN-25126 with the US National Cancer Institute.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Jones PA, et al. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. (2002) 3:415–428.[ISI][Medline]
  2. Dunn BK. Hypomethylation: one side of a larger picture. Ann. N. Y. Acad. Sci. (2003) 983:28–42.[Abstract/Free Full Text]
  3. Pereira MA, et al. Chemoprevention: mouse colon and lung tumor bioassay and modulation of DNA methylation as a biomarker. Exp. Lung Res. (2005) 31:145–163.[CrossRef][ISI][Medline]
  4. Wattenberg LW, et al. Chemoprevention of pulmonary carcinogenesis by aerosolized budesonide in female A/J mice. Cancer Res. (1997) 57:5489–5492.[Abstract/Free Full Text]
  5. Wattenberg LW, et al. Studies of chemopreventive effects of budenoside on benzo[a]pyrene-induced neoplasia of the lung of female A/J mice. Carcinogenesis (1997) 18:2015–2017.[Abstract/Free Full Text]
  6. Wattenberg LW, et al. Chemoprevention of pulmonary carcinogenesis by brief exposures to aerosolized budesonide or beclomethasone dipropionate and by the combination of aerosolized budesonide and dietary myo-inositol. Carcinogenesis (2000) 21:179–182.[Abstract/Free Full Text]
  7. Pereira MA, et al. Prevention of mouse lung tumors by budesonide and its modulation of biomarkers. Carcinogenesis (2002) 23:1185–1192.[Abstract/Free Full Text]
  8. Alyaqoub FS, et al. Prevention of mouse lung tumors and modulation of DNA methylation by combined treatment with budesonide and R115777 (ZarnestraMT). Carcinogenesis (2006) 27:1442–1447.
  9. Tao L, et al. Effect of budesonide on the methylation and mRNA expression of the insulin-like growth factor 2 and c-myc genes in mouse lung tumors. Mol. Carcinog. (2002) 35:93–102.[CrossRef][ISI][Medline]
  10. Pereira MA, et al. Modulation by budesonide of DNA methylation and mRNA expression in mouse lung tumors. Int. J. Cancer (2006) 27:2359–2362.
  11. Ge R, et al. Wy-14,643-induced hypomethylation of the c-myc gene in mouse liver. Toxicol. Sci. (2001) 62:28–35.[Abstract/Free Full Text]
  12. Szyf M. DNA methylation and demethylation as targets for anticancer therapy. Biochemistry (2005) 70:533–549.
  13. Ehrlich M. The controversial denouement of vertebrate DNA methylation research. Biochemistry (2005) 70:568–575.
  14. Zhu B, et al. 5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence. Proc. Natl Acad. Sci. USA (2000) 97:5135–5139.[Abstract/Free Full Text]
  15. Zhu B, et al. Overexpression of 5-methylcytosine DNA glycosylase in human embryonic kidney cells EcR293 demethylates the promoter of a hormone-regulated reporter gene. Proc. Natl Acad. Sci. USA (2001) 98:5031–5036.[Abstract/Free Full Text]
  16. Counts JL, et al. Hypomethylation of DNA: a nongenotoxic mechanism involved in tumor promotion. Toxicol. Lett. (1995) 82/83:663–672.[CrossRef]
  17. Goodman JI, et al. Altered DNA methylation: a secondary mechanism involved in carcinogenesis. Annu. Rev. Pharmacol. Toxicol. (2002) 42:501–525.[CrossRef][ISI][Medline]
  18. Pereira MA, et al. Prevention by methionine of dichloroacetic acid-induced liver cancer and DNA hypomethylation in mice. Toxicol. Sci. (2004) 77:243–248.[Abstract/Free Full Text]
  19. Gunning WT, et al. Chemoprevention of benzo(a)pyrene-induced lung tumors in mice by the farnesyltransferase inhibitor R115777. Clin. Cancer Res. (2003) 9:1927–1930.[Abstract/Free Full Text]
  20. Pereira MA, et al. Prevention of mouse lung tumors by targretin. Int. J. Cancer (2006) 118:2359–2362.[CrossRef][ISI][Medline]
  21. Pereira MA, et al. Modulation by celecoxib and difluoromethylornithine of the methylation of DNA and the estrogen receptor-{alpha} gene in rat colon tumors. Carcinogenesis (2004) 25:1917–1923.[Abstract/Free Full Text]
  22. Tao L, et al. Modulation of DNA hypomethylation as a surrogate endpoint biomarker for chemoprevention of colon cancer. Mol. Carcinog. (2004) 39:79–84.[CrossRef][ISI][Medline]
Received November 8, 2006; revised February 23, 2007; accepted March 1, 2007.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
28/7/1499    most recent
bgm056v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Li, L.
Right arrow Articles by Pereira, M. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, L.
Right arrow Articles by Pereira, M. A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?