Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Joint Metastasis Research Society-AACR Conference on Metastasis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Belinsky, S. A.
Right arrow Articles by Baylin, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Belinsky, S. A.
Right arrow Articles by Baylin, S. B.
[Cancer Research 63, 7089-7093, November 1, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Inhibition of DNA Methylation and Histone Deacetylation Prevents Murine Lung Cancer1

Steven A. Belinsky2, Donna M. Klinge, Christine A. Stidley, Jean-Pierre Issa, James G. Herman, Thomas H. March and Stephen B. Baylin

Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108 [S. A. B., D. M. K., T. H. M.]; University of New Mexico, Albuquerque, New Mexico 87131 [C. A. S.]; M. D. Anderson Cancer Center, Houston, Texas 77030 [J-P. I.]; and Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland 21231 [J. G. H., S. B. B.]


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Disruption of one allele for the cytosine-DNA methyltransferase 1 (DNMT1) gene in mice with a germ-line mutation in a tumor suppressor gene was shown previously to reduce tumor formation in juvenile animals. This effect is now reproduced in our studies of mature mice where this genetic DNMT1 reduction leads to a 50% decrease in tobacco carcinogen-induced lung cancer and a similar reduction in DNMT activity in type II pneumocytes that give rise to the tumors. Short-term treatment of DNMT wild-type female mice with low doses of the demethylating agent 5-aza-2'-deoxycytidine decreased the incidence of neoplasms by 30%. Importantly, when 5-aza-2'-deoxycytidine was combined with the histone deacetylase inhibitor sodium phenylbutyrate, lung tumor development was significantly reduced by >50%; no effect was seen with phenylbutyrate alone. This identical combination of inhibitors also acts synergistically to cause re-expression of densely hypermethylated and transcriptionally silenced tumor suppressor genes in human cancer cells. Thus, reduction in DNMT and histone deacetylase activities that likely block epigenetically mediated gene silencing might provide a novel clinical strategy to help prevent the leading cause of cancer death in the United States.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Lung cancer is the leading cause of cancer mortality in both men and women in the United States and will soon reach epidemic proportion worldwide (1) . A recent comparison of four chemotherapy regimens considered the "best approach" revealed no difference in survival, leading investigators of one study to conclude "that chemotherapy in non-small cell lung cancer has reached a therapeutic plateau" (2 , 3) . For persons at high risk for lung cancer, chemopreventive agents that modulate genes or pathways that are dysregulated in the premalignant cell could significantly affect the morbidity and mortality from this disease.

Transcriptional silencing by CpG island hypermethylation now rivals genetic changes that affect coding sequence as a critical trigger for neoplastic development and progression (4 , 5) . Genes responsible for all aspects of normal cellular function are targeted for inactivation by methylation. The fact that pharmacological agents can reverse this epigenetically mediated process makes it an ideal target for prevention. Gene silencing through hypermethylation is mediated by a series of events that include methylation of cytosines within the gene promoter and the establishment of heterochromatin in which the histone tails are modified through effects on acetylation, phosphorylation, methylation, and ubiquitylation (6 , 7) . The cytosine DNMT3 genes play a critical role in the establishment of the transcriptionally repressive complex. They function as de novo methylases to affect the methylation status of normally unmethylated CpG sites and to recruit HDACs to chromatin (8, 9, 10, 11, 12) . Treatment of cells with the demethylating agent DAC leads to re-expression of genes silenced by promoter methylation (7) . DAC inhibits DNA methylation by reducing the DNMT enzymatic activity via the formation of a stable complex between the enzyme and DAC-substituted DNA (13) . One difficulty in using demethylating agents such as DAC in vivo is the ability to achieve pharmacologically active doses without systemic toxicity. A more effective approach for modulating aberrant promoter methylation therapeutically was endorsed through a study by Cameron et al. (14) . These investigators observed that treatment of colorectal tumor cell lines with trichostatin A, an inhibitor of histone deacetylation, did not transcriptionally reactivate genes silenced by promoter hypermethylation. However, when the same cells were exposed to trichostatin A and a low dose of DAC that alone results in only minimal gene re-expression, synergistic re-expression was seen with the two-drug combination.

Laird et al. (15) , who examined the effect of reduced DNMT1 activity on APCMin-induced intestinal neoplasia, first demonstrated the in vivo modulation of DNMT activity as a target for cancer prevention. Min mice contain a mutation of the APC tumor suppressor gene and develop multiple intestinal adenomas within the first few months of life. The effect of DNMT levels on intestinal polyp formation was examined in an F1 mouse generated by crossing the C57BL/6 APCMin/+ mouse with a 129/Sv Dnmts/+ mouse that contains one mutationally inactive allele of the DNMT1 gene. The development of intestinal adenomas was reduced by 50% in this F1 mouse. Adenoma formation was virtually abolished in F1 mice after treatment with DAC (1 mg/kg weekly) beginning 7 days after birth for 14 weeks. These results indicate that DNMT activity during embryogenesis and juvenile development, in concert with loss of a critical tumor suppressor gene, contributes markedly to intestinal adenoma development.

Our studies additionally substantiated a true functional role for DNMT in neoplasia by demonstrating that target cells for the ensuing cancer exhibited a change in enzyme activity after exposure to carcinogen. DNMT activity was examined in alveolar type II (target) and Clara (nontarget) cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation (16) . After treatment of mice with the tobacco specific carcinogen NNK, DNMT activity increased only in the target alveolar type II cells of the susceptible A/J mouse (16) . Enzyme activity also increased incrementally during lung cancer progression and coincided with increased expression of the DNMT1 gene in hyperplasias, adenomas, and carcinomas. Lantry et al. (17) have demonstrated that chronic treatment with DAC starting before carcinogen exposure reduced tumor multiplicity in C3H/HeJ x A/J F1 mice, thus supporting the premise of targeting the DNMTs for lung cancer prevention. We have now extended these studies to address whether short-term treatment with low-dose DAC combined with the HDAC inhibitor sodium phenylbutyrate can prevent lung tumor development initiated by a tobacco-specific carcinogen in the A/J mouse. In addition, the effect of gene dosage for DNMT1 on tumor development in adult mice and response to DAC alone or combined with sodium phenylbutyrate was also determined.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Animals and Genotyping.
C57BL/6 mice containing one inactivated allele of the DNMT1 gene were obtained as a gift from the Whitehead Institute for Biomedical Research (Boston, MA). This allele was inactivated originally by homologous insertion of a 1.8 kb PGK-neo-poly(A) cassette into the 5' end of the DNMT1 gene in J1 embryonic stem cells from a male agouti 129/terSV embryo (15) . A/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice were bred with A/J mice; the resulting F1 hybrid was genotyped for the A/J K-ras allele and the inactivated form of the DNMT1 allele by PCR. DNA was isolated from tail biopsies by standard procedures. PCR primers and conditions used to amplify the knockout versus wild-type allele for the DNMT1 gene have been described (18) . PCR primers were designed to amplify the portion of K-ras intron 2 containing the 37-bp repeat. Primer sequences used were as follows: forward, (5'-GTTCCAAGGCTTTAAACTGGG-3') and reverse, (5'-AAAATGCAAATACAAAGCACG-3'). The cycling parameters for amplification of the K-ras intron 2 were as follows: 94°C for 10 min; then 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a 5-min final extension at 72°C. All of the PCR amplifications were performed using a Biometra T3 thermocycler and Taq Gold polymerase (Perkin-Elmer). A/J x C57 hybrid mice containing the A/J K-ras intron 2 and mutated allele of DNMT1 were then backcrossed into the A/J mouse for six generations.

Animal Treatment, Cell Isolation, and Histopathology.
DNMT+/+ and DNMT+/- mice (6–8 weeks old) were treated three times (every other day, 50 mg/kg, i.p.) with NNK (Chemsyn Science Laboratories, Lenexa, KS) dissolved in saline or with saline alone (0.1 ml). To isolate alveolar type II cells, mice were sacrificed 7 days after treatment with NNK. Type II cells were obtained by centrifugal elutriation after protease digestion of the lungs that were pooled from 6 mice for cell isolation (16) . Three to 5 groups of mice comprised each group. The purity of the type II cells was 73% ± 4% with small cells (primarily endothelial cells and lymphocytes) and macrophages comprising the major contaminating cells.

One week after treatment with NNK, DNMT+/+ and DNMT+/- mice were divided into 4 groups (10–20 mice/group, male and female). Sample sizes between groups varied attributable to the availability of DNMT+/- mice from a single breeding within the 6–8-week age range. Mice were treated on Tuesday, Wednesday, and Thursday for 4 weeks with saline, DAC (0.25 for DNMT+/- mice or 0.5 mg/kg for DNMT+/+ mice, i.p.; Sigma, St. Louis, MO), sodium phenylbutyrate (300 mg/kg, i.p.; Scandinavian Formulas, Perkasie, PA), or DAC and sodium phenylbutyrate. Mice were then held after cessation of treatment for 36 weeks.

Mice were sacrificed by exsanguination; lungs were inflated and fixed with 4% buffered paraformaldehyde for 18–24 h and then transferred to 70% ethanol for routine histological processing and staining of paraffin sections with H&E. A single standardized section was prepared from all lungs, which included all five of the lung lobes. Pulmonary lesions were classified as hyperplasia or neoplasia (adenoma or carcinoma) as described (19) . Hyperplastic lesions were microscopic and involved a minimum of 5–10 alveoli in a focus lined by hyperplastic type II epithelial cells. Adenomas were characterized by a monomorphic growth pattern and were generally composed of well-differentiated cells. Carcinomas were composed of cells with various degrees of differentiation and were characterized by complete loss of normal architecture.

DNMT Enzyme Assay.
The assay used to determine cytosine DNMT activity has been described (16) . Briefly, protein concentration was determined by the Bradford assay. Cell lysates containing 5 µg of protein were incubated for 2 h at 37°C with a dI-dC template (Amersham) and [3 H]S-adenosyl methionine (Amersham). Reactions were stopped, protein extracted, and dI-dC template recovered by ethanol precipitation. RNA was removed by resuspension of the precipitates in NaOH; DNA was spotted on Whatman filters, dried, and then washed with TCA followed by 70% ethanol. Filters were placed in scintillation mixture and enzyme activity determined by scintillation counting. Results were expressed as dpm/µg protein. All of the assays were performed in duplicate. Limit of detection was 20 dpm above background levels (determined in assays in which the dI-dC template had been omitted).

Statistics.
The Student t test was used to determine whether differences in methyltransferase enzyme activity differed between groups and treatment. The primary analyses for effect of tumor development compared the mean and median numbers of hyperplasias, neoplasms, and total lesions observed. ANOVA was conducted using design factors of DAC, phenylbutyrate, gender, and DNMT1 status. These analyses were conducted both on the counts and on ranks because of asymmetric distribution of counts. The final analyses and summaries were done by gender and DNMT1 status because of the main effects and interactions between gender and DNMT1 status. The Wilcoxon rank sum test was used for pairwise comparisons between sham and each treatment group. Fisher’s exact test was used to assess differences in proportion with tumors.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Development of an A/J DNMT1 Heterozygote Mouse.
The A/J mouse is a highly sensitive strain for lung cancer induction by carcinogens such as NNK, making it ideal for testing the efficacy of preventive agents (19 , 20) . To determine the effect of specifically reducing the DNMT1 activity on lung tumor multiplicity, and sensitivity to DAC and sodium phenylbutyrate, an A/J DNMT1 heterozygote (DNMT+/-) mouse was generated. The 129/Sv DNMTs/+ mouse had been crossed with a C57BL/6J mouse and then backcrossed for 10 generations to obtain this mutation in a complete C57 genome. Because the C57 mouse is resistant to carcinogen-induced lung tumor formation, we crossed the C57 DNMT+/- with the A/J mouse and then backcrossed the hybrid mouse into the A/J mouse for six generations (20) . Susceptibility has been linked to the K-ras gene that is mutated frequently in chemically induced A/J mouse lung tumors (21) . A repetitive element polymorphism within the second intron of the K-ras gene was subsequently linked to the difference in susceptibility among mouse strains (22) . Lung tumor-resistant strains have a 37-bp repetitive element polymorphism, whereas lung tumor-sensitive strains lack one 37-bp repetitive element within the second intron. Therefore, the initial backcrossed mice were genotyped through analysis of tail DNA for both the A/J K-ras allele and the presence of the inactivated DNMT1 allele (18 , 22) . This allowed subsequent backcrosses with hybrid mice containing the K-ras susceptibility allele and the inactivated allele of the DNMT1 gene.

Knocking Out the DNMT 1 Allele Affects Cytosine DNMT Activity.
DNMT1 accounts for >90% of the enzymatic activity measured by incorporation of 3 H-labeled S-adenosylmethionine onto a deoxyinosine-deoxycytosine template (23) . Therefore, the effect of knocking out one allele of the DNMT1 gene on endogenous DNMT activity and activity after treatment with NNK was examined in alveolar type II cells. A 50% reduction in enzyme activity compared with DNMT+/+ was seen in type II cells from DNMT+/- mice (Table 1)Citation . Enzyme activity was increased by ~100% in wild-type mice 1 week after treatment with NNK, a response that corroborated our previous studies (Ref. 16 ; Table 1Citation ). In contrast, only a 50% increase in DNMT activity was seen in type II cells from heterozygous mice. These results support a biochemical effect of cellular DNMT activity that correlates with gene copy number for DNMT1.


View this table:
[in this window]
[in a new window]
  		Table 1 Cytosine-DNA methyltransferase activity in type II cells from DNMT+/+ and DNMT+/- A/J mice

 
Reduction in DNMT1 Gene Copy Number Prevents Tumor Development.
Studies were then initiated to determine the effect of DNMT1 gene copy number and DAC treatment alone or combined with the HDAC inhibitor sodium phenylbutryate on tumor development in mice exposed to NNK. DNMT+/+ and DNMT+/- mice were treated with NNK (50 mg/kg x 3 doses, over 1 week). One week after the last treatment with NNK, DNMT+/+ and DNMT+/- mice were treated with vehicle, 0.25 (DNMT+/- mice), or 0.5 mg/kg DAC alone (DNMT +/+ mice); 0.25 (DNMT+/-) or 0.5 mg/kg (DNMT+/+) DAC +300 mg/kg sodium phenylbutyrate; or 300 mg/kg sodium phenylbutyrate alone. Mice were treated for 4 consecutive weeks (3 consecutive days/week, Tuesday through Thursday) and then held until sacrifice at 42 weeks after initiation of exposure to carcinogen. We initiated treatment with DAC and sodium phenylbutyrate 1 week after NNK treatment, because this time point represented the peak for increased DNMT activity in type II cells after carcinogen exposure (16) . DNMT+/- mice were treated with a lower dose of DAC, because pilot studies demonstrated severe toxicity manifested by weight loss and premature mortality. Finally, DAC and sodium phenylbutyrate treatments were given sequentially (morning and afternoon) to approximate the in vitro design used by Cameron et al. (14) . In those studies (14) cells were treated sequentially with the demethylating agent followed by the HDAC inhibitor. The doses and schedule used for these studies (Fig. 1)Citation did not adversely affect the mice, i.e., no weight loss or premature mortality.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 1. A, the timing and duration of treatment of mice with NNK, DAC, and phenylbutyrate. This line drawing depicts the order and duration for treatment with NNK followed by the preventive agents DAC and phenylbutyrate. B, effect of gender and DNMT1 gene dosage on pulmonary lesions. Mice were treated with NNK as shown in A and sacrificed 42 weeks after initiation of treatment. Pulmonary lesions (mean) were decreased significantly (P < 0.05) when comparing male to female mice (a) and in DNMT+/- compared with DNMT+/+ mice (b and c); bars, ±SE.

 
The results are depicted with outcomes denoted as the mean number of hyperplasias, neoplasms, and total lesions (hyperplasias + neoplasms) seen on a standard tissue section containing all five of the lung lobes (Table 2)Citation . Several significant effects were observed in this study. First, a gender effect not reported previously was observed for both wild-type and heterozygote mice with tumor multiplicity 3-fold greater in females than males (Fig. 2)Citation . Therefore, subsequent analysis of gene dosage and treatment effects was conducted separately for male and female mice. Second, and most important, the mean number of neoplasms and total lesions were reduced 65 and 47% respectively, in vehicle-treated female DNMT+/- mice compared with DNMT+/+ mice. Hyperplasias and neoplasms were both reduced in number by 60% in male mice lacking one copy of the DNMT gene compared with wild-type (Fig. 2Citation ; Table 2Citation ).


View this table:
[in this window]
[in a new window]
  		Table 2 Effect of DACa and sodium phenylbutyrate on tumor formation in DNMT+/+ and DNMT+/- A/J mice treated with NNK

 


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Effect of DAC and phenylbutyrate treatment on the mean number of pulmonary lesions. Female A/J mice were treated with NNK (50 mg/kg), DAC (0.5 mg/kg for DNMT+/+ mice and 0.25 mg/kg for DNMT+/- mice), and phenylbutyrate (300 mg/kg) as depicted in Fig. 1Citation A. The number of pulmonary lesions (mean) was decreased significantly (*, P < 0.05) in DNMT+/+ mice treated with the combination of DAC + sodium phenylbutyrate; bars, ±SE. DAC treatment alone significantly (*, P < 0.05) diminished the incidence of lesions in lungs from the DNMT+/- mice. Treatment with sodium phenylbutyrate alone had no effect on the number of pulmonary lesions.

 
Phenylbutyrate Synergizes with DAC to Prevent Lung Tumor Formation.
Significant effects of DAC combined with sodium phenylbutyrate were observed for female DNMT+/+ mice. DAC treatment alone decreased both total lesions and neoplasms by ~30%, whereas combining DAC with sodium phenylbutyrate caused a significant decrease of >50% (Fig. 2Citation ; Table 2Citation ). In contrast, treatment with sodium phenylbutyrate alone did not affect tumor multiplicity. In female DNMT+/- mice, treatment with DAC alone markedly (65–80%) decreased hyperplasias and neoplasms (Table 2)Citation . Because of the magnitude of this response, no additional effect by sodium phenylbutyrate was observed. Similarly, in male DNMT+/- mice where tumor multiplicity was already affected by gender and gene dosage, there was only a trend toward additional reduction after treatment with DAC or DAC and sodium phenylbutyrate (Table 2)Citation . However, the number of mice with tumors, i.e., tumor incidence, was reduced from 100% in male DNMT+/- vehicle-treated mice to 40% in mice treated with DAC and sodium phenylbutyrate (P = 0.03). This was the only group where a significant effect on tumor incidence was seen.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The results from these in vivo studies are the first to demonstrate that sodium phenylbutyrate can synergize with DAC to prevent lung tumor development. Our findings parallel those observed in tissue culture for re-expression of genes silenced by aberrant promoter hypermethylation. In that setting, low-dose DAC had minimal effect, whereas the addition of a HDAC inhibitor markedly increased re-expression of genes such as p16 (14) . Furthermore, as in the in vitro studies for gene re-expression, we observed no effect of phenylbutyrate alone on tumor development. A prominent gene dosage effect was seen for tumor development that correlated in magnitude with the decrease in both endogenous enzyme activity and activity after carcinogen treatment. This finding substantiates the important role of DNMT in de novo methylation changes that follow exposure of adult mice to carcinogen and affect tumor development. Finally, whereas some minimal gender differences have been reported between mouse strains, this study is the first to show a significant difference in sensitivity to NNK (20 , 24) . Interestingly, some epidemiology studies suggest that women who smoke are at a greater risk than men for lung cancer (25 , 26) .

A major goal for these studies was to provide proof-of-concept that combining low doses of a demethylating agent with a HDAC inhibitor could effectively prevent tumor development after initiation with a potent tobacco-specific carcinogen. Therefore, in this experimental setting, our end point was tumor development rather than the ability of this treatment protocol to reverse or impede progression of existing cancer. Whereas those studies will be important and will also entail examining specific gene targets that are methylated in murine lung cancer, we focused on first demonstrating the efficacy of combined treatment in a pure prevention model. Our studies extend those of Lantry et al. (17) who demonstrated that treatment with a higher dose of DAC (1 mg/kg) starting before carcinogen exposure and continuing for 24 weeks reduced tumor multiplicity in C3H/HeJ x A/J F1 mice, a hybrid with considerably less sensitivity to lung cancer than the A/J mouse. The start of the preventive intervention in our studies after carcinogen exposure eliminated any indirect effects of treatment on NNK activation. Finally, our studies also demonstrate that only 4 weeks of treatment can profoundly effect tumor development.

DNMT1 is considered a maintenance cytosine DNMT because of its high affinity for hemimethylated DNA template (27) . DNMT3a and 3b are thought to constitute the major de novo methylases that affect the methylation status of normally unmethylated CpG sites (9 , 10) . In addition to affecting the methylation status of cytosine, all three of the DNMTs bind HDACs and mediate the formation of heterochromatin surrounding the aberrantly methylated promoter region (8, 9, 10, 11, 12 , 28) . The stoichiometric reduction in tumor multiplicity as a function of gene dose implies a very tight regulation for DNMT1 activity in the cell. This situation was first implied in studies where only a 2-fold overexpression of the DNMT1 gene in NIH3T3 cells resulted in a marked increase in overall DNA methylation and tumorigenic transformation (29) . In vitro methylation assays have shown that DNMT3a and 3b could cooperate with DNMT1 to extend methylation within the Micrococus luteus genome (30) . However, in the presence of reduced DNMT1 activity, these de novo methylases appear unable to compensate for this loss and to facilitate tumor development.

Our studies indicate that DNMT1 plays a pivotal role in the development of lung cancer in this murine model. Our findings recapitulate the effects of simultaneous DNMT and HDAC inhibition on gene re-expression seen with cell lines from human tumors and substantiate that targeting aberrant methylation could be an effective approach for preventing human lung cancer. DAC and 5-azacytidine are being tested in clinical trials as cancer chemotherapeutic agents for treatment of both leukemia and solid tumors (31 , 32) . A recent study by Gaudet et al. (33) showed that mice carrying a hypomorphic DNMT1 allele that reduced expression to 10% were smaller at birth and developed T-cell lymphoma at 4–8 months of age. Whereas this study clearly speaks to the importance of the DNMT1 gene during development, in an adult setting such as ours, the inhibition of this enzyme did not result in the development of lymphomas. Therefore, the therapeutic strategies that are being evaluated currently are unlikely to increase the risk for cancer in other tissues, and none in fact has been reported. More importantly, the instability of these nucleoside analogues in neutral aqueous solution and their side effects could limit use in cancer prevention trials in high-risk, but cancer-free people where oral administration is the preferred delivery route. However, other agents and small molecules that target the DNMTs such as zebularine could prove an effective alternative for use in prevention (34) . In addition, L-selenomethionine, a nutrient demonstrated to reduce by half the incidence of expected lung cancer, may act in part through inhibition of DNMTs (35 , 36) . Thus, our studies provide a new avenue for cancer prevention through the modulation of critical proteins involved in establishing repressed chromatin to silence tumor suppressor genes.


   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 the Lung Specialized Program of Research Excellence Grant P50-CA-58184 and P20-ES-09871 in facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Back

2 To whom requests for reprints should be addressed, at Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE., Albuquerque, NM 87108-5127. Phone: (505) 348-9465; Fax: (505) 3489-4990; E-mail: sbelinsk{at}LRRI.org Back

3 The abbreviations used are: DNMT, cytosine-DNA methyltransferase; DAC, 5-aza-2'-deoxycytidine; HDAC, histone deacetylase; APC, adenomatosis polyposis coli; NNK, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone; dI-dC, deoxyinsosine-deoxycytidine. Back

Received 5/28/03. Accepted 8/11/03.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Greenlee R. T., Hill-Harmon M. B., Murray T., Thun M. Cancer statistics 2001. CA Cancer J. Clin., 51: 15-36, 2001.[Abstract/Free Full Text]
  2. Schiller J. H., Harrington D., Belani C. P., Langer C., Sandler A., Krook J., Zhu J., Johnson D. H. Comparison of four chemotherapy regimens for advanced non-small cell lung cancer. N. Engl. J. Med., 346: 92-98, 2002.[Abstract/Free Full Text]
  3. Scagliotti G. V., De Marinis F., Rinaldi M., Crino L., Gridelli C., Ricci S., Tonato M. Phase III randomized trial comparing three platinum-based doublets in advanced non-small cell lung cancer. J. Clin. Oncol., 20: 4285-4291, 2002.[Abstract/Free Full Text]
  4. Baylin S. B., Herman J. G. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet., 16: 168-174, 2000.[Medline]
  5. Jones P. A., Laird P. W. Cancer epigenetics comes of age. Nat. Genet., 21: 163-167, 1999.[Medline]
  6. Kelly W. K., O’Connor O. A., Marks P. A. Histone deacetylase inhibitors: from target to clinical trials. Exp. Opin. Investig. Drugs, 11: 1695-1713, 2002.
  7. Jones P. A., Baylin S. B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet., 3: 415-428, 2002.[Medline]
  8. Szyf M., Kaplan F., Mann V., Giloh H., Kedar E., Razin A. Cell cycle-dependent regulation of eukaryotic DNA methylase level. J. Biol. Chem., 260: 8653-8656, 1985.[Abstract/Free Full Text]
  9. Robertson K. D., Uzvolgyi E., Liang G., Talmadge C., Sumegi J., Gonzales F. A., Jones P. A. The human DNA methyltransferases (DNMTs) 1, 3a and 3b: Coordinate mRNA expression in normal tissues and overexpression in tumors. Nucleic Acids Res., 27: 2291-2298, 1999.[Abstract/Free Full Text]
  10. Okano M., Bell D. W., Haber D. A., Li E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99: 247-257, 1999.[Medline]
  11. Roundtree M. R., Bachman K. E., Baylin S. B. DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci. Nat. Genet., 25: 269-277, 2000.[Medline]
  12. Fuks F., Burgers W. A., Brehm A., Hughes-Davies L., Kouzarides T. DNA methyltransferase Dnmt1 associates with histone deacetylase activity. Nat. Gen., 24: 88-91, 2000.[Medline]
  13. Santi D. V., Garrett C. E., Barr P. J. On the mechanism of inhibition of DNA-cytosine methyltransferase by cytidine analogs. Cell, 83: 9-10, 1983.
  14. Cameron E. E., Bachman K. E., Myohanen S., Herman J. G., Baylin S. B. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nat. Gen., 21: 103-107, 1999.[Medline]
  15. Laird P. W., Jackson-Grusby L., Fazeli A., Dickinson S. L., Jung W. E., Li E., Weinberg R. A., Jaenisch R. Suppression of intestinal neoplasia by DNA hypomethylation. Cell, 81: 197-205, 1995.[Medline]
  16. Belinsky S. A., Nikula K. J., Baylin S. B., Issa J-P. Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer. Proc. Natl. Acad. Sci. USA, 93: 4045-4050, 1996.[Abstract/Free Full Text]
  17. Lantry L. E., Zhang Z., Crist K. A., Wang Y., Kelloff G. J., Lubet R. A., You M. 5-Aza-2'-deoxycytidine is chemopreventive in a 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone-induced primary mouse lung tumor model. Carcinogenesis (Lond.), 20: 343-346, 1999.[Abstract/Free Full Text]
  18. Eads C. A., Nickel A. E., Laird P. W. Complete genetic suppression of polyp formation and reduction of CpG-island hypermethylation in Apc(Min/+) DNMT1-hypomorphic mice. Cancer Res., 62: 1296-1299, 2002.[Abstract/Free Full Text]
  19. Belinsky S. A., Devereux T. R., Foley J. F., Maronpot R. R., Anderson M. W. Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylinitrosamino)-1-(3-pyridyl)-1-butanone in the A/J mouse. Cancer Res., 52: 3164-3173, 1992.[Abstract/Free Full Text]
  20. Malkinson A. M. The genetic basis of susceptibility to lung tumors in mice. Toxicology, 54: 349-362, 1989.
  21. Malkinson A. M., Nesbitt M. N., Skamene E. Susceptibility to urethan-induced pulmonary adenomas between A/J and C57BL/6J mice: Use of AxB and BxA recombinant inbred lines indicating a three-locus genetic model. J. Natl. Cancer Inst., 75: 971-974, 1985.
  22. You M., Wang Y., Stoner G., You L., Maronpot R., Reynolds S. H., Anderson M. Parental bias of Ki-ras oncogenes detected in lung tumors from mouse hybrids. Proc. Natl. Acad. Sci. USA, 89: 5804-5808, 1992.[Abstract/Free Full Text]
  23. Rhee I., Bachman K. E., Park B. H., Jair K. W., Yen R. W. C., Schuebel K. E., Cul H., Feinberg A. P., Lengauer C., Kinzler K. W., Baylin S. B., Vogelstein B. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. Nature (Lond.), 416: 552-556, 2002.[Medline]
  24. Festing M. F., Lin L., Devereux T. R., Gao F., Yang A., Anna C. H., White C. M., Malkinson A. M., You M. At least four gene loci and gender are associated with susceptibility to the chemical induction of lung adenomas in A/J X BALB/c mice. Genomics, 15: 129-136, 1998.
  25. Risch H. A., Howe G. R., Jain M., Burch J. D., Holoway E. J., Miller A. B. Are female smokers at higher risk for lung cancer than male smokers?A case-control analysis by histologic type. Am. J. Epidemiol., 138: 281-293, 1993.[Abstract/Free Full Text]
  26. Zang E. A., Wynder E. L. Differences in lung cancer risk between men and women: examination of the evidence. J. Natl. Cancer Inst., 88: 183-192, 1996.
  27. Bestor T. H. Activation of mammalian DNA-methyltransferase by cleavage of a Zn binding regulatory domain. EMBO J., 11: 2611-2617, 1992.[Medline]
  28. Bachman K. E., Rountree M. R., Baylin S. B. Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterchromatin. J. Biol. Chem., 276: 32282-32287, 2001.[Abstract/Free Full Text]
  29. Wu J., Issa J-P., Herman J., Bassett D. E., Jr., Nelkin B. D., Baylin S. B. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells. Proc. Natl. Acad. Sci. USA, 90: 8891-8895, 1993.[Abstract/Free Full Text]
  30. Kim G. D., Ni J., Kelesogiu N., Roberts R. J., Pradhan S. Co-operation and communication between the human maintenance and de novo DNA (cytosine-5) methyltransferases. EMBO J., 21: 4183-4195, 2002.[Medline]
  31. Wijermans P., Lubbet M., Verhoef G., Bosly A., Ravoet C., Andre M. Low dose 5-aza-2'-deoxycytidine, a DNA hypomethylating agent, for the treatment of high-risk myelodysplastic syndromes: a multicenter phase II study in elderly patients. J. Clin. Oncol., 18: 956-962, 2000.[Abstract/Free Full Text]
  32. Johnstone R. W. (2002) Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat. Rev., 1: 287-299, 2002.
  33. Gaudet F., Hodgson J. G., Eden A., Jackson-Grusby L., Dausman J., Gray J. W., Leonhardt H., Jaenisch R. Induction of tumors in mice by genomic hypomethylation. Science (Wash. DC), 300: 489-492, 2002.
  34. Cheng J. C., Matsen C. B., Gonzales F. A., Ye W., Greer S., Marquez V. E., Jones P. A., Selker E. U. Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J. Natl. Cancer Inst., 95: 399-409, 2003.[Abstract/Free Full Text]
  35. Clark L. C., Combs G. F., Turnbull B. W., Slate E. H., Chalker D. K., Chow J., Taylor J. R. Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. JAMA, 276: 1957-1963, 1996.[Abstract]
  36. Fiala E. S., Staretz M. E., Pandya G. A., El-Bayoumy K., Hamilton S. R. Inhibition of DNA cytosine methyltransferase by chemopreventive selenium compounds, determined by an improved assay for cytosine methyltransferase and DNA cytosine methylation. Carcinogenesis (Lond.), 19: 597-604, 1998.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Mol Cancer ResHome page
K. Yamashita, M. S. Kim, H. L. Park, Y. Tokumaru, M. Osada, H. Inoue, M. Mori, and D. Sidransky
HOP/OB1/NECC1 Promoter DNA Is Frequently Hypermethylated and Involved in Tumorigenic Ability in Esophageal Squamous Cell Carcinoma
Mol. Cancer Res., January 1, 2008; 6(1): 31 - 41.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
M. Fabbri, R. Garzon, A. Cimmino, Z. Liu, N. Zanesi, E. Callegari, S. Liu, H. Alder, S. Costinean, C. Fernandez-Cymering, et al.
MicroRNA-29 family reverts aberrant methylation in lung cancer by targeting DNA methyltransferases 3A and 3B
PNAS, October 2, 2007; 104(40): 15805 - 15810.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
J.-P. J. Issa
DNA Methylation as a Therapeutic Target in Cancer
Clin. Cancer Res., March 15, 2007; 13(6): 1634 - 1637.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
G. Egger, A. M. Aparicio, S. G. Escobar, and P. A. Jones
Inhibition of Histone Deacetylation Does Not Block Resilencing of p16 after 5-Aza-2'-Deoxycytidine Treatment
Cancer Res., January 1, 2007; 67(1): 346 - 353.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
A. H. Ting, K. M. McGarvey, and S. B. Baylin
The cancer epigenome--components and functional correlates
Genes & Dev., December 1, 2006; 20(23): 3215 - 3231.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T.-Y. Kim, S. Zhong, C. R. Fields, J. H. Kim, and K. D. Robertson
Epigenomic profiling reveals novel and frequent targets of aberrant DNA methylation-mediated silencing in malignant glioma.
Cancer Res., August 1, 2006; 66(15): 7490 - 7501.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
G. J. Kelloff, S. M. Lippman, A. J. Dannenberg, C. C. Sigman, H. L. Pearce, B. J. Reid, E. Szabo, V. C. Jordan, M. R. Spitz, G. B. Mills, et al.
Progress in Chemoprevention Drug Development: The Promise of Molecular Biomarkers for Prevention of Intraepithelial Neoplasia and Cancer--A Plan to Move Forward
Clin. Cancer Res., June 15, 2006; 12(12): 3661 - 3697.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
X. Cui, T. Wakai, Y. Shirai, K. Hatakeyama, and S. Hirano
Chronic Oral Exposure to Inorganic Arsenate Interferes with Methylation Status of p16INK4a and RASSF1A and Induces Lung Cancer in A/J Mice
Toxicol. Sci., June 1, 2006; 91(2): 372 - 381.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
B. R. Vuillemenot, J. A. Hutt, and S. A. Belinsky
Gene Promoter Hypermethylation in Mouse Lung Tumors
Mol. Cancer Res., April 1, 2006; 4(4): 267 - 273.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
Y. Tada, R. M. Brena, B. Hackanson, C. Morrison, G. A. Otterson, and C. Plass
Epigenetic modulation of tumor suppressor CCAAT/enhancer binding protein alpha activity in lung cancer.
J Natl Cancer Inst, March 15, 2006; 98(6): 396 - 406.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. T. McCabe, J. A. Low, S. Daignault, M. J. Imperiale, K. J. Wojno, and M. L. Day
Inhibition of DNA Methyltransferase Activity Prevents Tumorigenesis in a Mouse Model of Prostate Cancer
Cancer Res., January 1, 2006; 66(1): 385 - 392.
[Abstract] [Full Text] [PDF]

Home page
 ASH Education BookHome page
H. Fathallah and G. F. Atweh
Induction of Fetal Hemoglobin in the Treatment of Sickle Cell Disease
Hematology, January 1, 2006; 2006(1): 58 - 62.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. H. Lee, S. Yegnasubramanian, X. Lin, and W. G. Nelson
Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1
J. Biol. Chem., December 9, 2005; 280(49): 40749 - 40756.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
W. J. Petty, N. Li, A. Biddle, R. Bounds, C. Nitkin, Y. Ma, K. H. Dragnev, S. J. Freemantle, and E. Dmitrovsky
A Novel Retinoic Acid Receptor {beta} Isoform and Retinoid Resistance in Lung Carcinogenesis
J Natl Cancer Inst, November 16, 2005; 97(22): 1645 - 1651.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
P. Vineis and K. Husgafvel-Pursiainen
Air pollution and cancer: biomarker studies in human populations
Carcinogenesis, November 1, 2005; 26(11): 1846 - 1855.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. R. Acharya, A. Sparreboom, J. Venitz, and W. D. Figg
Rational Development of Histone Deacetylase Inhibitors as Anticancer Agents: A Review
Mol. Pharmacol., October 1, 2005; 68(4): 917 - 932.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
S. A. Belinsky
Silencing of genes by promoter hypermethylation: key event in rodent and human lung cancer
Carcinogenesis, September 1, 2005; 26(9): 1481 - 1487.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
K. N. Bhalla
Epigenetic and Chromatin Modifiers As Targeted Therapy of Hematologic Malignancies
J. Clin. Oncol., June 10, 2005; 23(17): 3971 - 3993.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
F. R. Hirsch and S. M. Lippman
Advances in the Biology of Lung Cancer Chemoprevention
J. Clin. Oncol., May 10, 2005; 23(14): 3186 - 3197.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. T. Agoston, P. Argani, S. Yegnasubramanian, A. M. De Marzo, M. A. Ansari-Lari, J. L. Hicks, N. E. Davidson, and W. G. Nelson
Increased Protein Stability Causes DNA Methyltransferase 1 Dysregulation in Breast Cancer
J. Biol. Chem., May 6, 2005; 280(18): 18302 - 18310.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. T. McCabe, J. N. Davis, and M. L. Day
Regulation of DNA Methyltransferase 1 by the pRb/E2F1 Pathway
Cancer Res., May 1, 2005; 65(9): 3624 - 3632.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
P. W. Laird
Cancer epigenetics
Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R65 - R76.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
J. L. Mauderly, A. P. Gigliotti, E. B. Barr, W. E. Bechtold, S. A. Belinsky, F. F. Hahn, C. A. Hobbs, T. H. March, S. K. Seilkop, and G. L. Finch
Chronic Inhalation Exposure to Mainstream Cigarette Smoke Increases Lung and Nasal Tumor Incidence in Rats
Toxicol. Sci., October 1, 2004; 81(2): 280 - 292.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
B. R. Vuillemenot, L. C. Pulling, W. A. Palmisano, J. A. Hutt, and S. A. Belinsky
Carcinogen exposure differentially modulates RAR-{beta} promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis
Carcinogenesis, April 1, 2004; 25(4): 623 - 629.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar