This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, H. Articles by Kim, D.-H. Search for Related Content PubMed PubMed Citation Articles by Kim, H. Articles by Kim, D.-H.

Tumor-Specific Methylation in Bronchial Lavage for the Early Detection of Non-Small-Cell Lung Cancer

From the Division of Pulmonary and Critical Care Medicine, Department of Thoracic Surgery, and Department of Pathology, Samsung Medical Center, Sungkyunkwan University, School of Medicine; Center for Genome Research, Samsung Biomedical Research Institute, Seoul; and Department of Molecular Cell Biology, Sungkyunkwan University, School of Medicine, Suwon, Korea

Address reprint requests to Duk-Hwan Kim, MD, Center for Genome Research, Samsung Biomedical Research Institute, Rm B155, #50 Ilwon-dong, Kangnam-Ku, Seoul, Korea, 135-710; e-mail: dukhwan{at}samsung.co.kr

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
PURPOSE: The aim of this study was to identify tumor-specific methylation in bronchial lavage for the early detection of non-small-cell lung cancer (NSCLC) by differentiating the age-related methylation from the tumor-specific methylation in NSCLC.

PATIENTS AND METHODS: Eighty-five NSCLC patients and 127 cancer-free subjects participated in this study. Aberrant methylation at the promoters of the p16, Ras association domain family 1A (RASSF1A), fragile histidine triad (FHIT), H-cadherin, and retinoic acid receptor ß (RARß) genes were evaluated in the resected tumor tissues and bronchial lavage samples of NSCLC patients and in the bronchial lavage samples of cancer-free subjects by methylation-specific polymerase chain reaction.

RESULTS: Of the 127 cancer-free samples, methylation was detected in 6% for p16, 13% for RARß, 3% for H-cadherin, 4% for RASSF1A, and 28% for FHIT. Hypermethylation of the p16, RARß, H-cadherin, and RASSF1A genes was not associated with patient age and smoking, whereas hypermethylation of the FHIT promoter occurred more frequently in older patients (P = .02) and was associated with exposure to tobacco smoke (P = .001). A strong correlation between age and smoking was found in patients with hypermethylation of the FHIT gene (r = 0.36; P = .03). A total of 68% of the bronchial lavage samples from the 85 NSCLC patients showed methylation of at least one of p16, RARß, H-cadherin, and RASSF1A genes.

CONCLUSION: Our study suggests that tumor-specific methylation of the p16, RASSF1A, H-cadherin, and RARß genes may be a valuable biomarker for the early detection of NSCLC in bronchial lavage, and that the age-related methylation of FHIT gene in the normal bronchial epithelium is related to the exposure to tobacco smoke.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Lung cancer is the most frequent cause of cancer-related death in the world, and causes over 1 million deaths worldwide each year.¹ Despite new diagnostic techniques, most lung cancers are detected late, and consequently, most patients present with advanced disease, with the overall 5-year survival rates remaining below 16% and 6% in non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), respectively.² The poor prognosis of lung cancer patients is largely a result of the occult metastatic dissemination, which appears in approximately two-thirds of patients at the time of diagnosis.² Thus, the development of efficient diagnostic methods to enable the early detection of cancer in these patients is clearly imperative.

Lung cancer screening by chest x-ray and conventional sputum cytology has not been proven effective at improving overall survival.³ One promising approach is the identification of lung cancer-specific biomarkers at an early stage. The de novo methylation of CpG islands within the promoters of tumor suppressor genes is one of frequent mechanisms of gene inactivation in neoplastic cells and is one of the most frequently acquired epigenetic changes during the pathogenesis of lung cancer.⁴ Therefore, it may become a good biomarker for the early detection of lung cancer.⁵

However, methylation of a tumor supressor gene does not necessarily indicate that it is tumor-specific, because CpG island hypermethylation of some tumor suppressor genes occurs after the onset of neoplastic evolution, and others become hypermethylated initially in normal epithelial cells by environmental factors such as exposure to tobacco and aging. Thus, such factors could be the source of false-positives in the study of tumor-specific methylation. The critical factor for the early detection of lung cancer is to differentiate age- or environmental factor-related methylation in patients who do not develop cancer from the tumor-specific methylation in cancer patients.

A number of studies have shown the effects of smoking on the aberrant methylation of tumor suppressor genes in primary lung cancers and in the bronchial epithelium of heavy smokers.^6-9 Hypermethylation of the p16 promoter was reported in lung cancer induced by the inhalation of cigarette smoke in F344/N rats and in the bronchial epithelium and sputum of smokers.^6-7 The hypermethylation of p16 was also associated with the duration of smoking in primary NSCLC.⁸ Belinsky et al⁹ reported on the hypermethylation of p16 in rat adenocarcinoma induced by tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

In addition, age-related methylation has also been reported for several genes.^10-16 Age-related changes in methylation were first reported in the promoter of the estrogen receptor (ER) gene in normal colon mucosa and in colon cancer.¹⁰ The insulin-like growth factor 2 gene was also found to show extensive methylation of the originally unmethylated paternal allele with age in colon cancer.¹¹ Ahuja et al¹² reported on the age-related methylation of the N33 and myogenic transcription factor (MYOD) genes, but not of p16, THBS1 (thromospondin), and HIC-1 (hypermethylated in cancer) in normal colon mucosa, suggesting that age-related methylation is gene-specific. In glioblastoma multiforme, a strong association was found between ER and N33 methylation and age.¹³ Moreover, age-related methylation of the hMLH1 (human MutL homologue) promoter occurs in gastric cancer and in normal colonic mucosa.^14-15 Issa et al¹⁶ also demonstrated widespread abnormalities in the age-related methylation of the ER and MYOD genes in ulcerative colitis patients with high-grade dysplasia or colon cancer.

p16, retinoic acid receptor ß (RARß), H-cadherin (CDH13), Ras association domain family 1A (RASSF1A), and fragile histidine triad (FHIT) genes are important in the pathogenesis of lung cancer and are frequently inactivated by aberrant methylation of their promoter regions.⁵ To identify tumor-specific methylation useful for the early detection of NSCLC, we first investigated the aberrant methylation of these genes by methylation-specific polymerase chain reaction (MSP) in the tumor tissues and bronchial lavage samples of NSCLC patients. We next determined the methylation status of the same genes in bronchial lavage samples from cancer-free individuals to discriminate between age- or smoking-related methylation and tumor-specific methylation. We studied age- or smoking-related methylation in the bronchial epithelium of normal individuals rather than in the corresponding normal tissues of cancer patients to eliminate any possibility of false-positive methylation by contamination of nonmalignant lung tissues with adjacent tumor cells.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Study Population
Samples were obtained with written informed consent from 85 NSCLC patients treated by surgical resection and from 127 cancer-free subjects who underwent fiberoptic bronchoscopy at the Samsung Medical Center (Seoul, Korea) between March 2002 and January 2004. This study was approved by our institutional review board. The cancer-free subjects were recruited from among patients that had received primary care for other diseases than NSCLC at the Division of Pulmonary and Critical Care Medicine. All cancer-free subjects were clinically free of any cancer at the time of bronchoscopy and their chest x-rays showed no evidence of lung cancer. Patients with an abnormal cytologic result, such as dysplasia or metaplasia, were excluded from the methylation analysis. Individuals with a prior history of any cancer were also excluded.

Information on sociodemographic characteristics was obtained using an interviewer-administered questionnaire. Patients who had never smoked or current smokers who had smoked within 12 months of bronchoscopy participated in this study. Those who had stopped smoking more than 12 months before bronchoscopy were excluded, since the effect of quitting smoking on age-related methylation is not clear.

Bronchoscopy and DNA Extraction
Flexible fiberoptic bronchoscopy (Olympus, Tokyo, Japan) via the oral route was performed under local anesthesia using 2% xylocaine. After the bronchoscope (Olympus, Tokyo, Japan) had been located at the segmental bronchi of the pulmonary lobe, 10 mL of sterile warm saline was instilled and then retrieved by medical suction. The bronchoalveolar lavage fluid so obtained was transported to the laboratory on ice. The cell suspension was then centrifuged at 2,500 rpm for 5 minutes at 4°C, cell pellets were washed with 2 mL of 1 x phosphate-buffered saline twice, and an aliquot of material was stored at –80°C until required. Cell pellets were then resuspended once in phosphate-buffered saline and digested with Protease (Qiagen, Valencia, CA). DNA was extracted using a QIAamp DNA extraction kit according to the manufacturer's instructions (Qiagen).

DNA Extraction From Paraffin Block
Formalin-fixed, paraffin-embedded tissue blocks containing at least 75% neoplastic tissue were used for this study. Serial 10 µm tissue sections were cut from each paraffin block and transferred to slides. Sections were stained with hematoxylin and eosin to locate tumor areas before DNA extraction. Areas corresponding to tumor were carefully microdissected from the surrounding normal stromal tissues, placed in an Eppendorf tube, deparaffinized overnight at 63°C in xylene, and then vortexed vigorously. After centrifuging at full speed for 5 minutes, the supernatant was removed, and ethanol was added to remove residual xylene, and subsequently removed by centrifugation. After evaporating the ethanol, the tissue pellet was resuspended in lysis buffer ATL (Qiagen), and the DNA was isolated according to the manufacturer's instructions.

Methylation Analysis
The methylation status of the p16, RARß, H-cadherin, RASSF1A, and FHIT promoters was analyzed by MSP (Fig 1), as previously described by Herman et al.¹⁷ Two sets of primers were designed for each gene, one specific for DNA methylated at the promoter region and the other specific for unmethylated DNA. The primers used for MSP are shown in Table 1.

View larger version (52K): [in this window] [in a new window]   Fig 1. Methylation analysis of the p16, retinoic acid receptor ß (RARß), H-cadherin, Ras association domain family 1A (RASSF1A), and fragile histidine triad (FHIT) genes in 127 cancer-free subjects and in 85 non-small-cell lung cancer patients. The numbers shown are sample identification numbers.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
Clinicopathologic Characteristics
Eighty-five NSCLC patients and 127 cancer-free subjects participated in this study. Of the 85 NSCLC patients, 52 patients had stage I disease and 33 had stage II. There were 31 adenocarcinomas, 43 squamous cell carcinomas, and 11 other cell types. The mean age of the NSCLC patients was 63 ± 17 years, and 57 were males. The average number of pack-years (56 ± 39) of the NSCLC patients was higher than that of the cancer-free subjects (37 ± 27), and the difference was statistically significant (P = .001).

The 127 cancer-free subjects did not show a visible evidence of tumor and were normal by cytologic examination. The cancer-free subjects examined included 83 with chronic obstructive pulmonary disease, 27 with pulmonary tuberculosis, and 17 with pneumonia without evidence of lung cancer by cytologic examination. The cancer-free subjects consisted of 84 males (66%) and 43 females (34%), and patient age ranged from 26 to 82 years. The mean age of the men was 62 ± 14 years and women 60 ± 15 years (data not shown). Of the 127 subjects, 17 subjects were never-smokers and 110 were current smokers.

Prevalence of Aberrant Methylation in Cancer-Free Subjects and NSCLC Patients
We determined the prevalence of the methylation of the p16, RARß, H-cadherin, RASSF1A, and FHIT genes in 85 NSCLC patients and 127 cancer-free subjects by MSP (Fig 1). Methylation in tumor tissues was detected in 28% for p16, 36% for RARß, 34% for H-cadherin, 38% for RASSF1A, and 33% for FHIT gene (Table 2). Approximately 30% to 65% of the patients showing hypermethylation in tumor tissues had promoter hypermethylation in the bronchial lavage at each promoter region (Table 2). In addition, hypermethylation in the bronchial lavage of patients without hypermethylation in tumor tissues was very rare (Table 2), suggesting that hypermethylation in bronchial lavage may be a surrogate marker for methylation status in tumor tissues.

View larger version (27K): [in this window] [in a new window]   Fig 2. Prevalence of hypermethylation according to (A) age and (B) exposure to tobacco smoke (pack-years) in 127 cancer-free subjects. RARß, retinoic acid receptor ß; RASSF1A, Ras association domain family 1A; and FHIT, fragile histidine triad.

Multivariate Logistic Regression Analysis
Multivariate logistic regression was conducted to estimate the relationship between hypermethylation of the FHIT gene and the covariates of age, sex, and exposure to tobacco smoke, and to calculate the corresponding odds ratios. Table 6 shows that patient age and exposure to tobacco smoke were significantly associated with hypermethylation of the FHIT promoter. Hypermethylation of the FHIT gene occurred at a 9.01 times (95% CI, 1.19 to 79.42; P = .03) higher prevalence in subjects over 60 years of age than in those less than 40 years of age, after adjusting for sex and exposure to tobacco smoke. Subjects who had smoked for more than 40 pack-years were 13.10 times (95% CI, 4.94 to 63.96; P = .001) more likely to show hypermethylation of the FHIT gene than those who had smoked for less than 20 pack-years. Based on these observations, it seems reasonable to suggest that hypermethylation of the FHIT gene was not tumor-specific.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

Whereas the hypermethylations of the p16, RARß, H-cadherin, and RASSF1A promoters in our study were not associated with patient age and smoking, the hypermethylation of the FHIT promoter was significantly associated with age and smoking. These findings suggest that age- and smoking-related methylation in the normal bronchial epithelium is gene-specific. At present, the molecular mechanisms responsible for differential susceptibility to hypermethylation during aging remain unclear, but are probably related to several factors; namely, de novo DNA methyltransferase activity, modulating factors such as smoking, different degrees of protection against methylation, and local triggering factors, such as an unusual secondary structure around a methylation center.

Aberrant de novo methyltransferase activity could play a role in age-related methylation, as maintenance methyltransferase activity decreases during cellular senescence, but de novo methyltransferase activity continues to increase in aged cells.¹⁸ Thus, an increased de novo methyltransferase activity with age may partially explain the observed age-related methylation of the FHIT promoter. Although there is no direct evidence that smoking induces DNA methylation, recent reports have suggested that smoking increases DNA methyltransferase activity and that DNA hypermethylation is associated with exposure to tobacco smoke.^6-9 The increased prevalence of the hypermethylation of the FHIT promoter in heavy smokers, found in the present study, may result from increased methyltransferase activity by smoking.

What then is responsible for the gene-specificity of age-related methylation? It is likely that factors other than increased DNA methyltransferase activity are involved. Different levels of protection against methylation could be one of the factors responsible for gene-specificity of age-related methylation. The distance from a methylation center to a CpG island center,^19-20 preexisting methylation on the CpG island around each gene, and binding sites for trans-activating factors around each gene may affect the degree of protection afforded against de novo CpG island methyl-ation.^21-22 Therefore, if a FHIT promoter is weakly protected, it may be disrupted by increased DNA methyltransferase activity during aging. It is also possible the FHIT promoter loses its protection from de novo methylation during aging.

Another possible factor responsible for the gene-specificity of age-related methylation concerns the chromatin structure. In present study, only the FHIT promoter showed age-related methylation in the normal bronchial epithelium. The main difference between the sequence of the FHIT gene and the p16, RARß, RASSF1A, and H-cadherin genes is that the FHIT gene contains chromosomal fragile sites that exhibit several features characteristic of a highly unstable region. In addition, the FHIT gene contains numerous short- and long- repeat sequences, such as long interspersed nuclear elements (LINE) and short interspersed nuclear elements (Alu and MIR).^23-26

Repeat sequences, repetitive elements, and unusual structures are known to be involved in the hypermethylation of CpG islands.^27-33 Several reports have suggested that repeat sequences or repetitive elements are associated with unusual secondary structures and that these secondary structures can signal de novo methylation in adjacent regions by attracting DNA methyltransferase into a de novo methylation center. Repetitive elements such as B1 or Alu form unusual secondary structures.^27-28 Yates et al²⁹ reported that tandem B1 elements located in a methylation center provide a signal for de novo methylation. Each subunit of Alu repeats folds independently, to form characteristic secondary structures. Trinucleotide repeats also form hairpin structures.^30-32 Smith et al³³ reported that DNA methyltransferase identifies preferentially a three-nucleotide recognition motif within the CpG dinucleotide pair and suggested that unusual DNA structures may provide signals for DNA methylation in vivo.

Based on these observations, it is possible that numerous repetitive sequences and unstable structures in the FHIT gene form an unusual secondary structure that facilities hypermethylation by attracting DNA methyltransferase, and that this process is increased by age and exposure to tobacco smoke. In the colon, age-related methylation is considered to result in a hyperproliferative state, which is thought to precede tumor formation. However, the role of age-related methylation in the normal bronchial epithelium remains to be elucidated. More study is needed to understand the mechanisms of age-related changes in methylation in normal lung tissues, and the effects of age- or smoking-related methylation on tumor formation in the lung.

The hypermethylation of several genes in the bronchial epithelium and sputum of cancer-free subjects has also been reported in other races.^5,7,34-36 At present, the molecular mechanisms of aberrant methylation in cancer-free subjects are not clear and require further investigation. The aberrant methylation of genes is seldom observed in normal DNA samples. Accordingly, the prevalence of these markers in the bronchial lavage from cancer-free subjects may reflect a latent period before clinical tumor detection or a high-risk status. The observation³⁵ that hypermethylation of p16 was detected in sputum samples taken from cancer-free smokers up to 3 years before clinical detection of lung cancer supports the possibility that the cancer-free subjects with positive methylation in the bronchoalveolar lavage samples actually have the lung cancer. Hence, longitudinal prospective studies are needed to monitor closely the change of methylation status in cancer-free patients and to determine whether cancer-free subjects with hypermethylation at each locus subsequently develop overt disease. In addition, the present study was limited by a small sample size and by the use of standard MSP. Quantitative information about the numbers of hypermethylated alleles may be useful to clearly discriminate tumor and nontumor lesions, given the significant prevalence of methylation markers in cancer-free subjects and the possible dose effect of methylated alleles in malignant transformation. By implementing a longitudinal prospective study and a quantitative assay, more accurate risk models for the early detection of NSCLC can be developed, and the false-positive rate reduced. In addition, the timing of aberrant methylation of each gene during carcinogenesis should also be investigated.

In conclusion, our study suggests that tumor-specific methylation of the p16, RASSF1A, CDH13, and RARß genes may be a useful marker for the early detection of NSCLC in bronchial lavage, and the age-related methylation of the FHIT gene in the normal bronchial epithelium is gene-specific and is related to the exposure to tobacco smoke.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
The authors indicated no potential conflicts of interest.

	Acknowledgment

 
The authors gratefully acknowledge Jin-Hyuk Kim for manuscript reading. We also thank Eun-Kyung Kim for assistance with the data collection and management.

	NOTES

 
Supported by the Samsung Biomedical Research Institute, Seoul; and Samsung Advanced Institute of Technology, Suwon, Korea.

Authors' disclosures of potential conflicts of interest are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... REFERENCES

 
1. Parkin DM, Bray F, Perlay J, et al: Estimating the world cancer burden: GLOBOCAN 2000. Int J Cancer 94:153–156, 2001[CrossRef][Medline]

2. Ries LAG, Kosary CL, Hankey BF, et al: SEER Cancer Statistics Review, 1973-1996. National Cancer Institute, Bethesda, MD, NIH Pub No. 99-2789, 1999

3. Marcus PM: Lung cancer screening: An update. J Clin Oncol 19:83S–86S, 2001 (suppl 18)

4. Baylin SB, Herman JG, Graff JR, et al: Alterations in DNA methylation: A fundamental aspect of neoplasia. Adv Cancer Res 72:141–196, 1998[Medline]

5. Zöchbauer-Müller S, Minna JD, Gazdar AF: Aberrant DNA methylation in lung cancer: Biological and clinical implications. Oncologist 7:451–457, 2002[Abstract/Free Full Text]

6. Swafford DS, Middleton SK, Palmisano WA, et al: Frequent aberrant methylation of p16^INK4A in primary rat lung tumors. Mol Cell Biol 17:1366–1374, 1997[Abstract]

7. Belinsky SA, Palmisano WA, Gilliland FD, et al: Aberrant promoter methylation in bronchial epithelium and sputum from current and former smokers. Cancer Res 62:2370–2377, 2002[Abstract/Free Full Text]

8. Kim DH, Nelson HH, Wiencke JK, et al: P16^INK4a and histology-specific methylation of CpG islands by exposure to tobacco smoke in non-small cell lung cancer. Cancer Res 61:3419–3424, 2001[Abstract/Free Full Text]

9. Belinsky SA, Nikula KJ, Palmisano WA, et al: Aberrant methylation of p16^INK4A is an early event in lung cancer and a potential marker for early diagnosis. Proc Natl Acad Sci U S A 95:11891–11896, 1998[Abstract/Free Full Text]

10. Issa JP, Ottaviano YL, Celano P, et al: Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 7:536–540, 1994[CrossRef][Medline]

11. Issa JP, Vertino PM, Boehm CD, et al: Switch from monoallelic to biallelic human IGF2 promoter methylation during aging and carcinogenesis. Proc Natl Acad Sci U S A 93:11757–11762, 1996[Abstract/Free Full Text]

12. Ahuja N, Li Q, Mohan AL, et al: Aging and DNA methylation in colorectal mucosa and cancer. Cancer Res 58:5489–5494, 1998[Abstract/Free Full Text]

13. Li Q, Jedlicka A, Ahuja N, et al: Concordant methylation of the ER and N33 genes in glioblastoma multiforme. Oncogene 16:3197–3202, 1998[CrossRef][Medline]

14. Nakajima T, Akiyama Y, Shiraishi J, et al: Age-related hypermethylation of the hMLH1 promoter in gastric cancers. Int J Cancer 94:208–211, 2001[CrossRef][Medline]

15. Nakagawa H, Nuovo GJ, Zervos EE, et al: Age-related hypermethylation of the 5' region of MLH1 in normal colonic mucosa is associated with microsatellite-unstable colorectal cancer development. Cancer Res 61:6991–6995, 2001[Abstract/Free Full Text]

16. Issa JP, Ahuja N, Toyota M, et al: Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res 61:3573–3577, 2001[Abstract/Free Full Text]

17. Herman JG, Graff JR, Myohanen S, et al: Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93:9821–9826, 1996[Abstract/Free Full Text]

18. Lopatina N, Haskell JF, Andrews LG, et al: Differential maintenance and de novo methylating activity by three DNA methyltransferase in aging and immortalized fibroblasts. J Cell Biochem 84:324–334, 2002[CrossRef][Medline]

19. Mummaneni P, Bishop PL, Turker MS: A cis-acting element accounts for a conserved methylation pattern upstream of the mouse adenine phosphoribosyltransferase gene. J Biol Chem 268:552–558, 1993[Abstract/Free Full Text]

20. Mummaneni P, Walker KA, Bishop PL, et al: Epigenetic gene inactivation induced by a cis-acting methylation center. J Biol Chem 270:788–792, 1995[Abstract/Free Full Text]

21. Macleod D, Charlton J, Mullins J, et al: SP1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev 8:2282–2292, 1994[Abstract/Free Full Text]

22. Brandeis M, Frank D, Keshet I, et al: SP1 elements protect a CpG island from de novo methylation. Nature 371:435–438, 1994[CrossRef][Medline]

23. Inoue H, Ishii H, Alder H, et al: Sequence of the FRA3B common fragile region: Implications for the mechanism of FHIT deletion. Proc Natl Acad Sci U S A 94:14584–14589, 1997[Abstract/Free Full Text]

24. Boldog F, Gemmill RB, West J, et al: Chromosome 3p14 homozygous deletions and sequence analysis of FRA3B. Hum Mol Genet 6:193–203, 1997[Abstract/Free Full Text]

25. Mimori K, Druck T, Inoue H, et al: Cancer-specific chromosome alterations in the constitutive fragile region FRA3B. Proc Natl Acad Sci U S A 96:7456–7461, 1999[Abstract/Free Full Text]

26. Corbin S, Neilly ME, Espinosa III R, et al: Identification of unstable sequences within the common fragile site at 3p14.2: Implications for the mechanism of deletions within fragile histidine triad gene/common fragile site at 3p14.2 in tumors. Cancer Res 62:3477–3484, 2002[Abstract/Free Full Text]

27. Labuda D, Sinnett D, Richer C, et al: Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved. J Mol Evol 32:405–414, 1991[CrossRef][Medline]

28. Sinnett D, Richer C, Deragon JM, et al: Alu RNA secondary structure consists of two independent 7 SL RNA-like folding units. J Biol Chem 266:8675–8678, 1991[Abstract/Free Full Text]

29. Yates PA, Burman RW, Mummaneni P, et al: Tandem B1 elements located in a mouse methylation center provide a target for de novo methylation. J Biol Chem 274:36357–36361, 1999[Abstract/Free Full Text]

30. Chen X, Mariappan SVS, Catasti P, et al: Hairpins are formed by the single DNA strands of the fragile X triplet repeats: Structure and biological implications. Proc Natl Acad Sci U S A 92:5199–5203, 1995[Abstract/Free Full Text]

31. Gacy AM, Goellner G, Juranic N, et al: Trinucleotide repeats that expand in human disease form hairpin structures in vitro. Cell 81:533–540, 1995[CrossRef][Medline]

32. Nadel Y, Weissman-Shomer P, Fry M: The fragile X syndrome single strand d(CGG)n nucleotide repeats readily fold back to form unimolecular hairpin structure. J Biol Chem 270:28970–28977, 1995[Abstract/Free Full Text]

33. Smith SS, Kan JLC, Baker DJ, et al: Recognition of unusual DNA structures by human DNA (cytosine-5) methyltransferase. J Mol Biol 217:39–51, 1991[CrossRef][Medline]

34. Kersting M, Friedl C, Kraus A, et al: Differential frequencies of p16(INK4a) promoter hypermethylation, p53 mutation, and K-ras mutation in exfoliative material mark the development of lung cancer in symptomatic chronic smokers. J Clin Oncol 18:3221–3229, 2000[Abstract/Free Full Text]

35. Palmisano WA, Divine KK, Saccomanno G, et al: Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res 60:5954–5958, 2000[Abstract/Free Full Text]

36. Soria JC, Rodriguez M, Liu DD, et al: Aberrant promoter methylation of multiple genes in bronchial brush samples from former cigarette smokers. Cancer Res 62:351–355, 2002[Abstract/Free Full Text]

Submitted October 10, 2003; accepted March 30, 2004.

This article has been cited by other articles:

				S. Umemura, N. Fujimoto, A. Hiraki, K. Gemba, N. Takigawa, K. Fujiwara, M. Fujii, H. Umemura, M. Satoh, M. Tabata, et al. Aberrant promoter hypermethylation in serum DNA from patients with silicosis Carcinogenesis, September 1, 2008; 29(9): 1845 - 1849. [Abstract] [Full Text] [PDF]

				F. Barlesi, G. Giaccone, M. I. Gallegos-Ruiz, A. Loundou, S. W. Span, P. Lefesvre, F. A.E. Kruyt, and J. A. Rodriguez Global Histone Modifications Predict Prognosis of Resected Non Small-Cell Lung Cancer J. Clin. Oncol., October 1, 2007; 25(28): 4358 - 4364. [Abstract] [Full Text] [PDF]

				C. A. Righini, F. de Fraipont, J.-F. Timsit, C. Faure, E. Brambilla, E. Reyt, and M.-C. Favrot Tumor-Specific Methylation in Saliva: A Promising Biomarker for Early Detection of Head and Neck Cancer Recurrence Clin. Cancer Res., February 15, 2007; 13(4): 1179 - 1185. [Abstract] [Full Text] [PDF]

				S. De Flora, F. D'Agostini, A. Izzotti, N. Zanesi, C. M. Croce, and R. Balansky Molecular and Cytogenetical Alterations Induced by Environmental Cigarette Smoke in Mice Heterozygous for Fhit Cancer Res., February 1, 2007; 67(3): 1001 - 1006. [Abstract] [Full Text] [PDF]

				M. J. Worsham, K. M. Chen, V. Meduri, A. O. H. Nygren, A. Errami, J. P. Schouten, and M. S. Benninger Epigenetic events of disease progression in head and neck squamous cell carcinoma. Arch Otolaryngol Head Neck Surg, June 1, 2006; 132(6): 668 - 677. [Abstract] [Full Text] [PDF]

				J. S. Kim, J. W. Kim, J. Han, Y. M. Shim, J. Park, and D.-H. Kim Cohypermethylation of p16 and FHIT Promoters as a Prognostic Factor of Recurrence in Surgically Resected Stage I Non-Small Cell Lung Cancer. Cancer Res., April 15, 2006; 66(8): 4049 - 4054. [Abstract] [Full Text] [PDF]

				F. D'Agostini, A. Izzotti, R. Balansky, N. Zanesi, C. M. Croce, and S. De Flora Early loss of fhit in the respiratory tract of rodents exposed to environmental cigarette smoke. Cancer Res., April 1, 2006; 66(7): 3936 - 3941. [Abstract] [Full Text] [PDF]

				M. O. Hoque, E. Rosenbaum, W. H. Westra, M. Xing, P. Ladenson, M. A. Zeiger, D. Sidransky, and C. B. Umbricht Quantitative Assessment of Promoter Methylation Profiles in Thyroid Neoplasms J. Clin. Endocrinol. Metab., July 1, 2005; 90(7): 4011 - 4018. [Abstract] [Full Text] [PDF]

				H. Izumi, J. Inoue, S. Yokoi, H. Hosoda, T. Shibata, M. Sunamori, S. Hirohashi, J. Inazawa, and I. Imoto Frequent silencing of DBC1 is by genetic or epigenetic mechanisms in non-small cell lung cancers Hum. Mol. Genet., April 15, 2005; 14(8): 997 - 1007. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, H. Articles by Kim, D.-H. Search for Related Content PubMed PubMed Citation Articles by Kim, H. Articles by Kim, D.-H.