This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Toyooka, S. Articles by Gazdar, A. F. Search for Related Content PubMed PubMed Citation Articles by Toyooka, S. Articles by Gazdar, A. F.

Hamon Center for Therapeutic Oncology Research [S. T., K. O. T., R. M., A. K. V., L. G., K. M., K. H., J. D. M., A. F. G.] and Departments of Pathology [A. K. V., A. F. G.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], University of Texas Southwestern Medical Center, Dallas, Texas 75390-8563; Division of Molecular Oncology, Aichi Cancer Center, Nagoya 464-8681, Japan [Y. A., T. T.]; Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan [K. S.]; Laboratoire de Pathologie Cellulaire, Centre Hospitalier Regional Universitaire, Grenoble 38043, France [E. B.]; and Department of Pathology, M. D. Anderson Cancer Center, Houston, Texas 77030 [M. G.]

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Aberrant methylation of CpG islands in promoter regions of tumor cells is one of the major mechanisms for silencing of tumor suppressor genes. We determined the frequency of aberrant promoter methylation of the p16, adenomatous polyposis coli (APC), H-cadherin (CDH13), glutathione S-transferase P1 (GSTP1), O⁶-methylguanine-DNA-methyltransferase (MGMT), retinoic acid receptor ß-2 (RARß), E-cadherin (CDH1), and RAS association domain family 1A (RASSF1A) genes in 198 tumors consisting of small cell lung cancers [SCLCs (n = 43)], non-small cell lung cancers [NSCLCs (n = 115)], and bronchial carcinoids (n = 40). The profile of methylated genes in the two neuroendocrine tumors (SCLC and carcinoids) were very different from that of NSCLC. However, whereas the overall pattern of aberrant methylation of carcinoids was similar to that of SCLC, carcinoids had lower frequencies of methylation for some of the genes tested. There were also significant differences in the methylation profiles between the two major types of NSCLC, adenocarcinoma and squamous cell carcinoma. We performed cluster analysis and found that SCLCs clustered with other SCLCs and carcinoids but not with NSCLCs, whereas the NSCLCs tended to cluster together. Within NSCLCs, adenocarcinomas and squamous cell carcinomas clustered with their respective histological types. Finally, we compared the methylation profiles of SCLC and NSCLC tumors and their respective cell lines (n = 44). In general, methylation frequencies were higher in tumor cell lines, but these differences were seldom significant. Thus, tumor cell lines appear to be suitable models to study aberrant DNA methylation. We conclude that SCLC, carcinoids, squamous cell carcinomas, and adenocarcinomas of the lung have unique profiles of aberrant methylation. Our findings should help us understand differences in the pathogenetic mechanisms of lung cancers.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lung cancer is the leading cause of cancer mortality in the United States and in the world (1, 2). Lung cancer is divided into two major histological categories, SCLC³ and NSCLC. SCLC accounts for 20–25% of all bronchogenic malignancies and follows a highly aggressive clinical course. Metastases (clinically apparent or occult) are usually present at diagnosis of SCLC, and the treatment of choice is usually combination chemotherapy. Less than 5% of patients currently survive 5 years past the initial diagnosis (3), whereas the 5-year survival rate for patients diagnosed with NSCLC is 15% (4). SCLC is initially highly responsive to radiation and systemic chemotherapeutic regimes. NSCLC is less sensitive to chemotherapy, and curative intent surgical resection is the treatment of choice (5). Bronchial carcinoids are tumors of the lung of low-grade malignancy, although a subtype, the atypical carcinoid, follows a more aggressive course (6). However, the overall survival of patients with carcinoids is far better than that of patients with SCLC. Carcinoids and SCLC are neuroendocrine tumors, although their pathogenesis is believed to be very different. Carcinoids occur at an earlier age and are not smoking related. In contrast to SCLC, they are highly resistant to chemotherapy, and most carcinoids are cured by surgical resection. There are both similarities and differences in the molecular changes present in SCLC and carcinoid (7, 8). In particular, mutations of the menin gene, characteristic of many carcinoids, are absent in high-grade neuroendocrine tumors including SCLC (9).

Whereas most lung cancers are smoking related, there are major somatically acquired genetic differences between SCLC and NSCLC (5, 10-14) and, to a lesser extent, between squamous cell carcinomas and adenocarcinomas (5, 15, 16). Aberrant methylation of the promoter region of tumor suppressor genes and the resultant gene silencing play an important role in the pathogenesis of most if not all types of human cancer (17, 18). Individual tumor types frequently have their characteristic pattern of acquired aberrant methylation (18, 19). We and others have identified several genes methylated frequently in NSCLC (19-25). Whereas several of these reports also describe gene promoter methylation in SCLC cell lines, there is very little published information about SCLC tumors (26) or the subtypes of NSCLC, and there is no published information about bronchial carcinoids. In this study, using eight genes previously studied for DNA methylation in lung cancers, we compare and contrast the aberrant methylation profiles of the neuroendocrine tumors SCLC and carcinoid and the squamous cell carcinomas and adenocarcinoma types of NSCLC. In addition, because tumor cell lines are frequently used as models to study DNA methylation, we compared the patterns of SCLC and NSCLC tumors with those of their respective cell lines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sample Collection. A total of 198 tumors consisting of 43 SCLCs (mean age, 64.4 years; age range, 43–78 years; male:female ratio = 4.0), 115 NSCLCs (77 adenocarcinomas and 38 squamous cell carcinomas; mean age, 60.3 years; age range, 32–87 years; male:female ratio = 2.0), and 40 bronchial carcinoids (33 typical and 7 atypical carcinoids; mean age, 54.2 years; age range, 31–73 years; male:female ratio = 1.4) were obtained from multiple centers. All NSCLC cases were from the United States, most carcinoid cases were from France, and most SCLC tumors were from Japan. With the exceptions noted later, the tumors were from previously untreated cases receiving curative intent surgical resections. Eleven of the SCLC tumors were recurrences occurring after prior chemotherapy. We also studied 20 SCLC and 24 NSCLC cell lines initiated from United States cases in the laboratory of A. F. G. and J. D. M. (27).

DNA Extraction. Genomic DNA was isolated from frozen tissues or cell pellets by homogenization, SDS/proteinase K (Life Technologies, Inc., Rockville, MD) digestion, phenol-chloroform extraction, and ethanol precipitation. (28).

MSP. Aberrant methylation of p16, APC, CDH13, GSTP1, MGMT, RARß, CDH1, and RASSF1A was determined by MSP using primers specific for the methylated and unmethylated alleles of each gene after treatment of the DNA with sodium bisulfate (22-24, 29-33). Briefly, 1 µg of genomic DNA was denatured by NaOH and modified by sodium bisulfite, which converts all unmethylated cytosine to uracil, whereas methylated cytosines remain unchanged. The modified DNA was purified using the Wizard DNA purification kit (Promega, Madison, WI), treated again with NaOH to desulfonate it, precipitated with ethanol, and resuspended in water. PCR amplification was done with treated DNA as template as described previously using specific primer sequences for the methylated and unmethylated forms of each gene. DNA from peripheral blood lymphocytes and scraped buccal mucosa (from 10 healthy subjects each) was used as negative control for the methylated genes along with water blanks. Aberrant methylation of nonmalignant lung tissue was, at most, 14%, as reported previously (20). DNA from lymphocytes of healthy volunteers treated with Sss1 methyltransferase (New England BioLabs) and then subjected to bisulfite treatment was used as a positive control for methylated alleles. PCR products were visualized on 2% agarose gels stained with ethidium bromide.

Cluster Analysis. Clustering analysis was done as described previously (13) using a Visual Basic program (available on request).

Data Analysis. Frequencies of methylation of two groups were compared using Fisher exact tests. The MI, a reflection of the methylation status of all of the genes tested, is defined as the total number of genes methylated divided by the total number of genes analyzed. The MIs for each case were determined, and the median MI for each tumor group was calculated. The MIs of different groups were compared using the Mann-Whitney nonparametric U test. For all tests, probability values of P < 0.05 were regarded as statistically significant. All statistical tests were two-sided.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Methylation Profiles of Lung Tumor Types. The methylation profiles of the different lung cancer groups were determined by testing the methylation status of the eight genes. Examples of the bands obtained by MSP are illustrated in Fig. 1. Fig. 2 shows complete data for 242 tumor and tumor cell line DNAs sorted using a clustering algorithm, and the frequency of aberrant methylation is summarized in Fig. 3. The mean MIs are tabulated in Table 1. Tumor tissues consist of mixtures of tumor and nonmalignant cells, and the unmethylated forms of all genes were present in all tumor samples. The presence of the unmethylated bands confirmed the integrity of DNA in all tumor samples. Whereas the unmethylated bands could come from the tumor, noninvolved tissue, or both, in our prior studies of tumor cell lines, we usually found only methylated or unmethylated alleles (21-24). We first compared the methylation profile of neuroendocrine tumors (SCLCs and carcinoids) with that of NSCLC tumors (squamous cell carcinomas and adenocarcinomas; Fig. 3A). There was no significant difference in the mean MI values of the two groups. The frequency of methylation of RASSF1A was significantly higher in the neuroendocrine tumors than in NSCLC tumors (P < 0.0001). By contrast, the frequencies of methylation of p16, APC, and CDH13 were significantly higher in NSCLC than in the neuroendocrine tumors (P < 0.0001 for all three genes). The methylation profiles of untreated SCLC tumors were similar to those of tumors that had received prior cytotoxic therapy; the exception was RARß, which was higher in the group that had received prior cytotoxic therapy (7 of 11 cases; 64%) than in the nontreated group (13 of 32 cases; 40%; P = 0.18).

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Representative examples of the MSP assay. Examples from one tumor sample each of SCLC, adenocarcinoma, squamous cell carcinoma, and bronchial carcinoid are illustrated. Results of testing for the methylated forms of p16, CDH13, CDH1, RARß, and RASSF1A are illustrated. The unmethylated form of p16 was run as a control for DNA integrity. U, unmethylated form; M, methylated form; Lane P, positive control; Lane N, negative control (water blank).

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Clustering analysis of DNA methylation profiles for 242 thoracic malignancies and tumor cell lines. Identical methylation patterns are clustered and separated by horizontal lines. Generally, neighboring clusters have similar methylation patterns. Sky blue, methylated; yellow, unmethylated. SCLC, red; SCLC cell lines, pink; carcinoid, cherry; adenocarcinoma, dark blue; squamous cell carcinoma, orange; NSCLC cell lines, purple. The right panel is a continuation of the left panel.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Comparison of aberrant methylation profiles of lung cancers (A, comparison between NSCLC and neuroendocrine tumors; B, comparison between SCLC and carcinoids; C, comparison between squamous cell carcinomas and adenocarcinomas). n = sample number examined. P is shown when there is a significant difference between two groups.

We compared the methylation profiles of the two major forms of NSCLC, adenocarcinoma and squamous cell carcinoma (Fig. 3C). Aberrant methylation of p16 was more frequent in squamous cell carcinoma than in adenocarcinoma (P = 0.02), whereas methylation frequencies of APC and CDH13 were greater in adenocarcinoma (P < 0.0001 for both genes).

We also compared the methylation profiles of NSCLC and SCLC tumors with those of tumor cell lines derived from these subtypes (Fig. 4). The overall patterns of tumors and their respective cell lines were similar. There was no significant difference between the mean MIs of tumors and their respective cell lines. Whereas the frequencies of methylation of several genes were higher in the tumor cell lines, these differences were significant in only two cases (MGMT in SCLC and RASSF1A in NSCLC).

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Comparison of the pattern of aberrant methylation profiles of SCLC (A) and NSCLC (B) tumors and lung tumor cell lines. P is shown when there is a significant difference between two groups.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our results demonstrate important differences in the methylation profiles of the major types of lung tumors. In a comparison of neuroendocrine and NSCLC tumors, the frequency of RASSF1A methylation was significantly higher in the neuroendocrine tumors, and the frequencies of p16, APC, and CDH13 methylation were significantly higher in NSCLC tumors. These findings are consistent with our previous studies using SCLC cell lines (21, 23, 24). They also confirm the findings of Dammann et al. (26) and Burbee et al. (24), who both found a high frequency of RASSF1A methylation in a few uncultured SCLC tumors. Cell cycle perturbations in lung tumors usually involve abnormalities of the p16 gene in NSCLC and the RB gene in SCLC (11). The p16 gene can be inactivated by multiple mechanisms in NSCLC, including methylation (34), but inactivation of p16 in SCLC is rare (11, 35). Our findings of frequent p16 methylation in NSCLC tumors combined with infrequent methylation in neuroendocrine tumors are consistent with these reports.

SCLC and bronchial carcinoids share expression of neuroendocrine features but have different pathogenetic mechanisms and clinical courses. Carcinoids are not associated with smoking, have a limited metastatic potential, and are chemoresistant. However, the patterns of methylation of these two very different neuroendocrine tumors of the lung were more similar to each other than to NSCLCs, although the frequencies of methylation of RARß, CDH1, and RASSF1A were higher in SCLC. Although conclusions must be tempered by the small number of samples available for study, there were no significant differences between previously treated and untreated SCLC tumors. In addition, whereas atypical carcinoids had a higher frequency of methylation of RASSF1A, similar to that of SCLC, there were no significant differences in the methylation frequencies of any individual gene or of the mean MIs between typical and atypical carcinoids. In a comparison of the methylation profiles of squamous cell carcinomas and adenocarcinomas, p16 was methylated more frequently in squamous cell carcinomas, as has been reported by others (36), whereas APC and CDH13 methylation was more frequent in adenocarcinomas. With rare exceptions, the profiles of NSCLC and SCLC tumors and their respective cell lines for the individual genes and the mean MIs were not significantly different. These findings suggest that cell lines are useful models for studying methylation. Of interest, the pattern of aberrant methylation in bladder cell lines is similar to that in the tumors from which they originated (37).

SCLC tumors are seldom resected in the United States, and carcinoids are relatively rare tumors. For these reasons, the methylation profiles of uncultured specimens from these tumors have not been determined. To obtain enough tumors for our studies, we had to obtain SCLC samples from Japan, where resection of localized SCLC tumors is more common than in the United States and Europe. In addition, the numbers were supplemented with tumors that had recurred after combination chemotherapy. There are no squamous cells in the normal bronchus, and squamous cell carcinomas are believed to arise from metaplastic cells frequently present in the bronchi of smokers (38). By contrast, most adenocarcinomas are peripherally located and may express features of Clara cells or type II pneumocytes. In addition, adenocarcinomas are the most frequent histological type of lung cancer arising in women and never smokers (39, 40). These findings demonstrate the very different origins of the two major forms of NSCLC. Whereas both squamous cell carcinomas and SCLCs are strongly related to smoking and usually arise from the bronchial tree, the molecular changes present in the bronchial epithelium of SCLC patients are much more extensive than those in the mucosa adjacent to squamous cell carcinomas (16). Thus, we have suggested that SCLC may arise directly from bronchial mucosa that demonstrates extensive molecular changes but few histological changes, whereas squamous cell carcinomas arise after a defined sequence of molecular and histological changes (16).

Our findings are consistent with these differences in pathogenesis, and clustering analysis further confirmed these results by showing tumors of the same histological type to have similar methylation patterns. Together, they demonstrate important differences in the methylation profile of the four types of lung cancer studied, as well as similarities within each subtype, and indicate that lung cancer cell lines are appropriate models for studying methylation.

Understanding the methylation profiles of tumors may impact on several clinical parameters. Methylation of some genes, such as Deach-associated protein kinase in lung, APC in breast tumors, and several genes as well as the mean MI in bladder cancer, correlates with survival or other parameters of poor prognosis cancers (23, 41).⁴ Treatment with demethylating agents results in temporary restoration of gene expression and may be a potential method of tumor therapy or chemoprevention (42, 43). We have suggested that loss of expression of the RARß gene by methylation in smoking-damaged epithelium may be the cause of failure of chemoprevention by its ligands (44). Thus, our findings may help stratify lung cancer patients into clinically important subsets and lead to improved chemoprevention strategies.

	Acknowledgments

 
We thank Dr. Keizo Sugimachi (Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan) for providing some of the SCLC tumor samples.

	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 grants from the Early Detection Research Network (5U01CA8497102) and the University of Texas Specialized Program of Research Excellence in Lung Cancer (P50CA70907), National Cancer Institute (Bethesda, MD).

² To whom requests for reprints should be addressed, at Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-8563. Phone: (214) 648-4921; Fax: (214) 648-4940; E-mail: adi.gazdar{at}utsouthwestern.edu

³ The abbreviations used are: SCLC, small cell lung cancer, NSCLC, non-small cell lung cancer; MSP, methylation-specific PCR; MI, methylation index.

⁴ R. Maruyama, S. Toyooka, K. O. Toyooka, K. Harada, A. K. Virmani, S. Zochbauer-Muller, A. Farines, E. Vakar-Lopez, A. Sagalowsky, B. Czerniak, and A. F. Gazdar. Aberrant promoter methylation profile of bladder cancer and its relationship to clinico-pathologic feature, submitted for publication.

Received 8/10/01; revised 8/29/01; accepted 9/ 7/01.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Greenlee, R. T., Hill-Harmon, M. B., Murray, T., and Thun, M. Cancer statistics, 2001. CA Cancer J. Clin 51:15 –36,2001 .[Abstract/Free Full Text]
2. Parkin, D. M., Pisani, P., and Ferlay, J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int. J. Cancer 80:827 –841,1999 .[CrossRef][Medline]
3. Chute, J. P., Kelley, M. J., Venzon, D., Williams, J., Roberts, A., and Johnson, B. E. Retreatment of patients surviving cancer-free 2 or more years after initial treatment of small cell lung cancer. Chest 110:165 –171,1996 .[Abstract/Free Full Text]
4. Wingo, P. A., Ries, L. A., Parker, S. L., and Heath, C. W., Jr. Long-term cancer patient survival in the United States, Cancer Epidemiol. Biomark. Prev 7:271 –282,1998 .[Abstract]
5. Sekido, Y., Fong, K. M., and Minna, J. D. Progress in understanding the molecular pathogenesis of human lung cancer. Biochim. Biophys. Acta 1378:F21 –F59,1998 .[Medline]
6. Travis, W. D., Rush, W., Flieder, D. B., Falk, R., Fleming, M. V., Gal, A. A., and Koss, M. N. Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid. Am. J. Surg. Pathol 22:934 –944,1998 .[CrossRef][Medline]
7. Onuki, N., Wistuba, I. I., Travis, W. D., Virmani, A. K., Yashima, K., Brambilla, E., Hasleton, P., and Gazdar, A. F. Genetic changes in the spectrum of neuroendocrine lung tumors. Cancer (Phila.) 85:600 –607,1999 .[CrossRef][Medline]
8. Kovatich, A., Friedland, D. M., Druck, T., Hadaczek, P., Huebner, K., Comis, R. L., Hauck, W., and McCue, P. A. Molecular alterations to human chromosome 3p loci in neuroendocrine lung tumors. Cancer (Phila.) 83:1109 –1117,1998 .[CrossRef][Medline]
9. Haruki, N., Yatabe, Y., Travis, W. D., Nomoto, S., Osada, H., Nakamura, S., Nakao, A., Fujii, Y., and Takahashi, T. Characterization of high-grade neuroendocrine tumors of the lung in relation to menin mutations. Jpn. J. Cancer Res 91:317 –323,2000 .[Medline]
10. Yuan, J., Knorr, J., Altmannsberger, M., Goeckenjan, G., Ahr, A., Scharl, A., and Strebhardt, K. Expression of p16 and lack of pRB in primary small cell lung cancer. J. Pathol 189:358 –362,1999 .[CrossRef][Medline]
11. Otterson, G. A., Kratzke, R. A., Coxon, A., Kim, Y. W., and Kaye, F. J. Absence of p16^INK4 protein is restricted to the subset of lung cancer lines that retains wildtype RB. Oncogene 9:3375 –3378,1994 .[Medline]
12. Williams, C. L. Basic science of small cell lung cancer. Chest Surg. Clin. N. Am 7:1 –19,1997 .[Medline]
13. Girard, L., Zochbauer-Muller, S., Virmani, A. K., Gazdar, A. F., and Minna, J. D. Genome-wide allelotyping of lung cancer identifies new regions of allelic loss, differences between small cell lung cancer and non-small cell lung cancer, and loci clustering. Cancer Res 60:4894 –4906,2000 .[Abstract/Free Full Text]
14. Virmani, A. K., Fong, K. M., Kodagoda, D., McIntire, D., Hung, J., Tonk, V., Minna, J. D., and Gazdar, A. F. Allelotyping demonstrates common and distinct patterns of chromosomal loss in human lung cancer types. Genes Chromosomes Cancer, 21:308 –319,1998 .
15. Slebos, R. J., and Rodenhuis, S. The ras gene family in human non-small-cell lung cancer. Monogr. Natl. Cancer Inst 13:23 –29,1992 .
16. Wistuba, I. I., Barry, J., Behrens, C., Maitra, A., Shivapurkar, N., Milchgrub, S., Mackay, B., Minna, J. D., and Gazdar, A. F. Molecular changes in the bronchial epithelium of patients with small cell lung cancer. Clin. Cancer Res 6:2604 –2610,2000 .[Abstract/Free Full Text]
17. Baylin, S. B., Herman, J. G., Graff, J. R., Vertino, P. M., and Issa, J. P. Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res 72:141 –196,1998 .[Medline]
18. Costello, J. F., Fruhwald, M. C., Smiraglia, D. J., Rush, L. J., Robertson, G. P., Gao, X., Wright, F. A., Feramisco, J. D., Peltomaki, P., Lang, J. C., Schuller, D. E., Yu, L., Bloomfield, C. D., Caligiuri, M. A., Yates, A., Nishikawa, R., Su Huang, H., Petrelli, N. J., Zhang, X., O’Dorisio, M. S., Held, W. A., Cavenee, W. K., and Plass, C. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat. Genet 24:132 –138,2000 .[CrossRef][Medline]
19. Esteller, M., Corn, P. G., Baylin, S. B., and Herman, J. G. A gene hypermethylation profile of human cancer. Cancer Res 61:3225 –3229,2001 .[Abstract/Free Full Text]
20. Zochbauer-Muller, S., Fong, K. M., Virmani, A. K., Geradts, J., Gazdar, A. F., and Minna, J. D. Aberrant promoter methylation of multiple genes in non-small cell lung cancers. Cancer Res 61:249 –255,2001 .[Abstract/Free Full Text]
21. Virmani, A. K., Rathi, A., Zochbauer-Muller, S., Sacchi, N., Fukuyama, Y., Bryant, D., Maitra, A., Heda, S., Fong, K. M., Thunnissen, F., Minna, J. D., and Gazdar, A. F. Promoter methylation and silencing of the retinoic acid receptor-ß gene in lung carcinomas. J. Natl. Cancer Inst. (Bethesda) 92:1303 –1307,2000 .[Abstract/Free Full Text]
22. Toyooka, K. O., Toyooka, S., Virmani, A. K., Sathyanarayana, U. G., Euhus, D. M., Gilcrease, M., Minna, J. D., and Gazdar, A. F. Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas. Cancer Res 61:4556 –4560,2001 .[Abstract/Free Full Text]
23. Virmani, A. K., Rathi, A., Sathyanarayana, U. G., Padar, A., Huang, C. X., Cunnigham, H. T., Farinas, A. J., Milchgrub, S., Euhus, D. M., Gilcrease, M., Herman, J., Minna, J. D., and Gazdar, A. F. Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas. Clin. Cancer Res 7:1998 –2004,2001 .[Abstract/Free Full Text]
24. Burbee, D. G., Forgacs, E., Zochbauer-Muller, S., Shivakumar, L., Fong, K., Gao, B., Randle, D., Kondo, M., Virmani, A., Bader, S., Sekido, Y., Latif, F., Milchgrub, S., Toyooka, S., Gazdar, A. F., Lerman, M. I., Zabarovsky, E., White, M., and Minna, J. D. Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J. Natl. Cancer Inst. (Bethesda) 93:691 –699,2001 .[Abstract/Free Full Text]
25. Toyooka, S., Pass, H. I., Shivapurkar, N., Fukuyama, Y., Maruyama, R., Toyooka, K. O., Gilcrease, M., Farinas, A., Minna, J. D., and Gazdar, A. F. Aberrant methylation and SV40 Tag sequences in malignant mesothelioma. Cancer Res 61:5727 –5730,2001 .[Abstract/Free Full Text]
26. Dammann, R., Takahashi, T., and Pfeifer, G. P. The CpG island of the novel tumor suppressor gene RASSF1A is intensely methylated in primary small cell lung carcinomas. Oncogene 20:3563 –3567,2001 .[CrossRef][Medline]
27. Phelps, R. M., Johnson, B. E., Ihde, D. C., Gazdar, A. F., Carbone, D. P., McClintock, P. R., Linnoila, R. I., Matthews, M. J., Bunn, P. A., Jr., Carney, D., Minna, J. D., and Mulshine, J. L. NCI-Navy Medical Oncology Branch cell line data base. J. Cell Biochem. Suppl 24:32 –91,1996 .[Medline]
28. Herrmann, B. G., and Frischauf, A. M. Isolation of genomic DNA. Methods Enzymol 152:180 –183,1987 .[Medline]
29. Cote, S., Sinnett, D., and Momparler, R. L. Demethylation by 5-aza-2'-deoxycytidine of specific 5-methylcytosine sites in the promoter region of the retinoic acid receptor ß gene in human colon carcinoma cells. Anticancer Drugs 9:743 –750,1998 .[CrossRef][Medline]
30. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93:9821 –9826,1996 .[Abstract/Free Full Text]
31. Esteller, M., Corn, P. G., Urena, J. M., Gabrielson, E., Baylin, S. B., and Herman, J. G. Inactivation of glutathione S-transferase P1 gene by promoter hypermethylation in human neoplasia. Cancer Res 58:4515 –4518,1998 .[Abstract/Free Full Text]
32. Graff, J. R., Herman, J. G., Myohanen, S., Baylin, S. B., and Vertino, P. M. Mapping patterns of CpG island methylation in normal and neoplastic cells implicates both upstream and downstream regions in de novo methylation. J. Biol. Chem 272:22322 –22329,1997 .[Abstract/Free Full Text]
33. Esteller, M., Hamilton, S. R., Burger, P. C., Baylin, S. B., and Herman, J. G. Inactivation of the DNA repair gene O⁶-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 59:793 –797,1999 .[Abstract/Free Full Text]
34. Gazzeri, S., Gouyer, V., Vour’ch, C., Brambilla, C., and Brambilla, E. Mechanisms of p16^INK4A inactivation in non-small-cell lung cancers. Oncogene 16:497 –504,1998 .[CrossRef][Medline]
35. Kelley, M. J., Nakagawa, K., Steinberg, S. M., Mulshine, J. L., Kamb, A., and Johnson, B. E. Differential inactivation of CDKN2 and Rb protein in non-small-cell and small-cell lung cancer cell lines. J. Natl. Cancer Inst. (Bethesda) 87:756 –761,1995 .[Abstract/Free Full Text]
36. Kim, D. H., Nelson, H. H., Wiencke, J. K., Zheng, S., Christiani, D. C., Wain, J. C., Mark, E. J., and Kelsey, K. T. 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]
37. Markl, I. D., Cheng, J., Liang, G., Shibata, D., Laird, P. W., and Jones, P. A. Global and gene-specific epigenetic patterns in human bladder cancer genomes are relatively stable in vivo and in vitro over time. Cancer Res 61:5875 –5884,2001 .[Abstract/Free Full Text]
38. Auerbach, O., Stout, A. P., Hammond, E. C., and Garfinkel, L. Changes in bronchial epithelium in relation to smoking and cancer of the lung. N. Engl. J. Med 265:253 –267,1961 .
39. Gazdar, A. F., and Minna, J. D. Cigarettes, sex, and lung adenocarcinoma. J. Natl. Cancer Inst. (Bethesda) 89:1563 –1565,1997 .[Free Full Text]
40. Travis, W. D., Travis, L. B., and Devesa, S. S. Lung cancer. Cancer (Phila.) 75:191 –202,1995 .[CrossRef][Medline]
41. Tang, X., Khuri, F. R., Lee, J. J., Kemp, B. L., Liu, D., Hong, W. K., and Mao, L. Hypermethylation of the death-associated protein (DAP) kinase promoter and aggressiveness in stage I non-small-cell lung cancer. J. Natl. Cancer Inst. (Bethesda) 92:1511 –1516,2000 .[Abstract/Free Full Text]
42. Schwartsmann, G., Schunemann, H., Gorini, C. N., Filho, A. F., Garbino, C., Sabini, G., Muse, I., DiLeone, L., and Mans, D. R. A Phase I trial of cisplatin plus decitabine, a new DNA-hypomethylating agent, in patients with advanced solid tumors and a follow-up early Phase II evaluation in patients with inoperable non-small cell lung cancer. Invest New Drugs 18:83 –91,2000 .[CrossRef][Medline]
43. Momparler, R. L., Eliopoulos, N., and Ayoub, J. Evaluation of an inhibitor of DNA methylation, 5-aza-2'-deoxycytidine, for the treatment of lung cancer and the future role of gene therapy. Adv. Exp. Med. Biol 465:433 –446,2000 .[Medline]
44. Gazdar, A. F., Zochbauer-Moller, S., Virmani, A., Kurie, J., Minna, J. D., and Lam, S. Response re: promoter methylation and silencing of the retinoic acid receptor-ß gene in lung carcinomas. J. Natl. Cancer Inst. (Bethesda) 93:67 –68,2001 .[Free Full Text]

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]

				M. V. Brock, C. M. Hooker, E. Ota-Machida, Y. Han, M. Guo, S. Ames, S. Glockner, S. Piantadosi, E. Gabrielson, G. Pridham, et al. DNA Methylation Markers and Early Recurrence in Stage I Lung Cancer N. Engl. J. Med., March 13, 2008; 358(11): 1118 - 1128. [Abstract] [Full Text] [PDF]

				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]

				S. Toyooka, M. Tokumo, H. Shigematsu, K. Matsuo, H. Asano, K. Tomii, S. Ichihara, M. Suzuki, M. Aoe, H. Date, et al. Mutational and Epigenetic Evidence for Independent Pathways for Lung Adenocarcinomas Arising in Smokers and Never Smokers Cancer Res., February 1, 2006; 66(3): 1371 - 1375. [Abstract] [Full Text] [PDF]

				A. M. Safar, H. Spencer III, X. Su, M. Coffey, C. A. Cooney, L. D. Ratnasinghe, L. F. Hutchins, and C.-Y. Fan Methylation Profiling of Archived Non-Small Cell Lung Cancer: A Promising Prognostic System Clin. Cancer Res., June 15, 2005; 11(12): 4400 - 4405. [Abstract] [Full Text] [PDF]

				A. Agathanggelou, W. N. Cooper, and F. Latif Role of the Ras-Association Domain Family 1 Tumor Suppressor Gene in Human Cancers Cancer Res., May 1, 2005; 65(9): 3497 - 3508. [Abstract] [Full Text] [PDF]

				C. M. Lewis, L. R. Cler, D.-W. Bu, S. Zochbauer-Muller, S. Milchgrub, E. Z. Naftalis, A. M. Leitch, J. D. Minna, and D. M. Euhus Promoter Hypermethylation in Benign Breast Epithelium in Relation to Predicted Breast Cancer Risk Clin. Cancer Res., January 1, 2005; 11(1): 166 - 172. [Abstract] [Full Text] [PDF]

				J. Wang, J. J. Lee, L. Wang, D. D. Liu, C. Lu, Y.-H. Fan, W. K. Hong, and L. Mao Value of p16INK4a and RASSF1A Promoter Hypermethylation in Prognosis of Patients with Resectable Non-Small Cell Lung Cancer Clin. Cancer Res., September 15, 2004; 10(18): 6119 - 6125. [Abstract] [Full Text] [PDF]

				I. Krop, A. Player, A. Tablante, M. Taylor-Parker, J. Lahti-Domenici, J. Fukuoka, S. K. Batra, N. Papadopoulos, W. G. Richards, D. J. Sugarbaker, et al. Frequent HIN-1 Promoter Methylation and Lack of Expression in Multiple Human Tumor Types Mol. Cancer Res., September 1, 2004; 2(9): 489 - 494. [Abstract] [Full Text] [PDF]

				J. Mazieres, B. He, L. You, Z. Xu, A. Y. Lee, I. Mikami, N. Reguart, R. Rosell, F. McCormick, and D. M. Jablons Wnt Inhibitory Factor-1 Is Silenced by Promoter Hypermethylation in Human Lung Cancer Cancer Res., July 15, 2004; 64(14): 4717 - 4720. [Abstract] [Full Text] [PDF]

				M. Suzuki, N. Sunaga, D. S. Shames, S. Toyooka, A. F. Gazdar, and J. D. Minna RNA Interference-Mediated Knockdown of DNA Methyltransferase 1 Leads to Promoter Demethylation and Gene Re-Expression in Human Lung and Breast Cancer Cells Cancer Res., May 1, 2004; 64(9): 3137 - 3143. [Abstract] [Full Text] [PDF]

				O. Topaloglu, M. O. Hoque, Y. Tokumaru, J. Lee, E. Ratovitski, D. Sidransky, and C.-s. Moon Detection of Promoter Hypermethylation of Multiple Genes in the Tumor and Bronchoalveolar Lavage of Patients with Lung Cancer Clin. Cancer Res., April 1, 2004; 10(7): 2284 - 2288. [Abstract] [Full Text] [PDF]

				S. Yegnasubramanian, J. Kowalski, M. L. Gonzalgo, M. Zahurak, S. Piantadosi, P. C. Walsh, G. S. Bova, A. M. De Marzo, W. B. Isaacs, and W. G. Nelson Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer Cancer Res., March 15, 2004; 64(6): 1975 - 1986. [Abstract] [Full Text] [PDF]

				H. Dote, S. Toyooka, K. Tsukuda, M. Yano, M. Ouchida, H. Doihara, M. Suzuki, H. Chen, J.-T. Hsieh, A. F. Gazdar, et al. Aberrant Promoter Methylation in Human DAB2 Interactive Protein (hDAB2IP) Gene in Breast Cancer Clin. Cancer Res., March 15, 2004; 10(6): 2082 - 2089. [Abstract] [Full Text] [PDF]

				S. Toyooka, K. O. Toyooka, K. Miyajima, J. L. Reddy, M. Toyota, U. G. Sathyanarayana, A. Padar, M. S. Tockman, S. Lam, N. Shivapurkar, et al. Epigenetic Down-Regulation of Death-associated Protein Kinase in Lung Cancers Clin. Cancer Res., August 1, 2003; 9(8): 3034 - 3041. [Abstract] [Full Text] [PDF]

				U. G. Sathyanarayana, S. Toyooka, A. Padar, T. Takahashi, E. Brambilla, J. D. Minna, and A. F. Gazdar Epigenetic Inactivation of Laminin-5-encoding Genes in Lung Cancers Clin. Cancer Res., July 1, 2003; 9(7): 2665 - 2672. [Abstract] [Full Text] [PDF]

				S. Toyooka, Y. Fukuyama, I. I. Wistuba, M. S. Tockman, J. D. Minna, and A. F. Gazdar Differential Expression of FEZ1/LZTS1 Gene in Lung Cancers and Their Cell Cultures Clin. Cancer Res., July 1, 2002; 8(7): 2292 - 2297. [Abstract] [Full Text] [PDF]

				S. Toyooka, K. O. Toyooka, K. Harada, K. Miyajima, P. Makarla, U. G. Sathyanarayana, J. Yin, F. Sato, N. Shivapurkar, S. J. Meltzer, et al. Aberrant Methylation of the CDH13 (H-cadherin) Promoter Region in Colorectal Cancers and Adenomas Cancer Res., June 1, 2002; 62(12): 3382 - 3386. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Toyooka, S. Articles by Gazdar, A. F. Search for Related Content PubMed PubMed Citation Articles by Toyooka, S. Articles by Gazdar, A. F.