An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor ␤-2 (RAR␤-2), CDH13 (H-cadherin), p16 INK4a (p16), RASSF1A (RAS association domain family I) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. Key words: aberrant methylation; methylation-specific PCR; cigarette smoking; preneoplastic lesion; lung cancerSquamous cell and small cell carcinomas of the lung are strongly associated with smoking and most arise from large and medium-sized bronchi (central tumors). Adenocarcinomas of the lung are less strongly associated with smoking, are more frequent in women and most arise peripherally from the smaller airways (bronchioles and alveoli). 1,2 In addition, there are important differences in the molecular changes present in the major forms of lung cancer. 3,4 These findings suggest that the pathogenesis of central and peripheral lung tumors may differ. Because smoking damages the entire upper aerodigestive tract, 5,6 it has been proposed that changes in the epithelium of surrogate organs such as the oropharynx may reflect molecular damage occurring in the target organ for lung cancer (i.e., the epithelium of the central and peripheral airways). 7 Aberrant methylation (referred to as methylation) of normally unmethylated CpG islands in gene promoter regions is an important method for silencing tumor suppressor genes (TSGs). 8,9 Methylation results in transcriptional inactivation of several TSGs in human cancer and serves as an alternative for the genetic loss of gene function by deletion or mutation. 8 -10 Methylation of the genes retinoic acid receptor ␤-2 (RAR␤-2), CDH13 (H-cadherin), p16 INK4a (p16), RAS association domain family I (RASSF1A) and FHIT occurs frequently in primary nonsmall cell lung cancers (NSCLC). [11][12][13][14][15][16][17][18] In addition, methylation of RAR␤-2 and RASSF1A has frequently been found in small cell lung cancers (SCLC) and their cell lines. 11,16,18,19 Moreover, Esteller et al. 20 demonstrated that methylation can also be detected in serum DNA samples from lung cancer patients. p16 methylation commences during the multistage pathogenesis of lung cancers, 21 and its deletion in sputum may predict the later onset of invasive cancer. 22 So ...

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TSLC1 (tumor suppressor in lung cancer-1, IGSF4) encodes a member of the immunoglobulin superfamily molecules, which is involved in cell-cell adhesion. TSLC1 is connected to the actin cytoskeleton by DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3) and it directly associates with MPP3, one of the human homologues of a Drosophila tumor suppressor gene, Discs large. Recent data suggest that aberrant promoter methylation is important for TSLC1 inactivation in lung carcinomas. However, little is known about the other two genes in this cascade, DAL-1 and MPP3. Thus, we investigated the expression and methylation patterns of these genes in lung cancer cell lines, primary lung carcinomas and nonmalignant lung tissue samples. By reverse transcription-polymerase chain reaction, loss of TSLC1 expression was observed in seven of 16 (44%) non-small-cell lung cancer (NSCLC) cell lines and in one of 11 (9%) small-cell lung cancer (SCLC) cell lines, while loss of DAL-1 expression was seen in 14 of 16 (87%) NSCLC cell lines and in four of 11 (36%) SCLC cell lines. By contrast, MPP3 expression was found in all tumor cell lines analysed. Similar results were obtained by microarray analysis. TSLC1 methylation was seen in 13 of 39 (33%) NSCLC cell lines, in one of 11 (9%) SCLC cell lines and in 100 of 268 (37%) primary NSCLCs. DAL-1 methylation was observed in 17 of 39 (44%) NSCLC cell lines, in three of 11 (27%) SCLC cell lines and in 147 of 268 (55%) primary NSCLCs. In tumors of NSCLC patients with stage II-III disease, DAL-1 methylation was seen at a statistically significant higher frequency compared to tumors of patients with stage I disease. A significant correlation between loss of expression and methylation of the genes in lung cancer cell lines was found. Overall, 65% of primary NSCLCs had either TSLC1 or DAL-1 methylated. Methylation of one of these genes was detected in 59% of NSCLC cell lines; however, in SCLC cell lines, methylation was much less frequently observed. The majority of nonmalignant lung tissue samples was not TSLC1 or DAL-1 methylated. Re-expression of TSLC1 and DAL-1 was seen after treatment of lung cancer cell lines with 5-aza-2 0 -deoxycytidine. Our results suggest that methylation of TSLC1 and/or DAL-1, leading to loss of their expression, is an important event in the pathogenesis of NSCLC. Oncogene (2006) 25, 959-968.

BACKGROUND. Silencing of tumor suppressor genes (TSG) by aberrant methylation (referred to as methylation) contributes to the pathogenesis of various human malignancies. However, little is known about the methylation of known and putative TSGs in monoclonal gammopathies. Thus, the authors investigated the methylation frequencies of 10 genes in patients with monoclonal gammopathies. METHODS. The methylation patterns of the genes p16 INK4a (p16), tissue inhibitor of metalloproteinase 3 (TIMP3), p15 INK4b (p15), E-cadherin (ECAD), death-associated protein kinase (DAPK), p73, RAS-association domain family 1A (RASSF1A), p14, O 6-methylguanine DNA methyltransferase (MGMT), and retinoid acid receptor ␤2 (RAR␤) were determined in patients with monoclonal gammopathy of undetermined significance (MGUS; n ϭ 29), smoldering multiple myeloma (SMM; n ϭ 5), multiple myeloma (MM; n ϭ 113), or plasma cell leukemia (PCL; n ϭ 7) by methylation-specific polymerase chain reaction analysis.

Recently, the human SRBC (hSRBC) gene, a candidate tumor suppressor gene (TSG), has been mapped to the chromosomal region 11p15.5-p15.4 where frequent allele loss has been described in lung cancer. Aberrant methylation (referred to as methylation) of the promoter region of TSGs has been identified as an important mechanism for gene silencing. Loss of hSRBC protein expression occurs frequently in lung cancer cell lines and sodium bisulfite sequencing of the promoter region of hSRBC in several lung cancer cell lines suggested that methylation plays an important role in inactivating hSRBC. To determine the methylation status of hSRBC in a large collection of primary lung cancer samples, corresponding nonmalignant lung tissues and lung cancer cell lines (N ¼ 52), we designed primers for a methylation-specific PCR assay. Methylation was detected in 41% of primary non-smallcell lung cancers (NSCLC) (N ¼ 107) and in 80% of primary small-cell lung cancers (SCLC) (N ¼ 5), but was seen only in 4% of corresponding nonmalignant lung tissues (N ¼ 103). In all, 79% of lung cancer cell lines were methylated and the frequency of hSRBC methylation was significantly higher in SCLC (100%) than in NSCLC (58%) cell lines. Normal hSRBC protein expression was detected in only 18% of primary NSCLCs (N ¼ 93) by immunostaining and a significant association between loss of protein expression and methylation was found. hSRBC re-expression was observed after treatment of lung cancer cells with the demethylating agent 5-aza-2 0 -deoxycytidine. In addition, 45% of the 76 hSRBC immunostainingnegative NSCLCs did not have hSRBC promoter methylation, indicating that other mechanisms of hSRBC expression silencing also exist. Both hSRBC immunostaining and methylation results did not correlate with clinicopathological characteristics of these patients. Our findings suggest that hSRBC is a candidate TSG involved in lung cancer pathogenesis, where expression is frequently inactivated by methylation and other mechanisms.