sodium-bisulfite and Lung-Neoplasms

The diagnosis of microscopic lymph node metastasis in lung cancer is challenging despite the constant advances in tumor staging. The analysis of the methylation status of certain genes in lymph node samples could improve the diagnostic capability of conventional cyto-histological methods. The aim of this study was to demonstrate the feasibility of methylation studies using cytological lymph node samples.. Prospective study including 88 patients with a diagnosis or strong suspicion of non-small cell lung cancer, in which an echobronchoscopy was performed on mediastinal or hilar lymph nodes for diagnostic and/or staging. DNA was extracted from cytological lymph node samples and sodium bisulfite modification was performed. Methylation studies for p16/INK4a and SHOX2 were accomplished by MS-qPCR and pyrosequencing.. The methodology used in our study yielded optimal/good DNA quality in 90% of the cases. No differences in DNA concentration were observed with respect to the lymph node biopsied and final diagnosis. Methylation analyses using MS-qPCR and pyrosequencing were not possible in a small number of samples mainly due to low DNA concentration, inadequate purity, fragmentation and/or degradation as a consequence of bisulfite conversion.. Methylation quantification using MS-qPCR and pyrosequencing of cytological lymph node samples obtained using echobronchoscopy is feasible if an appropriate DNA concentration is obtained, notably contributing to the identification of epigenetic biomarkers capable of improving decision-making for the benefit of potentially curable lung cancer patients.

Topics: Aged; Biopsy, Needle; Bronchoscopy; Carcinoma, Non-Small-Cell Lung; CpG Islands; DNA Methylation; DNA, Neoplasm; Endosonography; Feasibility Studies; Female; Genes, p16; Homeodomain Proteins; Humans; Lung Neoplasms; Lymph Nodes; Lymphatic Metastasis; Male; Mediastinum; Middle Aged; Neoplasm Proteins; Prospective Studies; Sequence Analysis, DNA; Sulfites; Ultrasonography, Interventional

Sulfur dioxide (SO(2)) is a major air pollutant suspected to act as a promoter or co-carcinogen. The present study was designed to investigate whether SO(2) derivatives (bisulfite and sulfite) had effects on the expression of several proto-oncogenes and tumor suppressor genes in cultured human bronchial epithelial (BEP2D) cells. The mRNA and protein levels were measured by real-time RT-PCR and western blotting, respectively, following exposure to differing SO(2)-derivative concentrations and exposure times. SO(2) derivatives caused mRNA and protein over-expression of c-fos, c-jun, and c-myc at all tested doses (0.001-2mM). Over-expression of H-ras and p53 were observed in cells receiving the highest concentration (0.1-2mM), as well as the under-expression of p16 and Rb. The over-expression of c-fos and c-jun was observed after 24h recovery. The expression of c-myc and H-ras decreased to base line levels while the p53 expression decreased compared with control after 24h recovery. The mRNA and protein expression of p16 and Rb remained at initial levels after 24h recovery. The data support the hypothesis that SO(2) derivatives could cause the activation of proto-oncogenes and inactivation of tumor suppressor genes and SO(2) derivatives may play a role in the pathogenesis of SO(2)-associated lung cancer.

Topics: Bronchi; Cell Line; Dose-Response Relationship, Drug; Epithelial Cells; Genes, p16; Genes, p53; Genes, ras; Genes, Tumor Suppressor; Humans; Lung Neoplasms; Oncogenes; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Proto-Oncogene Proteins c-myc; Retinoblastoma Protein; RNA, Messenger; Sulfites

Aberrant promoter methylation is a major mechanism for silencing tumor suppressor genes in cancer. Detection of hypermethylation is used as a molecular marker for early cancer diagnosis, as a prognostic index, or to define therapeutic targets for reversion of aberrant methylation. We report on a novel signal generation technology for real-time PCR to detect gene promoter methylation.. FLAG (fluorescent amplicon generation) is a homogeneous signal generation technology based on the exceptionally thermostable endonuclease PspGI. FLAG provides real-time signal generation during PCR by PspGI-mediated cleavage of quenched fluorophores at the 5' end of double-stranded PCR products. Methylation-specific PCR (MSP) applied on bisulfite-treated DNA was adapted to a real-time format (methylation-specific FLAG; MS-FLAG) for quantifying methylation in the promoter of CDKN2A (p16), GATA5, and RASSF1. We validated MS-FLAG on plasmids and genomic DNA with known methylation status and applied it to detection of methylation in a limited number of clinical samples. We also conducted bisulfite sequencing on these samples.. Real-time PCR results obtained via MS-FLAG agreed with results obtained via conventional, gel-based MSP. The new technology showed high specificity, sensitivity (2-3 plasmid copies), and selectivity (0.01% of methylated DNA) on control samples. It enabled correct prediction of the methylation status of all 3 gene promoters in 21 lung adenocarcinoma samples, as confirmed by bisulfite sequencing. We also developed a multiplex MS-FLAG assay for GATA5 and RASSF1 promoters.. MS-FLAG provides a new, quantitative, high-throughput method for detecting gene promoter methylation and is a convenient alternative to agarose gel-based MSP for screening methylation. In addition to methylation, FLAG-based real-time signal generation may have broad applications in DNA diagnostics.

Topics: Adenocarcinoma; CpG Islands; Cyclin-Dependent Kinase Inhibitor p16; DNA Methylation; Fluorescence; GATA5 Transcription Factor; Humans; Lung Neoplasms; Polymerase Chain Reaction; Promoter Regions, Genetic; Sensitivity and Specificity; Sulfites; Tumor Suppressor Proteins

It is known that chromium is one of the important inhaled carcinogens that cause lung cancer. Our previous studies revealed a variety of genetic changes in lung cancers from chromate-exposed workers (chromate lung cancer). However, the epigenetic effects of chromium are not understood.. We investigated the methylation of the p16 gene using a methylation-specific PCR method in 30 chromate lung cancers and 38 non-chromate lung cancers, and the expression of the p16 protein using immunohistochemistry in 25 chromate lung cancers.. Ten (33%) chromate lung cancers showed methylation of the p16 promoter region. On the other hand, 10 (26%) of the non-chromate lung cancers also showed it. The frequency of p16 methylation in non-chromate lung cancer was 0%, 33% and 30% for low (< or =600), moderate (<600, >1000) and high (> or =1000) Brinkman indexes, respectively. However, the frequency of p16 methylation in chromate lung cancer was constant, irrespective of the Brinkman index. In chromate lung cancer, patients with chromate exposure of less than 15 years never had p16 methylation, while 40% (> or =25 years) or 43% (> or =15, <25 years) of patients with chromate exposure of more than 15 years did. In chromate lung cancer, chromate exposure, not smoking, mainly influenced the p16 methylation. Most of the chromate lung cancers with p16 methylation (85.7%) showed repression of the p16 protein.. We speculate that not only genetic but also epigenetic alterations are involved in the carcinogenesis due to chromium.

Topics: Adult; Aged; Chromates; Chromium; Cyclin-Dependent Kinase Inhibitor p16; DNA; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Middle Aged; Occupational Exposure; Sulfites

Aberrant methylation of CpG islands is an important pathway for regulation of gene expression. Recent data suggest that epigenetic abnormalities may occur very early in lung carcinogenesis. We studied the promoters of the four genes, HOX A9, p16(INK4a) (p16), MAGE A1 and MAGE B2 by methylation-specific PCR in matched normal tissue, tumour, and cytological negative sputum samples obtained from 22 patients with non-small cell lung cancer (NSCLC). We further report methylation abnormalities in sputum samples from 56 smokers with differential cytology readouts (negative, inflammatory changes, suspicious, and cancer). Our method was successfully performed on formalin-fixed and paraffin-embedded (FFPE) samples, and was fit to study only few cells obtained by a convenient non-invasive sputum collection and handling. The promoters of MAGE A1 and MAGE B2 had abnormal methylation patterns in, respectively, 50% and 41% of the cytologically negative sputum samples from NSCLC patients, whereas methylation abnormalities of p16 was observed in 27% of negative sputum samples. Interestingly, 95.5% (21 of 22) of the cytologically negative sputum samples from NSCLC patients had abnormal methylation in at least one of the four genes indicating a high sensitivity of this marker system. Moreover, a higher frequency of methylation abnormalities was observed in sputum samples from smokers with high cytological grade compared to low cytological grade. We conclude that the identification of abnormal gene methylation of a limited number of markers in FFPE sputum samples is feasible and may be investigated as a potential system for population-based screening of early stages of lung cancer.

Topics: Aged; Carcinoma, Non-Small-Cell Lung; DNA Methylation; DNA Probes; Female; Humans; Lung Neoplasms; Male; Middle Aged; Mutagens; Paraffin Embedding; Promoter Regions, Genetic; Sensitivity and Specificity; Sputum; Sulfites

Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.

Topics: Adenocarcinoma; Animals; Azacitidine; CpG Islands; Cyclin-Dependent Kinase Inhibitor p16; Decitabine; DNA Methylation; Fibroblasts; Gene Silencing; Histiocytoma, Benign Fibrous; Lung Neoplasms; Neoplasms; Osteosarcoma; Promoter Regions, Genetic; Rats; RNA, Messenger; Sulfites; TATA Box; Tumor Cells, Cultured

Hypermethylation of the O(6)-methylguanine DNA methyltransferase (MGMT) promoter region leads to transcriptional repression of the MGMT gene and is a common event in primary human neoplasia. The purpose of this study was to determine the frequency and clinical relevance of MGMT gene promoter hypermethylation in curatively resected non-small cell lung cancer (NSCLC).. MGMT hypermethylation, expressed as the ratio between methylated MGMT to unmethylated MYOD1 in genomic DNA, was analyzed in normal and matching tumor tissue from 90 patients with NSCLC, and a control group of 10 patients without cancer using a methylation-specific fluorogenic Real-Time PCR (Taqman) system.. Hypermethylation of the MGMT promoter was detected in 34 of 90 (38%) tumor specimens and 16 of 90 (18%) matching normal lung tissues of patients with NSCLC, and in 0 (0%) cases of the control group without lung cancer. The mean MGMT methylation level was significantly higher in tumor than in matching normal tissue (P < 0.001). MGMT methylation in normal tissue was always accompanied with MGMT methylation in matching tumor tissue. Patients without MGMT promoter hypermethylation showed a significantly better survival than patients with MGMT promoter hypermethylation (P = 0.017). Multivariate analysis revealed MGMT promoter methylation as an independent unfavorable prognostic factor (P = 0.030).. MGMT promoter hypermethylation is a common event in patients with primary NSCLC. This epigenetic alteration is associated with inferior survival, suggesting that MGMT promoter hypermethylation might be an important biomarker for a biological aggressive disease in NSCLC.

Topics: Adult; Aged; Aged, 80 and over; Carcinoma, Non-Small-Cell Lung; Female; Humans; Lung Neoplasms; Male; Methylation; Middle Aged; Multivariate Analysis; O(6)-Methylguanine-DNA Methyltransferase; Promoter Regions, Genetic; Proportional Hazards Models; Reverse Transcriptase Polymerase Chain Reaction; Sulfites; Time Factors; Treatment Outcome

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT-PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Topics: Aged; Blotting, Southern; Bronchi; Carcinoma, Squamous Cell; Cell Line; Cells, Cultured; CpG Islands; DNA; DNA Methylation; Endothelin-1; Epithelial Cells; Female; Gene Silencing; Humans; Immunohistochemistry; Introns; Lung Neoplasms; Male; Middle Aged; Models, Genetic; Physical Chromosome Mapping; Promoter Regions, Genetic; Receptor, Serotonin, 5-HT1B; Receptors, Serotonin; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Sulfites; Transcription, Genetic; Tumor Cells, Cultured