NAA10 Hypomethylation is associated with particulate matter exposure and worse prognosis for patients with non-small cell lung cancer.

Airborne particulate matter (PM) is a major health hazard worldwide and is a key factor in lung cancer, which remains the most common type of malignancy and the leading cause of cancer-related deaths. DNA methylation is a critical mechanism underlying the detrimental effects of PM, however, the molecular link between PM exposure and lung cancer remains to be elucidated. N-α-acetyltransferase 10 (NAA10) is involved in the cell cycle, migration, apoptosis, differentiation, and proliferation. In order to investigate the role of NAA10 in PM-induced pathogenesis processes leading to lung cancer, we determined the expression and methylation of NAA10 in normal human bronchial epithelial (NHBE) cells treated with PM10, PM10-polycyclic aromatic hydrocarbons (PAH), and PM2.5 and evaluated the prognostic value of the NAA10 methylation status in lung cancer patients. Exposure to all PM types significantly increased the expression of NAA10 mRNA and decreased the methylation of the NAA10 promoter in NHBE cells compared with the mock-treated control. NAA10 hypomethylation was observed in 9.3% (13/140) of lung cancer tissue samples and correlated with NAA10 transcriptional upregulation. Univariate and multivariate analyses revealed that NAA10 hypomethylation was associated with decreased survival of patients with lung cancer. Therefore, these results suggest that PM-induced hypomethylation of the NAA10 may play an important role in the pathogenesis of lung cancer and may be used as a potential prognostic biomarker for lung cancer progression. Further studies with large numbers of patients are warranted to confirm our findings.

Promoter hypermethylation of the CFTR gene and clinical/pathological features associated with non-small cell lung cancer.

BACKGROUND AND OBJECTIVE: The exact role of the cystic fibrosis transmembrane conductance regulator (CFTR) in pathophysiology, and the mechanisms regulating its expression are poorly understood. The CFTR gene is known to be genetically or epigenetically associated with several cancers. In the present study, the methylation status of the promoter region of the CFTR gene and its expression in primary non-small cell lung cancer (NSCLC) were investigated. METHODS: The methylation status of the promoter region of the CFTR gene in NSCLC tissue was assessed by pyrosequencing and methylation-specific PCR. Expression of the CFTR gene was analysed by real-time PCR, and CFTR gene reactivation was investigated using 5-aza-2'-deoxycytidine. The correlation between methylation of the CFTR gene and the clinical features of the patients was assessed. RESULTS: Methylation of the CFTR gene in NSCLC was quantitatively high by pyrosequencing analysis and qualitatively frequent by methylation-specific PCR analysis. Expression of the CFTR gene was significantly lower in NSCLC compared with normal lung tissue. In addition, the demethylating agent 5-aza-2'-deoxycytidine increased CFTR gene expression. Methylation of the CFTR gene was significantly greater in squamous cell carcinomas than in adenocarcinomas. CFTR gene methylation was associated with significantly poorer survival in young patients, but not in elderly patients. CONCLUSIONS: These findings suggest that DNA methylation may be important for downregulation of CFTR gene expression in lung cancer. Promoter hypermethylation of the CFTR gene may be an important prognostic factor in younger patients with NSCLC.

Infrequent hypermethylation of the PTEN gene in Korean non-small-cell lung cancers.

CpG islands (CGIs) hypermethylation is implicated in the pathogenesis of many cancers, including lung cancer. The phosphate and tension homolog (PTEN) is a tumor suppressor that controls a variety of biological processes including cell proliferation, growth, migration, and death. The defects in PTEN regulation have a profound impact on carcinogenesis. Herein, we have examined the methylation status of the human PTEN gene in 137 primary non-small-cell lung cancers (NSCLCs) by using a methylation-specific PCR and correlated the results with clinicopathological features. Promoter methylation of the PTEN gene was observed in 5.1%, 2.9%, and 0.0% of three different CpG regions, which were localized at -1460 to -1263, -984 to -848, and -300 to -128 nucleotides upstream of the translation start site, respectively. Reverse transcription-PCR and immunohistochemical analysis showed the methylation of the CGI region at -984 to -848 correlated more accurately with PTEN expression. In addition, no significant correlation was found between PTEN methylation and clinicopathological factors, including the survival rates. These findings suggest that promoter methylation is not an important mechanism for PTEN deregulation in NSCLCs from Koreans.

Epigenetic inactivation of Homeobox A5 gene in nonsmall cell lung cancer and its relationship with clinicopathological features.

Promoter methylation is an important mechanism in gene silencing and is a key epigenetic event in cancer development. Homeobox A5 (HOXA5) is a master regulator of the morphogenesis and cell differentiation to be implicated as a tumor suppressor gene in breast cancer, but its role in lung cancer is still unknown. In this study, we have investigated the methylation status of the promoter region of the HOXA5 gene in nonsmall cell lung cancers (NSCLCs) using nested and standard methylation-specific PCR (MSP) and correlated the methylation status with clinicopathological features. With standard MSP analysis, HOXA5 methylation were found in 113 (81.3%) of 139 NSCLCs and 72 (51.8%) in their corresponding nonmalignant lung tissues. RT-PCR and immunohistochemical analysis showed that HOXA5 methylation correlates with gene expression. Moreover, in the patients with stage I disease, HOXA5 methylation was more frequent in smokers than in never-smokes (P = 0.01). There was no influence of HOXA5 methylation on survival in all NSCLCs or at stages II-IV. However, in the patients with stage I disease, HOXA5 methylation was associated with a borderline significantly worse survival (P = 0.09). These findings suggest that downregulation of the HOXA5 gene by aberrant promoter methylation occurs in the vast majority of NSCLCs and that it may play a role in the pathogenesis of NSCLC. Additional studies with larger sample sizes are required to evaluate the prognostic value of HOXA5 methylation in patients with stage I NSCLC.

Epigenetic deregulation of the human Oct4 promoter in mouse cells.

To examine whether the epigenetic status of the human Oct4 promoter is similarly regulated in mouse cells, we engineered a human bacterial artificial chromosome to express green fluorescent protein under the control of the hOct4 promoter and stably integrated it into mouse embryonic stem cells (mESCs), NIH3T3, and 293T cells. The hOct4 promoter is unmethylated in mESCs and it undergoes methylation during retinoic acid-induced differentiation. However, the hOct4 promoter is demethylated in NIH3T3 cells even though it is fully methylated in 293T cells. Methylation status of the hOct4 promoter is associated with green fluorescent protein expression at transcription level. Our findings indicate that the hOct4 promoter is differently regulated in mouse cells.

Hypermethylation of the Ras association domain family 1A (RASSF1A) gene in gallbladder cancer.

The tumor suppressor gene Ras association domain family 1A (RASSF1A) is highly methylated in a wide range of human sporadic tumors. The current study investigated the hypermethylation of RASSF1A, the expression of RASSF1A protein, and the correlation between these and the clinicopathological features of gallbladder (GB) cancer in Korean patients. Formalin-fixed, paraffin-embedded tumors and non-neoplastic GB tissues (22 carcinomas, 8 adenomas, 26 normal epithelia) were collected from patients who had undergone surgical resection. The methylation status of two regions of the RASSF1A CpG island was determined by methylation-specific PCR (MSP), and the expression of RASSF1A protein was examined by immunohistochemistry using tissue microarrays. The K-RAS mutation was analyzed by direct sequencing. Methylation of the RASSF1A promoter (region 1) was detected in 22.7% (5/22) of carcinomas, 12.5% (1/8) of adenomas, and 0% (0/26) of normal gallbladder epithelia (P = 0.025). Methylation of the first exon (region 2) was found in 36.4% (8/22) of carcinomas, 25.0% (2/8) of adenomas, and 8.0% (2/26) of normal gallbladder epithelia (P = 0.038). K-RAS mutations were present in 4.5% (1/22) of carcinomas and 25% (2/8) of adenomas. RASSF1A methylaton was not associated with clinicopathological factors or K-ras mutation. Reduction or loss of RASSF1A expression was observed in most methylated adenocarcinomas. Three RASSF1A-expressing human biliary tract cancer cell lines examined contained unmethylated promoters and exons 1. These results suggest that downregulation of RASSF1A expression by DNA hypermethylation may be involved in GB carcinogenesis.

Associations between XPC expression, genotype, and the risk of head and neck cancer.

Squamous cell carcinoma of the head and neck (SCCHN) is a relatively rare cancer with a poor prognosis. In the present study, we investigated susceptibility biomarkers for SCCHN in 155 Koreans (73 SCCHN cases and 82 controls). Expression of the xeroderma pigmentosum group C (XPC) DNA-repair gene was measured by TaqMan fluorogenic real-time RT-PCR using RNA isolated from peripheral blood samples. In addition, the XPC-PAT genotype [an intronic and biallelic poly(AT) insertion/deletion polymorphism] was determined by PCR of peripheral blood DNA. Among known environmental and physical risk factors, age, gender, tobacco smoking, and alcohol consumption were associated with the SCCHN risk (P < 0.01). Lower XPC expression levels were found for the SCCHN cases, particularly in the larynx, than for the controls (P < 0.05); the geometric means (standard deviations) of XPC expression normalized by 18S rRNA in the cases and controls were 5.89 (8.13) and 15.14 (9.77), respectively. However, the distributions of the XPC-PAT genotypes were similar in the cases and controls. In addition, there were no associations between the expression of XPC and the PAT genetic polymorphism. XPC expression was not affected by age, gender, tobacco smoking, or alcohol consumption. After adjusting for SCCHN-associated lifestyle factors, the effective strength of XPC expression on SCCHN risk was weaker (0.05 < P < 0.1) than those of tobacco smoking and age (P < 0.05). There was a positive association between XPC mRNA expression in blood and SCCHN tissues (n = 9; P < 0.01); therefore, peripheral blood appears to be a reasonable surrogate tissue for XPC expression in tumor tissue. In conclusion, the XPC-PAT polymorphism had no effect on XPC expression or SCCHN risk. However, XPC expression may influence SCCHN risk.

Sources of polycyclic aromatic hydrocarbon exposure in non-occupationally exposed Koreans.

Urinary 1-hydroxypyrene (1-OHP), an exposure biomarker for polycyclic aromatic hydrocarbons (PAHs), was used to identify potential sources of PAH exposure for 660 Koreans who were not occupationally exposed to PAHs (65% male; 35% female; mean age, 36.5 +/- 11.1 years). In this study, 74% of subjects had detectable levels of urinary 1-OHP, with a concentration range of 0.001-3.796 microg/L (median, 0.079 microg/L). A backward elimination was conducted: five variables were selected with a significance level for removal of P < or = 0.1. The results of this study showed that residence in areas with relatively poor environmental conditions (Seoul and Suwon) was strongly associated with high concentrations of urinary 1-OHP (P = 0.007), while consumption of fried chicken and length of time spent outdoors had marginal positive associations with urinary 1-OHP levels (P = 0.06 and P = 0.09, respectively). Compared with the above three factors, tobacco smoking and urinary cotinine levels were poorly associated with urinary 1-OHP (P = 0.16 and 0.23, respectively). Pear consumption had an inverse association with urinary 1-OHP levels (P < 0.01). Individual variations in urinary 1-OHP concentrations were evaluated by considering the subjects' age, sex, and genetic polymorphisms in enzymes involved in the metabolism of PAHs. Among the individual variations, GSTT1-present subjects showed higher 1-OHP levels than GSTT1-absent subjects in cities having 10-microm particulate matter (PM(10)) levels and population density lower than those of Seoul and Suwon (P < 0.05). These epidemiological results suggest that the above factors that should be considered in preventing PAH exposure.

Unmethylation of the CHRNB4 gene is an unfavorable prognostic factor in non-small cell lung cancer.

OBJECTIVES: Lung cancer is the leading cause of cancer-related deaths and is currently a major health problem owing to difficulties in diagnosis at the early stage of the disease. Changes in DNA methylation status have now been identified as a critical component in the initiation of lung cancer, and the detection of DNA methylation is expected to be an important method for the early diagnosis of lung cancer. Nicotine, the principal tobacco alkaloid, directly contributes to lung carcinogenesis through the activation of nicotinic acetylcholine receptors (nAchRs). MATERIALS AND METHODS: To investigate the role of the CHRNB4 gene, which encodes the nAchR ß4 subunit that is ubiquitously expressed on lung epithelial cells, we analyzed its methylation status in 266 patients with non-small cell lung cancer (NSCLC) by using methylation-specific polymerase chain reaction and compared it with clinicopathological parameters. RESULTS AND CONCLUSION: The frequency of CHRNB4 unmethylation was 13.5% and 8.3% in malignant and nonmalignant tissues, respectively. CHRNB4 demethylation was associated with upregulation of its mRNA expression and was more frequent in squamous cell carcinoma and pathological stages II-IIIA disease than in adenocarcinoma and pathological stage I disease, respectively (P=0.003 and P=0.01, respectively). Univariate and multivariate analyses showed that CHRNB4 unmethylation was significantly associated with unfavorable overall survival in the entire patient group as well as in men and ever-smokers. These results suggest that epigenetic regulation of CHRNB4 may affect tumor progression and survival in patients with NSCLC. Further investigation into the molecular basis of the role of CHRNB4 in the progression of NSCLC is warranted.

Wif1 hypermethylation as unfavorable prognosis of non-small cell lung cancers with EGFR mutation.

Lung cancer is a leading cause of cancer-related mortality across the world and tobacco smoking is the major risk factor. The Wnt signaling pathway is known to be involved in smoke-induced tumorigenesis in the lung. Promoter hypermethylation of Wnt inhibitory factor 1 (Wif1) has become a common event in a number of human tumors. Using a methylation-specific PCR, hypermethylation of the Wif1 gene promoter was evaluated in 139 primary nonsmall cell lung cancers (NSCLCs) and its correlation with clinicopathological and prognostic parameters was evaluated. Methylation of Wif1 was observed in 47.5% and 20.9% of neoplastic and adjacent normal lung tissues, respectively. Its methylation rate tended to be higher in stage I than stages II-IIIA. Results of Kaplan-Meier analysis showed no significant difference in overall survival according to Wif1 methylation status. However, Wif1 methylation showed an association with unfavorable prognosis of adenocarcinoma (AC) patients with EGFR mutation. According to our current findings, Wif1 promoter methylation is an early, frequent event as an epigenetic field manner and could be considered as a useful prognostic marker for AC patients with EGFR mutation. Further investigation into the therapeutic potential of this finding is warranted.

Genetic and epigenetic alterations of the LKB1 gene and their associations with mutations in TP53 and EGFR pathway genes in Korean non-small cell lung cancers.

INTRODUCTION: Liver kinase 1 (LKB1) plays a critical barrier role in lung tumorigenesis by controlling initiation, differentiation and metastasis. We searched for genetic and epigenetic alterations of the LKB1 gene in Korean non-small cell lung cancers (NSCLCs) and correlated the results with clinicopathological features. We also investigated the relationship between genetic and epigenetic alterations of LKB1 and mutations in the TP53 gene and epidermal growth factor receptor (EGFR) pathway genes. METHODS: A total of 159 NSCLCs were analyzed for loss of heterozygosity (LOH) at microsatellite loci D19S886, and D19S878. Mutations and methylation status of LKB1 were examined by direct sequencing and a methylation-specific polymerase chain reaction, respectively. RESULTS: A somatic mutation was found in one of the 159 tumors. LOH and promoter methylation was detected in 19.5% (31/159) and 13.2% (21/159) of the tumors, respectively. Four of the 159 tumors had concomitant LOH and methylation of LKB1. In total, 30.2% of the 159 NSCLCs harbored LKB1 LOH or promoter methylation, which were correlated with down-regulation of gene expression. LKB1 LOH was more frequent in males, smokers, and tumors with a TP53 mutation than in females, never-smokers, and tumors without a TP53 mutation, respectively. However, no significant correlation between LKB1 alterations and mutations in EGFR pathway genes was found. CONCLUSION: These results suggest that the prevalence of LKB1 genetic and epigenetic alterations in NSCLCs vary depending on patient ethnicity. Our results show that LKB1 alterations often occur simultaneously with mutations in EGFR pathway genes.

Methylation of TMEFF2 gene in tissue and serum DNA from patients with non-small cell lung cancer.

Lung cancer remains a global health problem with a high mortality rate. CpG island methylation is a common aberration frequently associated with gene silencing in multiple tumor types, emerging as a highly promising biomarker. The transmembrane protein with a single EGF-like and two follistatin domains (TMEFF2) is epigenetically silenced in numerous tumor types, suggesting a potential role as a potential tumor suppressor. However, the role of TMEFF2 in lung cancer remains to be fully elucidated. We explored the methylation status of TMEFF2 gene in 139 patients with non-small cell lung cancer (NSCLC) and the feasibility of detecting circulating methylated DNA as a screening tool for NSCLC using methylation-specific PCR in 316 patients and 50 age-matched health controls. TMEFF2 methylation in tumor tissues was found in 73 of the 139 NSCLCs (52.5%) and was related to gene expression. The frequency of TMEFF2 methylation was higher in females and never-smokers than in males and smokers with borderline significance (65.8% vs 47.8%, p = 0.06; 65.7% vs 48.1%, p = 0.07). Notably, in adenocarcinomas, TMEFF2 methylation was significantly more frequent in tumors without EGFR mutation than those with EGFR mutation (adjusted odds ratio = 7.13, 95% confidence interval = 2.05-24.83, P = 0.002). Furthermore, TMEFF2 methylation was exclusively detected in the serum of NSCLC patients at a frequency of 9.2% (29/316). These findings suggest that methylation-associated down-regulation of TMEFF2 gene may be involved in lung tumorigenesis and TMEFF2 methylation can serve as a specific blood-based biomarker for NSCLC.

Promoter methylation of Wnt antagonist DKK1 gene and prognostic value in Korean patients with non-small cell lung cancers.

Dickkopf-1 (DKK1) is known as a negative regulator of the Wnt signaling pathway, which plays a crucial role in carcinogenesis. However, aberrant expression and the role of DKK1 in human cancers remain controversial. To estimate the role of DKK1 and its prognostic potential in lung cancer, promoter methylation of DKK1 was evaluated in 139 primary non-small cell lung cancers (NSCLCs) by methylation-specific PCR and its association with clinical and prognostic parameters. DKK1 hypermethylation was detected in 48.9% of neoplastic lung tissues and was significantly more frequent in stage I than the more advanced stages II-IIIA (P=0.04). Additionally, patients with DKK1 methylation had a better overall survival than those with no methylation under univariate analysis. When stratified by clinicopathologic features, DKK1 methylation was significantly associated with a favorable survival in a subset of patients. The current findings suggested that DKK1 promoter methylation may be a tumor-associated event in the early stage of NSCLC and could also be useful prognostic indicator for NSCLC. Further work may clarify the molecular basis of DKK1 action in progression of NSCLC.

Quantitative promoter hypermethylation analysis of RASSF1A in lung cancer: comparison with methylation-specific PCR technique and clinical significance.

Lung cancer is the major health problem and leading cause of cancer-related deaths worldwide owing to late diagnosis and poor prognosis. Aberrant promoter methylation is an important mechanism for silencing tumor-suppressor genes in cancer and a promising tool for the development of molecular biomarkers. Ras association domain family 1A (RASSF1A), a pivotal gatekeeper of cell cycle progression, is highly methylated in a wide range of human sporadic tumors, including lung cancer. However, no significant prognostic implications have been observed in most studies qualitatively analyzed by methylation-specific PCR (MSP). We found that the RASSF1A promoter was aberrantly methylated in 44.7 and 37.4% of the tumors by pyrosequencing (PS) and MSP methods, respectively. RASSF1A methylation evaluated by the two methods was more frequent in ever-smokers and tumors with TP53 mutation than in never-smokers and tumors without TP53 mutation, respectively. Univariate and multivariate analyses revealed that strong methylation was an unfavorable prognostic factor with stage I (adjusted HR, 2.25; 95% CI 1.03-4.90; P=0.003) and squamous cell carcinoma patients (adjusted HR=2.25, 95% CI 1.03-4.90, P=0.042). Taken together, these results suggested that quantitative PS could gain wider applications in clinical samples as a promising method for early detection screening and prognosis compared with MSP.

Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

Promoter methylation of the RGC32 gene in nonsmall cell lung cancer.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. Epigenetic inactivation of certain genes by aberrant promoter methylation is recognized as a crucial component in the initiation and progression of lung cancer. Response gene to complement 32 (RGC32) has been identified as a cell cycle regulator induced by activation of complements; however, its role in carcinogenesis is still controversial. METHODS: The authors examined the methylation status in the promoter region of RGC32 gene in nonsmall cell lung cancers (NSCLCs) using a methylation-specific PCR and correlated the results with clinicopathological features. RESULTS: RGC32 methylation was found in 45 of 173 NSCLCs (26.0%) and was related to the gene expression. RGC32 methylation was more frequent in females than in males (P<0.05). RGC32 methylation was not significantly associated with the prognosis of patients; however, when the patients were categorized by TP53 mutational status, the effect of RGC32 methylation on prognosis was significantly different between those with and without TP53 mutations (P = .005 [test for homogeneity]). Specifically, RGC32 methylation was associated with significantly worse survival in the cases with wild-type TP53, whereas it exhibited a better survival outcome in the cases with TP53 mutations. CONCLUSIONS: The current findings suggest that methylation-associated down-regulation of RGC32 plays an important role in the pathogenesis of NSCLC, particularly in females. However, further studies with a large number of cases are needed to confirm the authors' findings.

Hypomethylation of the thymosin ß(10) gene is not associated with its overexpression in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide and is usually associated with a late diagnosis and a poor prognosis. Thymosin ß(10) (TMSB10) is a monomeric actin sequestering protein that regulates actin cytoskeleton organization. The aberrant TMSB10 expression has been implicated in the pathogenesis of human cancers. However, its role in carcinogenesis is still controversial. To better understand the role of TMSB10 in lung tumorigenesis and its regulatory mechanism, we examined the methylation status and expression of the TMSB10 gene in non-small cell lung cancers (NSCLCs) using methylation-specific PCR (MSP) and immunohistochemistry (IHC), respectively. MSP analysis showed that the TMSB10 promoter was already unmethylated in most tumor tissues and became demethylated in 20 (14.4%) of the 139 NSCLCs. TMSB10 hypomethylation was not significantly correlated with the clinicopathological features. IHC showed that the TMSB10 protein was strongly expressed in the cytoplasm of malignant cells and its overexpression was detected in 50.0% of the tumor tissues compared to normal tissues. TMSB10 overexpression was frequently observed in sqaumous cell carcinomas compared to adenocarcinomas with border line significance (P = 0.072). However, TMSB10 methylation status was not linked to its overexpression. Collectively, these results suggest that TMSB10 hypomethylation may be a frequent event in NSCLCs, but it may not be a common mechanism underlying TMSB10 overexpression. However, further studies with large numbers of patients are needed to confirm our findings.

Epigenetic inactivation of retinoid X receptor genes in non-small cell lung cancer and the relationship with clinicopathologic features.

Retinoid X receptors (RXRs) are nuclear receptors for retinoids that play a critical role in the regulation of growth and differentiation in normal and tumor cells. Deregulation of RXR expression has been reported in non-small cell lung cancer (NSCLC); however, the mechanism underlying the impaired expression of RXRs in lung cancer is not known. Aberrant methylation of promoter CpG islands is known to be a major mechanism for inactivation of tumor suppressor genes. We investigated the methylation status of the RXR genes in 139 surgically resected NSCLCs and correlated the results with the clinicopathologic characteristics of the patients. Methylation in the tumors was detected in all three genes: RXRA, 5.7%; RXRB, 4.3%; RXRG, 23.7%. Reverse transcriptase-polymerase chain reaction analysis showed that RXRG methylation correlates with mRNA expression. Methylation of the RXRG gene was not significantly associated with the prognosis of patients. When the patients were categorized by smoking status, however, the effect of RXRG methylation on prognosis was significantly different between never- and ever-smokers (P=0.003, test for homogeneity). Specifically, RXRG methylation was associated with a significantly worse survival in never-smokers; a trend to better survival outcome was observed for ever-smokers, although not statistically significant. This finding suggests that methylation-associated downregulation of the RXRG gene may play a differential role in the carcinogenesis of NSCLCs according to smoking status, but further studies are needed to confirm this.

Hypermethylation of growth arrest DNA-damage-inducible gene 45 in non-small cell lung cancer and its relationship with clinicopathologic features.

The growth arrest DNA-damage-inducible protein 45 (GADD45) can serve as a key coordinator of the stress response by regulating cell cycle progression, genomic stability, DNA repair, and other stress-related responses. Although deregulation of GADD45 expression has been reported in several types of human tumors, its role in lung cancer is still unknown. DNA hypermethylation of promoter CpG islands is known to be a major mechanism for epigenetic inactivation of tumor suppressor genes. We investigated the methylation status of GADD45 family genes (GADD45A, B, and G) in 139 patients with non-small cell lung cancer (NSCLC) using methylation-specific PCR (MSP) and correlated the results with clinicopathologic features of the patients. Methylation frequencies in tumors were 1.4% for GADD45A, 7.2% for GADD45B, and 31.6% for GADD45G. RT-PCR and MSP analysis showed that promoter methylation of the GADD45G gene resulted in downregulation of its mRNA expression. GADD45G methylation was significantly more frequent in female patients than male patients (P = 0.035). This finding suggests that methylation-associated down-regulation of the GADD45G gene may be involved in lung tumorigenesis.

Obox4 regulates the expression of histone family genes and promotes differentiation of mouse embryonic stem cells.

Obox genes are preferentially expressed in the ovary, testis and oocyte, and play important roles in many developmental processes. In this study, we report that Obox4 and Obox6 are expressed in mouse embryonic stem cells (mESCs) and that Obox4 regulates histone family gene expression in mESCs. Obox4 protein expressing mESCs formed colonies with a flattened and irregular morphology, and exhibited decreased expression levels of self-renewal related proteins, such as Oct4 and Sox2, as well as reduced alkaline phosphatase activity. The results of microarray analysis and siRNA mediated knockdown experiments suggest that Obox4 is an upstream regulator of the histone gene family.