Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas

Abstract

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′-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.

Introduction

Inactivation of tumor suppressor genes (TSGs) plays a major role in cancer. Besides mutations and deletions, DNA methylation of CpG islands in the promoter region of cancer-related genes is a frequently acquired epigenetic event in the pathogenesis of many human malignancies leading to gene silencing. So far, aberrant methylation (referred to as methylation) has been described for several genes in various malignancies including lung cancer (Burbee et al., 2001; Esteller et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b; Toyooka et al., 2003).

TSLC1 (tumor suppressor in lung cancer-1, IGSF4) is a TSG that was identified on chromosome 11q23.2 by functional complementation through suppression of tumorigenicity in nude mice (Murakami et al., 1998; Kuramochi et al., 2001). Recently, it has been demonstrated that re-expression of TSLC1 in the non-small-cell lung cancer (NSCLC) cell line A549 induces apoptosis and inhibits tumor growth (Mao et al., 2004). Methylation was observed in a high percentage of primary NSCLCs (Kuramochi et al., 2001; Fukami et al., 2003). TSLC1 encodes a 75 kDa N-linked glycoprotein that mediates intracellular adhesion through homophilic interactions in a Ca2+/Mg2+-independent manner (Masuda et al., 2002). This 75 kDa N-linked glycoprotein comprises three Ig-like C2-type domains, a single hydrophobic membrane-spanning α-helix and a short cytoplasmic domain of 46 amino acids that contains a protein 4.1-binding motif just adjacent to the transmembrane domain and a PDZ-binding motif (class II) at the carboxy-terminal end (Songyang et al., 1997; Fukuhara et al., 2001). The 4.1 binding motif at the juxtamembrane region is essential for the interaction with the tumor suppressor protein in lung cancer, DAL-1 (differentially expressed in adenocarcinoma of the lung-1, EPB41L3), which is localized at the cytoplasmic side of the cell membrane (Tran et al., 1999). DAL-1 belongs to the 4.1 protein family and acts as an anchoring protein connecting TSLC1 to the actin cytoskeleton (Yageta et al., 2002). DAL-1 is localized in the chromosomal region 18p11.3, which is affected by LOH in different cancer types including NSCLCs (Tran et al., 1998). Tran et al. (1999) reported reduced expression of DAL-1 in >50% of primary NSCLCs and Kittiniyom et al. (2004) recently observed that DAL-1 mutations are rare in NSCLCs, suggesting other mechanisms important for inactivation of this gene.

The PDZ-binding motif of TSLC1 interacts with the Drosophila discs large tumor suppressor homologue MPP3 (Fukuhara et al., 2003). Human MPP3 is a cytoplasmic protein of 585 amino acids and belongs to the MAGUK (membrane-associated guanylate kinase) family molecules. MPP3 interacts with the cytoskeleton and regulates cell proliferation, signaling pathways and intracellular junctions (Anderson, 1996). It has been demonstrated that MPP3 is colocalized with TSLC1 at cell–cell attachment sites, suggesting that TSLC1 and MPP3 are cooperatively involved in normal cell adhesion (Fukuhara et al., 2003).

In conclusion, these findings suggest that DAL-1 and MPP3 interact with TSLC1 at the cell–cell attachment site and that these genes may play an important role in a TSG cascade that regulates cell growth (Fukuhara et al., 2003). Moreover, loss of function of the TSLC1 cascade may promote metastasis as it has been suggested recently (Yageta et al., 2002; Ito et al., 2003).

In this study, we investigated the expression of TSLC1, DAL-1 and MPP3 in various specimen types by microarray analysis and reverse transcription–polymerase chain reaction (RT–PCR). The methylation status of TSLC1 and DAL-1 was determined in a large number of lung cancer cell lines, primary lung carcinomas of different histologies and in corresponding nonmalignant lung tissue samples. In addition, our results were compared with clinicopathological characteristics from the lung carcinoma patients and with methylation results of other genes that had been investigated in a subset of tumors in previous studies (Burbee et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b). We found that methylation of either TSLC1 or DAL-1 leading to loss of expression is a frequent event in lung cancer pathogenesis.

Results

Expression of TSLC1, DAL-1 and MPP3

We examined the expression of TSLC1, DAL-1 and MPP3 in lung cancer cell lines, primary lung tumors, nonmalignant lung tissue samples and human bronchial epithelial cell lines by microarray analysis (Figure 1). Loss of TSLC1 expression occurs frequently in NSCLC cell lines but was not detected in small-cell lung cancer (SCLC) cell lines. DAL-1 expression was lost in SCLC cell lines and in an even higher percentage in NSCLC cell lines. MPP3 was expressed in all NSCLC and SCLC cell lines. In primary lung tumors and nonmalignant lung tissue samples, loss of expression of these genes was either not observed or in a low percentage. In HBECs, loss of expression of DAL-1 and MPP3 was frequently observed. Detailed results of the expression microarrays are shown in Table 1. In lung cancer cell lines, the expression of these genes was also determined by RT–PCR. Examples are shown in Figure 2. The expression of TSLC1 was lost in seven of 16 (44%) NSCLC and in one of 11 (9%) SCLC cell lines; however, the frequency of loss of TSLC1 expression in NSCLC compared to SCLC cell lines did not reach statistical significance (P=0.053). Loss of DAL-1 expression was found in 14 of 16 (87%) NSCLC and in four of 11 (36%) SCLC cell lines. The frequency of loss of DAL-1 expression was significantly higher in NSCLC compared to SCLC cell lines (P=0.006). MPP3 expression was detected in all tumor cell lines analysed. A statistically significant correlation between loss of TSLC1 and loss of DAL-1 expression was observed (r=0.55; P=0.014). Detailed results of the expression analysis of TSLC1 and DAL-1 by RT–PCR are summarized in Table 2. Overall, the expression results in lung cancer cell lines obtained by microarray analysis are similar to those found by RT–PCR.

Figure 1
figure1

Range of array expression data for the various sample categories and for the three genes TSLC1, DAL-1 and MPP3. Expression values are in a range of 0–100 (see Materials and methods) with signals <20 arbitrarily considered turned off. 1, SCLC cell lines; 2, NSCLC cell lines; 3, primary lung tumors; 4, nonmalignant lung tissue; 5, HBECs

Table 1 Frequency of lossa of TSLC1, DAL-1 and MPP3 by expression microarrays
Figure 2
figure2

RT–PCR analysis of TSLC1, DAL-1 and MPP3 expression in a sampling of lung cancer cell lines

Table 2 Expression pattern of TSLC1 and DAL-1 in lung cancer cell lines by RT–PCR

Methylation analysis of the 5′ region of TSLC1 and DAL-1

Methylation analysis was performed by bisulfite genomic sequencing and methylation-specific PCR (MSP). CpG islands of TSLC1 and DAL-1 have been localized using MethPrimer programme as described in Materials and methods (Li and Dahiya, 2002). Bisulfite genomic sequencing of a 282 bp fragment of the TSLC1 promoter region and of a 251 bp fragment of the DAL-1 promoter region in lung cancer cell lines lacking expression of these genes showed that cytosines within CpG dinucleotides were methylated. Representative sequencing results are shown in Figure 3. Based on this methylation pattern, primer sequences for the MSP assay for TSLC1 and DAL-1 were designed and MSP was performed in all samples. Examples are shown in Figure 4. Methylation of TSLC1 was detected in 13 of 39 (33%) NSCLC and in one of 11 (9%) SCLC cell lines (Table 3) and was present in all NSCLC cell lines lacking TSLC1 expression except in the tumor cell line NCI-H2073, which showed both TSLC1 expression and methylation indicating monoallelic silencing of TSLC1. All tumor cell lines that expressed TSLC1 were unmethylated. The correlation between loss of TSLC1 expression and TSLC1 methylation was statistically significant (r=0.81; P=0.0003). DAL-1 methylation was observed in 17 of 39 (44%) NSCLC and in three of 11 (27%) SCLC cell lines (Table 3). We found three tumor cell lines (NCI-2009, NCI-H211, NCI-1299) that showed loss of DAL-1 expression in the absence of methylation, suggesting an additional mechanism(s) for silencing DAL-1 besides methylation. Moreover, the tumor cell line HCC515 was DAL-1 methylated and expressed DAL-1, indicating again monoallelic silencing of this gene. The correlation between loss of DAL-1 expression and DAL-1 methylation was statistically significant (r=0.68; P=0.002). In addition, we observed a correlation between TSLC1 and DAL-1 methylation in lung cancer cell lines (r=0.318; P=0.004).

Figure 3
figure3

(A) Representative bisulfite sequencing results of the TSLC1 (1) and DAL-1 (2) 5′ region in lung cancer cell lines that showed loss of expression of these genes. Sequences from bisulfite-modified DNA are compared to sequences of untreated DNA in the top line. Asterisks indicate methylated cytosine residues at the CpG sites. (B) Schematic description of the methylation status of CpG sites in the 5′ region of TSLC1 (1) and DAL-1 (2) in lung cancer cell lines (a), primary NSCLCs (b) and nonmalignant lung tissues (c). The regions analysed contain 10 CpG sites (−482 to −310 bp from the transcription start site; GenBank accession # NM_014333) in the case of TSLC1 and 11 CpG sites (−140 to −30 bp from the transcription start site; GenBank accession # NM_012307) in the case of DAL-1. Black and white circles represent methylated and unmethylated CpG sites, respectively, and gray circles represent overlapping ‘C’ and ‘T’ peaks, which indicate partial or heterogenous methylation. Lung cancer cell lines NCI-H1299, NCI-H1573, NCI-H1607, NCI-H1915 and NCI-H1993 were TSLC1 methylated by MSP and did not express TSLC1 by RT–PCR. Lung cancer cell line NCI-H2073 was also TSLC1 methylated, however, it showed expression of this gene. DAL-1 methylation and loss of DAL-1 expression was found in lung cancer cell lines NCI-H157, NCI-H460 and NCI-H1915 by MSP and RT–PCR, respectively. Lung cancer cell lines NCI-H211, NCI-H1299 and NCI-H2009 were not DAL-1 methylated and did not express this gene. Primary NSCLC samples P7, P25, P30, P120, P227 and P320 were TSLC1 methylated by MSP and samples P7, P38, P188, P235, P244 and P370 were DAL-1 methylated. Nonmalignant lung tissue samples P6, P26, P228 and P321 were found to be not TSLC1 methylated; however, methylation of TSLC1 was observed in samples P105 and P236. In the case of DAL-1, nonmalignant lung tissue samples P6, P245 and P371 were not methylated; however, methylation was detected in samples P105, P233 and P236

Figure 4
figure4

Representative samples of TSLC1 and DAL-1 MSP analysis in lung cancer cell lines (a) and primary lung tumors (b). U, results with primers specific for unmethylated sequences; M, results with primers specific for methylated sequences. P, patient

Table 3 Frequency of TSLC1 and DAL-1 methylation in various specimen types

Moreover, TSLC1 and DAL-1 methylation was analysed in a total of 272 primary lung carcinomas. TSLC1 methylation was found in 100 (37%) primary NSCLCs. A total of 42 (32%) adenocarcinomas and 50 (46%) squamous cell carcinomas were TSLC1 methylated. The frequency of TSLC1 methylation was significantly higher in squamous cell carcinomas compared to adenocarcinomas (P=0.023). However, TSLC1 methylation was not found in any of the primary SCLCs. In addition, two of 30 (7%) corresponding nonmalignant lung tissue samples were TSLC1 methylated. DAL-1 methylation was found in 147 (55%) primary NSCLCs. A total of 75 (56%) adenocarcinomas and 60 (55%) squamous cell carcinomas showed DAL-1 methylation. Again, all primary SCLCs were DAL-1 unmethylated. DAL-1 methylation was found in 10% of the corresponding nonmalignant lung specimens. Also four HBEC lines were studied for the methylation status of TSLC1 and DAL-1 genes. However, these cell lines were not methylated for TSLC1 and/or DAL-1. Bands for the unmethylated allele of TSLC1 and DAL-1 were detected in all primary tumors because the tumors were not microdissected and thus, consist of a mixture of nonmalignant cells and malignant cells. Detailed results are shown in Table 3. Taken together, 174 of 268 (65%) primary NSCLC patients showed at least one of the two genes methylated. The comparison between TSLC1 and DAL-1 methylation in primary NSCLCs is shown in Table 4. A correlation between TSLC1 and DAL-1 methylation was observed (r=0.28; P=0.000004).

Table 4 Comparison of TSLC1 and DAL-1 methylation in primary NSCLCs

Gene re-expression by 5-aza-2′-deoxycytidine treatment

To confirm that methylation contributes to the lack of TSLC1 and DAL-1 expression in the TSLC1- and DAL-1-nonexpressing cell lines NCI-H1915 and NCI-H1993, we investigated the effect of 5-aza-2′-deoxycytidine (5-aza-dC), a drug that inhibits DNA methylation. Re-expression of TSLC1 and DAL-1 in the lung cancer cell lines NCI-H1915 and NCI-H1993 was observed after the exposure of cells to 5-aza-dC, suggesting that methylation is an important mechanism to inactivate TSLC1 and DAL-1 (Figure 5). However, we also found very faint bands in the expression analysis when the cell lines NCI-H1915 and NCI-H1993 were treated with trichostatin A (TSA) alone.

Figure 5
figure5

Re-expression of TSLC1 and DAL-1 after treatment of lung cancer cells with 5-aza-dC. The cell lines NCI-H1915 and NCI-H1993, which lack TSLC1 and DAL-1 expression, were grown in the presence (+ lanes) and absence (− lanes) of 0.5 μ M 5-aza-dC for 6 days. Afterwards, RT–PCR was performed for TSLC1 and DAL-1. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a control

Comparison of TSLC1 and DAL-1 methylation with clinicopathological characteristics of the lung cancer patients

The methylation results were compared with certain clinicopathological characteristics of the lung cancer patients (Table 5). Data about histology, sex, age and stage of disease were available from all patients, while data about smoking history and overall survival were available only from a subset of patients. DAL-1 methylation was more frequently found in patients with lymph node metastasis than in patients without lymph node involvement (P=0.005). In addition, NSCLC patients with stage II–III disease showed a significantly higher frequency of DAL-1 methylation than patients with stage I disease (P=0.002). No statistically significant correlation between TSLC1 methylation and clinicopathological characteristics was observed. Furthermore, we compared the clinicopathological characteristics of patients who showed at least one of the two genes methylated with those who were not methylated for TSLC1 and/or DAL-1. Methylation correlated with both lymph node involvement (P=0.008) and stage of disease (P=0.004). We did not observe geographic differences in the methylation pattern between tumors from patients from Australia and Austria. Additionally, our methylation results were compared with methylation results of the genes RARβ-2, FHIT, RASSF1A, TIMP-3, p16, MGMT, DAPK, ECAD, p14 and GSTP1 in a subset of patients who had been investigated in previous studies (Burbee et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b). However, no statistically significant correlation of TSLC1 or DAL-1 and methylation of these genes was detected.

Table 5 Clinicopathological characteristics of NSCLC patients and methylation results of TSLC1 and DAL-1

Discussion

In our study, we analysed the expression of three genes that are involved in the TSLC1 cell adhesion cascade by microarray analysis and RT–PCR. Overall, the expression results in lung cancer cell lines obtained by microarray analysis and RT–PCR are similar. By RT–PCR, 87% of NSCLC cell lines did not express TSLC1 and/or DAL-1, suggesting that inactivation of the TSLC1 cell adhesion cascade is an important event in the pathogenesis of NSCLCs. Kuramochi et al. (2001) observed loss of TSLC1 expression in four of 12 (33%) NSCLC cell lines and Fukami et al. (2003) reported that TSLC1 is lost in two of 10 (20%) SCLC cell lines. While the percentage of loss of TSLC1 expression in NSCLC cell lines is slightly higher (44%) in our study, we observed a lower percentage of loss of TSLC1 expression in SCLC cell lines (9%). We found DAL-1 expression to be lost in 87% of NSCLC cell lines, which is very similar to the results of Tran et al. (1999), who reported loss of DAL-1 expression in 80% of NSCLC cell lines, and is slightly higher compared to the 64% of loss of DAL-1 expression reported by Kikuchi et al. (2005). While Tran et al. (1999) found DAL-1 expressed in all six SCLC cell lines, Kikuchi et al. (2005) reported loss of DAL-1 expression in one of seven (14%) SCLC cell lines and we observed loss of DAL-1 expression in four of 11 (36%) of SCLC cell lines. This difference might be explained by the different sample sizes in these studies. Similar to the data of Fukuhara et al. (2003), who found loss of MPP3 expression in only one of nine NSCLC cell lines, we detected MPP3 expression in all tumor cell lines analysed. These data suggest that MPP3 inactivation does not play a role in the pathogenesis of lung cancer.

TSLC1 methylation was observed at a statistically significant higher frequency in NSCLC compared to SCLC cell lines. Also in the case of DAL-1, the frequency of methylation was higher in NSCLC compared to SCLC cell lines, although the difference did not reach statistical significance. We observed TSLC1 methylation in 37% of primary NSCLCs, which is similar to the frequency of 44% reported by Fukami et al. (2003). While they did not observe differences in the frequency of methylation between the histologic subtypes, we found primary squamous cell carcinomas more frequently methylated than primary adenocarcinomas (P=0.028). In addition, Kuramochi et al. (2001) found 11 of 14 primary NSCLC tumors with LOH on 11q23.2, the locus of TSLC1, to have TSLC1 methylation. Similar to our results, Kikuchi et al. (2005) reported 57% of primary NSCLCs to be DAL-1 methylated by bisulfite single strand conformational polymorphism analysis (SSCP). Although the sample size was small, methylation of TSLC1 and DAL-1 was not found in primary SCLCs. These findings are in agreement with data reported by Toyooka et al. (2001) and Virmani et al. (2002), who found that the methylation pattern differs between histologic lung cancer subtypes. We observed a statistically significant correlation between TSLC1 and DAL-1 methylation in primary NSCLCs. Our data suggest that methylation of TSLC1 or DAL-1 is not a mutually exclusive event and we believe that methylation of these genes may work together to inactivate the pathway involved in the pathogenesis of lung cancer. In contrast, we did not find a correlation between methylation of TSLC1 or DAL-1 and methylation of the other genes previously investigated (Burbee et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b).

Treatment with 5-aza-dC restored TSLC1 and DAL-1 expression in the tumor cell lines NCI-H1993 and NCI-H1915, further confirming that promoter methylation is a major mechanism for silencing these genes. Very faint bands were observed in the expression analysis of TSA alone treated NCI-H1915 and NCI-H1993 cells compared to the bands observed after treatment of these cells with 5-aza-dC. This finding can be explained by the recently reported data from Xiong et al. (2005), who demonstrated that TSA is capable of inducing DNA demethylation even in the absence of 5-aza-dC.

Methylation of TSLC1 and DAL-1 was not observed in the majority of nonmalignant lung tissue samples. The detection of methylation in a small number of nonmalignant lung tissue samples might be explained by the occurrence of methylation in preneoplastic/preinvasive and even histologically normal smoking-damaged epithelium, although contamination of some samples with tumor cells cannot be excluded. Although a significant correlation between loss of expression and methylation of TSLC1 and DAL-1 in lung cancer cell lines was observed, loss of TSLC1 and DAL-1 expression was also detected in lung cancers without methylation of these genes. Moreover, four HBEC lines, which did not express DAL-1, were not DAL-1 methylated. These data suggest that one or more additional mechanisms for silencing of TSLC1 and DAL-1 also exist. In addition, it cannot be excluded that the HBEC lines acquired some changes during the immortalization process leading to gene silencing.

Cell adhesion molecules are important participants in cell–cell interaction and interactions between cells and components of the extracellular matrix. Tumor cells often show a decrease in cell–cell or cell–matrix adhesion, which correlates with tumor invasion and metastasis (Cavallaro and Christofori, 2001). TSLC1 is a TSG involved in cell–cell adhesion and cell motility and it induces apoptosis in NSCLCs (Kuramochi et al., 2001; Masuda et al., 2002; Mao et al., 2004). The role of TSLC1 in metastasis and invasion was demonstrated by the fact that invasion of the lung adenocarcinoma cell line A549, which lacks TSLC1, was inhibited in vivo after expression of exogenous TSLC1. These data suggest that loss of TSLC1 leads to impaired cell adhesion. Through its cytoplasmic domain, TSLC1 is associated with DAL-1. Although DAL-1 itself is not an adhesion molecule, Charboneau et al. (2002) demonstrated that re-expression of DAL-1 in a DAL-1 lacking cell line increased cellular adhesion. Thus, it may be hypothesized that, like β-catenin, which links the plasma membrane-bound E-cadherin to the α-catenin/actin cytoskeleton complex, DAL-1 regulates intercellular adhesion by connecting TSLC1 to the actin cytoskeleton. Loss of DAL-1 expression could therefore lead to decreased cell adhesion. Interestingly, we observed that DAL-1 was more frequently methylated in NSCLC patients with nodal involvement compared to patients without lymph node metastasis, suggesting that inactivation of DAL-1 by methylation may promote the spread of tumor cells. Further, it has been suggested that TSLC1 and DAL-1 are components of a cascade that participates in organizing the actin cytoskeleton by constructing stable adhesion between adjacent cells (Yageta et al., 2002). Inactivation of one of the two genes may disrupt cell adhesion and links between the membrane and the cytoskeleton, resulting in a malignant phenotype, invasion and metastasis. This theory is supported by our observation that methylation of at least one of the two genes occurred at a statistically significant higher frequency in patients with stage II–III disease compared to patients with stage I disease. However, additional studies are necessary to prove this concept. In conclusion, our results demonstrate that TSLC1 and DAL-1 are frequently epigenetically silenced in NSCLCs, which is important for the pathology of this tumor type.

Materials and methods

Tissue samples and tumor cell lines

Tissue samples were collected after obtaining appropriate Institutional Review Board permission and informed consent from the patients. For methylation analysis, primary NSCLC tumors (N=268) and corresponding nonmalignant lung tissue specimens (N=30) were obtained from patients who had received surgical resections at the Prince Charles Hospital, Brisbane, Australia (N=187) or at the General Hospital of Vienna, Austria (N=81). In addition, four SCLC samples were obtained. There were 191 male and 77 female NSCLC patients, ages 28–81 years (mean 61 years) at diagnosis. A total of 145 patients had stage I disease, 72 patients had stage II disease, 43 patients had stage IIIA disease and eight patients had stage IIIB disease. Histological subtypes of primary NSCLCs included 133 adenocarcinomas, 109 squamous cell carcinomas, 18 adenosquamous carcinomas and eight large cell carcinomas. In a subset of patients who had been investigated in previous studies (Burbee et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b), data about the smoking history (N=177) and survival of 5 or more years (N=187) were available. A total of 176 patients were ever smokers (range 3–135 pack-years, mean 40 pack-years) and one patient was a never smoker. The SCLC patients included four males, ages 56–72 years (mean 62 years), one patient had stage I disease, one patient had stage II disease and two patients had stage IIIA disease. Primary lung cancers (N=50) and nonmalignant lung tissue samples (N=9) for expression microarrays were obtained from a Hong Kong population (Lam et al., 2005). Cell lines used for microarray analysis included 55 NSCLC, 21 SCLC cell lines and four HBEC lines (normal lung lines immortalized with CDK4 and hTert) with their various transfected derivatives (25 in total) (Ramirez et al., 2004). For RT–PCR, 16 NSCLC and 11 SCLC cell lines were used. The methylation pattern of TSLC1 and DAL-1 was investigated in a total of 50 lung cancer cell lines.

Gene expression by microarray analysis and RT–PCR

Expression of TSLC1, DAL-1 and MPP3 was analysed by microarray analysis in lung cancer cell lines, primary lung tumors, nonmalignant lung tissue samples and HBECs and by RT–PCR in lung cancer cell lines.

For microarray analysis, RNAs were labeled and hybridized to Affymetrix HG-U133-Plus2 GeneChips (54 675 genes; 29 180 unique genes) or HG-U133A and HG-U133B (44 928 genes; 23 583 unique genes) according to the manufacturer's protocol (http://www.affymetrix.com). Softwares used were Affymetrix MicroArray Suite 5.0 and MATRIX 1.26 (Girard and Minna, 2005). Briefly, array data were median-normalized and log-transformed onto a scale of values from 0 to 100. In this range, 0 is the lowest (null) expression, 100 is the highest expression and 50 corresponds to the average ‘present’ signal across all array data (in Affymetrix nomenclature, present refers to detection P-value <0.05). Multiple Affymetrix array studies we have performed on lung cancer cell lines show that there is a correlation coefficient of >0.95 (usually >0.98) for replicate array analysis using the same RNAs. All primary array data are available in a MIAME compliant database at http://microarray.swmed.edu/smcdb/swarray_db.html.

For RT–PCR, total cellular RNA was isolated using TRIZOL (Gibco BRL, Carlsbad, CA, USA) according to the manufacturer's instructions. Total RNA (2 μg) was reverse transcribed using the Superscript II RNase H Reverse Transcriptase kit (Gibco BRL, Carlsbad, CA, USA). Primer sequences for RT–PCR are shown in Table 6. Primers for glyceraldehyde-3-phosphate dehydrogenase were used to confirm RNA integrity (Virmani et al., 2000).

Table 6 Oligonucleotide primer sequences for RT–PCR and MSP of TSLC1, DAL-1 and MPP3

5-aza-2′-deoxycytidine and trichostatin A treatment of lung cancer cell lines

For gene reactivation, NCI-H1915 and NCI-H1993 cells (2 × 105/ml) were seeded in 10% RPMI and treated with 0.5 μ M 5-aza-dC for 6 days. In addition, NCI-H1915 and NCI-H1993 cells (2 × 105/ml) were treated with 100 ng/ml TSA for 24 h. Gene reactivation was tested by RT–PCR.

Nucleic acid isolation, bisulfite sequencing and methylation-specific PCR

Genomic DNA was isolated from tumor cell lines, frozen lung tumors and normal lung tissues by digestion with proteinase K, followed by standard phenol–chloroform extraction and ethanol precipitation as reported previously (Sambrook et al., 1989). Afterwards, genomic DNA was modified by treatment with sodium bisulfite. For the chemical modification of DNA, 1 μg of genomic DNA was used as reported previously (Herman et al., 1996). We performed a search for CpG islands in the promoter region of TSCL1 and DAL-1 using the MethPrimer programme (Li and Dahiya, 2002) and found CpG islands in the promoter region of both genes. In the case of TSLC1 (GenBank accession # NM_014333), the length of the CpG island is 803 bp and it is located from nt −730 to nt +72 from the transcription start site. In the case of DAL-1 (GenBank accession # NM_012307), the CpG island is 1947 bp long and it is located from nt −178 to nt +1809 from the transcription start site. The methylation status of the region 5′ to the coding sequence of TSLC1 and DAL-1 of several cell lines lacking TSLC1 and DAL-1 expression was analysed by bisulfite genomic sequencing. PCR primers for bisulfite sequencing are listed in Table 6. The PCR products were sequenced using the 3100 Genetic Analyzer (PE Applied Biosystems). The methylation pattern obtained by sequencing was used to design primers for the MSP assay as described previously (Clark et al., 1994; Herman et al., 1996). The primers used for methylation-specific PCR to determine the methylation status of TSLC1 are located between nt −482 and nt −463 (forward primer) and between nt −332 and nt −310 (reverse primer) in the 5′ region of TSLC1. The primers to detect DAL-1 methylation are located between nt −140 and nt −120 (forward primer) and between nt −51 and nt −30 (reverse primer) in the 5′ region of DAL-1. Primer sequences for the methylated and the unmethylated allele of TSLC1 and DAL-1 are summarized in Table 6. The MSP products were then separated in 2% agarose gels stained with ethidium bromide and visualized under UV spectrophotometry. DNA extracted from normal B-lymphocytes treated with Sss1 methyltransferase (New England Biolabs, Beverly, MA, USA) was used as a positive control for methylated alleles. Water blanks were used as negative controls.

Statistical analysis

The statistical analysis of the microarray data was performed as described in ‘Gene expression by microarray analysis and RT–PCR’. Comparison of methylation results with certain clinicopathological characteristics (age, sex, TNM stage, histology, smoking history) from the patients and previously determined methylation patterns of 10 genes (RARβ-2, FHIT, RASSF1A, TIMP-3, p16, MGMT, DAPK, ECAD, p14, GSTP1) in a subset of tumors was performed using χ2 test for differences between groups and t-tests between means (Burbee et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b). Correlation coefficients between expression and methylation of genes and between methylation patterns of genes were estimated by Kendall's tau-b method. Overall survival was calculated using Kaplan–Meier log-rank testing.

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Acknowledgements

This study was supported by grants from the Austrian Federal Ministry of Education, Science and Culture (GZ 200.062/2-VI/1/2002), by the Medical-Scientific Fund of the Mayor of the Federal Capital Vienna, by an award from the ‘Fonds der Stadt Wien für Innovative Interdisziplinäre Krebsforschung’ and Lung Cancer SPORE P50 CA70907.

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Correspondence to Sabine Zöchbauer-Müller.

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Heller, G., Fong, K., Girard, L. et al. Expression and methylation pattern of TSLC1 cascade genes in lung carcinomas. Oncogene 25, 959–968 (2006). https://doi.org/10.1038/sj.onc.1209115

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Keywords

  • tumor suppressor gene
  • TSLC1
  • DAL-1
  • MPP3
  • methylation
  • lung cancer

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