Expression of the candidate tumor suppressor gene hSRBC is frequently lost in primary lung cancers with and without DNA methylation

Abstract

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=52), we designed primers for a methylation-specific PCR assay. Methylation was detected in 41% of primary non-small-cell lung cancers (NSCLC) (N=107) and in 80% of primary small-cell lung cancers (SCLC) (N=5), but was seen only in 4% of corresponding nonmalignant lung tissues (N=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=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′-deoxycytidine. In addition, 45% of the 76 hSRBC immunostaining-negative 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.

Introduction

Frequent allele loss at chromosomal regions is a strong evidence for the presence of one or more tumor suppressor genes (TSG) in these regions. Chromosome 11p15.5–p15.4 has been recognized as such a region since allele loss at this region was frequently observed in a variety of malignant diseases including lung cancer (Vandamme et al., 1992; Winqvist et al., 1995; Baffa et al., 1996; Tran and Newsham, 1996). Recently, the human SRBC (hSRBC) gene has been mapped to this chromosomal region (Xu et al., 2001). A cDNA clone encoding human SRBC (serum deprivation response factor (sdr)-related gene product that binds to c-kinase) was isolated when a normal mammary epithelial cDNA library was screened by the yeast two-hybrid method using a recombinant protein containing amino acids 1–304 of the breast cancer susceptibility gene BRCA1 as the ‘bait’ (Xu et al., 2001). The hSRBC gene encodes an open-reading frame of 261 amino acids with a leucine zipper-like motif in its amino-terminal region (GenBank accession number AF408198). As a BRCA1-interacting protein, hSRBC may also be involved in DNA damage response and participate in BRCA1-mediated tumor suppression pathways. hSRBC protein expression is frequently lost in breast and lung cancer cell lines compared to normal mammary and lung epithelial cells, suggesting that hSRBC is a candidate TSG involved in the pathogenesis of breast and lung carcinomas (Xu et al., 2001). This loss was associated with tumor cell line acquired promoter methylation. Aberrant methylation of normally unmethylated CpG islands located in or near the promoter region of cancer-related genes has been associated with transcriptional inactivation of these gene in human cancer (Jones and Baylin, 2002). It is becoming increasingly apparent that aberrant methylation (referred to as methylation) of the promoter region of genes is the main mechanism for gene silencing in tumors (Jones and Baylin, 2002). Bisulfite genomic sequencing of several breast and lung cancer cell lines revealed that the promoter region of hSRBC is frequently methylated in these cell lines. Moreover, hSRBC expression in the breast cancer cell line MCF7 was partially restored by treatment with the deacetylase inhibitor trichostatin A, whereas treatment with both trichostatin A and, additionally, the demethylation reagent 5-aza-2′-deoxycytidine (5-aza-dC) induced hSRBC expression in MCF7 cells (Xu et al., 2001). Thus, there is a strong evidence that tumor acquired promoter region methylation is an important mechanism for inactivating hSRBC in some human tumors.

To further investigate the role of hSRBC in cancer pathogenesis, we developed a methylation-specific PCR (MSP) assay, which is a very suitable method to investigate methylation in a large number of primary tumor samples. Using MSP, we analysed the methylation status of the promoter region of hSRBC in a total of 267 samples consisting of primary lung carcinomas, corresponding nonmalignant lung tissues and lung cancer cell lines. In addition, immunostaining of hSRBC was performed in primary lung carcinomas and lung cancer cell lines, and these results were compared with the methylation pattern of hSRBC. Furthermore, the results from both the methylation analysis and the immunostaining were compared with clinicopathological characteristics of these patients.

Results

hSRBC expression by RT–PCR and immunostaining

hSRBC expression was investigated in 18 lung cancer cell lines by RT–PCR. The expression of this gene was observed only in eight (44%) lung cancer cell lines. Eight of 13 (62%) non-small-cell lung cancer (NSCLC) cell lines expressed hSRBC, while none of the five (0%) small cell cancer (SCLC) cell lines expressed this gene.

In 101 primary NSCLC samples and in 11 lung cancer cell lines, hSRBC protein expression was investigated by immunostaining. In all, 18 (18%) primary NSCLCs demonstrated a membranous staining pattern in the neoplastic cells and were scored as positive for hSRBC protein expression (Figure 1). However, complete loss of hSRBC protein expression was observed in 62 (61%) (Figure 1) and a heterogeneous staining pattern was observed in 15 (15%) of the primary NSCLCs (Figure 2). In some cases with a heterogeneous pattern, the areas of positive staining were randomly distributed throughout the carcinoma (Figure 2). In other cases, most of the staining in the positive areas was localized to the periphery of tumor nests. Samples with a heterogeneous staining pattern were combined with the group of samples with complete loss of protein expression resulting in 77 (76%) samples negative for hSRBC immunostaining. Six samples demonstrated only cytoplasmatic staining and were excluded from the analysis. Only two of 11 (18%) lung cancer cell lines expressed hSRBC protein by immunostaining (Figure 3). Interestingly, expression was observed in two of six (33%) NSCLC cell lines, but in none of the five (0%) investigated SCLC cell lines.

Figure 1
figure1

Examples of hSRBC immunostaining in primary NSCLCs. (a) Positive staining with distinct membrane hSRBC reactivity in all areas of the tumor (patient #76). (b) No hSRBC staining in tumor with preserved reactivity in admixed mesenchymal cells (patient #74)

Figure 2
figure2

Heterogeneous hSRBC expression pattern in the primary NSCLC sample from patient #123. (a) Area of hSRBC expression in the tumor sample and (b) area with loss of hSRBC expression in tumor cells of the same sample while the admixed stroma is positive

Figure 3
figure3

Examples of hSRBC immunostaining in lung cancer cell lines. (a) hSRBC is expressed in cells of cell line NCI-H1264 and (b) hSRBC expression is lost in cells of cell line NCI-H661

Frequency of hSRBC methylation by MSP

We performed genomic sequencing of the hSRBC 5′CpG island (GenBank accession number AF408198) after bisulfite treatment of DNA from five lung cancer cell lines, which did not express hSRBC by Western immunoblotting as described by Xu et al. (2001). The methylation pattern of the 5′CpG island of the hSRBC gene indicated that the region between nts 3520 and 3866 was highly methylated. In addition, bisulfite genomic sequencing was also performed in several primary tumor samples, confirming the results in tumor cell lines. An example is shown in Figure 4. Based on these results, we designed a set of PCR primers that distinguish between methylated and unmethylated DNA sequences in the 5′ region of the hSRBC gene. Using these primers in MSP assay, we determined the frequency of hSRBC methylation in primary NSCLC samples and corresponding nonmalignant lung tissues, primary SCLC specimens and lung cancer cell lines. The results are summarized in Table 1 and examples for MSP are shown in Figure 5. hSRBC promoter region methylation was frequent in primary lung tumors and rare in nonmalignant lung specimens. In addition, the unmethylated form of hSRBC was found in 100% of nonmalignant lung specimens and in 100% of primary tumors, which had been only macroscopically dissected, and thus all samples had some contamination with normal cells. By contrast, the lung cancer cell lines represent pure populations of tumor cells and we found these tumor lines to have either methylated or unmethylated hSRBC alleles.

Figure 4
figure4

Representative part of the 5′CpG island sequence of the hSRBC gene in lung cancer sample #34. Methylated cytosines in CpG sites are indicated with an arrow

Table 1 Frequency of hSRBC methylation in primary lung carcinomas, corresponding nonmalignant lung specimens and lung cancer cell lines
Figure 5
figure5

Examples of hSRBC MSP in primary NSCLCs. (a) The amplified product represents the methylated form of hSRBC with a product size of 347 bp (M). Samples whose lanes do not show a band are not methylated. (b) To confirm the presence and integrity of DNA, MSP was also performed with the primer pair specific for the unmethylated form of hSRBC and all primary NSCLC samples tested positive (U) because of contamination with normal cells

Comparison of hSRBC methylation with hSRBC expression by immunostaining

A statistical significant correlation between methylation of hSRBC and loss of hSRBC protein expression was found for both primary NSCLCs (P=0.00003) and lung cancer cell lines (P=0.01) (Table 2). All cases with methylation exhibited loss of hSRBC protein expression. Of interest, there were 34/76 (45%) of tumors that had lost hSRBC expression but were not methylated (Table 2). This indicates that there are other mechanisms besides tumor acquired promoter methylation leading to loss of hSRBC expression.

Table 2 Correlation between methylation of hSRBC and loss of hSRBC protein expression in primary NSCLCs and in lung cancer cell lines

hSRBC re-expression after treatment with 5-aza-dC

Loss of hSRBC expression was found in the lung cancer cell line NCI-H1993 by RT–PCR. In addition, this cell line was hSRBC methylated, making it a suitable candidate for treatment with the demethylating agent 5-aza-dC. Re-expression of hSRBC was seen after treatment of NCI-H1993 cells with 5-aza-dC, confirming the role of methylation in regulating hSRBC expression (Figure 6).

Figure 6
figure6

Re-expression of hSRBC after treatment of lung cancer cells with 5-aza-dC. The lung cancer cell line NCI-H1993 that lack hSRBC expression was grown in the presence (+lanes) and absence (−lanes) of 0.5 μ M 5-aza-dC for 6 days. Afterwards, RT–PCR was performed. GAPDH was used as a control

Comparison of hSRBC methylation and loss of protein expression with clinicopathological characteristics and molecular abnormalities

Both the methylation results and data about loss of hSRBC protein expression from primary NSCLC samples were compared with clinicopathological characteristics from these patients including sex, age, histology, TNM classification of the tumors, smoking history and overall survival of the patients. In addition, a comparison of our results with certain molecular abnormalities that had been investigated previously was performed. These abnormalities included K-ras codon 12, and p53 exons 5–8 mutations, allele loss at 1p (MYCL), 3p21 (D3S1029), 3p25.3–26.2 (D3S1038), 8p (LPL), 9p (IFNA, D9S126), 13q (RB, D13S260) and 18q (DCC), and the methylation status of the genes retinoic acid receptor β-2 gene (RARβ), RAS association domain family 1A (RASSF1A), FHIT, tissue inhibitor of metalloproteinase-3 (TIMP-3), p16INK4a (p16), O6-methylguanine-DNA-methyltransferase (MGMT), death-associated protein kinase (DAPK), E-cadherin (ECAD), p14ARF (p14), BLU and glutathione S-transferase P1 (GSTP1). No significant correlation between hSRBC methylation or loss of hSRBC protein expression and any of the clinicopathological characteristics was observed. However, a statistically significant association between hSRBC methylation and loss of heterozygosity at the MYCL locus at chromosome 1p was detected (P=0.005). Moreover, a correlation between methylation of hSRBC and methylation of the RARβ (P=0.04) was found. No correlation between hSRBC methylation and other investigated molecular abnormalities was observed.

Discussion

We studied the methylation status of the 5′CpG island of hSRBC in primary lung cancers, adjacent nonmalignant tissues and lung cancer cell lines. We first sequenced sodium bisulfite-treated DNA from lung cancer cell lines in the hSRBC 5′CpG island region as reported by Xu et al. (2001). Based on these results, we designed a set of primers for MSP. Using this primer set, we found a high percentage of primary lung cancer samples and lung cancer cell lines methylated for hSRBC. Interestingly, both primary SCLC samples and SCLC cell lines were methylated in a higher percentage compared to primary NSCLC samples and NSCLC cell lines. In concordance, loss of hSRBC expression was more frequently observed in SCLC compared to NSCLC cancer cell lines. While the comparison of hSRBC methylation between SCLC and NSCLC cell lines was statistically significant (P=0.0004), the comparison between primary SCLC samples and primary NSCLC specimens did not reach statistical significance because of the small number of primary SCLC samples. Different methylation frequencies between SCLC and NSCLC samples also have been reported for the genes p16, adenomatous polyposis coli (APC), H-cadherin (CDH13) and RASSF1A (Dammann et al., 2000, 2001; Burbee et al., 2001; Toyooka et al., 2001). In three of four nonmalignant lung samples where hSRBC methylation was detected, the corresponding tumor was also methylated for this gene, suggesting that there was some contamination with adjacent malignant cells. Another explanation for this finding might be that hSRBC methylation occurs already in premalignant changes as it has been demonstrated for other genes (Kersting et al., 2000; Belinsky et al., 2002; Soria et al., 2002; Zöchbauer-Müller et al., 2003).

We compared hSRBC methylation and loss of expression of hSRBC by immunostaining in primary NSCLC samples and lung cancer cell lines. A significant correlation between hSRBC methylation and loss of expression by immunostaining for both primary NSCLC samples and lung cancer cell lines was observed. The heterogeneity of hSRBC immunostaining in 15 primary NSCLC samples may be explained by tumor heterogeneity for methylation. Every case with hSRBC methylation exhibited loss of hSRBC protein expression, which is an evidence that methylation plays a major role in inactivating hSRBC. However, 45% of primary NSCLCs lacked hSRBC expression that did not show hSRBC methylation, and thus other mechanisms besides methylation are responsible for silencing hSRBC expression. Although Xu et al. (2001) detected hSRBC-coding region mutations in a few ovarian and lung cancer cell lines, they do not seem to play an important role in inactivating hSRBC. However, mutations as well as deletions may be responsible for the inactivation of hSRBC in some cases. Recently, DNA methylation and chromatin structure has been linked. Methylated DNA recruits methyl binding proteins, which attract a chromatin-remodeling complex along with proteins that modify histones by deacetylating them, thus closing down DNA to transcription (Jones and Baylin, 2002). The role of histone deacetylation in silencing hSRBC has not been determined yet and needs to be investigated in future studies.

We did not find any significant correlation between hSRBC methylation status and clinicopathological characteristics including sex, age, histology, TNM classification of the tumors, smoking history and overall survival of the NSCLC patients. However, we observed a strong correlation between hSRBC methylation and allele loss at the MYCL locus at chromosome 1p, which needs to be further evaluated.

In conclusion, we found frequent methylation of hSRBC associated with loss of hSRBC expression in primary lung cancers and lung cancer cell lines. Moreover, we were able to show that hSRBC methylation is reversible with 5-aza-dC. Our results support the hypothesis that inactivation of hSRBC is involved in the pathogenesis of lung cancer also providing evidence that hSRBC is functioning as a TSG. However, the function of hSRBC needs to be determined.

Materials and methods

Tissue samples

Tissues were collected after obtaining appropriate Institutional Review Board permission and informed consent from the patients. From NSCLC patients, tumor samples (N=107) and corresponding normal lung tissues (N=103) were obtained surgically in the Prince Charles Hospital in Brisbane, Australia. This cohort of patients had been investigated previously for various genetic abnormalities including LOH at multiple chromosomal sites, K-ras codon 12 mutations, p53 exons 5–8 mutations and methylation analysis of multiple genes (Fong et al., 1995a, 1995b, 1996; Zöchbauer-Müller et al., 2001a, 2001b; Burbee et al., 2001; Agathanggelou et al., 2003). There were 76 males and 31 females, age 28–81 (mean 61) years at diagnosis. In all, 61 patients had stage I, 21 stage II, 24 stage IIIA and one patient stage IIIB disease. Histological subtypes included 45 adenocarcinomas, 43 squamous cell carcinomas, 11 adenosquamous carcinomas, four large-cell carcinomas, three atypical carcinoids and one typical carcinoid. In all, 98 patients were ever smokers (consisting of current and former smokers) with mean exposure of 31 pack-years, and nine were never smokers. Survival data of 5 or more years were available for most patients. SCLC samples (N=5) were obtained from patients who underwent a surgical procedure because of suspect lung lesions in the University Hospital in Vienna, Austria. There were five males with an age of 54–76 (mean 63) years at diagnosis.

Lung cancer cell lines

In all, 52 lung cancer cell lines (24 NSCLC, 24 SCLC, one carcinoid and three not further specified NSCLC cell lines) which were generated by us and which have been previously described were analysed for the methylation status of hSRBC (Phelps et al., 1996; Wistuba et al., 1999). In 18 and in 11 lung cancer cell lines, expression analysis of hSRBC was performed by RT–PCR and immunostaining, respectively.

hSRBC expression by RT–PCR and 5-aza-dC treatment of lung cancer cell line NCI-H1993

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 the RT–PCR are IndexTerm5′-AGC TCC ACG TTC TGC TCT TCA-3′ (forward) and IndexTerm5′-GGC GTG AGT GCT ACA TTC TGA-3′ (reverse). Primers for glyceraldehyde-3-phosphate dehydrogenase were used to confirm RNA integrity (Virmani et al., 2000). For gene reactivation, NCI-H1993 cells (2 × 105/ml) were seeded in 10% RPMI and treated with 0.5 μ M 5-aza-dC (Sigma Chemical Co., St Louis, MO, USA) for 6 days. Gene reactivation was tested by RT–PCR.

Immunostaining

For immunohistochemical protein expression analysis, we used a previously described mouse monoclonal antibody developed in one of our laboratories (Xu et al., 2001). Deparaffinized and rehydrated formalin-fixed tissue sections were reacted with primary antibody at a concentration of 0.2 μg/ml for 2 h, following a 20 min antigen retrieval step in subboiling 0.1 M EDTA buffer (pH 8.0) and a 20 min blocking step in 0.3% H2O2. The antigen detection reaction utilized the mouse Envision kit from Dako (Carpinteria, CA, USA). Diaminobenzidine and hematoxylin were used as chromogen and counterstain, respectively. Nonspecific mouse IgG was used in lieu of the hSRBC-specific antibody in negative control reactions. Non-neoplastic lung tissue and a cell block of lung cancer cell line H2141 were used as positive and negative external controls, respectively (Xu et al., 2001). A staining reaction was scored as positive if there was distinct membrane reactivity in all areas of the tumor. A tumor was scored as negative if there was complete or, less commonly, partial loss of membrane staining with preserved reactivity in adjacent benign cells.

Methylation-specific PCR

DNA was prepared from tissue samples, cell lines by standard methods and bisulfite modification of genomic DNA was performed as reported by Herman et al. (1996). Treatment of genomic DNA with sodium bisulfite converts unmethylated but not methylated cytosines to uracil, which are then converted to thymidine during the subsequent PCR step giving sequence differences between methylated and unmethylated DNA. PCR primers that distinguish between methylated and unmethylated DNA were designed. Primer sequences were determined based on the sequence data of the 5′CpG island of the gene as described in the ‘Results’ section. Primer sequences for the methylated hSRBC reaction were IndexTerm5′-GTT TCG GGT TTT GAT AGT TCG CG-3′ (forward) and IndexTerm5′-CCT TCC GCT ATC CCG CGC CG-3′ (reverse), and primer sequences for the unmethylated hSRBC reaction were IndexTerm5′-GTT TTG GGT TTT GAT AGT TTG TG-3′ (forward) and IndexTerm5′-CCT TCC ACT ATC CCA CAC CA-3′ (reverse). The PCR mixture has been reported previously (Zöchbauer-Müller et al., 2001a, 2001b). Amplification was carried out in a 9700 Perkin-Elmer Thermal Cycler. DNA from peripheral blood lymphocytes treated with SssI methyltransferase (New England BioLabs Inc., Beverly, MA, USA) was used as a positive control for methylated alleles. Negative control samples without DNA were included for each set of PCR. PCR products were analysed on 2% agarose gels and visualized under UV illumination. The PCR reactions for all samples demonstrating methylation were repeated to confirm these results.

Other molecular markers

Available molecular markers from previous studies in the 107 NSCLC patients included K-ras codon 12, and p53 exons 5–8 mutations and allele loss at 1p (MYCL), 3p21 (D3S1029), 3p25.3–26.2 (D3S1038), 8p (LPL), 9p (IFNA, D9S126), 13q (RB, D13S260) and 18q (DCC) (Fong et al., 1995a, 1995b, 1996). In addition, data about the methylation status of the genes RARβ, RASSF1A, FHIT, TIMP-3, p16, MGMT, DAPK, ECAD, p14, BLU and GSTP1 were available from these patients (Burbee et al., 2001; Zöchbauer-Müller et al., 2001a, 2001b; Agathanggelou et al., 2003).

Statistics

Statistical analysis was performed using χ2 test for differences between groups and t-tests between means. Overall survival was calculated using Kaplan–Meier log-rank testing.

Accession codes

Accessions

GenBank/EMBL/DDBJ

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Acknowledgements

This work was supported by grants from the Austrian Science Foundation (J1658-MED, J1860-MED), the Austrian Federal Ministry of Education, Science and Culture (GZ 200.062/2-VI/1/2002), the Medical-Scientific Fund of the Mayor of the Federal Capital Vienna (project number 2089), Lung Cancer SPORE P50 CA70907, Department of Defense Grant DAMD170110422 and The Susan G Komen Foundation.

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Correspondence to Sabine Zöchbauer-Müller.

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Zöchbauer-Müller, S., Fong, K., Geradts, J. et al. Expression of the candidate tumor suppressor gene hSRBC is frequently lost in primary lung cancers with and without DNA methylation. Oncogene 24, 6249–6255 (2005). https://doi.org/10.1038/sj.onc.1208775

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Keywords

  • tumor suppressor gene
  • lung cancer
  • hSRBC
  • epigenetic
  • methylation-specific PCR

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