Aberrant methylation of N-methyl-D-aspartate receptor type 2B (NMDAR2B) in non-small cell carcinoma

Nine NSCLC cell lines (HCC193, HCC366, HCC515, HCC1171, NCI-H1395, NCI-H1770, NCI-H1993, NCI-H2126, and NCI-H2882) were examined in this study. These cell lines were established and kindly provided by Dr. Adi Gazdar of the University of Texas Southwestern Medical Center. Cell cultures were grown in RPMI-1640 medium (Life Technologies, Inc., Rockville, MD, USA) supplemented with 5% CO₂ at 37°C. Normal bronchial epithelial cells (NHBECs) were cultured as reported previously [8] and normal trachea RNA was obtained from Clontech (Palo Alto, CA, USA).

Surgically resected tumor samples and 120 non-malignant lung tissues were obtained from 216 unselected NSCLC patients who had not received any treatment prior to resection at the Chiba University Hospital, Chiba, Japan, from 1995 to 2000. NSCLC include adenocarcinoma (N = 116), squamous cell carcinoma (N = 76), large cell carcinoma (N = 12), and adeno-squamous cell carcinoma (N = 2). This study was approved by the Institutional Review Board, and written informed consent was obtained from each participant. All patients received curative intent surgery. Resected samples were immediately frozen and stored at -80°C until used. Methylation status was determined for each patient sample, and 20 of the 216 cases were randomly selected for immunohistochemical examination to compare expression and methylation in primary samples.

RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and reverse transcribed with SuperScript II reverse transcriptase (Invitrogen). GAPDH expression was used as an internal control to confirm the success of the reverse transcription reaction. The primer sequences used for reverse transcription of NMDAR2B and GAPDH are shown in Table 1. PCR products were analyzed on 2% agarose gels stained with ethidium bromide. NHBEC and normal trachea were used as normal controls for reverse transcriptase-PCR (RT-PCR).

Five tumor cell lines with negative gene expression were incubated in culture medium with 1 μM of the demethylating agent 5-aza-2'-deoxycytidine (Sigma-Aldrich, St. Louis, MO, USA) for six days, with medium changes on days one, three, and five. Cells were harvested and RNA was extracted on day six.

Genomic DNA was obtained from lung cancer cell lines, cultured nonmalignant cells, primary tumors and adjacent nonmalignant tissues after overnight digestion with sodium dodecyl sulfate and Proteinase K (Life Technologies, Inc.) at 37°C, followed by standard phenol chloroform (1:1) extraction and ethanol precipitation.

DNA was treated with sodium bisulfite as described previously [9]. DNA methylation patterns in the CpG island of the gene were determined using methylation specific real-time quantitative PCR (Taqman-MSP) [7]. All oligonucleotide primer pairs were purchased from Invitrogen, while Taqman probes were purchased from VWR (West Chester, PA, USA). Taqman primer and probe sequences for NMDAR2B and β-actin are listed in Table 1. Serial dilutions of human leukocyte genomic DNA, which was methylated in vitro, were used to construct a calibration curve (Figure 1) (Thermal Cycler Dice^® Real Time System TP 800); all reactions were performed in duplicate. The methylation ratio was defined as the quantity of fluorescence intensity derived from NMDAR2B promoter amplification divided by fluorescence intensity from β-actin amplification and multiplied by 100 [Taqman methylation value (TaqMeth V)]. Cut off value was defined as 1.0, based on previous reports [7].

Four micron sections were used for the tissue arrays. Tissue arrays were deparaffinized in xylene, rehydrated in graded alcohol, and transferred to PBS. Thereafter, antigen retrieval was carried out via a microwave in 0.01 M citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked by incubating sections in hydrogen peroxide (0.3%, v/v) for 15 min. Non-specific binding was blocked with 1% (w/v) BSA in PBS for one hr followed by incubation with anti-NMDAR2B polyclonal antibody (1:100) (Chemicon, Temecula, CA, USA) for 16 hr at 4°C. The primary antibody was detected using biotinylated secondary antibody and peroxidase-labeled streptavidin complex using the Dako LSAB Plus Kit (Dako, Glostrup, Denmark). Color was developed using the chromogen, diaminobenzidine (DAB). Finally, the slides were counter stained with Mayer's hematoxylin and mounted with D.P.X. Parallel sections in which the primary antibody was replaced by nonimmune rabbit IgG of the same isotype were examined to ensure specificity and exclude cross reactivity between the antibodies and conjugates used (negative control).

The staining intensity of NMDAR2B expression was graded semi-quantitatively into none, weak, moderate, and strong. Overexpression of NMDAR2B was considered positive if > 30% of tumor cells were moderately or strongly stained. Slides were blindly evaluated at three different times and the average levels were used by two pathologists (H.T. and Y.M.) for statistical analyses. IHC photoimaging was done by Carl Zeiss MicroImaging GmbH and a digital camera (Canon Power Shot A640).

The gene expression levels of NMDAR2B in NSCLC cell lines were assessed by RT-PCR (Figure 2). A comparison between tumor cell lines and NHBEC and trachea cells showed that five out of nine cell lines had lost NMDAR2B expression (Table 2). The non-expressing cell lines were treated with 5-aza-2'-deoxycytidine (5-Aza-CdR) to confirm that aberrant methylation was responsible for silencing NMDAR2B expression. NMDAR2B expression was upregulated by 5-Aza-CdR in five out of five lines (Figure 2, Table 2). Thus, aberrant methylation was found in five out of nine NSCLC cell lines and was inversely correlated with NMDAR2B expression (Table 2).

NMDAR2B methylation in primary tumors and non-malignant tissues was analyzed by Taqman MSP (Figure 3). Of 216 NSCLCs, 131 (61%) were methylated, while 5 (4%) of 120 corresponding non-malignant lung tissues were methylated, indicating that NMDAR2B methylation was a tumor-specific event (P < 0.0001).

The relationship between methylation and clinical features was examined. No significant associations among gender, age, smoking, histological type, or stage were observed (Table 3). In terms of survival, the presence of NMDAR2B methylation was significantly associated with a better prognosis in squamous cell carcinoma cases (P = 0.002), but not in adenocarcinoma cases (Figure 4). Cox proportional hazard regression analysis determined that NMDAR2B methylation is a prognostic factor independent of TNM staging (Table 4). Prognosis was significantly better for cases of squamous cell carcinoma in which NMDAR2B was methylated.

The typical immunostaining pattern for NMDAR2B in NSCLC is shown in Figure 5. NMDAR2B was expressed in bronchial epithelial cells. Using the criteria described in the Methods section, low NMDAR2B expression was found in 11 of 20 (55%) tumors, while moderate to strong expression was found in nine (45%) tumors. In eight of the 11 cases, there was low expression of NMDAR2B protein and methylation of NMDAR2B. The remaining nine cases exhibited moderate to strong expression of NMDAR2B and unmethylated NMDAR2B. Thus, there was a significant inverse correlation between the DNA methylation status of NMDAR2Band the level of protein expression (P = 0.0014).