Methylation associated inactivation of RASSF1A and its synergistic effect with activated K-Ras in nasopharyngeal carcinoma

Abstract

Background: Epigenetic silencing of tumor suppressor genes associated with promoter methylation is considered to be a hallmark of oncogenesis. RASSF1A is a candidate tumor suppressor gene which was found to be inactivated in many human cancers. Although we have had a preliminary cognition about the function of RASSF1A, the exact mechanisms about how RASSF1A functions in human cancers were largely unknown. Moreover, the effect of mutated K-Ras gene on the function of RASSF1A is lacking. The aim of this study was to investigate the expression profile and methylation status of RASSF1A gene, and to explore its concrete mechanisms as a tumor suppressor gene in Nasopharyngeal Carcinoma.

Methods: We examined the expression profile and methylation status of RASSF1A in two NPC cell lines, 38 primary nasopharyngeal carcinoma and 14 normal nasopharyngeal epithelia using RT-PCR and methylated specific PCR(MSP) respectively. 5-aza-dC was then added to confirm the correlation between hypermethylation status and inactivation of RASSF1A. The NPC cell line CNE-2 was transfected with exogenous pcDNA3.1(+)/RASSF1A plasmid in the presence or absence of mutated K-Ras by liposome-mediated gene transfer method. Flow cytometry was used to examine the effect of RASSF1A on cell cycle modulation and apoptosis. Meanwhile, trypan blue dye exclusion assays was used to detect the effect of RASSF1A transfection alone and the co-transfection of RASSF1A and K-Ras on cell proliferation.

Results: Promoter methylation of RASSF1A could be detected in 71.05% (27/38) of NPC samples, but not in normal nasopharyngeal epithelia. RASSF1A expression in NPC primary tumors was lower than that in normal nasopharyngeal epithelial (p < 0.01). Expression of RASSF1A was down-regulated in two NPC cell lines. Loss of RASSF1A expression was greatly restored by the methyltransferase inhibitor 5-aza-dC in CNE-2. Ectopic expression of RASSF1A in CNE-2 could increase the percentage of G0/G1 phase cells (p < 0.01), inhibit cell proliferation and induce apoptosis (p < 0.001). Moreover, activated K-Ras could enhance the growth inhibition effect induced by RASSF1A in CNE-2 cells (p < 0.01).

Conclusion: Expression of RASSF1A is down-regulated in NPC due to the hypermethylation of promoter. Exogenous expression of RASSF1A is able to induce growth inhibition effect and apoptosis in tumor cell lines, and this effect could be enhanced by activated K-Ras.

Figure 1

(a) Expression level of RASSF1A in NPC cell lines, normal nasopharyngeal epithelial and primary tumor biopsies by RT-PCR, T, primary nasopharyngeal tumor tissues; N, normal nasopharyngeal epithelial; M; marker I. GAPDH was amplified as an internal control. (b) Summary of overall expression of RASSF1A in 38 primary NPC tumors and 14 normal nasopharyngeal epithelial biopsies. RASSF1A expression was significantly down-regulated in NPC primary tumors compared with normal nasopharyngeal epithelial (p < 0.01, Mann-Whitney's U test).

Figure 2

(a) Methylation-specific PCR analysis of RASSF1A promoter region in NPC primary tumors and normal nasopharyngeal tissues. Three NPCs (T12, T22, T25) and two normal nasopharyngeal (N12, N10) were showed as examples. DNA modified by methylase SssI severed as a positive methylation control and water was included as blank control. M: methylated alleles; U: unmethylated alleles. (b) Methylation status of RASSF1A in NPC cell lines CNE-1 and CNE-2, DNAs from these two cell lines could be amplified with both methylated (M) and unmethylated (U) DNA-specific primers.

Figure 3

(a) Re-expression of RASSF1A by treatment with 5-aza-2'-deoxycytidine in CNE-2 cell lines at different concentration (0, 1, 3, 5, 7, 10 μmol/L), and GAPDH was amplified as an internal control. (b) Summary of RASSF1A expression in RASSF1A-methylated and--unmethylated NPC primary tumors. Inactivation of RASSF1A expression was significantly correlated with promoter hypermethylation of RASSF1A (p < 0.05, Mann-Whitney's U test). (c) The methylation status of RASSF1A after the treatment of 0, 1, 3, 5, 7, 10 μmol/L of 5-aza-2'-deoxycytidine in CNE-2 cells.

Figure 4

RASSF1A-mediated growth inhibition and cell death is enhanced by K-RasG12V. CNE-2 cells were transiently transfected with RASSF1A ± activated K-Ras. Trypan blue was added in situ after 48 h, and the dye uptake was quantitated. (a) Transfect efficiency of RASSF1A and K-RasG12V is confirmed by RT-PCR and western-blot. B: blank group, V: empty vector group, E: experimental group;(b) Cell death assays; up-panel: CNE-2 cells were transfected with RASSF1A ± K-RasG12V, phase contrast microscopic digital images were taken at 48 h post-transfection, RASSF1A promoted a modest growth inhibition that was enhanced by the presence of activated K-RasG12V; lower-panel: Trypan blue in situ staining, the dye uptake was enhanced when RASSF1A was co-expressed with activated K-Ras.

Figure 5

Quantification analysis of the result of cell death assay is the average of three experiments. *: vs Vector group, p < 0.001; (Black triangle): vs RASSF1A group, p < 0.01.

Figure 6

Ectopic expression of RASSF1A induces cell cycle arrest. (a) Cell cycle arrest effect of RASSF1A, the CNE-2 cells were transiently transfected with either empty vectors or RASSF1A-expression vectors, after 48 h, the CNE2-RASSF1A cells showed a 11% increase in G0/G1 phrase cells than CNE2-empty vector cells. (b) The statistical analysis of the cell cycle distribution. *: vs Vector group, p < 0.01.

Figure 7

RASSF1A promotes apoptosis that is enhanced by K-RasG12V. (a) CNE-2 cells were transiently transfected with empty vector and RASSF1A-expression vectors in the presence or absence of activated K-Ras, 48 h post transfection, empty vector cells showed 4.1% of apoptosis rate, RASSF1A expression cells was 22.7%, and RASSF1A + activated K-Ras expression CNE-2 cells showed 36.0% of apoptosis rate. (b) The statistical analysis of the apoptotic cells in each group. *: vs Vector group, p < 0.001; (Black triangle): vs RASSF1A group, p < 0.001.