Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 May;99(5):958-66.
doi: 10.1111/j.1349-7006.2008.00769.x. Epub 2008 Feb 24.

Epigenetic inactivation of RASSF2 in oral squamous cell carcinoma

Takashi Imai  1 , Minoru Toyota, Hiromu Suzuki, Kimishige Akino, Kazuhiro Ogi, Yohei Sogabe, Lisa Kashima, Reo Maruyama, Masanori Nojima, Hiroaki Mita, Yasushi Sasaki, Fumio Itoh, Kohzoh Imai, Yasuhisa Shinomura, Hiroyoshi Hiratsuka, Takashi Tokino
Affiliations

Epigenetic inactivation of RASSF2 in oral squamous cell carcinoma

Takashi Imai et al. Cancer Sci. 2008 May.

Abstract

Genetic and epigenetic alterations in tumor-suppressor genes play important roles in human neoplasia. Ras signaling is often activated in oral squamous cell carcinoma (OSCC), although Ras mutations are rarely detected in Japanese OSCC patients, and the mechanisms underlying the gene's activation remain unclear. Here, we examined the expression of Ras association family (RASSF) genes in a panel of OSCC cell lines and found that RASSF2 is often downregulated by DNA methylation in OSCC cells. In addition, aberrant methylation of RASSF2 was detected in 12 of 46 (26%) primary OSCC, and 18 (39%) of those OSCC showed methylation of at least one RASSF gene. Ectopic expression of RASSF2 in OSCC cells suppressed cell growth and induced apoptosis. A RASSF2 deletion mutant lacking the Ras-association domain, which was therefore unable to interact with Ras, exhibited less pro-apoptotic activity than the full-length protein, indicating that the pro-apoptotic activity of RASSF2 is related to its association with Ras. Genomic screening of genes regulated by RASSF2 showed that genes involved in immune responses, angiogenesis, and metastasis are suppressed by RASSF2. Our results suggest that epigenetic inactivation of RASSF2 plays an important role in OSCC tumorigenesis, and that RASSF2 may be a useful molecular target for the diagnosis and treatment of OSCC.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Analysis of RASSF gene expression in oral squamous cell carcinoma (OSCC) cell lines. (a) Reverse transcription–polymerase chain reaction analysis of RASSF1–6 in OSCC cell lines. cDNA was prepared from cells treated with either 2 µM 5‐aza‐dC (+) or mock (–). The integrity of the mRNA was confirmed by amplifying glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). The genes analyzed are shown on the left; the cell lines analyzed are shown above the columns. Corresponding negative controls (amplifying without reverse transcription) are shown as RT–. (b) Quantitative analysis of RASSF2 expression in OSCC cell lines treated with either mock (–) or 2 µM 5‐aza‐dC for 72 h (+). RASSF2 Expression was examined with real‐time polymerase chain reaction using a TaqMan expression system. The cell lines examined are shown below the column. The bars indicate means ± SD of three independent experiments. (c) Activation of p44/p42 and AKT in OSCC cell lines. Total cell lysates from the indicated cell lines were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis and western analysis using antibodies that recognize phosphorylated or total p44/p42 or AKT.
Figure 2
Figure 2
Analysis of RASSF gene methylation in oral squamous cell carcinoma (OSCC). (a) Combined bisulfite restriction analysis (COBRA) was carried out using DNA treated with Na‐bisulfite. The cell lines are shown at the top. DNA treated with SssI methylase served as a positive control. DNA from HCT116 cells in which the DNMT1 and DNMT3B loci were knocked out served as a negative control. M, methylated alleles. (b) Analysis of RASSF2 methylation in primary OSCC. COBRA was used to analyze RASSF2 methylation in primary OSCC specimens and corresponding samples of normal tissue. N, normal tissues adjacent to tumors; T, tumors. (c) Profiles of RASSF gene methylation and mutation of K‐ras and Ha‐ras in 46 primary OSCC. Cases in which methylation or mutation was detected are shown as solid boxes. The genes analyzed are shown at the top.
Figure 3
Figure 3
Bisulfite sequencing of RASSF2. (a) Schematic representation of the RASSF2A CpG island. CpG sites are indicated by vertical bars; the region analyzed by combined bisulfite restriction analysis (COBRA) and bisulfite sequencing is indicated by a solid bar. Arrows indicate the transcription start sites. Arrowhead indicates the CpG site analyzed by COBRA. The cell lines studied are shown below the columns. (b) Open squares indicate unmethylated CpG sites; filled squares indicate methylated CpG sites. (c) Demethylation of RASSF2 in OSCC cells treated with 2 µM 5‐aza‐dC for 72 h. The cell lines examined are shown below the column.
Figure 4
Figure 4
Suppression of cell growth by RASSF2. (a) Schematic diagrams of RASSF2 and the deletion mutants used. (b,c) Suppression of growth evaluated by assaying geneticin‐resistant colony formation. HSC4 and OSC19 cells were transfected with either pCDNA3.1 (control plasmid), RASSF2A, RASSF2‐ΔN, or RASSF2A‐ΔC and incubated with 0.6 mg/mL G418 in the RPMI‐1640 medium. After 14 days, plates were stained with Giemsa solution (b), and the colonies were counted (c). The bars indicate mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 versus vector control (pcDNA).
Figure 5
Figure 5
Induction of apoptosis by RASSF2. (a) Immunocytochemical analysis of RASSF2 in OSC19 cells infected with either Ad‐RASSF2 or Ad‐LacZ. RASSF2 was visualized using a fluorescein isothiocyanate (FITC)‐conjugated secondary antibody. Nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Ho‐1‐u‐1 and MoT cells were also examined as positive controls for RASSF2 expression. (b) Flow cytometric analysis of the pro‐apoptotic effect of RASSF2 in OSC19 cells. The incidence of sub‐G1 cells was determined 72 h after infection.
Figure 6
Figure 6
Identification of the genes regulated by RASSF2. Real‐time polymerase chain reaction analysis confirming the results of the microarray analysis. Expression of putative RASSF2 target genes was examined using cDNA from HSC4 and OSC19 cells infected with Ad‐RASSF2. HSC4 and OSC19 cells infected with Ad‐LacZ served as a control, and the Ad‐RASSF2/Ad‐LacZ ratio is shown on the y‐axis. The genes examined are shown below the columns; the bars indicate SE.
Figure 7
Figure 7
Suppression of nuclear factor (NF)‐κB activity by RASSF2. (A) HSC4 and OSC19 cells were infected with Ad‐RASSF2, and the transcriptional activity of NF‐κB was examined using a luciferase reporter plasmid. Ad‐lacZ served as the control. (B) Western analysis of nuclear NF‐κB/p50. Nuclear lysates from HSC4 and OSC19 cells infected with Ad‐RASSF2 were immunoblotted using anti‐p50 antibody. Cell lysates from cells infected with Ad‐LacZ served as the control. Levels of proliferating cell nuclear antigen (PCNA) protein were used as a loading control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005; 55: 74–108. - PubMed
    1. Gangane N, Chawla S, Subodh A, Gupta S, Sharma SM. Reassessment of risk factors for oral cancer. Asian Pac J Cancer Prev 2007; 8: 243–8. - PubMed
    1. Petti S, Scully C. Oral cancer: the association between nation‐based alcohol‐drinking profiles and oral cancer mortality. Oral Oncol 2005; 41: 828–34. - PubMed
    1. Chiba I, Shindoh M, Yasuda M et al . Mutations in the p53 gene and human papillomavirus infection as significant prognostic factors in squamous cell carcinomas of the oral cavity. Oncogene 1996; 12: 1663–8. - PubMed
    1. Ha PK, Califano JA. Promoter methylation and inactivation of tumour‐suppressor genes in oral squamous‐cell carcinoma. Lancet Oncol 2006; 7: 77–82. - PubMed

Publication types