Surgical oncology and reconstruction
Comprehensive Epigenetic Analysis Using Oral Rinse Samples: A Pilot Study

https://doi.org/10.1016/j.joms.2011.04.021Get rights and content

Purpose

To prove that chromatin immunoprecipitation assay can be performed with oral rinse samples and to develop a protocol for comprehensive analysis of functional interactions among DNA methylation, histone modification, and gene expression using such samples.

Materials and Methods

Eleven cancer cell lines and oral rinse samples from 10 patients with oral squamous cell carcinoma and 3 healthy subjects were examined. The expression of CDKN2A, a tumor suppressor gene, was determined by reverse transcription/polymerase chain reaction and immunohistochemistry. Promoter DNA methylation was assessed by methylation-specific polymerase chain reaction. Chromatin modifications were analyzed by a chromatin immunoprecipitation assay using antibodies for dimethylation and acetylation of lysine 9 of histone H3.

Results

Epigenetic control of CDK2NA was observed in vitro in 11 cancer cell lines. Using the present protocol, comprehensive epigenetic analysis could be successfully performed with oral rinse samples. All patients were comfortable using the prescribed amount (16 mL) of normal saline to rinse their mouths. Nine patients (90%) and 1 healthy subject (33%) showed dimethylation of lysine 9 of histone H3. Moreover, 8 patients (80%) showed hypoacetylation of lysine 9 of histone H3, which was not observed in healthy subjects.

Conclusions

The present study showed for the first time that chromatin modifications can be analyzed using oral rinse samples by chromatin immunoprecipitation analysis. To evaluate the contribution of histone modifications for carcinogenesis of oral squamous cell carcinoma, studies including a larger number of subjects should be conducted in the future.

Section snippets

Cell Lines

Human tongue SCC cell lines HSC-3 and HSC-4 and human gingival carcinoma cell line Ca9-22 were obtained from RIKEN BioResource Center (Ibaraki, Japan). Human breast cancer (MCF-7 and T-47D), human lung cancer (A427 and NCI-H292), human pancreatic carcinoma (BxPC3 and HPAFII), and human colon adenocarcinoma (LS174T and Caco2) cell lines were obtained from American Type Culture Collection (Manassas, VA). HSC-3, Ca9-22, MCF-7, A427, HPAFII, LS174T, and Caco2 cells were cultured in Eagle's Minimum

Expression and Epigenetic Status of CDKN2A in Cancer Cell Lines

To confirm the previously reported epigenetic mechanism of CDKN2A and establish an experimental method for further study using oral rinse samples, RT-PCR, MSP, and ChIP analyses were performed using 11 human cancer cell lines. HSC-3, HSC-4, and HPAFII cells expressed CDKN2A mRNA, as examined by RT-PCR analysis (Fig 1A), but LS174T cells showed relatively low expression. Ca9-22, MCF-7, T-47D, A427, NCI-H292, BxPC-3, and Caco2 cells did not show CDKN2A mRNA expression.

The status of DNA promoter

Discussion

DNA methylation and chromatin modification are essential for regulating gene activity.8 DNA methylation effectively downregulates gene activity by the addition of a methyl group to the 5-carbon of a cytosine base. Less specifically, modification of the chromatin structure can be achieved by multiple mechanisms that cause upregulation or downregulation of the associated gene.3, 21 Pathologic events such as carcinogenesis can be caused by a drastic alteration in established methylation patterns

Acknowledgments

This work was supported by Grants-in-Aid 19390521 (K. Sugihara), 19791533 (T. Hamada), 21792020 (T. Hamada) and 20014022 (S. Yonezawa) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the JSPS Fellowship Grant-in-Aid 219447 (N. Yamada).

References (29)

  • H. Shi et al.

    Triple analysis of the cancer epigenome: An integrated microarray system for assessing gene expression, DNA methylation, and histone acetylation

    Cancer Res

    (2003)
  • V. Mutskov et al.

    Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9

    EMBO J

    (2004)
  • J.T. DeAngelis et al.

    An overview of epigenetic assays

    Mol Biotechnol

    (2008)
  • P. Collas

    The state-of-the-art of chromatin immunoprecipitation

    Methods Mol Biol

    (2009)
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