title: Comprehensive epigenetic analysis using oral rinse samples: a pilot study.
abstract: OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Maxillofacial Diagnostic and Surgical Science, Field of Oral and Maxillofacial Rehabilitation, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kusumoto
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Hamada
authors.forename: Norishige
authors.initials: N
authors.lastname: Yamada
authors.forename: Satoshi
authors.initials: S
authors.lastname: Nagata
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kanmura
authors.forename: Izumi
authors.initials: I
authors.lastname: Houjou
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kamikawa
authors.forename: Suguru
authors.initials: S
authors.lastname: Yonezawa
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Sugihara
mesh_terms: D000107:Acetylation; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism; D016022:Case-Control Studies; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis / Q000235:genetics*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D019942:Genes, p16 / Q000502:physiology; D006657:Histones / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009061:Mouth Mucosa / Q000473:pathology*; D009062:Mouth Neoplasms / Q000235:genetics* / Q000378:metabolism; D010865:Pilot Projects; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D006657:Histones; D008239:Lysine