Hypermethylation of the retinoic acid receptor-beta(2) gene in head and neck carcinogenesis.
OBJECTIVE
Retinoic acid receptor-beta(2) (RAR-beta(2)) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR-beta(2) gene expression in such lesions can be silenced by promoter methylation.


METHODS
RAR-beta(2) methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR-beta(2) promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR-beta(2) hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates.


RESULTS
Significantly higher RAR-beta(2) hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P = 0.002). RAR-beta(2) methylation in the cell lines was correlated with loss of RAR-beta(2) expression (P = 0.013) and inversely related to the presence of mutated p53 (P = 0.025). The demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) restored RAR-beta(2) inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR-beta(2) promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA.


CONCLUSIONS
RAR-beta(2) silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR-beta(2) inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.
Youssef
Emile M
EM
Departments of Thoracic/Head and Neck Medical Oncology,The University of Texas, MD Anderson Cancer Center, Houston, Texas 77345, USA.
Lotan
Dafna
D
Issa
Jean-Pierre
JP
Wakasa
Kenichi
K
Fan
You-Hong
YH
Mao
Li
L
Hassan
Khaled
K
Feng
Lei
L
Lee
J Jack
JJ
Lippman
Scott M
SM
Hong
Waun K
WK
Lotan
Reuben
R
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000473:pathology; D002455:Cell Division; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D019175:DNA Methylation; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D020868:Gene Silencing; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D007972:Leukoplakia, Oral / Q000235:genetics / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009062:Mouth Neoplasms / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction; D011230:Precancerous Conditions / Q000235:genetics / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000235:genetics; D018168:Receptors, Retinoic Acid / Q000187:drug effects / Q000235:genetics*; D014212:Tretinoin / Q000494:pharmacology
D017931:DNA Primers; D004273:DNA, Neoplasm; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D014212:Tretinoin