Promoter methylation and silencing of the retinoic acid receptor-beta gene in lung carcinomas.
BACKGROUND
Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-beta (RARbeta) gene. Because receptor isoforms RARbeta2 and RARbeta4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARbeta gene in human lung tumors and cell lines.


METHODS
Methylation of the P2 promoter from small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cell lines and tumor samples was analyzed by the methylation-specific polymerase chain reaction (PCR). Expression of RARbeta2 and RARbeta4 was analyzed by reverse transcription-PCR. Loss of heterozygosity (LOH) was analyzed by PCR amplification followed by electrophoretic separation of PCR products. Statistical differences were analyzed by Fisher's exact test with continuity correction.


RESULTS
The P2 promoter was methylated in 72% (63 of 87) of SCLC and in 41% (52 of 127) of NSCLC tumors and cell lines, and the difference was statistically significant (two-sided P:<.001). By contrast, in 57 of 58 control samples, we observed only the unmethylated form of the gene. Four tumor cell lines with unmethylated promoter regions expressed both RARbeta2 and RARbeta4. Four tumor lines with methylated promoter regions lacked expression of these isoforms, but demethylation by exposure to 5-aza-2'-deoxycytidine restored their expression. LOH at chromosome 3p24 was observed in 100% (13 of 13) of SCLC lines and 67% (12 of 18) of NSCLC cell lines, and the difference was statistically significant (two-sided P: =.028).


CONCLUSIONS
Methylation of the RARbeta P2 promoter is one mechanism that silences RARbeta2 and RARbeta4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.
Virmani
A K
AK
Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75390-8593, USA.
Rathi
A
A
Zöchbauer-Müller
S
S
Sacchi
N
N
Fukuyama
Y
Y
Bryant
D
D
Maitra
A
A
Heda
S
S
Fong
K M
KM
Thunnissen
F
F
Minna
J D
JD
Gazdar
A F
AF
D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism*; D018288:Carcinoma, Small Cell / Q000235:genetics / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019656:Loss of Heterozygosity; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism*; D008745:Methylation; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta