Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas

Arvind K. Virmani, Asha Rathi, Sabine Zöchbauer-Müller, Nicoletta Sacchi, Yasuro Fukuyama, David Bryant, Anirban Maitra, Shashank Heda, Kwun M. Fong, Frederik Thunnissen, John D. Minna, Adi F. Gazdar

Research output: Contribution to journalArticle

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Abstract

Background: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-β (RARβ) gene. Because receptor isoforms RARβ2 and RARβ4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARβ 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 RARβ2 and RARβ4 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 RARβ2 and RARβ4. 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 RARβ P2 promoter is one mechanism that silences RARβ2 and RARβ4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.

Original languageEnglish (US)
Pages (from-to)1303-1307
Number of pages5
JournalJournal of the National Cancer Institute
Volume92
Issue number16
StatePublished - Aug 16 2000

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Retinoic Acid Receptors
Small Cell Lung Carcinoma
Tumor Cell Line
Methylation
Non-Small Cell Lung Carcinoma
Carcinoma
Lung Neoplasms
Polymerase Chain Reaction
Lung
Loss of Heterozygosity
decitabine
Genetic Promoter Regions
Genes
Protein Isoforms
Cytoplasmic and Nuclear Receptors
Tretinoin
Reverse Transcription
Chromosomes
Cell Line
Neoplasms

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Virmani, A. K., Rathi, A., Zöchbauer-Müller, S., Sacchi, N., Fukuyama, Y., Bryant, D., ... Gazdar, A. F. (2000). Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas. Journal of the National Cancer Institute, 92(16), 1303-1307.

Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas. / Virmani, Arvind K.; Rathi, Asha; Zöchbauer-Müller, Sabine; Sacchi, Nicoletta; Fukuyama, Yasuro; Bryant, David; Maitra, Anirban; Heda, Shashank; Fong, Kwun M.; Thunnissen, Frederik; Minna, John D.; Gazdar, Adi F.

In: Journal of the National Cancer Institute, Vol. 92, No. 16, 16.08.2000, p. 1303-1307.

Research output: Contribution to journalArticle

Virmani, AK, Rathi, A, Zöchbauer-Müller, S, Sacchi, N, Fukuyama, Y, Bryant, D, Maitra, A, Heda, S, Fong, KM, Thunnissen, F, Minna, JD & Gazdar, AF 2000, 'Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas', Journal of the National Cancer Institute, vol. 92, no. 16, pp. 1303-1307.
Virmani AK, Rathi A, Zöchbauer-Müller S, Sacchi N, Fukuyama Y, Bryant D et al. Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas. Journal of the National Cancer Institute. 2000 Aug 16;92(16):1303-1307.
Virmani, Arvind K. ; Rathi, Asha ; Zöchbauer-Müller, Sabine ; Sacchi, Nicoletta ; Fukuyama, Yasuro ; Bryant, David ; Maitra, Anirban ; Heda, Shashank ; Fong, Kwun M. ; Thunnissen, Frederik ; Minna, John D. ; Gazdar, Adi F. / Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas. In: Journal of the National Cancer Institute. 2000 ; Vol. 92, No. 16. pp. 1303-1307.
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abstract = "Background: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-β (RARβ) gene. Because receptor isoforms RARβ2 and RARβ4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARβ 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 RARβ2 and RARβ4 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 RARβ2 and RARβ4. 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 RARβ P2 promoter is one mechanism that silences RARβ2 and RARβ4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.",
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T1 - Promoter methylation and silencing of the retinoic acid receptor-β gene in lung carcinomas

AU - Virmani, Arvind K.

AU - Rathi, Asha

AU - Zöchbauer-Müller, Sabine

AU - Sacchi, Nicoletta

AU - Fukuyama, Yasuro

AU - Bryant, David

AU - Maitra, Anirban

AU - Heda, Shashank

AU - Fong, Kwun M.

AU - Thunnissen, Frederik

AU - Minna, John D.

AU - Gazdar, Adi F.

PY - 2000/8/16

Y1 - 2000/8/16

N2 - Background: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-β (RARβ) gene. Because receptor isoforms RARβ2 and RARβ4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARβ 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 RARβ2 and RARβ4 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 RARβ2 and RARβ4. 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 RARβ P2 promoter is one mechanism that silences RARβ2 and RARβ4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.

AB - Background: Retinoic acid plays an important role in lung development and differentiation, acting primarily via nuclear receptors encoded by the retinoic acid receptor-β (RARβ) gene. Because receptor isoforms RARβ2 and RARβ4 are repressed in human lung cancers, we investigated whether methylation of their promoter, P2, might lead to silencing of the RARβ 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 RARβ2 and RARβ4 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 RARβ2 and RARβ4. 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 RARβ P2 promoter is one mechanism that silences RARβ2 and RARβ4 expression in many lung cancers, particularly SCLC. Chemical demethylation is a potential approach to lung cancer therapy.

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