Related Experiment Video
Updated: Aug 14, 2026
Isolation of Lung Retinoid-Containing Cells by Cell Sorting
Published on: April 11, 2025
A novel retinoic acid receptor beta isoform and retinoid resistance in lung carcinogenesis
W Jeffrey Petty1, Na Li, Adrian Biddle
1Department of Pharmacology and Toxicology, Dartmouth College, Hanover, NH, USA. wpetty@wfubmc.edu
Background:
We previously reported that all-trans-retinoic acid (RA) treatment can prevent in vitro transformation of immortalized human bronchial epithelial (HBE) cells.
Methods:
To determine whether methylation inhibits RARbeta expression in HBE cells, we used sodium bisulfite sequencing to compare RARbeta P2 promoter methylation patterns in RA-sensitive (BEAS-2B) and RA-resistant (BEAS-2B-R1) HBE cells. Immunoblotting was used to assess induction of the RARbeta, placental transforming growth factor beta (PTGF-beta), Fos-related antigen 1 (Fra-1), and transglutaminase II (TGase II) proteins by RA following treatment with azacitidine, a DNA demethylating agent. The expression, transcriptional activity, and growth suppressive activity of RARbeta1', a novel RAR isoform, were evaluated in lung cancer cells transfected with RARbeta1', and expression was also studied in paired normal lung tissues and lung tumors. All statistical tests were two-sided.
Results:
Hypermethylation was observed in the 3' region of the RARbeta P2 promoter of BEAS-2B-R1 but not BEAS-2B cells. Azacitidine treatment of BEAS-2B-R1 cells restored RA-inducible RARbeta2 and PTGF-beta expression but not that of RARbeta1', Fra-1, or TGase II. RARbeta1' expression was repressed in RA-resistant BEAS-2B-R1 cells and in lung cancers, compared with adjacent normal lung tissues. BEAS-2B-R1 cells transiently transfected with RARbeta1' had increased RA-dependent activation of a retinoic acid receptor element (RARE)-containing reporter plasmid compared with vector control (mean = 3.2, 95% confidence interval [CI] = 3.1 to 3.3 versus mean = 1.4, 95% CI = 1.3 to 1.5; P<.001). In H358 lung cancer cells transiently transfected with RARbeta1', RA treatment restored target gene expression compared with that in vector-transfected cells and suppressed cell growth compared with that in untreated cells (4 microM; treated mean = 0.49 versus untreated mean = 1.0, difference = 0.51, 95% CI = 0.35 to 0.67, P = .003; 8 microM: treated mean = 0.50 versus untreated mean = 1.0, difference = 0.50, 95% CI = 0.26 to 0.74, P = .015).
Conclusion:
Restoration of RARbeta1' expression may overcome retinoid resistance in lung carcinogenesis.
Insights
Retinoid resistance in lung cancer may be overcome by restoring RARbeta1' expression. Studies show hypermethylation of the RARbeta P2 promoter in resistant cells, and RARbeta1' reintroduction suppresses tumor growth.
Area of Science:
- Oncology
- Molecular Biology
- Epigenetics
Background:
- All-trans-retinoic acid (RA) prevents in vitro transformation of human bronchial epithelial (HBE) cells.
- Retinoid resistance is a challenge in lung cancer treatment.
- Understanding the mechanisms of retinoid resistance is crucial for developing effective therapies.
Purpose of the Study:
- To investigate the role of RARbeta P2 promoter methylation in retinoid resistance in HBE cells.
- To determine if restoring RARbeta1' expression can overcome retinoid resistance in lung cancer cells.
- To evaluate the expression and function of the novel RAR isoform, RARbeta1', in lung carcinogenesis.
Main Methods:
- Sodium bisulfite sequencing to analyze RARbeta P2 promoter methylation in RA-sensitive and RA-resistant HBE cells.
- Immunoblotting to assess protein expression following azacitidine treatment (a demethylating agent).
- Transfection of lung cancer cells with RARbeta1' to evaluate its expression, transcriptional activity, and growth-suppressive effects.
Main Results:
- Hypermethylation of the RARbeta P2 promoter was observed in RA-resistant HBE cells (BEAS-2B-R1) but not in RA-sensitive cells (BEAS-2B).
- Azacitidine treatment restored RA-inducible expression of RARbeta2 and PTGF-beta in resistant cells.
- RARbeta1' expression was repressed in RA-resistant cells and lung cancers; its reintroduction in lung cancer cells restored RA-dependent gene expression and suppressed cell growth.
Conclusions:
- Hypermethylation of the RARbeta P2 promoter contributes to retinoid resistance in HBE cells.
- Restoration of RARbeta1' expression holds potential for overcoming retinoid resistance in lung carcinogenesis.
- RARbeta1' may serve as a therapeutic target for lung cancer treatment.
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