Role of p21/waf1, in lung fibroblast cell cycle regulation in the presence of hyperoxia, dexamethasone and retinoic acid

N. Hussain, L. Zhu, Mitchell Kresch

Research output: Contribution to journalArticle

Abstract

The cell cycle inhibitor, p21/waf1 regulates cell proliferation. Hyperoxia (Hx), dexamethasone (Dx) and all-trans-retinoic acid (tRa) are therapeutic agents that affect lung cells, specially, fibroblasts, but the role of p21 in mediating their effects are unknown. We hypothesized that these agents modify cell proliferation and apoptosis of lung fibroblasts through changes in p21 expression. Methods: Freshly isolated fetal rat lung fibroblasts were cultured in serum supplemented medium in the presence or absence of Hx (95% oxygen), Dx (0.1-10μM) or tRa (0.01-1μM). Total protein and mRNA were extracted at various time points. The steady state levels of p21 mRNA were determined by Northern analysis and densitometric quantification. Immunoblots from Western analysis of p21 protein were quantified by densitometry. Cell proliferation was assayed by 3H-thymidine incorporation and apoptosis identified by the TUNEL assay. Results: Compared to normal control (NI) conditions, all three agents tested, decreased 3H-thymidine incorporation from 6 h-120 h in fibroblasts (bombesin was used as a positive control). In parallel, the steady state levels of p21(inhibitor of cell cycle) mRNA were significantly increased in Hx and Dx but were unchanged with tRa. Similarly, p21 protein levels were significantly increased in Hx and Dx. There were no changes in p21 protein with tRa(0.01-1μM). Apoptosis (TUNEL) was seen in < 5% of cells after 7 days in culture with Hx, but ≤ 1% apoptosis was seen in controls and cells treated with Dx and tRa for 7 days. Conclusions: Hx and Dx induced suppression of fibroblast proliferation is associated with increased p21 expression at a pretranslational level but not associated with changes in apoptosis. tRA causes decrease in cell proliferation by a mechanism that does not involve increase in p21 or apoptosis. (Graph Presented).

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume47
Issue number2
StatePublished - Jan 1 1999

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Hyperoxia
Fibroblasts
Tretinoin
Dexamethasone
Cell Cycle
Cells
Cell proliferation
Apoptosis
Lung
Cell Proliferation
In Situ Nick-End Labeling
Thymidine
Messenger RNA
Proteins
Bombesin
Densitometry
Rats
Assays
Western Blotting
Oxygen

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

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title = "Role of p21/waf1, in lung fibroblast cell cycle regulation in the presence of hyperoxia, dexamethasone and retinoic acid",
abstract = "The cell cycle inhibitor, p21/waf1 regulates cell proliferation. Hyperoxia (Hx), dexamethasone (Dx) and all-trans-retinoic acid (tRa) are therapeutic agents that affect lung cells, specially, fibroblasts, but the role of p21 in mediating their effects are unknown. We hypothesized that these agents modify cell proliferation and apoptosis of lung fibroblasts through changes in p21 expression. Methods: Freshly isolated fetal rat lung fibroblasts were cultured in serum supplemented medium in the presence or absence of Hx (95{\%} oxygen), Dx (0.1-10μM) or tRa (0.01-1μM). Total protein and mRNA were extracted at various time points. The steady state levels of p21 mRNA were determined by Northern analysis and densitometric quantification. Immunoblots from Western analysis of p21 protein were quantified by densitometry. Cell proliferation was assayed by 3H-thymidine incorporation and apoptosis identified by the TUNEL assay. Results: Compared to normal control (NI) conditions, all three agents tested, decreased 3H-thymidine incorporation from 6 h-120 h in fibroblasts (bombesin was used as a positive control). In parallel, the steady state levels of p21(inhibitor of cell cycle) mRNA were significantly increased in Hx and Dx but were unchanged with tRa. Similarly, p21 protein levels were significantly increased in Hx and Dx. There were no changes in p21 protein with tRa(0.01-1μM). Apoptosis (TUNEL) was seen in < 5{\%} of cells after 7 days in culture with Hx, but ≤ 1{\%} apoptosis was seen in controls and cells treated with Dx and tRa for 7 days. Conclusions: Hx and Dx induced suppression of fibroblast proliferation is associated with increased p21 expression at a pretranslational level but not associated with changes in apoptosis. tRA causes decrease in cell proliferation by a mechanism that does not involve increase in p21 or apoptosis. (Graph Presented).",
author = "N. Hussain and L. Zhu and Mitchell Kresch",
year = "1999",
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language = "English (US)",
volume = "47",
journal = "Journal of Investigative Medicine",
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T1 - Role of p21/waf1, in lung fibroblast cell cycle regulation in the presence of hyperoxia, dexamethasone and retinoic acid

AU - Hussain, N.

AU - Zhu, L.

AU - Kresch, Mitchell

PY - 1999/1/1

Y1 - 1999/1/1

N2 - The cell cycle inhibitor, p21/waf1 regulates cell proliferation. Hyperoxia (Hx), dexamethasone (Dx) and all-trans-retinoic acid (tRa) are therapeutic agents that affect lung cells, specially, fibroblasts, but the role of p21 in mediating their effects are unknown. We hypothesized that these agents modify cell proliferation and apoptosis of lung fibroblasts through changes in p21 expression. Methods: Freshly isolated fetal rat lung fibroblasts were cultured in serum supplemented medium in the presence or absence of Hx (95% oxygen), Dx (0.1-10μM) or tRa (0.01-1μM). Total protein and mRNA were extracted at various time points. The steady state levels of p21 mRNA were determined by Northern analysis and densitometric quantification. Immunoblots from Western analysis of p21 protein were quantified by densitometry. Cell proliferation was assayed by 3H-thymidine incorporation and apoptosis identified by the TUNEL assay. Results: Compared to normal control (NI) conditions, all three agents tested, decreased 3H-thymidine incorporation from 6 h-120 h in fibroblasts (bombesin was used as a positive control). In parallel, the steady state levels of p21(inhibitor of cell cycle) mRNA were significantly increased in Hx and Dx but were unchanged with tRa. Similarly, p21 protein levels were significantly increased in Hx and Dx. There were no changes in p21 protein with tRa(0.01-1μM). Apoptosis (TUNEL) was seen in < 5% of cells after 7 days in culture with Hx, but ≤ 1% apoptosis was seen in controls and cells treated with Dx and tRa for 7 days. Conclusions: Hx and Dx induced suppression of fibroblast proliferation is associated with increased p21 expression at a pretranslational level but not associated with changes in apoptosis. tRA causes decrease in cell proliferation by a mechanism that does not involve increase in p21 or apoptosis. (Graph Presented).

AB - The cell cycle inhibitor, p21/waf1 regulates cell proliferation. Hyperoxia (Hx), dexamethasone (Dx) and all-trans-retinoic acid (tRa) are therapeutic agents that affect lung cells, specially, fibroblasts, but the role of p21 in mediating their effects are unknown. We hypothesized that these agents modify cell proliferation and apoptosis of lung fibroblasts through changes in p21 expression. Methods: Freshly isolated fetal rat lung fibroblasts were cultured in serum supplemented medium in the presence or absence of Hx (95% oxygen), Dx (0.1-10μM) or tRa (0.01-1μM). Total protein and mRNA were extracted at various time points. The steady state levels of p21 mRNA were determined by Northern analysis and densitometric quantification. Immunoblots from Western analysis of p21 protein were quantified by densitometry. Cell proliferation was assayed by 3H-thymidine incorporation and apoptosis identified by the TUNEL assay. Results: Compared to normal control (NI) conditions, all three agents tested, decreased 3H-thymidine incorporation from 6 h-120 h in fibroblasts (bombesin was used as a positive control). In parallel, the steady state levels of p21(inhibitor of cell cycle) mRNA were significantly increased in Hx and Dx but were unchanged with tRa. Similarly, p21 protein levels were significantly increased in Hx and Dx. There were no changes in p21 protein with tRa(0.01-1μM). Apoptosis (TUNEL) was seen in < 5% of cells after 7 days in culture with Hx, but ≤ 1% apoptosis was seen in controls and cells treated with Dx and tRa for 7 days. Conclusions: Hx and Dx induced suppression of fibroblast proliferation is associated with increased p21 expression at a pretranslational level but not associated with changes in apoptosis. tRA causes decrease in cell proliferation by a mechanism that does not involve increase in p21 or apoptosis. (Graph Presented).

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