Culture of conducting airway epithelial cells in serum-free medium

Robinson, Cynthia B.; Wu, Reen

doi:10.1007/bf01666138

Cited by 43 publications

(37 citation statements)

References 20 publications

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“…The protocol for obtaining human tissues has been approved and periodically reviewed by the campus Human Subject Review Committee. TBE cells were isolated from these tissues by a protease dissociation procedure and cultured in a serum-free hormonesupplemented medium as previously described (39). Briefly, cells were grown in 6-well tissue culture plates either without additional substratum (TC) or with collagen gel substratum (Cg) or in air-liquid biphasic culture insert chambers (Transwell™ chamber, Corning/Costar 3450, Corning, NY) without (Bi) or with collagen gel substratum (Bi-Cg).…”

Section: Methodsmentioning

confidence: 99%

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Soref¹,

Di²,

Hayden

et al. 2001

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Soref¹,

Di²,

Hayden

et al. 2001

Journal of Biological Chemistry

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“…The Human Subject Review Committee of the University of California at Davis approved all procedures involved in tissue procurement. Epithelial cell isolation and culture conditions were performed as described previously with some modification (19,20). Briefly, protease-dissociated primary TBE cells were cultured in Ham's F-12/Dulbecco's modified Eagle's medium (1:1) medium supplemented with six growth factors: insulin (5 g/ml), transferrin (5 g/ml), EGF (10 ng/ml), cholera toxin (10 ng/ml), dexamethasone (0.1 M), and bovine hypothalamus extract (15 g/ml), as well as 0.2% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

“…Jim Yankaskas (University of North Carolina at Chapel Hill) and J. F. Lechner (Wayne State University, Detroit, MI), respectively. These cell lines were maintained in serum-free F-12 medium supplemented with six hormonal supplements as described before (19,20). When appropriate conditions were applied, such as the addition of vitamin A or PMA/Ca 2ϩ (1.5 mM), HBE l cell line could express mucin synthesis and secretion, and the formation of cross-linked envelopes, respectively.…”

Section: Methodsmentioning

confidence: 99%

Distinct Roles for Amino- and Carboxyl-terminal Sequences of SPRR1 Protein in the Formation of Cross-linked Envelopes of Conducting Airway Epithelial Cells

Deng

Pan

2000

Journal of Biological Chemistry

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The small proline-rich protein, SPRR1, is a marker gene whose expression in conducting airway epithelium is elevated under a variety of conditions that enhance squamous differentiation. The purpose of this study is to elucidate the nature of the SPRR1 sequence involved in cross-linked envelope formation in a tissue/cell type, such as conducting airway epithelium, that normally does not express squamous function except after injury or maintenance in culture. For this, a Flag-SPRR1 fusion protein expression system has been developed. Using the liposome-mediated gene transfer technique on passage 1 culture of human tracheobronchial epithelial (TBE) cells, the Flag-SPRR1 fusion protein can be expressed and detected immunologically by both anti-Flag and anti-SPRR1 antibodies. The incorporation of Flag-SPRR1 fusion protein into cross-linked envelopes can be demonstrated when transfected human passage 1 TBE cultures are treated with phorbol 12-myristate 13-acetate and high calcium (1.5 mM). By deletion and sitedirected mutagenesis, two distinct roles of the aminoand carboxyl-terminal sequences of SPRR1 have been demonstrated. First, we demonstrated that the aminoterminal sequence of SPRR1 protein is required for the incorporation of the fusion protein into cross-linked envelopes, whereas a deletion on the carboxyl-terminal region or on the middle repetitive unit has no effect. Interestingly, insertion of a 24-amino acid peptide of monkey MUC2 repetitive sequence in the amino-terminus of SPRR1 protein had a stimulatory effect. Sitedirected mutagenesis on the following amino acid residues, Lys 7 , Gln 88 , and Lys 89 , which were found previously to participate in the cross-linked envelope formation of keratinocytes, had no detrimental effect on the incorporation. However, mutations on Gln clusters, such as Gln 4 -Gln 6 and Gln 22 -Gln 25 , had detrimental effects on the incorporation. These results suggest an amino-terminal sequence-dependent and multiple crosslinked sites for the incorporation of Flag-SPRR1 fusion protein into cross-linked envelopes of cultured human TBE cells. Second, we demonstrated that the carboxyl terminus of SPRR1 protein is required for a high level of Flag-fusion protein expression. A deletion in the carboxyl region or a mutation on the last lysine residue of the carboxyl end had a detrimental effect on the level of Flag-SPRR1 fusion protein expressed in transfected cells. In contrast, there was only a slight decrease in the level of expression if the amino-terminus was deleted. Interestingly, the efficiency for fusion protein to incorporate into cross-linked envelopes was elevated by the mutation at the carboxyl end. These results suggest distinct roles, perhaps coordinately, for both amino-and carboxyl-terminal sequences in the regulation of the life cycle of SPRR1 protein in cultured TBE cells.

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“…This cell line was routinely cultured in a serum-free hormone-supplemented medium [24], which was Ham's F 12 nutrient medium supplemented with insulin, 0014-5793/95/$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved.…”

Section: Cell Culturementioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells

et al. 1995

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Treatment of human respiratory tract epithelial cellswith H202 led to concentration-dependent DNA strand breakage that was highly-correlated with multiple chemical modifications of all four DNA bases, suggesting that damage is due to hydroxyl radical, OH'. However, the ma|or base damage occurred to adenine. Hence, conclusions made about the occurrence and the extent of oxidative DNA damage on the basis only of changes in 8-hydroxyguanine should be approached with caution.

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Culture of conducting airway epithelial cells in serum-free medium

Cited by 43 publications

References 20 publications

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

Distinct Roles for Amino- and Carboxyl-terminal Sequences of SPRR1 Protein in the Formation of Cross-linked Envelopes of Conducting Airway Epithelial Cells

DNA strand breakage and base modification induced by hydrogen peroxide treatment of human respiratory tract epithelial cells

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