Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts

Tarek Bisat, Terry R. Brown, Claude J. Migeon, Gary Berkovitz

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×106 cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×106 or at 0.25×106 cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution.

Original languageEnglish
Pages (from-to)806-812
Number of pages7
JournalIn Vitro Cellular & Developmental Biology
Volume25
Issue number9
DOIs
StatePublished - Sep 1 1989
Externally publishedYes

Fingerprint

Aromatase
Fibroblasts
Cell culture
Human Activities
Skin
Cell Culture Techniques
Androgen Receptors
Enzyme activity
Plating
Oxidoreductases
DNA
Enzymes
Culture Media
Cultured Cells
Estrogens
Proteins
Cell Count
Cells

Keywords

  • aromatase activity
  • cell culture
  • skin fibroblast

ASJC Scopus subject areas

  • Developmental Biology
  • Cell Biology
  • Clinical Biochemistry

Cite this

Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts. / Bisat, Tarek; Brown, Terry R.; Migeon, Claude J.; Berkovitz, Gary.

In: In Vitro Cellular & Developmental Biology, Vol. 25, No. 9, 01.09.1989, p. 806-812.

Research output: Contribution to journalArticle

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abstract = "Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1×106 cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1×106 or at 0.25×106 cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower V max . Preincubation of cells plated at one density with conditoned medium from cells plated at the other density did not change the relatve levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5α-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30{\%} of the in the latter group. DHT and dexamethasone receptor binding and 5α-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution.",
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