The effects of actinomycin D and cycloheximide upon the ultrastructural localization of3H-DOPA in differentiating chick neural crest melanocytes in vitro
✍ Scribed by Brumbaugh, J. A. ;Schall, D. G.
- Publisher
- John Wiley and Sons
- Year
- 1977
- Tongue
- English
- Weight
- 887 KB
- Volume
- 202
- Category
- Article
- ISSN
- 0022-104X
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✦ Synopsis
Abstract
Cultured neural crest melanocytes from chick embryos were treated with actinomycin D or cycloheximide. The effects of these inhibitors upon melanogenesis was determined by monitoring the incorporation of ^3^H‐DOPA into premelanosomes and melanosomes using high resolution autoradiography.
Seven‐day cultures were treated with actinomycin D (1 μm/ml) or cycloheximide (1 μm/ml) for five hours with the terminal three hours in labeled medium (0.5 m Ci/ml). They were compared to control cultures which were labeled three hours in the absence of inhibitors. Actinomycin D reduced the quantity of silver grains associated with melanogenic organelles to 39.7%‐62.5% (different determinations) of the control value. Similarly, cycloheximide reduced the grain quantity to 58.9% of the control value.
In a second type of experiment, 6.5‐day cultures that had been subcultured once were treated with cycloheximide (1 μgm/ml) for 11 hours with the terminal three hours in ^3^H‐DOPA. In this case, the quantity of silver grains associated with melanogenic organelles was reduced to 22.7% of the control value.
Actinomycin D reduces ^3^H‐DOPA incorporation by about one‐half in from four to seven hours, while cycloheximide takes from six to seven hours to achieve a similar reduction. Therefore, RNA synthesis is at least as rate limiting as protein synthesis with regard to melanogenesis.
Based on determinations from the literature regarding the minimal half‐lives of ribosomal and transfer RNA's in cultured cells it is possible to infer that the main effect of actinomycin D was upon messenger RNA synthesis. Therefore, the ability of differentiating chick neural crest melanocytes to incorporate ^3^H‐DOPA is under transcriptional control.