Effects of glucocorticosteroids on cultured human skin fibroblasts. V. Influence of anabolic steroids on the inhibitory effects of clobetasol-17-propionate on cell proliferation and collagen synthesis.

As previously found, the glucocorticosteroid clobetasol-17-propionate inhibits cell proliferation during the early growth stage of normal baby foreskin fibroblasts and collagen synthesis in confluent cultures of these cells. The degree of inhibition of cell proliferation decreases with increasing cell density and, moreover, is transient. The anabolic steroids nandrolone and nandrolone-phenyl-propionate have similar effects on these cells. Likewise the magnitude of the inhibition is dose-dependent. When present together the two types of drug do not act in an additive manner. Even at low concentrations the anabolic steroids abolish the inhibitory effect of the glucocorticosteroid on cell proliferation. Furthermore, in this case only the inhibitory effect of the glucocorticosteroid on collagen synthesis is found and there is no further increase in this effect due to the presence of the anabolic steroids. Our results imply that the use of low concentrations of anabolic steroids combined with glucocorticosteroids in topical application to the skin may abolish some of the undesirable side effects of the glucocorticosteroids.

Effects of glucocorticosteroids on cultured human skin fibroblasts. III. Transient inhibition of cell proliferation in the early growth stages and reduced susceptibility in later growth stages.

An immediate depression of the rate of cell proliferation occurred upon addition of glucocorticosteroids to cultures of human skin fibroblasts in the early growth stages. A reduced sensitivity or even insensitivity of the fibroblasts to growth inhibition inhibition was found upon the addition of the steroids at later stages of cell growth, when the cell density has increased. The inhibition in the early growth stages is transient and is most pronounced if the cultured medium is not renewed. This transient inhibition is not due to the development of steroid-resistant cell lines, and resembles the effect called tachyphylaxis, as is also observed in vasoconstriction tests.

Effects of glucocorticosteroids on primary human skin fibroblasts. I. Inhibition of the proliferation of cultured primary human skin and mouse L929 fibroblasts.

Various glucocorticosteroids were added to logarithmically growing cultures of primary human skin fibroblasts and of mouse L929 fibroblasts. These steroids inhibited proliferation of the human fibroblasts at concentrations which fall in a range expected to occur during the topical treatment of skin disorders. In terms of the concentrations required for the inhibition hydrocortisone was least and clobetasol-17-propionate most effective. All other steroids studied (hydrocortisone-17-butyrate, triamcinolone acetonide, betamethasone-17-valerate and hydrocortisone-21-acetate) showed medium effectiveness. Fluorination as such may not enhance the inhibitory effect. The inhibition was independent of the source (baby foreskin or adult arm skin) and passage number (7th to 13th or 15th and 16th passage, respectively) of the cells. The possible relationship between the inhibition of cell proliferation by such steroids and their therapeutic effect in psoriasis and their atrophic side effects is discussed. Mouse L929 fibroblasts were affected at 10(3)--10(4)-fold lower steroid concentrations and the range of the effective concentrations was 10(4)--10(5) times as wide as that for the primary human skin fibroblasts. It was concluded that these mouse fibroblasts are a poor model system for the study of in vivo effects of glucocorticosteroids in man.

Effects of glucocorticosteroids on primary human skin fibroblasts. II. Effects on total protein and collagen biosynthesis by confluent cell cultures.

Confluent cultures of normal primary human skin fibroblasts were incubated with various glucocorticosteroids which are in current use clinically for the treatment of various skin disorders. For all steroids concentrations were found at which collagen hydroxyproline formation was inhibited, while total protein synthesis was little affected. The concentration effective for inhibition was highest for hydrocortisone and lowest for clobetasol-17-propionate. All other steroids (hydrocortisone-17-butyrate, triamcinolone acetonide and betamethasone-17-valerate) showed medium effectiveness. Fluorination as such was not a factor in the degree of inhibition. The inhibition observed was shown to be independent of concomitant specific effects on cell proliferation or cell turnover. The possible implications of these findings on the therapeutic effects in psoriasis and the frequently occurring atrophic side-effects of these steroids are discussed.

Self complementarity in messenger RNA of collagen. I. Possible hairpin structures in regions coding for oligopeptides of glycine, proline (hydroxyproline) and alanine.

The periodic protein collagen is of special interest for the study of the relationship which exists between the structure of a protein and that of its mRNA, because oligopeptides containing glycine, proline (hydroxyproline) and alanine occur with great frequency in it. Collagen is particularly rich in these amino acids, which have codons containing only G and/or C in the obligatory first and second positions. If unlimited choice of codons for all amino acids were to occur, the stretch of mRNA coding for an alpha-chain should contain about 40% G and 31% C (Bachra et al., 1974). These high values suggest that a considerable degree of secondary structure will occur, unless selective codon use would result in the avoidance of G and C in optional third codon positions. In the present paper putative secondary structure formation in collagen mRNA was studied. This was done by studying the positions and frequencies of hairpin structures which could contain stem sections composed of the coding triplets of the above mentioned amino acids and hairpin sections of 4-40 bases. Calculation of the free energy contributions of such hairpin structures, using published values for the contributions of base-pair stacking, hairpin, bulge and interior loops and also taking into account the possible minimum number of base-pairs required for helix nucleation from a single-strand RNA (3 adjacent AU-pairs or 1 or 2 adjacent GC-pairs) led to the following conclusions. A considerable number of alternative, mutually exclusive hairpins can be constructed.