Ro 10-9359 Retinoid Inhibits Both in vitro Epidermal Cell Proliferation and Differentiation

Summary

The TMMP analog of retinoic acid (Ro 10-9359) is successfully used in the treatment of psoriasis, a hyperproliferative scaling disorder. Primary neonatal mouse keratinocyte cultures that grow at 32 °C were used to study the effect of the retinoid on epidermal cell proliferation and fibrous protein (keratin) synthesis. ³H-TdR uptake into isolated DNA fractions (cpm/µg DNA) was used to quantitate proliferation. ³H-histidine (³H-his) pulse-labelling of the cells followed by serial extraction with 4 buffers. Lowry protein analyses and SDS polyacrylamide gel electrophoresis (PAGE) were used to quantitate the synthesis of 5 categories of epidermal proteins. 8M urea + reducing agent soluble and insoluble fibrous proteins are two of these categories. The cultures were treated with Ro 10-9359 in 0.2% DMSO on day 3 after plating and with every medium change. The doses were: 12 µg/ml (34 µM), 6, 3, 0.05, 0.01 and 0.005 µg/ml (14 nM) (N =15). There was no change in cpm/µg DNA one or 5 days after treatment. After 9 days of growth in the analog, there occurred an approximately 50% decrease in ³H-TdR cpm/µg DNA with 12, 6 and 3 µg/ml of the retinoid. Thirty percent, 12% and 5% decreases in cpm/µg DNA were seen with 0.05, 0.01 and 0.005 µg/ml of the drug. No changes in total culture DNA were noted and the DMSO vehicle did not affect culture proliferation. Cultures treated with 12 µg/ml of the drug (day 3) were pulse-labelled for 4 h with 2 pCi/m1 ³H-his after 1, 5 and 9 days of drug treatment. Analyses of the different protein fractions showed an effect on day 1 by the Ro 10-9359: the synthesis of the 8M urea + reducing agent soluble fibrous protein fraction was inhibited approximately 50%. The inhibitory effect was also observed on the 5th and 9th day after treatment. PAGE showed no changes in the band patterns of the fractions and documented a decrease in normally synthesized fibrous proteins. The DMSO vehicle also affected the synthesis of these fibrous proteins but only at the later time points. We conclude that Ro 109359 can inhibit both the proliferation and fibrous protein synthesis (differentiation) of isolated epidermal cells in culture. Although the differentiation effect apparently occurred at an earlier time point than the proliferation effect, we do not know at present whether the two effects of this retinoid are independent or related in a cause-effect manner. It is possible that Ro 10-9359 is beneficial in psoriasis through similar proliferation and differentiation control mechanisms.