CHANGES IN ONCOGENE MESSENGER-RNA EXPRESSION DURING HUMAN KERATINOCYTE DIFFERENTIATION

The nuclear proto-oncogenes are involved in transcriptional regulation and control many cell processes. The role of changes in proto-oncogen e expression in controlling the balance between proliferation and diff erentiation was studied in cultured keratinocytes. Normal human kerati nocytes were grown in the serum-free medium MCDB153 with an extracellu lar calcium concentration of 70 mu M. After treatment with different d ifferentiation conditions, cellular RNA was size-fractionated on agaro se gels and transferred to nylon membranes which were subsequently hyb ridized with c-myc, c-jan, and H-ms P-32-labelled probes. Relative RNA loading was assessed using probes for beta-actin and ribosomal 18s RN A. Inducing differentiation by increasing the calcium concentration of the medium from 70 mu M to 1.5 mM resulted in a marked decrease in c- myc RNA levels to 26% of control levels within 8 h. After 48 h in 1.5 mM calcium, c-myc levels had recovered to approximately 50% of control levels. There was a gradual reduction in c-jun levels to 56% of contr ol levels by 4 days. Treatment with 10 nM TPA, which also induces kera tinocyte differentiation, reduced c-myc RNA levels to 70% of control l evels during the first 4 h, but thereafter c-myc levels remained appro ximately constant for a further 20 h. TGF beta (2 ng/ml), which inhibi ts keratinocyte growth without inducing differentiation, did not alter c-myc RNA levels over a 4-day period. There were no changes in c-myc levels following the addition of retinoic acid and none of the conditi ons altered H-ras levels. We conclude that c-myc levels are high in pr oliferating keratinocytes and decrease following differentiation stimu li, but further work is required to investigate the underlying molecul ar mechanisms of differentiation.