Modulation of involucrin and envelope competence in human keratinocytes by hydrocortisone, retinyl acetate, and growth arrest.

Involucrin accumulation and ionophore-assisted envelope formation, markers of keratinocyte differentiation, were found to be highly dependent on culture conditions in the malignant epidermal keratinocyte line, SCC-13, derived from a human squamous cell carcinoma. In confluent cultures, approximately one-half of the cells were competent to form envelopes when grown in medium without hydrocortisone or retinyl acetate supplementation. Addition of hydrocortisone to the medium during growth resulted in up to 90% competence, while addition of retinyl acetate instead resulted in as low as 10% competence. Hydrocortisone partially antagonized the effect of retinyl acetate when both agents were added together. Involucrin levels, measured by radioimmunoassay, were modulated essentially in parallel with envelope competence under the various conditions tested. When the cells were grown in medium supplemented with hydrocortisone, the levels shortly after confluence were over 50-fold higher than in sparse cultures. Regardless of hydrocortisone or retinyl acetate addition, less than 1% of the cells were competent in sparse cultures of growing cells, but up to 90% exhibited this property after growth arrest in serum-free medium containing hydrocortisone. High levels of competence were correlated with cessation of cell division but not with loss of colony-forming efficiency; under optimal conditions, two-thirds of the cells were capable of both envelope formation and colony initiation. Normal human epidermal cells showed a 4- to 5-fold increase in envelope competence from sparse to confluent culture but were insensitive to the suppressive effect of retinyl acetate. The results suggest that some potential differentiated character of malignant keratinocytes may be suppressed in vivo by physiological agents such as vitamin A.

Mutually antagonistic effects of hydrocortisone and retinyl acetate on envelope competence in cultured malignant human keratinocytes.

Serially propagated SCC-13 keratinocytes, derived from a human squamous cell carcinoma, are greatly influenced by culture conditions in their ability to form ionophore-inducible cross-linked envelopes. Supplementation of the growth medium with fetal bovine serum at concentrations ranging from 0.5 to 20 percent had little effect on competence to form envelopes in confluent cultures. At each serum concentration, however, addition of hydrocortisone to the medium led to an increase in competence of almost fourfold, from approximately 20 to nearly 80 percent. With the serum supplementation held at 5 percent, addition of retinyl acetate to the medium suppressed competence in a concentration-dependent manner over the range of 1 to 100 ng/ml. At the highest concentration employed, competence was reduced over fourfold in the presence of hydrocortisone and virtually eliminated in its absence. When the cells were grown using serum depleted of endogenous vitamin A, a majority were competent in the absence of hydrocortisone. Under this condition, retinyl acetate suppressed competence over fivefold in the absence of hydrocortisone, but not at all in its presence. We conclude that hydrocortisone stimulates envelope competence primarily by antagonizing the suppressive effect of vitamin A. The SCC-13 cell line may prove valuable in studying mechanisms of retinoid and corticosteroid therapeutic action on diseased human keratinocytes.