Vitamin A inhibition of keratinization in rat urinary bladder cancer cell line Nara Bladder Tumor No. 2 in meniscus gradient culture.

The Nara Bladder Tumor No. 2 cell line, established from a urinary bladder carcinoma in the Wistar rat, formed keratinizing cells and multicellular pearls in meniscus gradient culture when fed with a medium of 15 or 30% fetal calf serum in Eagle's minimal essential medium. Confluent monolayer cultures were first prepared with the tubes in conventional horizontal position. When cultures were then changed to a vertical position, stratification, piling up, and aggregation of cells were observed in a few days at the aerobic end of the gradient. Keratinization appeared 1 week after the tubes were placed in vertical position. Squamous differentiation proceeded to the formation of keratin pearls, a phenomenon never observed so distinctly and in such abundance in horizontal culture. Supplements of vitamin A, as low as 1 IU/ml, added to the medium did not inhibit piling up or aggregation but did prevent keratinization. Inhibition of keratinization by vitamin A was reversible. After vitamin A was removed from the medium, the cells in the aggregates progressed to keratinization.