Qualitative changes in the biologic characteristics of cultured fetal rat keratinizing epidermal cells during the process of malignant transformation after benzo[a]pyrene treatment

Abstract

In vitro malignant transformation of fetal rat keratinizing epidermal cells from inbred SD rats after benzo[a]pyrene (BP) treatment was analyzed from various biologic viewpoints. BP treatment directly and indirectly effected changes in cell growth characteristics, i.e., temperature dependence for growth, in vitro keratinization, chromosome structure, and the ability to form colonies on plastic substrate, on 0.57% agar medium layer, and in 0.33% soft agar medium. BP-treated cells at 30 degrees C remained in the premalignant stages and showed shifts in chromosome structure toward the hypodiploid range and parakeratotic changes in their keratinization process. However, the cells failed to form colonies even on a plastic substrate. BP-treated cell lines that adapted to temperatures of 35 and 37.5 degrees C remained in the premalignant stages; however, they acquired the ability to form colonies on plastic substrates during subcultivation. Malignantly transformed colonies appeared in these cell lines. In vitro keratinization processes were classified into nearly normal (diffuse lamellar, focal lamellar, and parakeratotic), intermediate, and atypical subtypes (columnar, spherical, and single-cell type). Cells of atypical keratinization subtypes and some of the intermediate subtypes formed squamous cell carcinomas in syngeneic hosts. Malignantly transformed cells showed shifts in chromosome structure toward the hypotetraploid range and colony formation on the 0.57% agar medium layer. However, they failed to form colonies in 0.33% soft agar medium. With the use of changes in biologic characteristics of the cells as indicators, fetal rat keratinizing epidermal cells in culture were classified into five stages. The appearance of stage III cells seemed to be the first key step in their malignant transformation.