In vitro transformation of human skin epithelial cells: Role of RAS oncogene in malignant progression

Immortalized human skin epithelial cell lines provide useful models to study progressive stages in human carcinogenesis. Alterations have been examined occurring with immortalization and malignant progression of the human keratinocyte cell line HaCaT, which developed spontaneously in a long-term culture from trunk skin keratinocytes. The cell line has maintained many features of epidermal growth and differentiation in vitro and has acquired clonogenicity. HaCaT cells exhibited a transformed phenotype (aneuploidy and clonogenicity in soft agar) but remained non-tumorigenic. On transplantation they formed normally structured and differentiating epithelia, but did not grow invasively. Following transfection with the c-Ha-ras oncogene (EJ) randomly selected (G418-resistant) clones exhibited different stages of tumour progression. They formed either (1) rapidly regressing cysts, as seen with the parental line, (2) slowly growing benign tumours or (3) progressively enlarging well differentiated carcinomas. Tumorigenic (benign and malignant) clones had higher levels of mRNA expression and produced mutated p21. However, no correlation existed between both parameters and malignant growth. Ras-transfected clones showed improved morphological differentiation in vitro, in transplants, and in tumours and expressed differentiation-specific keratins. Tumorigenic HaCaT-ras clones were clonogenic in serum-free medium but had lost their ability to grow in soft agar. Thus, c-Ha-ras oncogene expression initiated tumour progression in immortalized human keratinocytes by altering growth regulation in vitro and in vivo, but per se was insufficient for malignant transformation.