Abstract
INTRODUCTION: Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC) transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras. METHODS: HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT). The immortalized cells were passaged in vitro and studied by a combination of G-banding and Spectral Karyotyping (SKY). H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting. RESULTS: Immortal HMEC alone were not tumorigenic in gamma-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor. CONCLUSION: Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.