Chart 1. Derivation of transformed HMEC cultures. Primary cultures derived from reduction mammoplasties (RM), milk (MK) or non-tumor mastectomy tissues (P) were initiated in three different types of medium. Cells grown in any serum-containing medium all ceased proliferation at the stasis barrier. Exposure of pre-stasis cultures to various oncogenic insults (red above) induced cells to overcome or bypass stasis and become post-stasis by different means (e.g., the Extended Life cultures had been exposed to the chemical carcinogen benzo(a)pyrene; the post-selection cultures had been grown in a highly stressful serum-free medium). Further alterations were required to overcome the telomere dysfunction barrier, gain telomerase expression, and become immortal. For example, following unknown errors presumably resulting from the B(a)P exposure and the genomic instability at telomere dysfunction, the Extended Life cultures gave rise to rare clonal lines. Transduction with breast cancer-associated oncogenes or GSE22 increased the frequency of immortalization; transduction with c-myc gave uniform immortalization. Cells from post-selection cultures all ceased proliferation at the telomere dysfunction barrier. Transduction with breast cancer-associated oncogenes gave rise to rare clonal lines. Non-malignant immortal lines were no longer sensitive to OIS, and overexpression a number of different oncogenic genes conferred AIG (anchorage-independent growth). The greatest change in properties occurs when the finite cells become immortal. Non-malignant immortal lines cluster with tumor-derived lines and not the finite cells for many properties (e.g., gene expression, global promoter methylation).