Graphic relationship of cell lines profiled in this study. Cell lines characterized in this study are shown with reference to their stage in transformation. The pre-stasis HMEC used were cultured for 2–3 passages before analysis, and reach stasis by passages 3–5. Rare isolates of cells grown in serum-free media (MEBM) emerge spontaneously from stasis, associated with the absence of p16 expression due to promoter silencing, and continue growing as post-selection HMEC until reaching a second, proliferation barrier (telomere dysfunction). This barrier is highly stringent, and spontaneous immortalization has never been observed in cells that were not mutagenized or virally transduced during pre-stasis or post-selection growth. HMEC grown in MM do not spontaneously give rise to post-selection cells, however primary populations exposed to the chemical carcinogen benzo(a)pyrene (BaP) have produced rare clonal isolates with post-stasis growth, associated with absence of p16 expression due to mutation or promoter silencing. These non-spontaneously arising post-stasis cells are referred to as extended lifespan, and may harbor additional errors due to the carcinogen exposure. Overcoming the telomere dysfunction barrier is associated with reactivation of telomerase activity. The fully immortalized lines 184A1 and 184B5 were derived from extended lifespan post-stasis cells grown in MM and exposed to BaP in primary culture. Exposure of extended lifespan 184Aa cells to retroviral infection resulted in two cell lines that had lost both copies of the TP53 gene. The cell lines profiled in this study are shown relative to the profiling analyses performed. Comparisons used to analyze selection and immortalization, as well as the influence of p53 and ERBB2/Her2 status are shown by colored boxes and identified in the key at the lower left of the figure.