Activation of telomerase is sufficient for immortalization of some types of human cells but additional factors may also be essential. It has been proposed that stress imposed by inadequate culture conditions induces senescence due to accumulation of p16INK4a. Here, we present evidence that many human cell types undergo senescence by activation of the p16INK4a/Rb pathway, and that introduction of Bmi-1 can inhibit p16INK4a expression and extend the life span of human epithelial cells derived from skin, mammary gland and lung. Introduction of p16INK4a-specific short hairpin RNA, as well as Bmi-1, suppressed p16INK4a expression in human mammary epithelial cells without promoter methylation, and extended their life span. Subsequent introduction of hTERT, the telomerase catalytic subunit, into cells with low p16INK4a levels resulted in efficient immortalization of three cell types without crisis or growth arrest. The majority of the human mammary epithelial cells thus immortalized showed almost normal ploidy as judged by G-banding and spectral karyotyping analysis. Our data suggest that inhibition of p16INK4a and introduction of hTERT can immortalize many human cell types with little chromosomal instability.
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