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 p16(INK4a). Here, we present evidence that many human cell types 
undergo senescence by activation of the p16(INK4a)/Rb pathway, and that 
introduction of Bmi-1 can inhibit p16(INK4a) expression and extend the life span 
of human epithelial cells derived from skin, mammary gland and lung. 
Introduction of p16(INK4a)-specific short hairpin RNA, as well as Bmi-1, 
suppressed p16(INK4a) 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 p16(INK4a) 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 p16(INK4a) and 
introduction of hTERT can immortalize many human cell types with little 
chromosomal instability.