Genetic and epigenetic changes in human epithelial cells immortalized by telomerase

Exogenous expression of hTERT, the catalytic component of telomerase, is su fficient for the immortalization of human fibroblasts but insufficient for the immortalization of human foreskin keratinocytes (HFKs) and human mammar y epithelial cells (HMECs), These latter cell types can overcome senescence by coexpression of hTERT and human papillomavirus (HPV) E7 or by expressio n of hTERT and loss of p16(INK4a) expression, indicating that the retinobla stoma (Rb) pathway, along with a telomere maintenance pathway, plays a role in determining the life span of epithelial cells. In this study, we furthe r characterize hTERT-immortalized HFKs and human adenoid epithelial cells ( HAKs) for genotypic and phenotypic alterations that are associated with imm ortalization, Of five hTERT-immortalized HFK and HAK cell lines examined, f our exhibited repression of p16(INK4a) expression by promoter methylation o r specific large-scale deletion of chromosome 9p, the location of p16(INK4a ). Interestingly, one cell line exhibited complete down-regulation of expre ssion of p14(ARF), with Only slight down-regulation of expression of p16(IN K4a) Yet, all of the immortal cells lines exhibited hyperphosphorylated Rb. Cytogenetic analysis revealed clonal chromosome aberrations in three of th e five cell lines. All of the cell lines retained a growth block response w ith the expression of mutant ras, When grown on organotypic raft cultures, however, the hTERT-immortalized cells exhibited a maturation delay on termi nal differentiation. Our results indicate that immortalization of epithelia l cells may require both activation of telomerase and other genetic and/or epigenetic alterations that abrogate normal differentiation.