Abstract
We previously developed the technique of conditional reprogramming (CR), which allows primary epithelial cells from fresh or cryopreserved specimens to be propagated long-term in vitro, while maintaining their genetic stability and differentiation potential. This method requires a combination of irradiated fibroblast feeder cells and a Rho-associated kinase (ROCK) inhibitor. In the present study, we demonstrate increased levels of full-length p53 and its natural isoform, Δ133p53α, in conditionally reprogrammed epithelial cells from primary prostate, foreskin, ectocervical, and mammary tissues. Increased Δ133p53α expression is critical for CR since cell proliferation is rapidly inhibited following siRNA knockdown of endogenous Δ133p53α. Importantly, overexpression of Δ133p53α consistently delays the onset of cellular senescence of primary cells when cultured under non-CR conditions in normal keratinocyte growth medium (KGM). More significantly, the combination of Δ133p53α overexpression and ROCK inhibitor, without feeder cells, enables primary epithelial cells to be propagated long-term in vitro. We also show that Δ133p53α overexpression induces hTERT expression and telomerase activity and that siRNA knockdown of hTERT causes rapid inhibition of cell proliferation, indicating a critical role of hTERT for mediating the effects of Δ133p53α. Altogether, these data demonstrate a functional and regulatory link between p53 pathways and hTERT expression during the conditional reprogramming of primary epithelial cells.