Δ133p53α, a natural p53 isoform, contributes to conditional reprogramming and long-term proliferation of primary epithelial cells.

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.

Conditional cell reprogramming involves non-canonical β-catenin activation and mTOR-mediated inactivation of Akt.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing in vitro into an undifferentiated adult stem cell-like state that is characterized by long-term proliferation. This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions.

Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.

Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632).

ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes.

The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc.

Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells.

Both feeder cells and Rho kinase inhibition are required for the conditional reprogramming and immortalization of human epithelial cells. In the present study, we demonstrated that the Rho kinase inhibitor Y-27632, significantly suppresses keratinocyte differentiation and extends life span in serum-containing medium but does not lead to immortalization in the absence of feeder cells. Using Transwell culture plates, we further demonstrated that physical contact between the feeder cells and keratinocytes is not required for inducing immortalization and, more importantly, that irradiation of the feeder cells is required for this induction.