Rescue of Embryonic Epithelium Reveals That the Homozygous Deletion of the Retinoblastoma Gene Confers Growth Factor Independence and Immortality but Does Not Influence Epithelial Differentiation or Tissue Morphogenesis

Kathleen C Day,Michael T Mccabe,Xin Zhao,Yuzhuo Wang,Joanne N Davis,John Phillips,Marion Von Geldern,Thomas Ried,Mark A Kukuruga,Gerald R Cunha,Simon W Hayward,Mark L Day

doi:10.1074/jbc.m205361200

Abstract

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.

Highlights

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb؊/؊) fetal mice has allowed for the isolation and characterization of the first Rb؊/؊ prostate epithelial cell line
Genotype and Phenotype of PrE and RbϪ/ϪPrE Cells— Because the Rb gene was disrupted by the insertion of the neomycin resistance cassette (47), the use of cellular recombination (34), employing neomycin-sensitive, wild type rat urogenital mesenchyme (rUGM), and neomycin-resistant RbϪ/ϪPrE mouse epithelium allowed for the specific selection of RbϪ/ϪPrE from the wild type stroma
The lethal nature of the Rb knockout precluded the establishment of RbϪ/Ϫ epithelial cell lines, where definitive experiments to investigate physiological roles for Rb in specific epithelial populations could be performed

Summary

Introduction

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb؊/؊) fetal mice has allowed for the isolation and characterization of the first Rb؊/؊ prostate epithelial cell line. Rb Deletion and Epithelial Physiology and Differentiation the androgen receptor, transactivate or repress a number of transcriptional targets, including cell cycle regulatory genes. It was recently demonstrated (8, 22–24) that pRb is activated during androgen-stimulated epithelial proliferation and during androgen ablation-induced apoptosis. The use of viral oncogenes has provided substantial insight into the function of the Rb family members and their roles in regulating cell cycle, cell growth, and differentiation; viral oncogenes are promiscuous in their interactions with other cellular proteins and promote genomic instability making interpretation of these experimental models difficult. Models that target and inactivate Rb, which minimize complicating genetic alterations inherent with viral oncogenes, might provide novel insight into the physiologic role of Rb in epithelial cells

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