INDUCTION OF RETINOBLASTOMA GENE-EXPRESSION DURING TERMINAL GROWTH ARREST OF A CONDITIONALLY IMMORTALIZED FETAL-RAT LUNG EPITHELIAL-CELL LINE AND DURING FETAL LUNG MATURATION

The process by which fetal lung epithelial cells differentiate into ty pe 1 and type 2 cell is largely unknown. In order to study lung epithe lial cell proliferation and differentiation we have infected 20- day f etal lung epithelial cells with a retrovirus carrying a temperature-se nsitive SV40 T antigen (T Ag) and isolated several immortalized fetal epithelial cell lines. Cell line 20-3 has characteristics of lung epit helial cells including the presence of distinct lamellar bodies, tight junctions, keratin 8 and 18 mRNA, HFH8, and T1 alpha mRNA and low lev els of surfactant protein A mRNA. At 33 degrees C 20-3 grows with a do ubling time of 21 h. At 40 degrees C the majority of cells cease to pr oliferate. Growth arrest is accompanied by significant morphological c hanges including an increase in cell size, transition to a squamous ph enotype that resembles type 1 cells, and an increase in the number of multinucleated cells within the population. Greater than 95% of the ce lls incorporate [H-3]thymidine into DNA at 33 degrees C whereas at 40 degrees C label incorporation drops to less than 20%. When shifted dow n to 33 degrees C 40% of the cells remain terminally growth arrested. In addition, cells plated at 40 degrees C have a reduced ability to fo rm colonies when replated at 33 degrees C. Treatment with TGF-beta inc reases the percentage of cells that terminally growth arrest to greate r than 80%. Growth arrest is accompanied by an increase in the levels of c-jun, jun D, cyclin D1, C/EBP-beta, transglutaminase type II, and retinoblastoma (Rb) mRNA and an induction of p105, the hypophosphoryla ted, growth regulatory form of Rb. Evaluation of Rb mRNA in fetal lung indicates that it is induced 2.5-fold between 17 and 21 days of gesta tion. These studies indicate that 20-3 terminally growth arrests in cu lture at the nonpermissive temperature and that it may be useful in st udying changes in gene expression that accompany terminal growth arres t during lung development. (C) 1998 Academic Press.