DNA damage and consequent mutations initiate the multistep carcinogenic process. 
Differentiated cells have a reduced capacity to repair DNA lesions, but the 
biological impact of unrepaired DNA lesions in differentiated lung epithelial 
cells is unclear. Here, we used a novel organotypic human lung three-dimensional 
(3D) model to investigate the biological significance of unrepaired DNA lesions 
in differentiated lung epithelial cells. We showed, consistent with existing 
notions that the kinetics of loss of simple double-strand breaks (DSBs) were 
significantly reduced in organotypic 3D culture compared to kinetics of repair 
in two-dimensional (2D) culture. Strikingly, we found that, unlike simple DSBs, 
a majority of complex DNA lesions were irreparable in organotypic 3D culture. 
Levels of expression of multiple DNA damage repair pathway genes were 
significantly reduced in the organotypic 3D culture compared with those in 2D 
culture providing molecular evidence for the defective DNA damage repair in 
organotypic culture. Further, when differentiated cells with unrepaired DNA 
lesions re-entered the cell cycle, they manifested a spectrum of 
gross-chromosomal aberrations in mitosis. Our data suggest that downregulation 
of multiple DNA repair pathway genes in differentiated cells renders them 
vulnerable to DSBs, promoting genome instability that may lead to 
carcinogenesis.