Chromosome in situ hybridization and molecular genetic analyses of bronchial biopsies of smokers demonstrate genetic instability and clonal/subclonal bronchial cell outgrowths that continue despite tobacco cessation. To better understand the molecular underpinnings of genomic instability in the lungs of former smokers, an organotypic culture model was established whereby bronchial epithelial cells at different stages of lung tumorigenesis were plated onto transwell-clear culture filter inserts, allowed to reach confluence, and an air-liquid interface created. The cell types utilized included normal human bronchial epithelial cells (NHBE), NHBE cells immortalized (Jerry Shay) with hTERT and cdk4 (HccBE), NHBE cells immortalized (Klein-Szanto) with Ad12/SV40 (Beas2B), and immortalized (1799), transformed (1198), and tumorigenic (1170I) carcinogen-treated Beas2B cells. These cultures evolved into multi-layer cultures resembling normal (NHBE and HccBE) or dysregulated stratified bronchial epithelium (Beas2B, 1799, 1198, 1170I). Confocal microscopy of cultures after BrdU pulse labeling or phospho-histone H3 (P-H3) immunostaining demonstrated that NHBE cells preferentially undergo DNA synthesis and mitosis at the basal layer, HccBE cells proliferate at the basal and peribasal layers, whereas more advanced bronchial epithelial cells proliferate throughout the multi-layer cultures. Examination of anaphase cells in these populations by propidium iodide staining and confocal microscopy demonstrated that NHBE cells showed less than 2% anaphases with either lagging chromosomes or chromosome bridges compared to more than 10% in other cell types. Visualization of mitotic figures with P-H3 immunostaining demonstrated that P-H3 staining was strong at metaphase and uniformly disappeared during anaphase in cells at the basal layer, whereas P-H3 staining was not uniform in cells dividing away from the basal layer with lagging chromosomes and chromosome bridges differentially P-H3-positive in late anaphase. To determine when the chromosome regions involved in aberrations had been replicated, cultures were pulse-labeled with BrdU, harvested at various times following the pulse label, and analyzed for BrdU staining patterns of the lagging chromosomes and chromosome bridges. More than 90% of the aberrant chromosome regions were labeled in cells harvested 3-4 hours after BrdU labeling (i.e., replicated in late S phase) whereas less than 60% were labeled at 9-10 hours after BrdU labeling (i.e., replicated in early-mid S phase). These results suggest that proliferation away from the basal layer in these organotypic cultures is associated with increased genomic instability, possibly associated with dysregulated cell cycle control and abnormal mitotic progression. Supported in part by DAMD 17-02-1-0706 and W81XWH-04-1-01-42, and NIH/NCI P01 CA-91844 and EDRN CA-86390.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]