Enrichment of side population (SP) cells in established radioresistant (RR) and paclitaxel-resistant (CR) lung cancer cell lines. (A) and (B), H460, H460/RR, and H460/CR cells (A) or A549, A549/RR, and A549/CR cells (B) were labeled with Hoechst 33342 dye and the percentage of SP cells determined by flow cytometry before and after treatment with verapamil; all resistant cell lines demonstrated enrichment of SP cells. (C) Flow cytometric analysis of H460/RR and H460/CR SP cells after 7-10 days of culture followed by re-sorting (by flow cytometry) and a second 7- to 10-day culture revealed progressive enrichment of SP cells as well as the presence of non-SP cells. Data shown represent one of three independent experiments. (D) CAR expression measured by flow cytometry and specific fluorescein isothiocyanate conjugated anti-CAR antibodies revealed increased proportions of CAR-positive cells among the resistant variants but not in normal human bronchial epithelial cells (NHBE) or normal human fibroblasts (NHFB). IgG was used as a control. (E) CAR expression was increased in treatment-resistant H460 cells in the side population (SP) subset but not in the non-SP subset. (F) CAR expression was also increased in the resistant variants of A549 cells and in the SP subset Relative to the non-SP cells (G). Columns in (D) through (G) represent means of three independent experiments; bars are 95% confidence intervals.