Melphalan-resistant cells have reduced ICL formation and enhanced ICL removal. (A) The alkaline comet assay was used to detect melphalan-induced cross-links. When cells were treated with vehicle control without irradiation, no comet tail moment was observed. When cells were irradiated, the comet tail moment was increased to 50. (B) Within 3 hours following a 2-hour exposure to 25 μM, 50 μM, and 100 μM melphalan, fewer ICL formations were observed in 8226/LR5 and U266/LR6 compared with 8226/S and U266/S, respectively. ^*P < .05 (ANOVA). (C) Similar amounts of cross-links are formed in drug-sensitive and drug-resistant cells at 10 hours by increasing the dose of melphalan treatment for 8226/LR5 (50 μM) and U266/LR6 (60 μM) compared with 8226/S (25 μM) and U266/S (25 μM), respectively. Within 24 hours after treatment, the remaining ICLs in drug-resistant 8226/LR5 (30.4%) are significantly less than the percentage of drug-sensitive 8226/S cells (55.2% ICLs) within 10 hours. The remaining ICLs in U266/LR6 cells (54.9%) is significantly less compared with U266/S cells (75.6%). Student t test was used for statistical analysis. ^*P < .05. Error bars denote standard deviation.