Abstract
The human melanoma cell lines MM96L, A2058 and HT144 were examined for sensitivity to ionizing radiation and UVB radiation. HT144 demonstrated a significant increase in sensitivity to ionizing and UVB radiation compared with the MM96L and A2058 cells. Sensitivity to both agents was associated with susceptibility to apoptosis. Using a protein truncation assay, a mutation for the gene for ataxia telangiectasia (ATM) was identified in HT144 cells. This was confirmed to be a homozygous mutation by subsequent sequencing of the abnormal region. Protein truncation assay of the other two cell lines showed no abnormality. The results suggest that somatic mutation of the A-T gene may be important in determining tumour radiosensitivity.
Highlights
We have examined the mechanism in observed differences in radiosensitivity between three melanoma cell lines
The established human malignant melanoma lines MM96L, A2058 and HT144 were cultured as monolayers in 5% carbon dioxide/air at 37°C in RPMI-1640 culture medium supplemented with 10% (v/v) fetal calf serum (FCS), penicillin (100 IU ml-'), streptomycin (100 jig ml-') and Hepes (3 mM)
Levels of apoptosis reached 65% in the HT144 cells compared with 23.8% and 20.4% for the MM96L and A2058 (P < 0.05)
Summary
Cell linesThe established human malignant melanoma lines MM96L, A2058 and HT144 were cultured as monolayers in 5% carbon dioxide/air at 37°C in RPMI-1640 culture medium supplemented with 10% (v/v) fetal calf serum (FCS), penicillin (100 IU ml-'), streptomycin (100 jig ml-') and Hepes (3 mM). The HT144 cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were irradiated in standard culture medium at room temperature and were immediately returned to culture conditions. A clonogenic assay was used to assess survival and replicative potential of cells after single-fraction irradiation. Plating efficiencies were first determined to calculate the required plating densities for each cell line. Colony counts were performed on cells washed in PBS fixed and stained with crystal violet stain (5% in methanol) for 5 min. Colonies of greater than 50 cells were counted under a magnifying illuminator. The surviving fraction was calculated as the number of colonies counted divided by the number of cells plated multiplied by the inverse of the plating efficiency. Survival curves were plotted as a common logarithmic plot
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