Characterization of the Association of Radiolabeled Bleomycin A₂ with HeLa Cells

The association of [3H]bleomycin A2 and Cu(ll):[3H]bleomycin A2 with HeLa cells has been characterized. Under the conditions of our experiments, approximately 0.1% of the total drug in the medium associates with HeLa cells. Both forms of the drug bind to HeLa cells in a specific and saturable manner, with a Km of 20 μm and a Vmax of 2.5 pmol/min/106 cells. Scatchard analysis of the specific binding data demonstrates a single set of high-affinity binding sites. Cytotoxic activities of both forms of the drug are similar, with a 50% lethal dose of 0.5 μM at 48 hr. The specific binding in HeLa cells of either the labeled metal-free drug or its copper complex is reversible by a 100-fold excess of either unlabeled drug. Interaction of the drug with cells is temperature sensitive but is unaffected by metabolic poisons, suggesting that this process is not energy dependent. Isolation of DNA from HeLa cells incubated with the drug indicates that 1 mol of either [3H]bleomycin A2 or Cu(ll):[3H]bleomycin A2 binds per 108 nucleotides. Further studies with the radiolabeled drug are required to define precisely the mechanisms involved in bleomycin uptake and compartmentalization within the cell.