Immunohistochemical localization of actinomycin D in human melanoma tumor cells

Human melanoma tumor colonies and derived cell lines (81-46a) were exposed to Actinomycin D (Act D) in vitro in order to study subcellular sites of drug-cell interaction. Light microscopy of 13 day-old colonies previously treated for 24 hours with 0.01 microgram/ml, 0.1 microgram/ml, and 1 microgram/ml Act D revealed a decrease in colony size and organization, an increase in pyknotic nuclei, and an accumulation of cell debris concomitant with an increase in Act D concentration. The 81-46a cells were treated with 0.1 microgram/ml Act D for 15, 30, and 60 minute intervals, followed by Act D antibodies. Immunofluorescence microscopy revealed both cytoplasmic, vesicular, filamentous, and nuclear variations in drug distribution with increased exposure. Cell viability studies indicated a decrease in viable cells as exposure time to Act D increased.