title: An assay to measure adriamycin binding in osteosarcoma cells.
abstract: Adjuvant chemotherapy is currently employed in the treatment of patients with osteosarcoma, but the drug regimens, although effective in improving disease-free survival, are unsuccessful in 20-40% of patients and very toxic. It would be useful to know whether tumor cells are sensitive to a given drug prior to its use. To this end, we developed a method of assessing Adriamycin (doxorubicin) binding to tumor nuclei as a possible means of detecting sensitivity to the drug. Adriamycin-sensitive murine osteosarcoma cells were used to develop the assay. The in vitro conditions (drug concentration, duration of incubation, and temperature) were optimized with use of the murine osteosarcoma cells in culture. After the cells had been incubated with Adriamycin, cell viability was determined and Adriamycin fluorescence intensity was measured with a cytofluorometer. The optimal parameters for Adriamycin binding were found to be a 30-minute incubation in a 10 micrograms/ml concentration of Adriamycin at 37 degrees C; the frequency of cells that emitted Adriamycin fluorescence from the nucleus compared with the total number of living cells reached 100% under these conditions. In a murine leukemia cell line with known sensitivity to Adriamycin, the cells emitted red fluorescence from the nucleus and cytoplasm, whereas in a resistant line the cells emitted Adriamycin fluorescence from only the cytoplasm. We demonstrated that it is possible to differentiate nuclear from cytoplasmic concentration of Adriamycin in a tumor cell with use of a fluorescent microscope and that resistant cell lines can be distinguished from sensitive cell lines by this method.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Orthopaedic Oncology Unit, Massachusetts General Hospital, Boston 02114.
authors.forename: M C
authors.initials: MC
authors.lastname: Gebhardt
authors.forename: K
authors.initials: K
authors.lastname: Kusuzaki
authors.forename: H J
authors.initials: HJ
authors.lastname: Mankin
authors.forename: D S
authors.initials: DS
authors.lastname: Springfield
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D004317:Doxorubicin / Q000378:metabolism*; D004354:Drug Screening Assays, Antitumor; D005470:Fluorometry / Q000379:methods*; D051379:Mice; D012516:Osteosarcoma / Q000378:metabolism* / Q000473:pathology; D014407:Tumor Cells, Cultured
chemical_list: D004317:Doxorubicin