Measurements of daunorubicin uptake of Yoshida sarcoma cells in culture using flow cytofluorimetry.

Abstract

A comparative measurement of the transport and localisation of daunorubicin into Yoshida sarcoma cells, was undertaken by a biochemical extraction process and a flow cytometric method. An advantage of this latter procedure would be to identify subpopulation of cells which have enhanced or impaired daunorubicin incorporation as well as the ability to exclude any non-specific incorporation into cell debris, which would otherwise interfere with the overall estimation. It has been possible to use the Biophysics argon ion laser at a wavelength of 488 nm which coincides with the visible absorption bands of daunorubicin and doxorubicin (adriamycin) and the cytofluorimetric estimations of daunorubicin incorporation have now been compared with biochemically determined uptake in Yoshida cells. A high lethal dose of 10 microM was required to achieve the direct measurement by cytofluorimetry procedures on the Biophysics instrument. From cell fractionation and CHCl3/amyl alcohol extraction, it was possible to show that during a 5-h exposure period to daunorubicin (10 microM), the uptake into the nucleus was at first rapid and that into the cytoplasm was much slower. After about 3-h incubation, the level in the cytoplasm decreased, followed by a decrease from the nucleus 1 h later. This could be equated when observed microscopically to the gain in fluorescent cell debris. If all nuclear binding is to DNA, then at the level of (10 microM) concentration in the medium, the number of base pairs to daunorubicin would be 9 : 1, respectively. Cytofluorimetry showed a broad spread of intracellular daunorubicin fluorescence which increases with cell size. Increasing external concentration caused a more rapid incorporation as well as a quicker release from the cells.