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Drug-induced Changes in DNA Fluorescence Intensity Detected by Flow Microfluorometry and Their Implications for Analysis of DNA Content Distributions

Laboratory of Pathology [O. A., E. T., M. C. H., C. H.] and Pediatric Oncology Branch [I. M.], National Cancer Instititute, NIH, Bethesda, Maryland 20014

Chicken erythrocytes, which contain less DNA than mammalian diploid cells, were used as an internal standard to control instrumental and staining varlables during flow microfluorometric analysis. With the DNA stain, mithramycin, and with an EPICS II flow microfluorometer, ratios between the modal G₁ fluorescence of experimental cells and that of chicken erythrocytes were determined. The results indicate that unperturbed cell populations of L1210 and HeLa cells in vitro and L1210 ascites cells in vivo have relatively stable fluorescence ratios, although there is a significant difference between the ratios of one L1210 cell line in vitro and another in vivo.

In contrast, L1210 ascites treated in vivo with different schedules of cyclophosphamide and Adriamycin showed wide fluctuations in the fluorescence intensity ratios for 96 hr after treatment. Also, differences in the fluorescence ratios were observed between less advanced and more advanced L1210 ascites after treatment with the same schedule.

These effects indicate an alteration in DNA staining with mithramycin, brought about by drug treatment that could seriously affect the interpretation of DNA histogram data. Nevertheless, changes in mithramycin staining may prove to be a very important probe to detect persistent drug effects.

¹ To whom requests for reprints should be addressed, at Room 1A21, Building 10, National Cancer Institute, NIH, Bethesda, Md. 20014.

Received 9/16/77. Accepted 1/16/78.