Rhodamine 123 and flow cytometry to monitor the cytotoxic actions of nucleoside analogues in nondividing human lymphocytes.
We used human lymphocytes from the peripheral blood of normal individuals to compare the cytolytic actions of 2-chloro-2'-deoxyadenosine (2-Cl-dAdo) and 9-beta-D-arabinofuranosylguanine (ara-G). Membrane integrity was ascertained by flow cytometric quantification of the cells' uptake of the fluorescent mitochondria-specific probe rhodamine 123. Addition of 10 microM ara-G to lymphocyte cultures led to a progressive decline in the number of cells that could assimilate rhodamine 123, and after 2 days exposure to drug less than 50% of the cells showed normal staining compared to untreated cells. 2-Cl-dAdo had similar cytotoxic effects at a 0.1 microM concentration. Under the same conditions trypan blue revealed only a 10-20% toxic effect by these drugs. Using rhodamine 123 in combination with the vital stain propidium iodide, we found that 90% of the cells with abnormal rhodamine 123 uptake also stained with propidium iodide. The changes in membrane permeability to fluorescent dyes correlated with the loss of lymphocyte ability to respond to the plant mitogen phytohemagglutinin. Our data indicate that rhodamine 123 is a more sensitive probe than trypan blue for ascertaining early loss of the membrane integrity of dying lymphocytes. Combined with flow cytometry, rhodamine 123 uptake represents a simple and rapid method for identifying the important biochemical events associated with the cytocidal and cytostatic effect of drugs against normal and leukemic cells.
D001087:Arabinonucleosides / Q000494:pharmacology; D002463:Cell Membrane Permeability; D017338:Cladribine; D003839:Deoxyadenosines / Q000031:analogs & derivatives / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D009705:Nucleosides / Q000633:toxicity*; D020112:Rhodamine 123; D012235:Rhodamines; D014343:Trypan Blue; D014966:Xanthenes
D001087:Arabinonucleosides; D003839:Deoxyadenosines; D009705:Nucleosides; D012235:Rhodamines; D014966:Xanthenes; D020112:Rhodamine 123; C028771:9-arabinofuranosylguanine; D017338:Cladribine; D014343:Trypan Blue