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Effect of Adriamycin and Analogs on the Nuclear Fluorescence of Propidium Iodide-Stained Cells1

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Effect of Adriamycin and Analogs on the Nuclear Fluorescence of Propidium Iodide-Stained Cells1 [CANCER RESEARCH 38, 3656-3662, November 1978] 0008-5472/78/0038-OOOOS02.00 Effect of Adriamycin and Analogs on the Nuclear Fluorescence of Propidium Iodide-stained Cells1 Awtar Krishan, Ram N. Ganapathi, and Mervyn Israel Comprehensive Cancer Center for the State of Florida. University of Miami School of Medicine, Miami, Florida 33752 ¡A.K., R. N. G.¡,and Sidney Farber Cancer Institute, Harvard Medical School. Boston. Massachusetts 02115 [M. I.] ABSTRACT study chromosomal variations as small as the presence of an extra X (XXY) were rapidly identified. Adriamycin (ADR) and AMrifluoroacetyladriamycin-14- With proper instrumentation and precautions (e.g., uni valerate, respectively, inhibit and enhance the nuclear form flow rate and proper alignment of the flow channel), it fluorescence of cells stained with propidium iodide for is routinely possible to obtain highly reproducible quantita DNA per cell estimation by flow cytometry. In cells incu tion of per cell DNA content by FCM. However, it is conceiv bated with ADR, the reduction in fluorescence is gradually able that exposure of cells to other DNA-intercalating drugs manifested due to the slow intracellular drug transport. In (e.g., in cancer chemotherapy) may alter binding of PI to contrast the effect of N-trifluoroacetyladriamycin-14-val- DNA and thus yield erroneous results. This report describes erate on propidium iodide nuclear fluorescence is seen our observations on the effect of a number of cancer within 5 min of incubation. The effect of ADR on propidium chemotherapeutic agents (e.g., ADR, AD-32, ACT-D, meth- iodide nuclear fluorescence could be detected in vivo otrexate, vinblastine, vincristine), chelating agents (EDTA, even after 24 hr of ADR administration. sodium citrate), and other agents (e.g., heparin, trypsin, Tween 80) on the fluorescence of cells stained with PI. This INTRODUCTION study also shows the potential of this method for detecting the presence of a DNA-intercalating agent (e.g., ADR) in the FCM2 is an important technique for rapid quantitation of nuclei of single cells. cellular DNA content (6, 19). In this system single cells in a suspension are excited with a monochromatic light source MATERIALS AND METHODS (e.g., argon laser beam, 488 nm), and fluorescence of a DNA-dye complex is quantitated at the rate of ~105 cells/ Log-phase cultures of CCRF-CEM human lymphoblasts min. A number of fluorescent DMA-intercalating drugs (e.g., were grown in suspension cultures and nourished with ethidium bromide, PI, mithramycin) have been used for Eagle's minimal essential medium (for spinner cultures), quantitation of per cell DNA content by FCM (3, 4). The use supplemented with 10% fetal calf serum and the antibiotics, of these fluorochromes obviates the need for prior fixation penicillin and streptomycin. Human peripheral blood was and acid hydrolysis of the cells and has led to the extensive collected from healthy donors in blood collection tubes use of FCM for rapid cell cycle analysis and determination containing potassium EDTA as an anticoagulant. Mononu- of aneuploidy. Thus, FCM can provide information on the clear cells were separated by centrifugation over a Ficoll- proliferative status of a cell population within 15 min of Hypaque gradient, and washed twice in HBSS. Single-cell sample retrieval. suspensions were prepared from spleen and liver of DBA/2 PI, an analog of ethidium bromide, apparently binds to mice by mincing and filtering of the tissues through glass double-stranded RNA and DNA, primarily by intercalation, wool and 70 ¿¿mmonofilamentnylon cloth (Small Parts, and the quantum efficiency of the bound dye ethidium Inc., Miami, Fla.). bromide is enhanced 25-fold over that of the unbound dye MaleC57BL x DBA/2 F, mice were given i.p. injections of (3). We have described earlier a Pi-hypotonie citrate method 106 P388 mouse ascites cells maintained in DBA/2 mice by for per cell DNA quantitation by FCM (9). This method is weekly transplantation. Twenty-seven tumor-bearing mice rapid and reliable and can yield DNA distribution histo were divided at random in 3 groups of 9 each. Animals in grams similar to or better than those obtained by other Groups 1 and 2 were given i.p. injections of ADR, 1 or 4 methods (7, 14). In a recent report Callis and Hoehn (2) mg/kg body weight (average weight, 20 g) in 0.1 ml of have demonstrated the use of this staining method for rapid diluent (HBSS). The remaining 9 animals served as controls, diagnosis of aneuploidy in peripheral blood cells. In their and 2 of these were given injections of 2 and 8% Tween 80 diluted in HBSS, respectively. Animals (3 from each ADR group) were sacrificed after 1, 6, and 24 hr of drug admin ' These studies were supported by Grants CA-23688 and CA-19118 from istration. Tumor cells from 1 ml of ascites were retrieved by the National Cancer Institute, United States Department of Health, Education and Welfare. A preliminary report of this work was first presented at the 69th centrifugation and resuspended in 2 to 5 ml of PI solution Annual Meeting of the American Association for Cancer Research, Washing to give a final cell concentration of 1 to 2 x 106 cells/ml. ton, D. C., 1978 (10). This work was started at Sidney Farber Cancer Institute. Ascites cells from control animals were similarly treated. Boston and continued at University of Miami Medical School. ' The abbreviations used are: FCM. flow cytometry; PI. propidium iodide; PI was purchased from Calbiochem, San Diego, Calif., ADR, Adriamycin, AD-32, fV-trifluoroacetyladriamycin-14-valerate; ACT-D. actinomycin D; HBSS. Hanks' balanced salt solution; GDW. glass-distilled and staining solutions (henceforth referred to as PI solu water; CV, coefficient of variation. tion) were made by dissolving 5 mg of the dye in 100 ml of Received July 11, 1977; accepted July 27, 1978. GDW. ADR-HCL (NSC 123127) and AD-32 (NSC 246131) 3656 CANCER RESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. ADR and PI Nuclear Fluorescence were made as 1-mg/ml stock solutions in 10% Tween 80 ers, we have recently used a Coulter Electronics TPS I cell (ADR and AD-32) or ¡nHBSS (ADR) and further diluted in sorter fitted with a 4-watt Spectraphysics Model 164 laser to HBSS or GDW immediately before use. ACT-D (Cosmegen; analyze ADR-, AD-32-, and Pi-stained cells. DNA distribution Merck, Sharp and Dohme, West Point, Pa.), vinblastine and histograms of Pi-stained lymphoblasts analyzed in this vincristine sulfate (Eli Lilly and Co., Indianapolis, Ind.), and instrument at a laser power setting of 1 watt gave a CV of 3 methotrexate stock solutions were made in HBSS. In all to 4%. In 10 consecutive runs of Pi-stained lymphoblasts staining protocols an approximate cell concentration of 1 (100,000 each), the peak channel of G, DNA content did not to 2 x 106cells/ml was maintained unless stated otherwise. deviate by more than ±1channel. Fluorescence Microscopy. Cells stained with ADR, AD- 32, or PI were scanned for their fluorescence emission RESULTS spectra under a Zeiss fluorescence microscope fitted with a mercury light source (HBO-200), Zeiss III RS epiilluminator, Excitation and Emission Characteristics and a Nanometrics ultraspectrophotometer (Nanometrics Inc., Irvine, Calif.). Filters used were BG-38 and BG-12 Excitation spectra in Chart 1A show that the fluorescence of ADR, AD-32, and PI is maximally excited between 460 (peaktransmittance, 400 nm) for excitation and barrier filter and 480 nm. Emission spectra for ADR and AD-32 (Chart no. 53 (transmittance, 530 nm) for emission. 16) are similar with peaks at 550 and 590 nm. AD-32 in Fluorescence of single cells (or nuclei after hypotonie disruption of the cell membrane) stained with ADR, AD-32, equimolar concentration is less fluorescent than is ADR. PI and/or PI was measured either with a Zeiss 03 microscope (unbound) has an emission peak at 590 nm, but the overall photometer or under an Olympus BH/RFL quantitative fluorescence intensity of unbound PI (excitation at 488 nm) is less than that of ADR or AD-32. Addition of calf thymus microscope fitted with an epiilluminator for fluorescence. In the Olympus unit a high-pressure mercury light source DNA, 1 mg/ml, to ADR, AD-32, or PI solution has significant (HBO-100) was used with the following filter combination: effects on the fluorescence intensity. As seen in Chart 16, exciter, BG-12; dichoric mirror, DM-445; and barrier filter addition of calf thymus DNA to ADR resulted in pronounced transmitting above 590 nm. All measurements were made fluorescence quenching, whereas no effect was seen on under a x40oil immersion (nonfluorescing) Olympus objec the fluorescence of AD-32 (curve not shown). In contrast tive (N. A. 1.0) with a 30-fj.m illuminating spot. addition of calf thymus DNA to PI solutions enhanced the Excitation and emission spectra of ADR, AD-32, and PI fluorescence approximately 20-fold.3 solutions (with or without calf thymus DMA (1 mg/ml) were For measurement of the emission spectra of stained cells, obtained on a Hitachi Perkin-Elmer MPF-2A fluorescence PI and ADR dilutions were made in hypotonie GDW to spectrophotometer. For excitation spectra the emission facilitate diffusion across the cell membrane and nuclear monochromator was set at 580 nm for ADR, 590 nm for AD- binding. In view of the earlier reported (13) rapid appear ance and localization of AD-32 fluorescence in cytoplasm 32 and 590 nm for PI. For emission spectra the excitation (no detectable fluorescence in nuclei), AD-32 solutions monochromator was set at 488 nm.
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