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 Â¡A.K., R. N. G.Â¡,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 Â¿Â¿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 Â¡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 Â±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. Flow Analysis. Samples were analyzed on a Model 4801, were also made in isotonic HBSS. Cells stained with PI after Cytofluorograf (Bio/Physics Systems Inc., Mahopac, N. Y.) hypotonie disruption of the cell membrane have bright fitted with an improved flow channel, sheath water flow, nuclear fluorescence with a broad emission peak at 610 nm. Both ADR- and AD-32-stained cells have major emis and externally stabilized photomultiplier power supply. De tails of our instrumentation have been reported previously sion peaks at 580 nm and a smaller peak at 540 nm (Chart (14). Briefly, in this flow system, the fluorescence of cells 1C). However, Pi-stained single nuclei were approximately 20-fold more fluorescent than are nuclei stained with ADR individually excited by an argon laser beam (488 nm) is or cells stained with AD-32. quantitated and scaled in a pulse height distribution ana lyzer. The abscissae of the histograms generated are di Transport and Fluorescence of ADR, AD-32, and PI vided into 512 channels of increasing linear value, and the number of cells in each channel is recorded on the ordinate. CCRF-CEM and P388 cells incubated in isotonic solutions With our improved flow system, we are routinely obtaining of the 3 fluorochromes confirmed the earlier reported data a CV of 5 to 7% for the G, peak of Pi-stained human on the transport and localization of these agents (9,13,16). lymphoblasts. ADR (1 to 10 /*g/ml) slowly enters the cells, and nuclear Samples were incubated at an approximate cell concen and cytoplasmic fluorescence of ADR can be gradually tration of 106 cells/ml, and for FCM analysis the flow rate detected, reaching a peak after the second hrof incubation. was maintained between 1 and 2 x 103 cells/sec. Under Nuclei and chromatin are brightly fluorescent with faint red these conditions the CV of the G, peak in Pi-stained periph cytoplasmic fluorescence. Cells incubated in hypotonie eral blood mononuclear cells was 6.5%. In 10 consecutive solutions of ADR (GDW) show rapid appearance of fluores runs of 40,000 cells each, the mean G, peak channel cence and its localization in nuclei and in chromosomes. number was 124.72 Â±1.173(S.D.). In subsequent runs laser Fluorescence of cells stained with ADR (1 to 10 ^g/ml in power was increased to coincide the peak of Pi-stained GDW) and examined in the Cytofluorograf hardly can be mononuclear human cells with Channel 200 Â±2.2 of the detected even with maximum available laser power and pulse height distribution analyzer. P388 tumor cells in vivo amplification. Fluorescence of most of the population is were analyzed with the G, peak at Channel 150 Â±1.9. To confirm and compare these data obtained on the Bio/ 3These observations are similar to those of Dr. Harry A. Crissman, who Physics Cytofluorograf and at the suggestion of the review was generous enough to send us copies of his unpublished spectra on PI.

NOVEMBER 1978 3657

Effect of Diluent on Fluorescence Solutions of 10% Tween 80 had no measurable fluores cence when excited at 488 nm in the spectrofluorometer. Addition of 0.1 to 1% Tween 80 to PI staining solution had no effect on the amount of PI nuclear fluorescence in CCRF-CEM cells. Similarly, in animals given injections of 0.1 ml of 2 or 8% Tween 80, the PI nuclear fluorescence of P388 ascites cells was similar to that of cells from animals given injections of HBSS.

Photometric Measurement of Fluorescence

Â«2 UH Â«6 Â«8 50 52 5 Â« [UlUIIim Â»Â«VEIEK6TH i , li. â€¢¿r Although the Olympus BH/RFL and the Zeiss fluores cence use a similar mode of epiillumination and filter combinations, the 2 systems do differ in an important detail. In the Zeiss microscope-photometer, fluorescence of indi [BISSIÂ«ugotnDYTâ€”¿BOJÂ«- SPKTIÃŒ* vidual cells was measuring by moving the cell to the central EXCITATIONAT 438 nm photometer measuring spot. In the Olympus BH/RFL micro scope, a cell was located under tungsten lamp illumination and then illuminated with a narrow 30-/*m spot of excitation from the mercury source. Because PI nuclear fluorescence does not appreciably decay on excitation from a mercury source,4 no major differences were noted in measurements made with the 2 different methods of illumination. Reduc tion in the amount of PI nuclear fluorescence was seen in all types of cells (e.g., human peripheral blood lympho cytes, CCRF-CEM lymphoblasts, liver, and spleen cells) 50 52 5Â« 56 51 60 62 S Â« exposed to hypotonie solutions of ADR. Reduction in fluo mission Â«Â«vi11.Â»ti H (i unni rescence was proportional to the ADR concentration as well as to the length of exposure (in cells exposed to isotonic solutions of ADR). In contrast AD-32, which does not show nuclear fluorescence, slightly enhanced (10%) the fluores OliisiÂ« SKCTM STAIÂ«) CHIS cence of Pi-stained nuclei. Because of its rapid transport, EKCITATIM AT 4M nm tonicity of AD-32 solutions had no effect on the PI nuclear fluorescence. These observations are in contrast to those on ADR dissolved in isotonic solution in which reduction in PI fluorescence was gradually manifested and reached a maximum only after 3 hr of incubation at 37Â°(see next paragraph). Data in Table 1 show the fluorescence intensity values of cells (liver and spleen) resuspended in PI solution that contains various concentrations (1 to 10 and 100 /ug/ml) of 52 5Â» S( SI tO 12 M (6 (I 70 ADR, AD-32, or ACT-D. In both liver (2c, 4c DNA content) EmSSIOI HAÃ¯flfltlK (iDrnÃº and spleen cells, a maximum reduction (44 to 46% of Chart 1. A, excitation spectra of ADR, AD-32. and PI solutions; B, emis control) in PI nuclear fluorescence was seen after exposure sion spectra of unbound dye and dye after mixing with calf thymus DNA; C. to ADR (100 Â¿tg/ml).Exposure to 1 and 10 /ng/ml of ADR emission spectra of cells stained with the 3 fluorochromes. In C, PI fluores cence per cell was 20 times more than that of ADR or AD-32. caused a corresponding reduction in PI fluorescence. In spleen nuclei exposed to PI that contain ADR (1 /Â¿g/ml),we could not detect any reduction in fluorescence although recorded within the first 10 channels of the Cytofluorograf/ pulse height analyzer. In contrast to ADR, AD-32 fluores liver nuclei similarly treated had reduced fluorescence (85 to 86%). In contrast the effects of AD-32 on PI nuclear cence is rapidly (less than 5 min) detected in the cytoplasm fluorescence were less significant. Enhancement of PI nu- and does not appear in nuclei or on chromosomes. The tonicity of the drug diluent (isotonic HBSS or GDW) has no 4 Fluorescence of Pi-stained nuclei illuminated with a HBO-200 mercury effect on this rapid appearance or on its localization. AD-32 lamp (excitation filter BG-12, emission filter 590), under a Zeiss microscope fluorescence is also difficult to quantitate in the Cytofluo was measured with a Nanometrics ultraspectrophotometer and the Zeiss rograf. PI (isotonic solutions) also enters cells slowly, and photometer. Fluorescence values recorded after 1 min of constant illumina tion were approximately 2% lower than were those recorded at the start. fluorescence is localized mainly in nuclei with faint cyto- Most of this loss happened during the initial 30 sec. and the values remained plasic fluorescence. Hypotonie disruption of cytoplasm remarkably constant (less than 1% decay) on further illumination for up to 10 min. We presume that this indicates resistance of PI nuclear fluorescence to leads to rapid entry and localization of PI fluorescence in photodecay on illumination from a HBO-200 mercury source and the appro nuclei (9). priate filter combination.

3658 CANCER RESEARCH VOL. 38

Table 1 Effect of ADR, AD-32, and ACT-D on PI fluorescence of liver and spleen nuclei Fluorescence intensity units"

LiverControl

2c.8Â±.2Â± (PI)ADR100Â¿ig 4.1"Â±2.4Â± Â±2.2.2

PI Â±1.2 10Â¿igAD-32100+ PI 75.2 4.7 (70) .2 Â±5.3 (69) 62.4 Â±1.9 (75) +PI+ 90.9104.4Â±0.9Â±3.9(85)(97) 180198.8 Â±2.8,8Â± (86)(95) 8492.5Â±,6Â±2.12.5(102)(112)

Â¿ig PI 4.6 10Â¿igACT-D100+PI+ 107.086.8Â±4.3Â±(100)(81) 2081709 Â±9.8.9 (99)(81) 8263.8Â±.9Â±2.92.82.6(100)(46)(100)(77)

Â¿ig PI 3.3 Â±5.5 10 Â¿ig++ PI107.247.690.12cÂ±Â±2.5(100)c(44)(84)210951461754c.54 Â±5.3(100)(45)(83)8238 71Spleen.9Â±2.62.5 (87) " Measured with a Zeiss O3 microscope photometer. Â»Mean Â±S.D. '' Numbers in parentheses, percentage of control (cells stained with PI alone). clear fluorescence (112% of control) was seen in spleen nuclear fluorescence (peak at Channel 108, Histogram D). cells exposed to AD-32 (100 tig/ml); whereas liver cells AD-32 in Vitro. In contrast to the reduced amount of PI showed a small reduction in the fluorescence. In cells nuclear fluorescence in cells incubated with ADR as de exposed to ACT-D, a reduction in PI nuclear fluorescence, scribed above, cells incubated with the ADR analog AD-32 77 to 87% of control, was noted as shown in Table 1. showed an enhancement in the amount of PI nuclear However, this could not be confirmed by flow analysis on a fluorescence. Unlike ADR the effect of AD-32 on PI nuclear Cytofluorograf. fluorescence was seen within 5 min of incubation in the isotonic drug-containing medium. Flow Cytometry-Bio/Physics Cytofluorograph Chart 3 shows PI nuclear fluorescence peaks of human ADR in Vitro. CCRF-CEM lymphoblasts and peripheral peripheral blood lymphocytes incubated in PI staining so lution that contained ADR, 10 Â¿Â¿g/ml(A)(-41%) or 5 Â¿Â¿g/ml blood lymphocytes incubated with ADR (0.1 Â¿Â¿g/ml)(Â¡so- (B) (-28%), or stained with PI alone (C) (100%). These tonic), for up to 6 hr and subsequently stained with PI showed no apparent reduction in the amount of PI nuclear results are similar to those described previously for human lymphoblasts and P388 tumor cells. However, cells incu fluorescence. However, cells incubated with ADR concen bated with AD-32 (5 Â¿Â¿g/ml)andsubsequently stained with trations higher than 0.1 Â¿Â¿g/mlhada reduced amount of PI PI showed enhancement (+11%) in the amount of PI nuclear nuclear fluorescence, that in turn was related to ADR concentration and the length of incubation. In one set of fluorescence (channel peak at 219) as shown in Chart 3, Histogram D. experiments, we compared the above observations with those for human peripheral blood lymphocytes incubated Coulter Electronics TPS Cell Sorter with either ADR (5 to 10 Â¿Â¿g/ml)mixedin the PI solution (hypotonie) or with ADR (5 to 10 Â¿Â¿g/ml)dissolvedin GDW Mouse tumor ascites (P388) and human peripheral blood or HBSS (isotonic) prior to staining with PI. Exposure of lymphocytes exposed to various ADR concentrations, dis cells to hypotonie media resulted in cytoplasmic lysis and solved in PI solution, and analyzed on the Coulter Electron the rapid entry of ADR and PI into the nuclei. Histograms in ics TPS cell sorter confirmed the data obtained on the Bio- Chart 2 show the PI nuclear fluorescence peaks from this Physics Cytofluorograf and described above. In one set of series of experiments. As expected cells incubated in hy experiments, P388 cells incubated for 15 min in PI solution potonie ADR (5 M9/m|) Chart 2, (Histogram A) had a lower that contained ADR (1,10, and 100 Â¿Â¿g/ml)showed,respec amount of PI nuclear fluorescence (Channel 135, -32%) tively, 3.8, 32.6, and 48% reduction in the amount of PI than did the cells stained with PI after incubation in isotonic nuclear fluorescence (as measured by the shift of G, peak). ADR solution (Histogram B) for 2.5 hr (Channel 177, -11%) Similarly, P388 cells incubated for 30 min in various ADR or cells stained with PI alone (Histogram C, Channel 199, concentrations (GDW) followed by PI staining for 15 min 100%. Similarly, Histogram E (Chart 2) shows that exposure had a more pronounced reduction in PI nuclear fluores to higher ADR concentration (10 Â¿Â¿g/ml,HBSS)further cence. Thus, PI nuclear fluorescence in cells exposed to reduced the amount of PI nuclear fluorescence and that the ADR (10 and 100 /Â¿g/ml)was68 and 41% of control, respec peak shifted from a control (PI alone) value of 199 to 167 tively. In contrast to ADR cells exposed to AD-32 either (-16%). Cells incubated in hypotonie ADR solutions (GDW) concurrently with PI or first to AD-32 followed by PI showed for 15 min and subsequently resuspended in PI solution an enhancement of PI nuclear fluorescence. In one set of showed a further reduction (45.7%) in the amount of PI experiments analyzed on the TPS cell sorter, we recorded

NOVEMBER 1978 3659

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. A. Krishan et al. removed from animals after 24 hr of single ADR injection 1000- 135 (Table 2). Comparable results were obtained in cells ana lyzed in the Coulter Electronics TPS cell sorter (data not included here). ACT-D, Vinblastine, Vincristine and Methotrexate in Vi tro. CCRF-CEM cells incubated in various isotonic (HBSS) or hypotonie (GDW) concentrations of these drugs (1 to 10 Â¿Â¿g/ml)for 1 to 4 hr and subsequently resuspended in PI UJ solution did not show any shifting of the G, peak in the 500- Cytofluorograf. Chelating and Other Agents. EDTA (Na:l, Na,, and K3), X sodium citrate, and 1, 10-o-phenanthroline (1 to 10 /Â¿g/ml) o dissolved in HBSS or GDW had no detectable effect on PI (T nuclear fluorescence of human peripheral blood cells and LU CL CCRF-CEM lymphocytes incubated for 1 to 4 hr prior to co staining with PI. Similarly, no effect was seen in cells _l _l incubated directly in a mixture of these agents with PI LÃ¼ solution. In contrast cells incubated with heparin (sodium O heparin, 14 USP units/ml) and subsequently stained with PI U. did not always yield reproducible DMA distribution histo O 1000 199 tr grams although the channel position of the G, peak was LÃ¼ similar to that of cells stained with PI alone. We have m occasionally experienced similar problems with tissue cul S ture cells removed from the substrate by routine trypsiniza- 13 tion prior to their staining with PI. Microscopic examination of these preparations has ruled out the possibility that cytoplasmic remnants (containing RNA) were responsible for this effect. 500

DISCUSSION ADR, a DNA-intercalating antibiotic, inhibits RNA and DNA synthesis and blocks cell cycle traverse in G-, (5, 11,

Relative Amount of Fluorescence 117 LÃ¼IOOO Chart 2. PI nuclear fluorescence peaks of mononuclear cells from human peripheral blood incubated with ADR (5 Â¿ig/ml)and PI (A, Channel 135, -32%); ADR (5 Â¿ig/ml)in isotonic HBSS for 2.5 hr followed by staining with PI (B. Channel 177, - 11%); and in PIalone (C and F. Channel 199. 100%). In D and E, cells were incubated in ADR (10 M9/m|) dissolved in GDW (D, X Channel 108, -45%) or in isotonic HBSS (E, Channel 167, -16%). O

LJ 15, 19.6, and 27.4% increase in the fluorescence of P388 CL cells exposed to AD-32 (1, 10, and 100 /Â¿g/mlrespectively). (fi These observations confirm earlier data obtained on the 500 Bio/Physics Cytofluorograf. ADR in Vivo. In tumor-bearing animals given i.p. injec LÃ¼ U tions of various concentrations of ADR, we noticed a similar reduction in the amount of PI nuclear fluorescence (Table 2). In this series of experiments, P388 leukemia-bearing mice were given injections of ADR (1 and 4 mg/kg body weight), er Lu and ascites were removed at 1, 6, and 24 hr after drug CD injection. In samples retrieved after 1 hr, a small decrease in the amount of PI nuclear fluorescence (-3%) was noted. In samples analyzed after 6 hr of drug administration, there was a reduction (12%) in the amount of fluorescence, and Relative Amount of Fluorescence the peak had shifted from a control (PI alone) value of 150 Chart 3. Pi-nuclear fluorescence peaks of human peripheral blood mononuclear cells incubated with ADR (10 ^g/ml) and PI (A, Channel 117, -41%); to 134.4 (1 mg/kg) and 131.8 (4 mg/kg). A similar reduction ADR (5 ij.g/m\) and PI (B, Channel 142, -28%); PI alone (C, Channel 197, in PI nuclear fluorescence was evident in tumor cells 100%) and AD-32, (5 M9/ml); and PI (D, Channel219, +11%).

3660 CANCER RESEARCH VOL. 38

Table 2 Effect of ADR administration (in vivo) on the fluorescence of Pi-stained P388 tumor cells Measurements were made on a Bio/Physics Cytofluorograf. Model G, peak Channel no.

TimeControlADR1 hrÂ±1.9Â°Â± Â±.4 Â±.5Â±.9Â±hr2.2(100)3.3

Â¿Â¿g/t4bodywtI 1.6Â±0.8(100)"(97)(96)15013413161Â±.8Â±hr1.51.341.03(100)(89)(88)150129128245(86)1.9 ÃÂ¿9/ÃIbody wt150.5146.1145.01 (86) " Mean Â±S.D. of 10 consecutive histograms each containing 40,000 cells. 6 Numbers in parentheses, percentage of control value (100).

18). Meriwether and Bachur (16) have shown that ADR is rapid FCM analysis for aneuploidy determination. It is slowly transported across the cell membrane and reaches conceivable that, besides ADR, many other chemothera intracellular steady-state levels in 3 hr. AD-32, a recently peutic agents may alter the PI nuclear fluorescence and synthesized analog of ADR (8), has better chemotherapeutic thus give false results.5 efficacy in a number of animal tumor systems. In a recent study (13), we have shown that AD-32 fluorescence appears ACKNOWLEDGMENTS rapidly in cells as opposed to the slower appearance of ADR fluorescence. We also showed that the fluorescence We owe our thanks to Harry A. Crissman of Los Alamos Scientific of AD-32 is localized in the cytoplasm and does not appear Laboratory, Los Alamos, N. M . for sharing with us his unpublished data on PI fluorescence. To Peter T. Monachese of the Olympus Corporation of in nuclei or chromosomes (15). AD-32 resembles ADR in America, New York; we are thankful for the loan of the Olympus BH/RFL causing chromosomal damage, in inhibiting RNA and DMA quantitative microscope. Dr. Awtar Krishan owes his gratitude to Dr. Emil Frei, III, Director of the Sidney Farber Cancer Institute, for the loan of synthesis, and in causing a block of the cell cycle traverse equipment needed for the completion of this project. inG2(12). This study confirms the rapid intracellular appearance of REFERENCES AD-32 and furthermore shows differences in the effect of ADR and AD-32 on the fluorescence of Pi-stained nuclei. 1. Alabaster, O., Tannenbaum, E., Habbersett, M. C., Magrath, I., and Thus, ADR causes reduction in the amount of PI nuclear Herman, C. Drug-induced Changes in DNA Fluorescence Intensity De tected by Flow Microfluorometry and Their Implications for Analysis of fluorescence in contrast to AD-32, which enhances the DNA Content Distributions. Cancer Res., 38: 1031-1035, 1978. fluorescence of Pi-stained nuclei. These studies indicate: 2. Callis, J., and Hoehn, H. Flow-Fluorometric Diagnosis of Euploid and (a) the probable presence of AD-32 in nuclei; and (b) a Aneuploid Human Lymphocytes. Am. J. Human Genet., 28: 577-584, 1976. possible difference between AD-32 and ADR in their mode 3. Crissman. H. A., and Steinkamp, J. A. Rapid, Simultaneous, Measure of binding to nuclei. These observations are interesting in ment of DNA, Protein and Cell Volume in Single Cells from Large Mammalian Cell Populations. J. Cell Biol., 59: 766-771, 1973. view of our earlier observations on the lack of any visible 4. Crissman, H. A., and Tobey, R. A. Cell Cycle Analysis in 20 Minutes. detectable fluorescence in nuclei or chromosomes of cells Science, 784: 1297-1298, 1974. incubated with AD-32 (13) and the reported lack of its 5. DiMarco, A., Gaetani, M., and Scarpinato, B. Adriamycin (NSC-123127): A New Antibiotic with Antitumor Activity. Cancer Chemotherapy Rept., binding to calf thymus DMA (17). 53:33-37, 1969. Experiments in this report (see also Ref. 10) show the po 6. Dittrich, W.. and Gohde, W. Impulsfluorometrie bei Einzelzellen in tential of rapid FCM analysis for detecting the presence of a Suspensionen. Z. Naturforsch.,24b: 221-228,1969. 7. Fried, J., Perez, A. G., and Clarkson, B. D. Flow Cytofluorometric drug (e.g., ADR or AD-32) in nuclei from various tissues by Analysis of Cell Cycle Distributions Using Propidium Iodide. Properties measuring the effect of the drug on PI binding. As shown in of the Method and Mathematical Analysis of the Data. J. Cell Biol., 77: 172-181,1976. this study, it was possible to detect the interference on ADR 8. Israel, M., Modest, E. J. and Frei, E., III. N-Trifluoroacetyladriamycin-14- with PI nuclear fluorescence in tumor cells removed 24 hr Valerate, an Analog with Greater Experimental Antitumor Activity and after in vivo drug injection. Examination of these nuclei Less Toxicity than Adriamycin. Cancer Res., 35:1365-1368, 1975. 9. Krishan, A. Rapid Flow Cytofluorometric Analysis of Mammalian Cell under UV microscopy did not reveal any nuclear fluores Cycle by Propidium Iodide Staining. J. Cell Biol.,66: 188-193, 1975. cence; however, the FCM could detect the presence of the 10. Krishan, A. Detection of Adriamycin and Its Analogs in Cell Nuclei by bound drug. This technique could be used to detect the Flow-Cytometry. Proc. Am. Assoc. Cancer Res., 79: 182,1978. 11. Krishan, A., and Frei, E., III. Effect of Adriamycin on the Cell Cycle presence of the bound ADR in various animal tissues, and Traverse and Kinetics of Cultured Human Lymphoblasts. Cancer Res., studies are underway to analyze this method for scanning 36:143-150,1976. 12. Krishan, A., Frei, E., Ill, and Paika, K. Studies on the Biological Action of liver, bone marrow, and tumor cells in animals given injec Adriamycin Analog, N-Trifluoroacetyladriamycin (AD-32). Proc. Am. As tions of various anthracyclines (A. Krishan, unpublished soc. Cancer Res., 78: 188, 1977. observations). 13. Krishan, A., Israel, M., Modest, E. J., and Frei, E.. III. Differences in Cellular Uptake and Cytofluorescence of Adriamycin and N-Trifluoroac- This study also shows that the interference of ADR with etyladriamycin-14-valerate. Cancer Res., 36: 2114-2116, 1976. PI nuclear fluorescence varies from tissue to tissue. 14. Krishan, A., Pitman, S. W., Tattersall, M. H. N., Paika, K. D., Smith, D. Whether this is due to differential binding of ADR or to C., and Frei, E., III. Flow Microfluorometric Patterns of Human Bone differences in DMA configuration and availability of binding 'â€¢Inarecentreport Alabaster et al. (1) have demonstrated the interference sites remains to be elucidated. of cyclophosphamide with fluorescent binding of mithramycin in vivo to This study indicates the need for caution in the use of nuclei of L1210 cells.

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Marrow and Tumor Cells in Response to Cancer Chemotherapy. Cancer DMA-Binding Studies with Adriamycin (ADR), N-Trifluoroacetyladriamy- Res.,36: 3813-3820. 1976. cin-14-Valerate (AD-32) and Related Compounds. Proc. Am. Assoc. 15. Lin, C. C., and Vand De Sande, J. H. Differential Fluorescent Staining of Cancer Res., 17: 109. 1976. Human Chromosomes with Daunomycin and Adriamycin â€”¿TheD-Bands. 18. Skovsgaard, T. and Nissen, N. I. Adriamycin, an Antitumor Antibiotic: A Science. 190: 61-63, 1975. Review with Special Reference to Daunomycin. Danish Med. Bull., 22: 16. Meriwether, W. D , and Bachur, N. R. Inhibition of DMA and RNA 62-73,1975. Metabolism by Daunorubicin and Adriamycin in L-1210 Mouse Leuke 19. Van Dilla, M. A., Trujillo, T. T., Mullaney, P. F., and Coulter, J. R. Cell mia. Cancer Res.. 32. 1137-1142, 1972. Microfluorometry: A Method for Rapid Fluorescence Measurement. 17. Sengupta, S. Â«..Seshadri, R., Modest, E. J., and Israel, M. Comparative Science. 763: 1213-1214, 1969.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1978 American Association for Cancer Research. Effect of Adriamycin and Analogs on the Nuclear Fluorescence of Propidium Iodide-stained Cells

Awtar Krishan, Ram N. Ganapathi and Mervyn Israel

Cancer Res 1978;38:3656-3662.

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