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Differences in uptake of adriamycin and daunomycin by normal BM cells and acute leukemia cells determined by flow cytometry
Leukemia Research Vol. 5, No. 3. pp. 251 257, 1981 Printed in Great Britain.
0145-2126/81/030251-07502.00/0 © 1981 Pergamon Press Ltd.
DIFFERENCES IN UPTAKE OF ADRIAMYCIN AND DAUNOMYCIN BY NORMAL BM CELLS AND ACUTE LEUKEMIA CELLS DETERMINED BY FLOW CYTOMETRY* P. SONNEVELD a n d G. J. VAN DEN ENGH~ "tRadiobiological Institute TNO, 151 Lange Kleiweg, 2288 GJ Rijswijk, The Netherlands and ~Erasmus University Rotterdam, Medical Faculty, Rotterdam, The Netherlands. (Received 6 October 1980. Revised 6 February 1981. Accepted 10 February 1981) Abstract--Flow cytometry was used to measure the uptake of adriamycin (AM) and daunomycin (DM) by various cell types of the haemopoietic organs and by acute myeloid leukemia (AML) cells. For this purpose, the spontaneous fluorescence of the drugs upon excitation with laser light at 488 nm was measured. The difference in the uptake of these two drugs correlates with their cytotoxicity. Myeloid progenitor cells from mouse bone marrow (CFU-C) are ten times more sensitive for DM than AM I'EDto = 0.85/~g/ml (DM); 6.40/zg/ml (AM)]. The cytotoxicity of DM is correlated with a higher fluorescence intensity of blast cells, iymphocytes and granulocytes when treated with DM as compared to AM. In contrast to this, the fluorescence of AML blast cells is significantly lower after treatment with DM than with AM. The uptake of both drugs seems to occur following active membrane transport, although to a different extent. Key words: Flow cytometry, adriamyein, daunomycin, leukemia.
INTRODUCTION COMBINATION chemotherapy has led to considerable progress in remission induction rates and in increasing the duration of remission of acute myeloid leukemia (AML). Most studies include daunomycin (DM) or adriamycin (AM) for remission induction therapy [3]. Recent studies by Preisler suggested that the choice of the anthracycline (AM or DM) does not affect the remission induction rate or the duration of the remission period [7]. Still, in this and most other studies the clinical dosage of DM exceeds that of AM by 50~o. These results do not correlate with the observation in vitro that DM has a higher cytotoxicity than AM when added to leukemic cells or normal hemopoietic cells [-8, 11, 15]. Recently, Buick et al. reported higher in vitro cytotoxic activity of DM than of AM for myeloblastic leukemia progenitor cells and granulopoietic progenitor cells [1]. At present, no explanation has been provided for the discrepancy between the equal clinical effectiveness of AM and DM and their difference in in vitro activity. In the study reported here, it was investigated whether differences in the uptake of the two drugs by different cell types could explain the observed variations in drug sensitivity. For this purpose, a method was developed to determine intracellular AM and DM concentrations in a flow cytometer [13]. The uptake of the drugs in normal rat haemopoietic cells and in bone marrow from patients with and without acute myeloid leukemia was investigated. *This study was supported by the Konin#in Wilhelmina Fonds of the Dutch National Cancer League. Abbreviations: AM, adriamycin; A M L acute myeloid leukemia; CFU-C, colony forming units in culture; DM. daunomycin: FLS. forward light scatter. Correspondence and reprint requests to: Dr. P. Sonneveld, Radiobiological Institute TNO. 151 Lange Kleiweg, 2288 GJ Rijswijk. The Netherlands. 251
252
P. SONNEVELDand G. J. VAN DEN ENGH MATERIALS
AND
METHODS
Patients" samples Bone marrow specimens were obtained from 3 patients with AML at the time of presentation. The diagnosis had been made on the basis of morphologic criteria. Bone marrow specimens were also obtained from 4 patients without leukemia (immunocytoma, lymphoma, anemia). None of these patients had bone marrow involvement.
Cells Human bone marrow specimens were collected in Hanks' Balanced Salt Solution containing EDTA and filtered through cotton wool to remove erythrocytes. Rat thymocytes and bone marrow cells were suspended in Hanks' solution by repeatedly filtering the specimens through multiple layers of nylon gauze. Thymocytes were then centrifuged and rcsuspended in 96% ethanol.
Drugs Adriamycin and daunomycin were a gift from Farmitalia, Milan, Italy. Before each experiment the drugs were dissolved in 0.9% NaCI and added in varying concentrations to 106 nucleated cells in a final volume of 100 #1. Cell suspensions were incubated for 1 h at 37°C and directly afterwards washed twice with glacial saline to remove extracellular drug. The cells were resuspended in 1 ml saline and kept at 0°C until the anthracyclinerelated intracellular fluorescence was determined.
Measurement of intracellular anthrycycline content The AM and DM content of individual cells was measured in a FACS II flow cytometer (Becton and Dickinson, Mountain View, California) [13]. The method makes use of the fluorescent properties of the anthracyclines. In the FACS II systems, the cells are contained in a liquid jet. The cells traverse the light beam of a 5 Watt Argon ion laser (Spectra Physics 164-05). The laser is tuned at 488 nm (0.5 to 0.8 W). This wavelength is close to the absorption maximum of the anthracyclines. The fluorescence which is emitted by the cells upon excitation by the laser light, is registered on a photomultiplier. The scatter light is blocked by a cut on 520 nm glass filter. The signals from the photomultiplier are amplified and classified by a pulse height analyser (Nuclear Data ND 100). The intensity of the fluorescence, after correction for the background fluorescence, is proportional to the anthracycline content of the cells. The light scatter from the cells is measured on a separate detector and analysed in a similar manner. The intensity of this signal is related to cell cize [15]. This signal can be used to discriminate between the different cell types in the bone marrow. The speed of analysis is 103 to 2.10 a cells/s. The analysis method itself does not interfere with cell viability,
Assay for granulocytic colony (CFU-C)formation Granulopoietic colony formation of murine CFU-C was determined using the soft agar colony assay [12]. To determine the sensitivity to AM and DM, bone marrow cells were incubated with increasing quantities of drug. They wore subsequently washed and plated. The incubation procedure was similar to that described above. Granuiocytic colonies containing more than 50 cells were counted with the use of an inverted microscope.
RESULTS
1. Uptake of adriamycin and daunomycin by rat thymocytes F l o w c y t o m e t r y c a n be u s e d to m e a s u r e d r u g c o n c e n t r a t i o n s in i n d i v i d u a l cells after i n c u b a t i o n w i t h a n t h r a c y c l i n e s . V i a b l e a n d e t h a n o l fixed rat t h y m o c y t e s w e r e e x p o s e d to v a r y i n g d o s e s o f D M o r A M for 1 h. F i g u r e s 1 a n d 2 s h o w t h e f l u o r e s c e n c e i n t e n s i t y > 520 n m t h a t w a s m e a s u r e d after this t r e a t m e n t , I n b o t h figures t h e c h a n n e l n u m b e r o f t h e p e a k o f t h e f l u o r e s c e n c e d i s t r i b u t i o n is p l o t t e d a g a i n s t d r u g d o s e (Figs. 1 a n d 2). T h e t w o d r u g s e n t e r t h e fixed cells r e a d i l y a n d i n t e r c a l a t e w i t h D N A . A t t h e s a m e d o s e , t h e f l u o r e s c e n c e o f A M is t w o t i m e s m o r e i n t e n s e t h a n t h a t o f D M d e s p i t e t h e fact t h a t t h e f l u o r e s c e n c e efficiency o f t w o d r u g s is s i m i l a r ['9] ( F i g 1). I n c o n t r a s t , v i a b l e t h y m o c y t e s t a k e u p m u c h m o r e D M t h a n A M (Fig. 2). T h e f l u o r e s c e n c e o f D M in v i a b l e cells r e a c h e s h i g h e r levels t h a n t h a t o f fixed cells. T h i s s u g g e s t s t h a t t h e cells a c t i v e l y a c c u m u l a t e t h e d r u g . T h e y a r e a p p a r e n t l y n o t c a p a b l e o f d o i n g this w i t h A M s i n c e t h e f l u o r e s c e n c e i n t e n s i t y o f v i a b l e cells at all d o s e s r e m a i n s far b e l o w t h e f l u o r e s c e n c e i n t e n s i t y o f fixed cells. A c t i v e m e m b r a n e t r a n s p o r t m a y b e i n v o l v e d since the u p t a k e is t e m p e r a t u r e d e p e n d e n t (Fig. 2). A t 0 ° C v i r t u a l l y n o A M o r D M e n t e r s the cells.
253
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Drug related fluorescence of fixed rat thymocytes after treatment with adriamycin (--) or daunomycin ( ).
2. Uptake by subpopulations of haemopoietic cells When fluorescence measurements are combined with scatter measurements the anthracycline uptake of subpopulations of bone marrow cells can be studied. Using the forward light scatter (FLS) to measure cell size, 3 subpopulations of bone marrow cells can be distinguished (Fig. 3). By using three different windows along the FLS axis the drug uptake of erythrocytes, lymphocytes and blast cells can be measured separately. Figure 3 shows the relative uptake of lymphoeytes and blasts from rat marrow. The uptake of D M
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Fig. 2. lntracellular fluorescence of viable thymocytes treated with adriamycin or daunomycin in vitro
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254
P. SONNEVELD and G.J. VAN DEW ENGH
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by the two cell types is similar. Blast cells accumulate approximately three times as much as the smaller lymphocytes. This could be related to blast cells having a larger surface area. AM is almost entirely rejected by lymphocytes whereas blast cells accumulate the drug.
3. Correlation with cytotoxicity The difference in uptake correlates with the toxicity of AM and DM for myeloid progenitor cells in mouse bone marrow. These progenitor cells are found among the blast cells of the bone marrow. A suspension of mouse bone marrow cells was incubated with varying doses of the two drugs for 1 h at 37°C. Subsequently, the cells were assayed for their ability to form granulocyte-macrophage colonies in soft agar cultures in the presence of colony stimulating factor. The results are shown in Fig. 4. DM is 10 times more
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Anthracycllne ( mg/ml ) FIG. 4. Reduction of CFU-C following in t, itro incubation with daunomycin ( - - ) or adriamycin 1--). Expressed as a percentage of untreated controls (M + 2 S.E.t.
Flow cytometric determination of intracellular anthracycline levels
255
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FIG. 5. Drug related intracellular fluorescence of human normal myeloblasts and AML blast cells from three patients treated with adriamycin or daunomycin in ritro.
effective in abolishing colony formation than AM. This concurs with the difference in uptake by viable cells as observed in Fig. 2. 4. Clinical extrapolations It should be noted that the drug concentrations which have been measured in the bone marrow of patients during anthracycline therapy [5] are lower than the concentrations that were studied here. Therefore, we have compared the nuclear fluorescence of human AML and normal blast cells after in vitro incubation with either AM or DM at concentrations which average the in vivo obtained bone marrow concentrations. Figure 5 shows the anthracycline uptake by leukemic and normal blast cells. As with the higher concentration range (Fig. 3) the fluorescence of DM treated marrow blasts exceeds that of those treated with AM. A considerable difference can be observed between the uptake of DM by AML blasts and normal blasts. On contrast, the uptake of AM is equal by both cell types (EDso DM of AML blasts, 2.4/~g/ml; EDso AM of AML blasts, 0.5/~g/mi). However, more patients need to be investigated to draw conclusions and further investigations are required both in fresh and relapse AML patients. DISCUSSION It is assumed that AM and DM freely cross the membranes of ethanol fixed cells. The intensity of the fluorescence of fixed cells therefore represents the amount of AM or DM that is bound to the nuclear DNA when at equilibrium with the drug concentration in the buffer solution. It is evident from Fig. 1 that AM has a higher binding constant than
256
P. SONNEVELD and G. J. VAN DEN ENGH
DM. It should be noted that possible spectral changes and/or changes in the quantum efficiency of the fluorescence which may occur as a result of binding of the anthracyclines to DNA are neglected. Preliminary measurements or mixtures of purified DNA and anthracyclines indicate that spectral changes and fluorescence rendement are indeed of minor importance (Sonneveld and Van den Engh, unpublished). In the investigated concentration range, the concentration-fluorescence relationship is linear on a semilog scale for both drugs, which suggests that the binding of AM and DM to DNA follow firstorder kinetics. In contrast, the fluorescence of viable cells after incubation with DM greatly exceeds that of AM, indicating that active membrane transport mechanisms play a role for both drugs but at different rates. This is confirmed by the observation that after incubation at 0°C no differences of nuclear fluorescence can be detected between AM and DM. The rate of membrane transport was shown to be different for different cell types. DM is taken up similarly by lymphocytes and blast cells whereas AM enters blast cells far easier than lymphocytes or binds to more sites as their DNA. Krishan [4] has reported that the interference of AM with propidium iodide staining is less than that of DM. This may mean that AM binds less extensively to adenine-thymine base pairs in viable cells than DM. This can alternatively be explained by differences in the uptake rate of the two drugs by the cells. It might explain the consistently higher cytotoxicity of DM over AM, which has been reported by various authors [8, 11, 15] and was also found in this study. In view of these findings it cannot be understood why the clinical antileukemic activity of both agents is equal even with a clinically applied dose of DM 50~o higher than that of AM [7]. Although normal blast cells accumulate DM more readily than AM, the opposite seems to be the case for AML blast cells. Furthermore, the fluorescence of leukemic and normal blast cells is equal after treatment with AM, but with DM normal blast cells show a consistently higher fluorescence. This difference might explain why DM is more myelotoxic than AM. Alternatively, the sparing effect of AM on macrophages has been suggested as a possible cause for this discrepancy [6]. It is noteworthy that such a difference can also be detected with lymphocytes, which take up DM more readily than AM. Flow cytometry offers a reliable and sensitive method for the detection of fluorescent intercalating agents in subpopulations of normal and leukemic bone marrow cells. This method has revealed that differences in the rate of uptake of the anthracyclines exist in normal and leukemic blast cells. This may provide an explanation for the observed differences of in vitro cytotoxicity between both drugs. Future research will focus on the physiological differences which underly the observed patterns. REFERENCES 1. BUICK R. N., MESSNER H. A., TILL J. E. & McCuLLOCH E. A. (1979) Cytotoxicity of adriamycin and daunorubicin for normal and leukemia progenitor cells of man. J. HatH. Cancer. Inst. 62, 249. 2. COLLY L. P. (1980) Chemotherapy in a transplantable myeloid leukemia in Brown Norway rats. Thesis, University of Rotterdam, The Netherlands. 3. GALE R. P. (1979) Advances in the treatment of acute myelogenous leukemia. N. Enyl. J. Med. 300, 1189. 4. KRISHAN A. 8/. GANAPATHI R. (1979) Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry. J. Histochem. Cytochera. 27, 1655. 5. LEE Y. N., CHAN K. K., H^ggm P. A. & COHEN J. L. (1980) Distribution of adriamycin in cancer patients. Tissue uptakes, plasma concentration after i.v. and hepatic i.a. administration. Cancer 45, 2231. 6. MANTOVANIA., TAGLIABUE A., VECCHI A. gt SPREAFICO F. (1976) Effects of adriamycin and daunomycin on spleen cell populations in normal and tumor allografted mice. Eur. J. Cancer 12, 381. 7. PREISLER H. D., RUSTUM Y., HENDERSON E. S., BJORNSSON S., CREAVAN P. J., HIGBY D. J., FREEMAN A., GAILANI S. ,g" HAEHER C. (1979) Treatment of nonlymphocytic leukemia: use of anthracycline-cytosine arabinoside induction therapy and comparison of two maintenance regimens. Blood 53, 455.
Flow cytometric determination of intraceilular anthracycline levels
257
8. RAZEK A., VALERIOTEF. & VIETI"IT. (1972) Survival of hematopoietic and leukemic colony-forming cells in vivo following the administration of daunorubicin or adriamycin. Cancer Res. 32, 1496. 9. SCHWARTZ, H. S. (1973) A fluorometric assay for daunomycin and adriamycin in animal tissues. Biochera. Med. 7, 396. 10. SONNEVELD P. 11980) Pharmacokinetics of adriamycin in the rat. Thesis, University of Leiden. 11. TArSUMI K., NAKAMURAT. & WASKISAKAG. (1974) Comparative effect of daunomycin and adriamycin on nucleic acid metabolism in leukemic cells in vitro. Gann 65, 237. 12. VAN DEN ENGrl G. J. (1974) Quantitative in vitro studies on stimulation of murine haemopoietic cells by colony stimulating factor. Cell Tissue Kinet. 7, 537. 13. VAN DEN ENGH G. J., VZSSERJ., BOL S. & TRASK B. 11980) Concentration of hemopoietic stem cells using a light activated cell sorter. Blood Cells 6, 609. 14. VISSER J. W. M., CRAM L. S., MARTIN J. C., SALZMAN G. C. & PRICE B. J. 11978) Sorting of a murine granulocytic progenitor cell by use of laser light scattering measurements. In: Pulse cytophotometry, 3rd Int. Syrup., Vienna, 1977 (LUTZ D. Ed.), European Press, Ghent. 15. VISSERJ. W. M., VAN DEN ENGH G. J. & VAN BEKKU~I D. W. 11980) Light scattering properties of murine hemopoietic cells. Blood Cells 6, 391. 16. WANG J. J., CHERVINSKY D. S. & ROSEN J. M. (1972) Comparative biochemical studies of adriamycin and daunorubicin in leukemic cells. Cancer Res. 32, 511.
0145-2126/81/030251-07502.00/0 © 1981 Pergamon Press Ltd.
DIFFERENCES IN UPTAKE OF ADRIAMYCIN AND DAUNOMYCIN BY NORMAL BM CELLS AND ACUTE LEUKEMIA CELLS DETERMINED BY FLOW CYTOMETRY* P. SONNEVELD a n d G. J. VAN DEN ENGH~ "tRadiobiological Institute TNO, 151 Lange Kleiweg, 2288 GJ Rijswijk, The Netherlands and ~Erasmus University Rotterdam, Medical Faculty, Rotterdam, The Netherlands. (Received 6 October 1980. Revised 6 February 1981. Accepted 10 February 1981) Abstract--Flow cytometry was used to measure the uptake of adriamycin (AM) and daunomycin (DM) by various cell types of the haemopoietic organs and by acute myeloid leukemia (AML) cells. For this purpose, the spontaneous fluorescence of the drugs upon excitation with laser light at 488 nm was measured. The difference in the uptake of these two drugs correlates with their cytotoxicity. Myeloid progenitor cells from mouse bone marrow (CFU-C) are ten times more sensitive for DM than AM I'EDto = 0.85/~g/ml (DM); 6.40/zg/ml (AM)]. The cytotoxicity of DM is correlated with a higher fluorescence intensity of blast cells, iymphocytes and granulocytes when treated with DM as compared to AM. In contrast to this, the fluorescence of AML blast cells is significantly lower after treatment with DM than with AM. The uptake of both drugs seems to occur following active membrane transport, although to a different extent. Key words: Flow cytometry, adriamyein, daunomycin, leukemia.
INTRODUCTION COMBINATION chemotherapy has led to considerable progress in remission induction rates and in increasing the duration of remission of acute myeloid leukemia (AML). Most studies include daunomycin (DM) or adriamycin (AM) for remission induction therapy [3]. Recent studies by Preisler suggested that the choice of the anthracycline (AM or DM) does not affect the remission induction rate or the duration of the remission period [7]. Still, in this and most other studies the clinical dosage of DM exceeds that of AM by 50~o. These results do not correlate with the observation in vitro that DM has a higher cytotoxicity than AM when added to leukemic cells or normal hemopoietic cells [-8, 11, 15]. Recently, Buick et al. reported higher in vitro cytotoxic activity of DM than of AM for myeloblastic leukemia progenitor cells and granulopoietic progenitor cells [1]. At present, no explanation has been provided for the discrepancy between the equal clinical effectiveness of AM and DM and their difference in in vitro activity. In the study reported here, it was investigated whether differences in the uptake of the two drugs by different cell types could explain the observed variations in drug sensitivity. For this purpose, a method was developed to determine intracellular AM and DM concentrations in a flow cytometer [13]. The uptake of the drugs in normal rat haemopoietic cells and in bone marrow from patients with and without acute myeloid leukemia was investigated. *This study was supported by the Konin#in Wilhelmina Fonds of the Dutch National Cancer League. Abbreviations: AM, adriamycin; A M L acute myeloid leukemia; CFU-C, colony forming units in culture; DM. daunomycin: FLS. forward light scatter. Correspondence and reprint requests to: Dr. P. Sonneveld, Radiobiological Institute TNO. 151 Lange Kleiweg, 2288 GJ Rijswijk. The Netherlands. 251
252
P. SONNEVELDand G. J. VAN DEN ENGH MATERIALS
AND
METHODS
Patients" samples Bone marrow specimens were obtained from 3 patients with AML at the time of presentation. The diagnosis had been made on the basis of morphologic criteria. Bone marrow specimens were also obtained from 4 patients without leukemia (immunocytoma, lymphoma, anemia). None of these patients had bone marrow involvement.
Cells Human bone marrow specimens were collected in Hanks' Balanced Salt Solution containing EDTA and filtered through cotton wool to remove erythrocytes. Rat thymocytes and bone marrow cells were suspended in Hanks' solution by repeatedly filtering the specimens through multiple layers of nylon gauze. Thymocytes were then centrifuged and rcsuspended in 96% ethanol.
Drugs Adriamycin and daunomycin were a gift from Farmitalia, Milan, Italy. Before each experiment the drugs were dissolved in 0.9% NaCI and added in varying concentrations to 106 nucleated cells in a final volume of 100 #1. Cell suspensions were incubated for 1 h at 37°C and directly afterwards washed twice with glacial saline to remove extracellular drug. The cells were resuspended in 1 ml saline and kept at 0°C until the anthracyclinerelated intracellular fluorescence was determined.
Measurement of intracellular anthrycycline content The AM and DM content of individual cells was measured in a FACS II flow cytometer (Becton and Dickinson, Mountain View, California) [13]. The method makes use of the fluorescent properties of the anthracyclines. In the FACS II systems, the cells are contained in a liquid jet. The cells traverse the light beam of a 5 Watt Argon ion laser (Spectra Physics 164-05). The laser is tuned at 488 nm (0.5 to 0.8 W). This wavelength is close to the absorption maximum of the anthracyclines. The fluorescence which is emitted by the cells upon excitation by the laser light, is registered on a photomultiplier. The scatter light is blocked by a cut on 520 nm glass filter. The signals from the photomultiplier are amplified and classified by a pulse height analyser (Nuclear Data ND 100). The intensity of the fluorescence, after correction for the background fluorescence, is proportional to the anthracycline content of the cells. The light scatter from the cells is measured on a separate detector and analysed in a similar manner. The intensity of this signal is related to cell cize [15]. This signal can be used to discriminate between the different cell types in the bone marrow. The speed of analysis is 103 to 2.10 a cells/s. The analysis method itself does not interfere with cell viability,
Assay for granulocytic colony (CFU-C)formation Granulopoietic colony formation of murine CFU-C was determined using the soft agar colony assay [12]. To determine the sensitivity to AM and DM, bone marrow cells were incubated with increasing quantities of drug. They wore subsequently washed and plated. The incubation procedure was similar to that described above. Granuiocytic colonies containing more than 50 cells were counted with the use of an inverted microscope.
RESULTS
1. Uptake of adriamycin and daunomycin by rat thymocytes F l o w c y t o m e t r y c a n be u s e d to m e a s u r e d r u g c o n c e n t r a t i o n s in i n d i v i d u a l cells after i n c u b a t i o n w i t h a n t h r a c y c l i n e s . V i a b l e a n d e t h a n o l fixed rat t h y m o c y t e s w e r e e x p o s e d to v a r y i n g d o s e s o f D M o r A M for 1 h. F i g u r e s 1 a n d 2 s h o w t h e f l u o r e s c e n c e i n t e n s i t y > 520 n m t h a t w a s m e a s u r e d after this t r e a t m e n t , I n b o t h figures t h e c h a n n e l n u m b e r o f t h e p e a k o f t h e f l u o r e s c e n c e d i s t r i b u t i o n is p l o t t e d a g a i n s t d r u g d o s e (Figs. 1 a n d 2). T h e t w o d r u g s e n t e r t h e fixed cells r e a d i l y a n d i n t e r c a l a t e w i t h D N A . A t t h e s a m e d o s e , t h e f l u o r e s c e n c e o f A M is t w o t i m e s m o r e i n t e n s e t h a n t h a t o f D M d e s p i t e t h e fact t h a t t h e f l u o r e s c e n c e efficiency o f t w o d r u g s is s i m i l a r ['9] ( F i g 1). I n c o n t r a s t , v i a b l e t h y m o c y t e s t a k e u p m u c h m o r e D M t h a n A M (Fig. 2). T h e f l u o r e s c e n c e o f D M in v i a b l e cells r e a c h e s h i g h e r levels t h a n t h a t o f fixed cells. T h i s s u g g e s t s t h a t t h e cells a c t i v e l y a c c u m u l a t e t h e d r u g . T h e y a r e a p p a r e n t l y n o t c a p a b l e o f d o i n g this w i t h A M s i n c e t h e f l u o r e s c e n c e i n t e n s i t y o f v i a b l e cells at all d o s e s r e m a i n s far b e l o w t h e f l u o r e s c e n c e i n t e n s i t y o f fixed cells. A c t i v e m e m b r a n e t r a n s p o r t m a y b e i n v o l v e d since the u p t a k e is t e m p e r a t u r e d e p e n d e n t (Fig. 2). A t 0 ° C v i r t u a l l y n o A M o r D M e n t e r s the cells.
253
Flow cytometric determination of intracellular anthracycline levels 125-
0
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/
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50-
25-
C>.----O A d r i a m y e m Daunomycin
I
I
. . . . .
5
I
10
drugs ( p g / m l ) F I G . 1.
Drug related fluorescence of fixed rat thymocytes after treatment with adriamycin (--) or daunomycin ( ).
2. Uptake by subpopulations of haemopoietic cells When fluorescence measurements are combined with scatter measurements the anthracycline uptake of subpopulations of bone marrow cells can be studied. Using the forward light scatter (FLS) to measure cell size, 3 subpopulations of bone marrow cells can be distinguished (Fig. 3). By using three different windows along the FLS axis the drug uptake of erythrocytes, lymphocytes and blast cells can be measured separately. Figure 3 shows the relative uptake of lymphoeytes and blasts from rat marrow. The uptake of D M
1O0 37°C 0- ..... ._=
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Fig. 2. lntracellular fluorescence of viable thymocytes treated with adriamycin or daunomycin in vitro
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254
P. SONNEVELD and G.J. VAN DEW ENGH
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O@ blast, granulocytes 37°C zxlblast, granulocytes 0°C OII lymphocytes 37°C Fro. 3. Drug related fluorescence in various subpopulations of normal human bone marrow treated
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with adriamycin or daunomycin (O, @: 37°C, blasts, granulocytes; A, &: 0:C, blasts, granuloeytes; [], I1: 37°C, lymphocytes and related cells).
by the two cell types is similar. Blast cells accumulate approximately three times as much as the smaller lymphocytes. This could be related to blast cells having a larger surface area. AM is almost entirely rejected by lymphocytes whereas blast cells accumulate the drug.
3. Correlation with cytotoxicity The difference in uptake correlates with the toxicity of AM and DM for myeloid progenitor cells in mouse bone marrow. These progenitor cells are found among the blast cells of the bone marrow. A suspension of mouse bone marrow cells was incubated with varying doses of the two drugs for 1 h at 37°C. Subsequently, the cells were assayed for their ability to form granulocyte-macrophage colonies in soft agar cultures in the presence of colony stimulating factor. The results are shown in Fig. 4. DM is 10 times more
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Flow cytometric determination of intracellular anthracycline levels
255
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FIG. 5. Drug related intracellular fluorescence of human normal myeloblasts and AML blast cells from three patients treated with adriamycin or daunomycin in ritro.
effective in abolishing colony formation than AM. This concurs with the difference in uptake by viable cells as observed in Fig. 2. 4. Clinical extrapolations It should be noted that the drug concentrations which have been measured in the bone marrow of patients during anthracycline therapy [5] are lower than the concentrations that were studied here. Therefore, we have compared the nuclear fluorescence of human AML and normal blast cells after in vitro incubation with either AM or DM at concentrations which average the in vivo obtained bone marrow concentrations. Figure 5 shows the anthracycline uptake by leukemic and normal blast cells. As with the higher concentration range (Fig. 3) the fluorescence of DM treated marrow blasts exceeds that of those treated with AM. A considerable difference can be observed between the uptake of DM by AML blasts and normal blasts. On contrast, the uptake of AM is equal by both cell types (EDso DM of AML blasts, 2.4/~g/ml; EDso AM of AML blasts, 0.5/~g/mi). However, more patients need to be investigated to draw conclusions and further investigations are required both in fresh and relapse AML patients. DISCUSSION It is assumed that AM and DM freely cross the membranes of ethanol fixed cells. The intensity of the fluorescence of fixed cells therefore represents the amount of AM or DM that is bound to the nuclear DNA when at equilibrium with the drug concentration in the buffer solution. It is evident from Fig. 1 that AM has a higher binding constant than
256
P. SONNEVELD and G. J. VAN DEN ENGH
DM. It should be noted that possible spectral changes and/or changes in the quantum efficiency of the fluorescence which may occur as a result of binding of the anthracyclines to DNA are neglected. Preliminary measurements or mixtures of purified DNA and anthracyclines indicate that spectral changes and fluorescence rendement are indeed of minor importance (Sonneveld and Van den Engh, unpublished). In the investigated concentration range, the concentration-fluorescence relationship is linear on a semilog scale for both drugs, which suggests that the binding of AM and DM to DNA follow firstorder kinetics. In contrast, the fluorescence of viable cells after incubation with DM greatly exceeds that of AM, indicating that active membrane transport mechanisms play a role for both drugs but at different rates. This is confirmed by the observation that after incubation at 0°C no differences of nuclear fluorescence can be detected between AM and DM. The rate of membrane transport was shown to be different for different cell types. DM is taken up similarly by lymphocytes and blast cells whereas AM enters blast cells far easier than lymphocytes or binds to more sites as their DNA. Krishan [4] has reported that the interference of AM with propidium iodide staining is less than that of DM. This may mean that AM binds less extensively to adenine-thymine base pairs in viable cells than DM. This can alternatively be explained by differences in the uptake rate of the two drugs by the cells. It might explain the consistently higher cytotoxicity of DM over AM, which has been reported by various authors [8, 11, 15] and was also found in this study. In view of these findings it cannot be understood why the clinical antileukemic activity of both agents is equal even with a clinically applied dose of DM 50~o higher than that of AM [7]. Although normal blast cells accumulate DM more readily than AM, the opposite seems to be the case for AML blast cells. Furthermore, the fluorescence of leukemic and normal blast cells is equal after treatment with AM, but with DM normal blast cells show a consistently higher fluorescence. This difference might explain why DM is more myelotoxic than AM. Alternatively, the sparing effect of AM on macrophages has been suggested as a possible cause for this discrepancy [6]. It is noteworthy that such a difference can also be detected with lymphocytes, which take up DM more readily than AM. Flow cytometry offers a reliable and sensitive method for the detection of fluorescent intercalating agents in subpopulations of normal and leukemic bone marrow cells. This method has revealed that differences in the rate of uptake of the anthracyclines exist in normal and leukemic blast cells. This may provide an explanation for the observed differences of in vitro cytotoxicity between both drugs. Future research will focus on the physiological differences which underly the observed patterns. REFERENCES 1. BUICK R. N., MESSNER H. A., TILL J. E. & McCuLLOCH E. A. (1979) Cytotoxicity of adriamycin and daunorubicin for normal and leukemia progenitor cells of man. J. HatH. Cancer. Inst. 62, 249. 2. COLLY L. P. (1980) Chemotherapy in a transplantable myeloid leukemia in Brown Norway rats. Thesis, University of Rotterdam, The Netherlands. 3. GALE R. P. (1979) Advances in the treatment of acute myelogenous leukemia. N. Enyl. J. Med. 300, 1189. 4. KRISHAN A. 8/. GANAPATHI R. (1979) Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry. J. Histochem. Cytochera. 27, 1655. 5. LEE Y. N., CHAN K. K., H^ggm P. A. & COHEN J. L. (1980) Distribution of adriamycin in cancer patients. Tissue uptakes, plasma concentration after i.v. and hepatic i.a. administration. Cancer 45, 2231. 6. MANTOVANIA., TAGLIABUE A., VECCHI A. gt SPREAFICO F. (1976) Effects of adriamycin and daunomycin on spleen cell populations in normal and tumor allografted mice. Eur. J. Cancer 12, 381. 7. PREISLER H. D., RUSTUM Y., HENDERSON E. S., BJORNSSON S., CREAVAN P. J., HIGBY D. J., FREEMAN A., GAILANI S. ,g" HAEHER C. (1979) Treatment of nonlymphocytic leukemia: use of anthracycline-cytosine arabinoside induction therapy and comparison of two maintenance regimens. Blood 53, 455.
Flow cytometric determination of intraceilular anthracycline levels
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8. RAZEK A., VALERIOTEF. & VIETI"IT. (1972) Survival of hematopoietic and leukemic colony-forming cells in vivo following the administration of daunorubicin or adriamycin. Cancer Res. 32, 1496. 9. SCHWARTZ, H. S. (1973) A fluorometric assay for daunomycin and adriamycin in animal tissues. Biochera. Med. 7, 396. 10. SONNEVELD P. 11980) Pharmacokinetics of adriamycin in the rat. Thesis, University of Leiden. 11. TArSUMI K., NAKAMURAT. & WASKISAKAG. (1974) Comparative effect of daunomycin and adriamycin on nucleic acid metabolism in leukemic cells in vitro. Gann 65, 237. 12. VAN DEN ENGrl G. J. (1974) Quantitative in vitro studies on stimulation of murine haemopoietic cells by colony stimulating factor. Cell Tissue Kinet. 7, 537. 13. VAN DEN ENGH G. J., VZSSERJ., BOL S. & TRASK B. 11980) Concentration of hemopoietic stem cells using a light activated cell sorter. Blood Cells 6, 609. 14. VISSER J. W. M., CRAM L. S., MARTIN J. C., SALZMAN G. C. & PRICE B. J. 11978) Sorting of a murine granulocytic progenitor cell by use of laser light scattering measurements. In: Pulse cytophotometry, 3rd Int. Syrup., Vienna, 1977 (LUTZ D. Ed.), European Press, Ghent. 15. VISSERJ. W. M., VAN DEN ENGH G. J. & VAN BEKKU~I D. W. 11980) Light scattering properties of murine hemopoietic cells. Blood Cells 6, 391. 16. WANG J. J., CHERVINSKY D. S. & ROSEN J. M. (1972) Comparative biochemical studies of adriamycin and daunorubicin in leukemic cells. Cancer Res. 32, 511.