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[Cancer Research 60, 1901-1907, April 1, 2000]
© 2000 American Association for Cancer Research

Immunology

Anthracyclines Trigger Apoptosis of Both G0-G1 and Cycling Peripheral Blood Lymphocytes and Induce Massive Deletion of Mature T and B Cells1

Carole Ferraro2, Laurence Quemeneur2, Annie-France Prigent, Catherine Taverne, Jean-Pierre Revillard and Nathalie Bonnefoy-Berard3

Laboratory of Immunology, Institut National de la Santé et de la Recherche Médicale U503 UCBL, Hospital E. Herriot, 69437 Lyon Cedex 03 [C. F., L. Q., C. T., J-P. R., N. B-B.], and Laboratory of Biochemistry and Pharmacology, Institut National de la Santé et de la Recherche Médicale U352, INSA-Lyon, 69621 Villeurbanne Cedex [A-F. P.], France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNR4 and DOX are two anthracyclines that are widely used in clinical oncology, essentially in the treatment of acute leukemia, Hodgkin’s or non-Hodgkin’s lymphomas, but also in some solid tumors (breast, ovarian, or endometrial cancers; Ref. 1 ). Similar to other anthracyclines, they may also be used to intensify standard conditioning before bone marrow transplantation (2) . As DNA intercalating agents, anthracyclines were regarded as cell cycle-dependent cytotoxic drugs. Indeed, DNR and DOX were reported to inhibit cell proliferation and to trigger apoptosis of different cell lines, such as the leukemic T-cell line Jurkat and CEM (3 , 4) or the myelomonocytic cell lines HL60 and U937 (5) . However, in addition to their DNA intercalating properties, DNR and DOX were demonstrated to induce lipid peroxidation, generation of ROS, and to directly inhibit topoisomerase II (6) . How such properties account for the cytotoxic effect of theses two drugs is still not clearly defined. The apoptotic signaling pathways induced by anthracyclines have been investigated recently, and evidence for the implication of CD95-L/CD95 interaction has been reported in the Jurkat (4) and CEM (3) T-cell leukemia cell lines. Herr et al. (4) reported a CD95-L/CD95-dependent cell death consecutive to DOX-induced ceramide generation in Jurkat cell line. These results are in agreement with the capacity of DNR to stimulate neutral sphingomyelinase or ceramide synthase activity and subsequent ceramide generation in U937 or HL60 human leukemia cells (5 , 7, 8, 9) . Nevertheless, the involvement of CD95-L/CD95 interaction remains controversial (10 , 11) , and some Fas-resistant Jurkat cell lines were reported to undergo apoptosis after treatment with DOX (10) . DOX was reported to increase CD95-L mRNA expression in the HUT lymphoma and CEM cell lines but to induce apoptosis of these cells by a CD95-independent pathway (12 , 13) .

Thus far, investigations on the action of DNR and DOX have been essentially performed on leukemic T-cell lines. However, because of their extensive use in clinical oncology, it is essential to analyze the effect of anthracyclines on the immune response and more especially on T cells because of risk of long-lasting immune deficiency. Quite unexpectedly, when we investigated the mechanism of anthracycline cytotoxicity toward PBLs, we observed that nonactivated PBL cells in G0-G1 phases of the cell cycle were as susceptible as cycling mitogen-activated lymphoblasts. We report here that PBL death presents the main features of apoptosis, involves ceramide generation and caspase activation, and is independent from CD95/CD95-L or TNF/TNF-R interactions as well as from the oxidative properties of these two drugs. The clinical implications of these studies on T-cell depletion after anthracycline administration are discussed.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Preparation and Culture.
PBLs were collected from healthy donors in the presence of sodium citrate. Blood was defibrinated, and then mononuclear cells were isolated by centrifugation on a layer of Histopaque (Dutcher, Brumath, France). Those cell suspensions referred to as PBL contained 1.8 ± 0.4% monocytes and 4–11% B lymphocytes as defined by expression of CD14 and CD20, respectively. PBLs were resuspended in RPMI 1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 10% FCS, 2 mM L-glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin ). Cells (1 x 106/ml) were incubated in the presence of different mitogens. Cultures were maintained in a humid atmosphere containing 5% CO2 for 72 h. For proliferation assay, cells were pulsed during the indicated time with [3H]thymidine (Amersham France SA, Les Ulis, France) at 0.5 µCi/well. [3H]Thymidine uptake was measured using a Packard direct counter (Packard, Meriden, CT) after harvesting.

Isolation of Splenocytes, Thymocytes, and Lymph Node Cells.
BALB/c mice (4–5 weeks; obtained from IFFA Credo, Saint Germain sur l’arbresle, France) received injections i.p. with DNR, DOX, MTX, or solvent 0.15M NaCl alone. Nine or 36 h later, spleen, thymus, and mesenteric lymph nodes were removed and weighted. Cell suspensions were made by mashing the splenic capsule, the thymus, or mesenteric lymph nodes between frosted ends of glass slides, passage through nylon wool column, and centrifugation. Cells were resuspended in DMEM (Sigma) supplemented with 10% FCS, 5 x 10-5 M ß2-mercaptoethanol, 2 mM L-glutamine, and antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) and viable cells were counted by trypan blue dye exclusion.

Antibodies and Reagents.
DNR and DOX were purchased from ICN Biochemical (Costa Mesa, CA). ETO and MTX were from Sigma and dissolved in distilled water for in vitro studies or in 0.1% NaCl for in vivo studies. PHA, PMA, ionomycin, aphidicholin, glutathione, and N-acetyl-L-cysteine were purchased from Sigma. CsA was kindly supplied by Novartis Corporation. FK506 was purchased from Biomol (Plymouth Meeting, PA). FITC-conjugated CD25 mAb and FITC-conjugated CD69 mAb were obtained from Becton Dickinson (Pont de Claix, France). FITC-conjugated CD95 mAb was purchased from Immunotech (Marseille, France). Phycoerythrin-conjugated antimouse CD4, CD8, or B220 mAbs were purchased from Caltag Laboratories (Burlingame, CA). Purified anti-CD95 mAb agonist (IgM, clone 7C11) and antagonist (IgG1, clone ZB4) were purchased from Immunotech. Fas-Fc fusion protein and TNF-R p55 immunoglobulin fusion protein were kindly provided by Dr. D. Green (La Jolla, CA) and Dr. H. Waldman (Oxford, United Kingdom), respectively. The caspase inhibitory peptide zVAD-fmk was from Bachem (Voisins le Bretonneux, France).

Measurements of Cell DNA Content by Flow Cytometry.
For DNA content analysis, cells were washed in PBS and stained with 50 µg/ml propidium iodide in 0.1% Triton X-100, 0.1 mM EDTA, and 50 µg/ml RNase after fixation with 70% ethanol overnight at 4°C in the dark, as described previously (14) . Cell suspensions were analyzed with a FACScan flow cytometer using an argon laser ({lambda}. Ex.Max., 540 nm; {lambda}. Em.Max., 620 nm). The relative percentages of cells in G0-G1 or S-G2M phases of the cell cycle were determined using the Cell Fit software (Becton Dickinson).

Measurement of Cell Viability.
Cell death was measured by trypan blue exclusion. Viable and dead cells were counted by microscopy.

Measurement of Apoptosis.
Apoptotic cell death was measured by fluorescence microscopy after staining with Hoechst 33342 (Sigma) at 10 µg/ml following methods described previously (15) . Nuclear fragmentation and/or marked condensation of the chromatin with reduction of nuclear size were considered as typical features of apoptotic cells. On the basis of these measurements, results were expressed as a percentage of specific apoptosis according to the following formula:

Annexin V Binding.
Exposure of surface phosphatidylserine was quantified by surface annexin V staining as described previously (16) . Cells were resuspended in binding buffer and incubated with FITC-conjugated annexin V (Bender MedSystems, Vienna, Austria) for 5 min. Cells were analyzed by flow cytometry with the LYSIS II software.

TUNEL Assay.
DNA fragmentation was detected on 10% formaldehyde-fixed, paraffin-embedded tissue section by TUNEL assay with Apotag In Situ Apoptosis Detection kit and performed according to the manufacturer’s instructions (Intergen Company, Oxford, United Kingdom).

Determination of PARP Cleavage.
Cleavage of PARP was determined by Western blotting as described previously (17) .

Ceramide Measurement.
Ceramide was quantified by the diacylglycerol kinase assay as (32P) incorporated upon phosphorylation of ceramide to ceramide-1-phosphate by diacylglycerol kinase from Escherichia coli (Biomol, Plymouth Meeting, PA) as described previously (17) .

Measurement of DOX and DNR-induced Intracellular ROS Formation.
Production of ROS was detected with H2DCFDA (molecular probes; Interchim, Montlucon France), a nonfluorescent compound that freely permeates cells. Inside the cells, H2DCFDA is hydrolyzed to 2',7'-dichlorofluorescin and trapped. This molecule interacts with peroxides and gives rise to a fluorescent compound, the 2',7'-dichlorofluorescein, which can be detected by fluorescence-activated cell sorting analysis. Briefly, treated cells were washed in PBS and labeled with 5 µM H2DCFDA for 15 min at 37°C, and cell fluorescence was determined by flow cytometry with excitation and emission settings of 500 and 535 nm, respectively. The fluorescence intensity of each sample was expressed as MFI calculated from each peak. The extent of ROS production was indicated as the difference between MFIdrug and MFIcontrol. Results were expressed as an index according to the following formula:

Statistical Analysis.
Data are expressed as mean ± SE. Differences between data sets were evaluated by performing an unpaired Student’s t test. A level of P < 0.05 was accepted as statistically significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Anthracyclines Induce Apoptosis of Nonactivated Peripheral Lymphocytes.
We observed that DNR and DOX induced progressive cell loss in both resting and 3-day PHA-activated PBLs in a dose-dependent manner. The decrease of viable cell number was maximal after 48 h in activated PBLs and after 72 h in resting cells (data not shown). The decrease of cell viability in both types of PBLs was associated with an increased percentage of cells showing characteristic features of apoptotic cell death, such as nuclear condensation or fragmentation observed by transmission electronic microscopy, a decreased mitochondrial transmembrane potential ({Delta}{Psi}m) measured by 3,3'-dihexyloxacarbocyanine iodide (3) staining (data not shown), an externalization of phosphatidylserine measured by annexin V binding (Fig. 1Citation , left) and by nuclear condensation or fragmentation observed by fluorescence microscopy after Hoechst staining (Fig. 1Citation , right).



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  		Fig. 1. Kinetics of DNR- and DOX-induced apoptosis. Freshly purified PBLs (open symbols) or 3-day PHA-activated lymphoblasts (5 µg/ml; closed symbols) were cultured with DNR (2 µM) or DOX (2 µM). The percentage of apoptotic cells was measured at the indicated time by flow cytometry after annexin V labeling (left panel) or by fluorescence microscopy after staining with Hoechst 33342 (right panel). Results are expressed as the percentage of specific apoptosis as defined in "Materials and Methods." Values are the means of six and three independent experiments for PBLs and PHA-activated PBLs, respectively; bars, SE.

 
The kinetics of apoptosis induced by DNR (1 µM) or DOX (2 µM) was determined in parallel by annexin V binding and by Hoechst staining. Annexin V binding reached a maximum after 12 h of incubation with 3-day PHA-activated lymphocytes and after 24–48 h with nonactivated PBLs (Fig. 1Citation , left). As expected, the kinetics of apoptotic cells characterized by nuclear fragmentation or condensation after Hoechst staining were delayed as compared with annexin V staining with a maximum after 48 h for both nonactivated and activated cells (Fig. 1Citation , right).

Peripheral Lymphocytes in G0-G1 Are Susceptible to Apoptosis.
To further document that the transition from G1 to S phase of the cell cycle was not required for DNR- and DOX-triggered apoptosis, we investigated whether PHA-activated lymphoblasts blocked in G1 phase of the cell cycle were as susceptible to apoptosis as PHA-activated lymphocytes. Indeed, previous studies had shown that cell cycle-dependent drugs such as MTX did not induce apoptosis of activated T cells blocked in the G1 phase of the cell cycle (18) . To this end, PBLs were stimulated by PHA in the presence of CsA or FK506, which inhibit G1-S transition by transcriptional blockade of interleukin 2 (14 , 19) , or in the presence of aphidicolin, which inhibits DNA polymerases {alpha} and {delta} (20) and then inhibits the proliferative response by blocking the progression from the G1 to the S phase of the cell cycle. PHA-activated lymphocytes cultured in the presence of CsA expressed the G1 phase markers CD69, CD95, and CD25 (Table 1)Citation but the percentage of cells in S-G2 was markedly decreased as compared with PHA controls. Nevertheless, the level of apoptosis triggered by DNR and DOX was similar to that of unactivated or PHA-activated lymphocytes. By contrast, the topoisomerase II inhibitor ETO did not induce apoptosis of lymphocytes in the presence of CsA (Table 1)Citation . Similarly, incubation with FK506 or aphidicolin decreased ETO-induced but not DNR- and DOX-induced apoptosis (Fig. 2Citation ).


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  		Table 1 Activated T cells in G1 phase are susceptible to apoptosis

 


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  		Fig. 2. Effect of inhibition of the G1 to S phase transition on DNR- or DOX- induced apoptosis. PBLs were activated for 3 days with PHA (5 µg/ml) alone or in the presence of CsA (250 ng/ml), FK506 (10 nM), or aphidicolin (125 ng/ml). [3H]Thymidine uptake, measured on the last 12 h of activation, was 22609, 9948, 11043, and 9503 cpm, respectively. Seventy-five % of PHA-activated lymphoblasts expressed CD25, 58% with CsA, 62% with FK506, and 55% with aphidicolin. After removing dead cells, activated cells were treated by DNR (1 µM) or DOX (2 µM) for 20 h. The percentage of apoptotic cells was determined by fluorescent microscopy by Hoechst 33342 staining. Results are expressed as specific apoptosis as described in "Materials and Methods." Values are the means of four individual experiments for CsA and FK506 and three individual experiments for aphidicolin; bars, SE.

 
Anthracyclines Induce Ceramide Production in Both Resting and Cycling PBLs.
Knowing that DNR induces ceramide production in lymphoid and monocytic cell lines (5 , 7 , 21) , we investigated whether DNR and DOX would also induce ceramide production in lymphocytes. Lipids were extracted from nonactivated or 3-day PHA-activated lymphocytes treated with either DNR (2 µM) or DOX (2 µM), and endogenous ceramide production was measured by a diacylglycerol kinase assay. As shown in Fig. 3Citation , DNR and DOX induced a rapid and transient increase of ceramide production in both types of lymphocytes. Ceramide production after DNR treatment was maximal after 30 and 60 min in activated and nonactivated cells, respectively, and decreased rapidly to reach the basal level after 2–4 h (Fig. 3ACitation ). Similar kinetics was observed in activated cells treated with DOX, whereas in nonactivated cells, kinetics was delayed with an increased production of ceramide after 2 h, which returned to the basal level after 4 h (Fig. 3BCitation ).



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  		Fig. 3. Time course of ceramide production by DNR or DOX. Freshly purified PBLs (open symbols) or 3-day PHA-activated lymphoblasts (5 µg/ml; closed symbols) were incubated in the presence of medium alone, DNR (2 µM), or DOX (2 µM) for the indicated time. Quantification of ceramide production was performed by diacylglycerol kinase assay after lipid extraction as described in "Materials and Methods." Data from a representative experiment among three showing similar results.

 
After these experiments, we have tested the effect of exogenous ceramide on PBL. The cell-permeable ceramide analogue, C2-ceramide, induced apoptosis of both nonactivated and 3-day PHA-activated lymphocytes in a dose-dependent manner, with a maximum of 70 and 80% apoptotic cells observed in nonactivated and 3-day PHA-activated cells, respectively, after 20 h exposure to 100 µM C2-ceramide. The biologically inactive ceramide analogue, C2-dihydroceramide, had no toxic effect at the times and concentrations tested, either in resting lymphocytes or PHA-activated lymphoblasts (Fig. 4Citation ).



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  		Fig. 4. C2-ceramide-induced apoptosis of both nonactivated and activated lymphocytes. Freshly purified PBLs (open symbols) or 3-day PHA-activated lymphoblasts (5 µg/ml; closed symbols) were treated with C2-ceramide (C2-Cer; squares) or C2-dihydroceramide (DH-Cer; circles) in increasing concentrations (left panel). Right panel, freshly purified PBLs (open symbols) or 3-day PHA-activated lymphoblasts (5 µg/ml; closed symbols) were incubated with C2-ceramide (100 µM) for the indicated time. Then the percentage of apoptotic cells was determined by fluorescent microscopy after Hoechst 33342 staining. Results are expressed as specific apoptosis as described in "Materials and Methods." Values are the means of three individual experiments; bars, SE.

 
Anthracyclines and Exogenous Ceramide Induce Activation of Caspase-3 in Resting and Cycling Cells.
Activation of caspase-3 was assessed by the cleavage of one of its substrates, PARP, a Mr 116,000 protein, into its Mr 85,000 signature fragment. As shown in Fig. 5A,Citation PARP cleavage was observed after 12 h of treatment with DNR (2 µM) and DOX (2 µM) in unactivated lymphocytes and after 6 h in 3-day PHA-activated cells. As a control, we noted that the CD95 agonistic antibody 7C11 (1µg/ml) induced a rapid PARP cleavage in sensitive (activated cells) but not in resistant (nonactivated) cells. Treatment with exogenous C2-ceramide used at 100 µM, but not with its biologically inactive analogue C2-dihydroceramide, induced PARP cleavage in both types of cells (Fig. 5BCitation ). The inhibitory peptide zVAD-fmk at 100 µM decreased DNR- and DOX-induced apoptosis by {approx}80% in nonactivated cells and {approx}60% in activated cells (data not shown) and inhibited PARP cleavage completely in activated PBLs and partially in unactivated PBLs (Fig. 5BCitation ).



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  		Fig. 5. DNR, DOX, and exogenous ceramide induce PARP cleavage. A, freshly purified PBLs or 3-day PHA-activated lymphoblasts were treated for the indicated time with DNR (2 µM), DOX (2 µM), or the CD95 agonist mAb 7C11 (1 µg/ml). Cell extracts were processed as described in "Materials and Methods," subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antibodies that recognize PARP (Mr 116,000) and its signature fragment (Mr 85,000). B, freshly purified PBLs or 3-day PHA-activated lymphoblasts were cultured for 2 h in the presence of zVAD-fmk before being treated by DNR (2 µM), DOX (2 µM), or C2-ceramide (100 µM; C2-Cer) for 6 h (PHA-activated PBLs) or 12 h (Unactivated PBL). Cell extracts were processed as described above. DH-Cer, C2-dihydroceramide.

 
Apoptosis Induced by Anthracyclines Is Independent from CD95-L/CD95 and TNF/TNF-R Pathways.
Knowing that in some T-cell lines DNR- or DOX-triggered apoptosis is dependent on CD95/CD95-L interaction (3 , 4 , 22) , we have investigated whether such interaction was also involved in apoptosis of resting or activated lymphocytes. To this end, 3-day PHA-activated lymphocytes were preincubated for 1 h with a blocking Fas-Fc fusion protein or with the CD95 antagonist mAb ZB4 before addition of either DNR, DOX, the CD95 agonistic 7C11 mAb, or the combination of PMA plus ionomycin. As shown in Fig. 6Citation , DNR- or DOX-induced apoptosis was not inhibited by addition of either Fas-Fc or ZB4. However, in these experiments, apoptosis induced by either PMA plus ionomycin or 7C11 was markedly reduced by these two inhibitors. As expected, CD95 antagonists did not affect DNR- and DOX-induced apoptosis of unactivated lymphocytes, which are not sensitive to CD95-mediated apoptosis (Refs. 14 , 23, 24, 25 ; data not shown). We also tested whether DNR- and DOX-induced apoptosis required TNF-{alpha}/TNF-R interaction. Preincubation of lymphocytes with soluble p55 TNF-R-immunoglobulin fusion protein, which fully inhibited apoptosis induced by TNF-{alpha} or by PMA plus ionomycin, did not affect drug-induced apoptosis of PHA-activated cells (Fig. 6Citation ).



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  		Fig. 6. DNR- and DOX-induced apoptosis does not require CD95-L/CD95 or TNF/TNF-R interaction. Three-day PHA-activated lymphoblasts were incubated with Fas-Fc (20 µg/ml), CD95 antagonist mAb ZB4 (1µg/ml), or TNF-R p55 immunoglobulin (20 µg/ml) for 3 h. Then DNR (1 µM), DOX (2 µM), the CD95 agonistic mAb 7C11 (1 µg/ml), TNF-{alpha} (5 ng/ml), or PMA (10 ng/ml) plus ionomycin (0.5 µg/ml) were added for 20 h. The percentage of apoptotic cells was determined by fluorescent microscopy with Hoechst 33342. Results are expressed as specific apoptosis, as described in "Materials and Methods." Values are the means of three individual experiments; bars, SE.

 
Anthracycline-induced Oxidative Stress Is Not Required for Apoptosis Induction.
ROS have been involved in various signaling pathways leading to cell death, as for instance TNF-{alpha}/TNF-R and CD95-L/CD95 pathways (26 , 27) . Moreover, DNR and DOX side-effects on heart, skin, and kidney have often been associated with ROS production (6) . Therefore, we investigated whether DNR or DOX induced ROS production in PBLs. DNR (1 µM) and DOX (2 µM) significantly increased the amount of free radicals within unactivated but not activated lymphocytes (Fig. 7ACitation ). Maximal generation of ROS in nonactivated lymphocytes was detected 1–2 h after treatment with either DNR or DOX. Preincubation of nonactivated cells for 90 min with either N-acetyl-L-cysteine or glutathione (both used at 40 mM) inhibited DNR-, DOX-, or H2O2-induced ROS production (Fig. 7BCitation ). In these conditions, both N-acetyl-L-cysteine and glutathione inhibited H2O2-induced cell death, but these antioxidants failed to prevent DNR- or DOX-induced apoptosis (Fig. 7CCitation ). These results suggest that DNR and DOX induce apoptosis in a ROS-independent pathway.



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  		Fig. 7. Anthracycline-induced apoptosis is independent of ROS-generation. A, kinetics of ROS production induced by DNR and DOX. Freshly purified PBLs (open symbols) or 3-day PHA-activated lymphoblasts (closed symbols) were incubated with DNR (1 µM; circles) or DOX (2 µM; squares). At the indicated times, cells were analyzed for peroxide anion production using H2DCFDA as described in "Materials and Methods." Results were expressed as indices as defined in "Materials and Methods." Data are expressed as means obtained from three independent experiments (*, P < 0.05); bars, SE. Effect of antioxidants on DNR-and DOX-induced ROS generation (B) and apoptosis (C). Freshly purified PBLs or 3-day PHA-activated lymphoblasts were preincubated with N-acetyl-L-cysteine or glutathione for 90 min. H2O2 (200 µM), DNR (1 µM), or DOX (2 µM) were added for 30 min (H2O2), or 1 h (DNR and DOX). ROS production was analyzed as described in A, and apoptotic cells were measured with annexin V as described in "Materials and Methods." Data are expressed as means obtained from three independent experiments; bars, SE.

 
In Vivo Anthracyclines Induce Rapid and Massive T- and B-Cell Depletion in Mice.
We next asked whether the capacity of DNR and DOX to induce apoptosis of nonactivated lymphocytes in vitro could be associated with a T- and B-cell depleting effect in vivo. To this end, we injected BALB/c mice with DNR, DOX (10 mg/kg), or NaCl (0.5 mg/kg), and 9 or 36 h after drug administration, spleens, mesenteric lymph nodes, and thymuses were removed and assessed for the presence of apoptotic cells and for cell depletion. As shown in Fig. 8A,Citation cellular depletion was detectable in DNR-treated mice 9 h after injection, and the two drugs induced a 5-fold decrease in spleen cellular content 36 h after treatment and a significant decrease in spleen weight. Remarkably, MTX (7 mg/kg), which induces apoptosis of activated but not resting cells, did not induce substantial depletion. Lymphocyte depletion was also observed in mesenteric lymph nodes from DOX- and DNR-treated mice and in thymuses from DOX-treated mice (Table 2)Citation . The three subpopulations in spleens and lymph nodes were equally affected, as demonstrated by the unmodified percentages of CD4+, CD8+, and B220+ cells at 36 h (data not shown).



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  		Fig. 8. In vivo effects of DNR and DOX. BALB/c mice received injections i.p. with DNR (10 mg/kg), DOX (10 mg/kg), MTX (7 mg/kg), or NaCl (0.5 mg/kg). A, 9 ({square}) or 36 h () after the treatment spleens were removed and weighed (right), as described in "Materials and Methods." Viable cells were counted by trypan blue exclusion (left). Bars, SE. B and C, 16 h after treatment, spleens (B) or mesenteric lymph nodes (C) were fixed in 10% formaldehyde, and DNA fragmentation was detected by TUNEL assay as described in "Materials and Methods." B, x400; C, x200.

 

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  		Table 2 DNR- and DOX-induced cell depletion

 
To determine the contribution of apoptosis to cell depletion, spleens were removed 9 h after drug administration, i.e., before the occurrence of an effective cellular depletion, and splenocytes were further incubated ex vivo for 4 h; then apoptotic cells were identified by their capacity to bind annexin V. As shown in Table 3Citation , annexin V binding on CD4+, CD8+, and B220+ subpopulations was increased on splenocytes from DNR-treated mice as compared with controls. A similar trend was observed with DOX-treated mice. Of note, the higher percentage of apoptotic cells after DNR injection correlates with the more rapid cellular depletion in these mice (Fig. 8ACitation ). Presence of apoptotic cells in spleen or lymph node sections, including T-cell-rich areas, was further confirmed by TUNEL studies (Fig. 8, B and CCitation ).


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  		Table 3 Lymphocyte apoptosis induced by DNR and DOX in mice

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Anthracyclines such as DNR and DOX as well as more recent derivatives, such as idarubicin, are largely used in the treatment of leukemia and solid tumors. As is the case for most other chemotherapeutic molecules, their hematological toxicity is a major limiting side-effect in dose escalation, but anthracyclines also display additional toxic effects toward heart, kidney, and skin that may be related to their capacity to induce lipid peroxidation and ROS generation (6) . In this study, we have investigated the immunosuppressive properties of two anthracyclines, DNR and DOX. We show that these molecules trigger apoptosis of nonactivated peripheral lymphocytes, in contrast to other cycle-dependent chemotherapeutic agents thus far investigated. Such activity could not be anticipated from the DNA intercalating or DNA topoisomerase II inhibitory properties of anthracyclines, and it may have important clinical implications.

Convergent evidence indicates that nonactivated PBLs in the G0 or G1 phases of the cell cycle are as susceptible as cycling PHA-activated lymphoblasts to DNR- or DOX-triggered cell death. The effect of DNR and DOX on cells in the G0-G1 phase of the cell cycle is not associated with anthracycline-triggered activation of PBLs because expression of either CD69, CD95, or CD25 activation markers is not induced by these agents. Moreover, accumulating activated lymphocytes in the late G1 phase by using the DNA polymerase inhibitor aphidicolin or by blocking G1 to S phase transition using calcineurin inhibitors does not protect cells from DNR- or DOX-induced apoptosis, whereas such treatment completely abrogates apoptosis induced by ETO (Table 1)Citation , camptothecin (28) , and MTX (18) . The demonstration that anthracyclines are cytotoxic for both noncycling and cycling cells is reinforced by the observation that a single i.p. injection of either DNR or DOX in BALB/c mice induces a rapid and massive lymphocyte depletion in vivo, as documented by the decrease of cellular spleen and lymph node contents 36 h after injection. Such depletion is not observed in spleen from MTX-treated mice, in agreement with our results demonstrating that MTX-induced apoptosis is restricted to activated T cells in the S-G2 phase of the cell cycle (18) .5 CD4+, CD8+ T-cell subsets and B cells (B220+) were equally decreased in peripheral lymphoid tissues 36 h after a single injection of DNR and DOX. Furthermore, cell depletion was preceded by externalization of phosphatidylserine on the surface of CD4+, CD8+ and B220+ spleen cells from DNR-treated mice and to a lesser extent on those from mice treated with DOX. Interestingly, no major alteration of thymus cellularity and relative proportions of double- and single-positive CD4+ and CD8+ thymocytes was observed after DNR administration, whereas a proportional moderate decrease of all thymocyte subsets was induced by DOX. The rapid kinetics and high magnitude of cell depletion after a single drug injection clearly demonstrate the major contribution of mature T- and B-cell deletion and exclude a significant role for apoptosis or proliferation inhibition of cycling precursors.

Anthracycline-induced cell death of unactivated and activated lymphocytes can be classified as apoptosis on the following evidence: (a) typical nuclear alterations observed by transmission electronic microscopy or fluorescence microscopy after Hoechst staining; (b) early externalization of phosphatidylserine; (c) decrease of transmembrane mitochondrial potential; and (d) blockade by the caspase inhibitor zVAD-fmk. However, the apoptosis-signaling pathway triggered by anthracyclines still remains elusive. DNR and DOX were described previously to increase production of free radicals in different cell types such as cardiac myocytes (6) , kidney cells (29) or leukemic cell lines (30) .Several studies supported the view that oxidative stress, as evidenced by free radical production, lipid peroxidation, and decreased antioxidants and sulfhydryl groups may contribute to anthracycline cardiomyopathy (31) . However, a direct implication of ROS in anthracycline-induced cell death has only been documented in leukemic cell lines (30) . We did observe an increased production of free radicals in resting lymphocytes treated with DNR or DOX but not in PHA lymphoblasts, probably because of a high background level. However, complete inhibition of H2O2 synthesis by antioxidants failed to prevent apoptosis. Therefore, ROS production is not linked to apoptotic signaling in resting lymphocytes, at variance from its reported effect in leukemic cell lines (30) . Ceramide generated through both sphingomyelin hydrolysis (5) or ceramide synthase (7) has been implicated in leukemic cell lines undergoing apoptosis in response to DNR. Similarly, our results suggest that apoptosis of lymphocytes in response to DNR and DOX may be initiated by an early ceramide production, but the real contribution of ceramide to the apoptotic signaling remains poorly understood and controversial (32) . Ceramide synthesis was completely blocked in nonactivated PBLs by addition of fumonisin B1, a specific inhibitor of ceramide synthase (7) , whereas such inhibition was not observed in PHA-activated PBLs (data not shown). Hence, the two cell types may differ in respect to their initial signaling events triggered by anthracyclines. Finally, the CD95-L/CD95 pathway was reported to be involved in DNR- and DOX-induced apoptosis of leukemic cells (3 , 4 , 33) ; however, a contribution of this pathway was not expected in our model, because nonactivated T lymphocytes are resistant to CD95-induced cell death (14 , 23, 24, 25) . Nevertheless, one could hypothesize that the signaling pathway triggered by anthracyclines may differ between activated and nonactivated lymphocytes. However, using Fas-Fc molecules and CD95 antagonistic mAbs, we demonstrated that DNR- and DOX-induced apoptosis was independent of CD95-L/CD95 in the two cell types. Furthermore, anthracycline-induced apoptosis is also independent of the TNF/TNF-R pathway, because soluble TNF-RI-immunoglobulin fusion protein did not inhibit apoptosis.

The capacity of anthracyclines to induce rapid and massive peripheral T- and B-cell depletion as cell cycle-independent agents is not a common property of chemotherapeutic drugs. Comparable depletion of noncycling peripheral lymphocytes may be achieved by mAbs such as CDw52 (34) and polyclonal antithymocyte globulins (35) or by the Isaria sinclairii derivative FTY720 (36) as well as total body irradiation or total lymphoid irradiation (37 , 38) . The massive peripheral T-cell depletion can only be reversed by expansion of the residual T cells, which may be associated with major defects in the T-cell repertoire, and by repopulating by recent thymic emigrants, a process that progressively declines with aging (39) . Clinical implications of these data deal with chemotherapy and with conditioning for allogeneic stem cell transplantation. In cancer chemotherapy, the long-lasting immunosuppression will compromise the antitumoral T-cell cytotoxic responses that are required for eradication of the residual disease by spontaneous or vaccine-induced host responses (40 , 41) . The conditioning for hematopoietic stem cell transplantation was empirically designed as an antitumoral and immunosuppressive regimen aimed at destroying residual leukemic or tumoral cells and inducing the recipient’s T-cell depletion for the prevention of stem cell inoculum rejection. Distinct therapeutic agents may now achieve these two therapeutic objectives. In this respect, anthracyclines, besides their documented antitumoral activity, may represent a possible tool for conditioning recipients of allogeneic stem cell transplantation, among alternatives to total body irradiation. Our study proposes a rationale for the selection of new drugs susceptible to induce peripheral T-cell depletion. Anthracyclines induce a massive but incomplete lymphocyte depletion, and the phenotype of the resistant population remains to be identified. Further characterization of residual peripheral lymphocytes as well as the effect of a combination of different drugs would be helpful and should lead to optimize regimens based on drug associations for chemotherapy and for conditioning in allogeneic stem cell transplantation.

ACKNOWLEDGMENTS
We thank J. Margonari for technical assistance in tissue section, and X. Preville for advice in experiments with ROS.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Institut National de la Santé et de la Recherche Médicale, by Région Rhône Alpes Grant H098730000 (to J-P. R.), by Fondation pour la Recherche Médicale (to J-P. R.), and by Association pour la Recherche contre le Cancer Grant 9607 (to N. B-B.). CF is supported by an Association pour la Recherche contre le Cancer Fellowship CN1/98. Back

2 The first two authors contributed equally to this work and therefore share first authorship. Back

3 To whom requests for reprints should be addressed, at INSERM U503, Hospital E. Herriot, 69437 Lyon Cedex 03, France. Phone: 33-4-72-11-01-77; Fax: 33-4-72-33-00-44; E-mail: bonnefoy{at}lyon151.inserm.fr Back

4 The abbreviations used are: DNR, daunorubicin; DOX, doxorubicin or Adriamycin; CD95-L, CD95 ligand; CsA, cyclosporin A; ETO, etoposide; H2DCFDA, 6-carboxy-2'7'dichlorodihydrofluorescein diacetate; mAb, monoclonal antibody; MTX, methotrexate; PARP, poly(ADP-ribose) polymerase; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; PMA, phorbol 12-myristate acetate; ROS, reactive oxygen species; TNF-R, tumor necrosis factor receptor; TUNEL, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling; zVAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; MFI, mean fluorescence intensity. Back

5 K. Izeradjene, J. P. Revillard, and L. Genestier. Inhibition of thymidine synthesis by folate analogues induces a Fas/FasL-independent deletion of superantigen-activated peripheral T cells, submitted for publication. Back

Received 8/23/99. Accepted 2/ 3/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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