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Experimental Therapeutics |
Service d’Immuno-Hématologie and Laboratoire d’Immunopathologie [S. L., J-C. B., J-P. F.], Laboratoire de Biologie Cellulaire Hématopoïétique EP-107, Centre National de la Recherche Scientifique [P. R., J. L.], Laboratoire Central d’Hématologie [M-H. N.], and Laboratoire de Thérapie Cellulaire [J-P. M.], Hôpital Saint-Louis, 75475 Paris cedex 10, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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At pharmacological concentrations (10-8 to 10-6 mol/L), As2O3 and melarsoprol caused a dose- and time-dependent inhibition of survival and growth in myeloma cell lines that was, in some, similar to that of acute promyelocytic leukemia cells. Both arsenical compounds induced plasma cell apoptosis, as assessed by 4',6-diamidino-2-phenylindole staining, detection of phosphatidylserine at the cell surface using annexin V, and by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. As2O3 and melarsoprol also inhibited viability and growth and induced apoptosis in plasma-cell enriched preparations from the bone marrow or blood of myeloma patients. In nonseparated bone marrow samples, both arsenical compounds triggered death in myeloma cells while sparing most myeloid cells, as demonstrated by double staining with annexin V and CD38 or CD15 antibodies. In primary myeloma cells as in cell lines, interleukin 6 did not prevent arsenic-induced cell death or growth inhibition, and no synergistic effect was observed with IFN-
.
In contrast to As2O3, melarsoprol only slightly reduced the plasma cell differentiation of normal B cells induced by pokeweed mitogen. Both pokeweed mitogen-induced normal plasma cells and malignant plasma cells showed a normal nuclear distribution of PML protein, which was disrupted by As2O3 but not by melarsoprol, suggesting that the two arsenical derivatives acted by different mechanisms. These results point to the use of arsenical derivatives as investigational drugs in the treatment of multiple myeloma.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It has been suggested that As2O3 might induce apoptosis selectively in APL cells. Indeed, at pharmacological concentrations, it has no effect on the growth and survival of the leukemic myeloid cell lines U937 and HL60 (2)
. In addition, As2O3, like ATRA, leads to the degradation of the PML/RAR fusion protein generated by the specific t(15;17) translocation associated with APL (2)
. The mechanisms of the degradation of the fusion protein by the two drugs are, however, different (3, 4, 5)
, and whether PML/RAR degradation by As2O3 triggers apoptosis by itself or belongs to a spectrum of arsenic-induced cellular changes involved in programmed cell death remains open to question.
Recent preliminary reports suggest that the apoptotic effect of As2O3 is not specific for APL cells but can be observed in various cells lines of either myeloid or lymphoid origin (6, 7, 8) and in blast cells from patients with non-M3 acute myeloid leukemia (9) . Another arsenic-containing compound, the melaminyl-phenyl-arsenoxide melarsoprol, which is used in the treatment of human African trypanosomiasis, has a broad efficacy against leukemic cell lines of both lymphoid and myeloid lineage (10) . In particular, the organic arsenic melarsoprol inhibited the growth and promoted apoptosis in cell lines representative of chronic B-cell lymphoproliferative diseases, whereas the inorganic As2O3, at similar concentrations (10-8 to 10-6 mol/L), had no effect on either viability or growth in the three cell lines studied and a minor effect on freshly isolated cells from patients with B chronic lymphocytic leukemia (11) .
MM is a B-cell neoplasia due to the proliferation of clonal plasma cells. Remarkable progress has been made recently in the understanding of the pathophysiology of this disease (12) . Myeloma cells derive from B cells rescued in germinal centers by an antigen selection process before homing in the BM. Their survival and/or expansion depend on interactions with the BM environment and involve various cytokines, the most important of which is IL 6. Probably because of additional progressive oncogenic events, the disease can progress from an intramedullary to an extramedullary stage, with the occurrence of PCL and/or of visceral involvement. At this later stage, malignant cells may be propagated in vitro, giving rise to myeloma cell lines (13) . Since the introduction of melphalan, many therapeutic approaches have attempted to improve the survival rate of patients with MM. Although high-dose therapies with hematopoietic stem cell support can prolong duration of remission and survival in young patients, life expectancy remains on the order of a few years, and MM is still an incurable disease (14) . This outlines the need for new therapeutic strategies and/or of new active drugs.
We have investigated the effects of both As2O3 and melarsoprol on myeloma cell lines and on freshly isolated malignant plasma cells from patients with either the intramedullary or extramedullary stage of MM. We found that the two arsenical compounds, at therapeutical concentrations, markedly inhibit the viability and growth of most MM cells by triggering programmed cell death, with a preferential effect on tumoral cells in patients’ BM.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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were obtained from Diaclone (Besançon, France) and Hoffmann La Roche (Bale, Switzerland), respectively.
Cell Lines.
The human myeloma cell lines were purchased from DMS (Braunschweig, Germany; NCI H929 and LP1) and from American Type Culture Collections (Rockville, MD; OPM2 and U266). NCI, OPM2, and U266 (and all nonplasmacytic cell lines studied) were cultured in RPMI 1640 supplemented with 1% penicillin/streptomycin, 1 mmol/L L-glutamine, and 10% heat-inactivated FCS at 37°C, 5% CO2 in air. LP1 cells were cultured in similar conditions in Iscove’s medium.
Patient Samples.
Eleven patients with MM or PCL were studied after informed consent. Mononuclear cells were separated by Ficoll-Hypaque centrifugation from peripheral blood of 4 patients with PCL (including two with a primary PCL), from BM of six patients with MM (including four at diagnosis), and from the pleural effusion of a newly diagnosed myeloma patient. Plasma cell-enriched preparations were obtained by centrifugation of mononuclear cells on a Percoll gradient, as described previously (15)
. Plasma cell percentages were determined after intracytoplasmic staining using fluorescein-conjugated antibodies to human immunoglobulin heavy and light chains (from Nordic, Tilburg, the Netherlands). Short-term cultures of blood and BM mononuclear cells and of plasma cell-enriched preparations were performed in RPMI 1640 in similar conditions as cell lines.
PWM-induced Differentiation of Normal Lymphocytes.
Mononuclear blood cells of three normal donors were cultured in the presence of PWM (from Life Technology, Cergy Pontoise, diluted at 1/100) and of As2O3 or melarsoprol (5 x 10-7 or 10-6 mol/L). Plasma cells were numerated at day 7 after immunofluorescence staining with fluorescein-conjugated antibodies directed against human immunoglobulin chains. IgM concentrations in supernatants were also measured at day 5, by ELISA, as described previously (16)
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Viability, Cell Morphology, and Cell Proliferation.
Cells were incubated with or without 10- to 10-8 mol/L As2O3 or melarsoprol up to 96 h, without any adjustment after culture initiation. Cell number and viability were determined by trypan blue exclusion. Morphological analysis was performed using Wright-Giemsa and 4',6-diamidino-2-phenylindole staining on cytocentrifuge preparations. For proliferation assays, aliquots of 2 x 104 cells in triplicate were incubated in 96-well plates, and [3H]thymidine (1 µCi) was added for the final 8 h of the culture period. Cells were harvested onto glass filter, and [3H]thymidine incorporation was counted on a liquid scintillation counter.
Flow Cytometry Assay.
Detection of phosphatidylserine on the outer leaflet of apoptotic cells was performed using Annexin-V-FLUOS (Boehringer Mannheim, Mannheim, Germany) and PI according to the manufacturer’s recommendations. Expression of cell surface CD38, CD138 (BB-4), and CD15 antigens was studied using corresponding FITC or phycoerythrin-conjugated antibodies that were purchased from Diaclone (Besançon, France) and Becton Dickinson (San Jose, Ca.). Flow cytometry was performed with a FACScan apparatus (Becton Dickinson).
TUNEL Assay.
Apoptosis was also assessed by dUTP labeling of DNA nicks with terminal deoxynucleotidyl transferase. The TUNEL assay was performed by use of the In Situ Cell Death detection kit (Boehringer Mannheim). DNA strand breaks were identified on cytospin slides by labeling free 3'-OH termini with FITC-conjugated dUTP. Incorporated fluorescein was detected by anti-fluorescein antibody Fab fragments from sheep, conjugated with alkaline phosphatase. After substrate reaction, stained cells were analyzed under light microscope.
Immunofluorescence Study of PML.
Indirect immunofluorescence staining of PML was performed according to a method described previously (17)
using rabbit polyclonal antibodies specific to the NH2-terminal region of PML (kindly provided by H. de Thé, Saint Louis Hospital, Paris). In some cases, double intracytoplasmic stainings were performed using rabbit antibodies to PML and pig fluorescein-conjugated anti-rabbit immunoglobulin antibodies (Dako, Copenhagen, Denmark), followed by goat rhodamine-conjugated Fab'2 antibodies directed against human immunoglobulin (Nordic, Tilburg, the Netherlands).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Fifty % growth-inhibitory concentration was also on the order of 10-7 mol/L for OPM2 cells and was about 10-6 mol/L for U266 and LP1 cells.
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shows the [3H]thymidine uptake of the four plasma cell lines exposed to As2O3 10-6 mol/L over a time period of 96 h. The arsenical compound also led to a marked time-dependent inhibition of growth in NCI and OPM2 cells and to a significant but less important inhibition of growth in U266 and LP1 cells. As illustrated in Fig. 1
, results were similar using the organic arsenical salt melarsoprol instead of As2O3.
In addition to the plasma cell type, other hematopoietic cell types were also studied. The growth of the promyelocytic NB-4 cell line was inhibited, whereas inhibition of growth was marginal in T, myeloid, and epithelial cell lines. In contrast, B-EBV cell lines from normal donors were susceptible to both arsenical compounds. Indeed, at 10-6 mol/L and after 36 h, As2O3 (or melarsoprol) induced a reduction of thymidine incorporation of about 10–20% in Molt 4, HL60, and HELA cells, as compared with 
70% in EBV-immortalized B cells and with 100% in NB-4 and NCI cells.
As2O3 and Melarsoprol Reduce the Viability and Induce Apoptosis in Myeloma Cell Lines.
Treatment of plasma cell lines with As2O3 or melarsoprol led to a dose- and time-dependent inhibition of cell survival, as determined using trypan blue exclusion (Fig. 2)
. Cell death was mainly due to apoptosis, as shown by the characteristic morphological changes in nuclei as well as by the TUNEL method (Fig. 3)
. After 24 h of culture with 10-6 mol/L As2O3 or melarsoprol, a significant percentage of cells exhibiting DNA strand breaks (between 10 and 70% according to the studied cell line, as compared with 3% or less in control cultures) were detected by in situ terminal deoxynucleotidyl transferase assay. DNA fragmentation was confirmed by agarose gel electrophoresis of genomic DNA extracted from NCI cells treated with 10-6 mol/L As2O3 or melarsoprol, which showed typical DNA ladders corresponding to internucleosomal cleavage (not shown).
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, treatment of NCI cells with melarsoprol rapidly induced apoptosis, as demonstrated by an increasing percentage of annexin-positive, PI-negative apoptotic cells. During the same time, the number of double-positive necrotic cells increased, and at 18 h, only very few double-negative living cells were detectable. Exposure to As2O3 induced similar but delayed changes, whereas apoptosis was moderate in cells cultured with Dex (not shown) and in control culture (Fig. 4)
. Annexin-positive, PI-negative apoptotic U266 cells were also observed in the presence of melarsoprol (18% of residual cells after a 18-h culture) as well as in the presence of As2O3 (20.5% at 48 h).
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. In the three plasma-cell preparations that were cultured in the presence of different concentrations of both arsenical compounds, a dose-dependent inhibition of cell growth was observed, with a 50% inhibitory concentration on the order of 5 x 10-7 mol/L for As2O3 and of 10-7 mol/L for melarsoprol. A significant percentage of apoptotic cells (up to 50% and 90% at day 2 in the presence of 10-6 mol/L As2O3 or melarsoprol, respectively, as compared with 5–19% in control cultures) was demonstrated by the TUNEL method in all studied samples (n = 4).
Preferential Effect of As2O3 and Melarsoprol on Myeloma Cells in Patients’ BM.
To comparatively assess the effects of As2O3 and melarsoprol on myeloid and plasma cells, BM mononuclear cells obtained from patients with newly diagnosed MM were cultured for 5 days in the presence of 10-6 mol/L As2O3 or melarsoprol. FACS analysis of bone marrow cells after double staining with fluorescein-labeled annexin V and phycoerythrin-labeled CD38 or CD15 antibodies demonstrated that apoptotic cells were mainly myeloma cells. Indeed, in a representative study of a BM sample initially containing 30% plasma cells (CD38high positive), 12% of the latter expressed annexin V after 2 days of culture with 10-6 mol/L As2O3, whereas <1% of myeloid cells (CD38low, CD15 positive) were positive for the apoptosis marker. At day 5, the plasma cell number had decreased much more than the myeloid cell number, and viable cells (PI negative) contained 7.8% of CD38high positive cells, as compared with 22.5% in control culture, performed with medium alone (Fig. 5)
. Similar results were obtained in the presence of 10-6 mol/L melarsoprol (Fig. 5)
. CD15-positive myelomonocytic cell percentages were similar in arsenic-exposed cultures and in controls, with a decrease in the CD15 low positive fraction (Fig. 5)
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Did Not Influence the Inhibitory Effect of As2O3 and Melarsoprol.
The effect of IFN- was similarly studied. IFN- alone (100 or 1000 UI/ml) had a dose-dependent, antiproliferative effect on cell lines as well as on fresh plasma cells. In most cases, the combination of IFN- with As2O3 or melarsoprol had only a minimal effect on the growth inhibition induced by the arsenical compound alone (not shown). Similarly, no synergistic effect was observed by combining the arsenical derivatives and Dex (10-6 mol/L; not shown).
Differential Effect of As2O3 and Melarsoprol on the Plasma Cell Differentiation of Normal B Cells.
Mononuclear cells purified from peripheral blood of normal donors were incubated with PWM in the presence of two different concentrations (10-6 mol/L and 5 x 10-7 mol/L) of the organic or of the inorganic arsenical. Fig. 6
shows the percentages of viable plasma cells and the amounts of secreted IgM obtained in a representative experiment. In all cases, melarsoprol, including at 10-6 mol/L, only slightly inhibited the plasma cell differentiation induced by the mitogen. In contrast, the number of plasma cells and the amount of secreted IgM were markedly reduced in cultures performed with PWM plus As2O3, as compared with PWM alone.
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(100 or 1000 UI/ml) and/or of As2O3 or melarsoprol. In medium alone, the four cell lines showed a typical nuclear speckled staining pattern, which was brighter after a short exposure to IFN- (Fig. 7A)
. Treatment with As2O3 (10-6 mol/L) or As2O3 plus IFN- for 24 h led to a marked decrease of the number of PML bodies (Fig. 7B)
. In contrast, melarsoprol did not affect the PML nuclear localization (Fig. 7C)
. Immunofluorescence analysis of the subcellular distribution of PML was also performed in normal peripheral blood mononuclear cells exposed to PWM for 5 days with or without 10-6 mol/L As2O3 or melarsoprol. To analyze PML in plasma cells, slides were costained with rhodamine-conjugated antihuman immunoglobulin. As in plasma cell lines, the nuclear speckled pattern characteristic of PML disclosed in normal plasma cells was disrupted by As2O3 but not by melarsoprol (Fig. 7
, D–F).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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These in vitro data may prompt study of the clinical efficacy of the two arsenical derivatives in patients with MM, as is the case for As2O3 in APL (1)
. Indeed, As2O3 and melarsoprol were active at concentrations ranging from 10-6 to 10-8 mol/L, which correspond to the in vivo pharmacological concentrations of both drugs (1
, 20)
, and some malignant plasma cells were as sensitive to the arsenicals as promyelocytic cells. In contrast, at pharmacological concentrations of As2O3 and melarsoprol, reduction in survival and growth of other tested cell types, either lymphoid, myeloid, or epithelial, although significant in some, was less marked than in promyelocytic and plasma cells. The selective effect on myeloma cells of As2O3 and melarsoprol was illustrated by experiments performed in BM mononuclear cells freshly harvested from patients with MM, which showed a marked decrease in myeloma cell viability with, conversely, only minimal changes in myeloid cells (Fig. 5)
. These were mainly a decrease in the number of CD38low, CD15low-positive cells, which include monocytes and myeloid progenitors (21)
. Of note, semisolid culture assays performed in BM cells from normal donors in the presence of As2O3 (up to 10-6 mol/L) did not show any inhibition of the number of granulocytic colony-forming cells (data not shown), and no significant myelosuppression was observed in patients treated with As2O3 for APL (1)
. Similarly, treatment by melarsoprol in patients with African trypanosomiasis does not induce significant cytopenias (22)
.
König et al. (11)
recently reported a differential apoptotic effect in EBV or non-EBV cell lines derived from chronic lymphocytic leukemia patients that were sensitive to melarsoprol but resistant to As2O3. No similar differential effect was observed in all cell types we studied, including in EBV-transformed B-cell lines derived from normal donors. Results were also similar in plasma cell lines and primary plasma cells, although some (such as LP1 cells) appeared more sensitive to the inorganic arsenical, whereas others (such as NCI cells and most freshly isolated myeloma cells) responded better to the organic one. By contrast, plasma cell differentiation induced in normal B cells by PWM was inhibited markedly in the presence of As2O3 and only slightly in the presence of melarsoprol (Fig. 6)
. This suggests that the two arsenicals may have different mechanisms of action.
A different mechanism of action is also suggested by another striking difference between As2O3 and melarsoprol-treated cells, i.e., the nuclear distribution of PML protein. This zinc finger phosphoprotein is expressed in the NBs of normal and malignant plasma cells (Fig. 7)
, as well as in many normal and neoplastic human tissues (23)
. In APL cells, the PML/RAR fusion protein generated by the t(15;17) translocation leads to an abnormal micropunctuated distribution of NB proteins that is restored during induction of terminal differentiation by retinoic acid (24)
. Modifications of PML localization and PML/RAR degradation also occur during As2O3-induced apoptosis (4
, 5)
. Similarly, in non-APL cells studied thus far, As2O3 recruits the nucleoplasmic form of NB proteins onto NB and induces the degradation of PML (4)
. As demonstrated here, As2O3-induced apoptosis of plasma cells is also associated with changes in PML nuclear localization. By contrast, melarsoprol does not affect the targeting of PML onto nuclear bodies in plasma cell lines as well as in PWM-induced normal plasma cells. Similar results were reported recently in HL60 and U937 myeloid cell lines (25)
. Accordingly, PML does not appear to be involved in melarsoprol-induced apoptosis, whereas modifications of PML localization might play a role in programmed cell death induction by As2O3 (26)
. It has been shown recently, however, that not only melarsoprol but also As2O3 can inhibit growth and induce apoptosis in fibroblasts and BM progenitors in which the PML gene has been inactivated by homologous recombination (25)
.
Arsenic-induced apoptosis of plasma cells was not rescued by IL-6. This cytokine supports the expansion of myeloma cells not only by stimulating cell growth but also by preventing programmed cell death, as demonstrated in several apoptotic conditions such as serum starvation or treatment with dexamethasone or anti-Fas antibodies (19 , 27 , 28) . In addition, blockade of the IL-6 signal, for example using IL-6 receptor (IL-6R) antagonists, can led to apoptosis in myeloma cell lines (29) . Similarly, ATRA can act as an apoptotic factor, by down-modulating the IL-6 receptor gp80 and gp130 chain expression and by decreasing IL-6 production by MM cells and BM environment (30 , 31) . Whether As2O3 or melarsoprol could also interfere at any level of the IL-6 pathway remains to be determined. This appears, however, unlikely for melarsoprol because it only slightly inhibits PWM-induced plasma cell differentiation of normal B cells, which requires intact IL-6 circuits (32) .
Studies of the effect of arsenical compounds on the bcl-2 apoptosis regulatory family, which includes cell death inhibitors such as bcl-2 and apoptosis inducers such as Bax, provided variable results. Down-regulation of bcl-2 gene expression was observed either without simultaneous modification of Bax and bcl-X (2) or with down-regulation of other cell death inhibitors (and of some apoptosis inducers) (33) . Melarsoprol was also shown to down-regulate bcl-2 in B-cell leukemia lines, both at the mRNA and protein level (11) . By contrast, no changes in bcl-2 expression were observed in several cell lines representative of myeloid and lymphoid leukemias in the presence of apoptotic doses of both arsenical compounds (8 , 10) . Similarly, we found that As2O3 and melarsoprol-induced apoptosis of NCI and U266 cells occurred in the absence of any modification in the constitutive expression of the bcl-2 protein (data not shown). These differences may be related to the complexity of the regulation of apoptosis, which depends not only on the expression but also on the phosphorylation and dimerization of the different members of the bcl-2 family (34) .
The reactive form of arsenic trioxide is very likely the arsenite (As3+) ion, a sulfhydryl reagent that may interact with free -SH groups on various components of intracellular signaling pathways, as demonstrated for tyrosine phosphatases (35) . Arsenical compounds can also produce other reactive agents, such as the arsenate (As5+) ion, which is similar in structure to phosphate and can interfere with phosphorylation-dephosphorylation biochemical reactions. Of note, melarsoprol (C12H15AsN6OS2), the intracellular metabolism of which is not known, contains only one molecule of arsenic, whereas it is active in myeloma cells at similar molar concentrations as As2O3. This additionally argues in favor of different biochemical mechanisms for apoptosis between the two drugs.
Refractoriness of MM cells to presently available therapeutic strategies, even those including high-dose regimens, remains a major problem. Increased intracellular detoxification, mainly by the glutathione system, is a limiting factor for the efficacy of alkylating agents and expression of the Mr 170,000 P-glycoprotein encoded by the multiple drug resistance gene is correlated with resistance to anthracycline and/or vincristine-containing regimens (36 , 37) . Of note, treatment of cells with As3+ diminishes the intracellular level of reduced glutathione (38) . In addition, As2O3 and melarsoprol may be active on MDR-expressing cells, as suggested by recent preliminary data concerning doxorubicin-resistant cells, including in the doxorubicin-resistant variant of the RPMI 8266 human myeloma cell line (33) .
Although the in vitro effects of As2O3 and melarsoprol might indicate a clinical efficacy of both drugs in patients with MM, this must be confirmed in vivo. Indeed, differential in vitro and in vivo effects of drugs have been described. For example, the growth of human myeloma cells was inhibited in vitro in the presence of ATRA, whereas ATRA was ineffective in the treatment of severe combined immunodeficiency mice transplanted with the same cells (39) . We are presently studying As2O3 and melarsoprol using similar severe combined immunodeficiency models, and preliminary results appear to confirm in vivo the inhibitory effect of both the organic and the inorganic arsenical on MM cell growth. These results additionally point to the use of arsenical derivatives as investigational drugs in MM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant 9413 from Association pour la Recherche sur le Cancer. 
2 To whom requests for reprints should be addressed, at Service d’Immuno-Hématologie, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, 75475 Paris cédex 10, France. Phone: 33 1 42 49 96 92; Fax: 33 1 42 49 40 40; E-mail: jc.brouet{at}chu-stlouis.fr
3 The abbreviations used are: APL, acute promyelocytic leukemia; ATRA, all-trans-retinoic acid; RAR, retinoic acid receptor; MM, multiple myeloma; BM, bone marrow; IL, interleukin; PCL, plasma cell leukemia; PWM, pokeweed mitogen; PI, propidium iodide; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; Dex, dexamethasone; NB, nuclear body; FACS, fluorescence-activated cell sorter.
Received 8/18/98. Accepted 12/31/98.
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). IgM concentrations in supernatants were also measured at day 5, by ELISA (
). PWM-induced plasma cell differentiation was inhibited slightly by melarsoprol and more markedly by As2O3. Similar results were obtained in blood mononuclear cells from 3 other normal donors.