Experimental Hematology 30 (2002) 1293–1301
Ultrasonic low-energy treatment: A novel approach to induce apoptosis in human leukemic cells Laurence Lagneauxa, Eric Cordemans de Meulenaerb, Alain Delforgea, Marielle Dejeneffea, Martine Massya, Carine Moermanb, Baudouin Hannecartb, Yves Canivetb, Marie-Françoise Lepeltierb, and Dominique Brona a
Laboratoire d’Hématologie Expérimentale, Institut Jules Bordet, Université Libre de Bruxelles, Brussels, Belgium; bSonoxide, Nivelles, Belgium (Received 4 January 2002; revised 2 July 2002; accepted 15 July 2002)
Objective. We evaluated the cytotoxic effect of ultrasonic irradiation at low energy on the viability of normal and leukemic cells and the potential mechanisms of action inducing this cytotoxicity. Materials and Methods. Human leukemia cell lines (K562, HL-60, KG1a, and Nalm-6), primary leukemic cells, and normal mononuclear cells are treated by ultrasound at a frequency of 1.8 MHz during various exposure times (acoustical power of 7 mW/mL) and immediately tested for cell viability by the trypan blue exclusion assay. Apoptosis is evaluated by cell morphology, phosphatidylserine exposure, and DNA fragmentation. The mitochondrial potential, glutathione content, caspase-3 activation, PARP cleavage, and bcl-2/bax ratio are tested by flow cytometry. Cloning efficiency is evaluated by assays in methylcellulose. Results. The technique we describe here, using minute amounts of energy and in the absence of any chemical synergy, specifically triggers apoptosis in leukemic cells while necrosis is significantly reduced. Ultrasonic treatment of 20 seconds’ duration induces a series of successive phases showing the characteristic features of apoptosis: mitochondrial transmembrane potential disturbances, loss of phosphatidylserine asymmetry, morphological changes, and, finally, DNA fragmentation. In contrast to K562 cells, for which a significant reduction of cloning efficiency is observed, the growth of hematopoietic progenitors is totally unaffected. Ultrasound treatment is also associated with depletion of cellular glutathione content, suggesting a link with the oxidative stress. Moreover, the fact that active oxygen scavengers reduce ultrasonicinduced apoptosis suggests a sonochemical mechanism. Conclusion. The cell damage observed after exposure of leukemic cells to ultrasound is associated with the apoptotic process and may be a promising tool for a smooth, specific, and effective ex vivo purging of leukemic cells. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.
Cell-culture suspensions can be damaged by exposure to the therapeutic level of ultrasound [1]. Ultrasound may cause irreversible cell damage and induce important cell membrane modifications [2–3]. Several reports have suggested that cavitation resulting from the collapse of gas bubbles generated by acoustic pressure fields may be the cause for cell damage following ultrasonic irradiation [4–5]. It has also been suggested that cavitation induces single-strand breaks in DNA by the action of residual hydrogen peroxide [6]. Offprint requests to: Laurence Lagneaux, Ph.D., Institut Jules Bordet, Hématologie Expérimentale, 1, rue Héger-Bordet, 1000 Bruxelles, Belgium; E-mail:
[email protected]
The use of ultrasound in cancer therapy has become an important issue [7–9]. Ultrasound has been used in conjunction with hyperthermia, and photo-, radio-, and chemotherapy [10]. Malignant cells are known to be more susceptible to these combined methods than their normal counterparts [11]. The effect of a direct irradiation (e.g., ultrasound, laser, light) on certain molecules (porphyrins, psoralenes, and anthracyclines) is to generate highly active oxygen species such as singlet oxygen, superoxyde radicals, or fatty acid radicals, which can play an important role in cancer treatment, acting selectively on malignant cells [12–13]. According to the origin of the photons, the therapy is called PDT (photodynamic therapy) or, if by sonoluminescence, SDT
0301-472X/02 $–see front matter. Copyright © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(02)0 0 9 2 0 - 7
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(sonodynamic therapy). The effects generated by SDT and PDT are different in terms of cell viability; both SDT (specifically related to the ultrasonic cavitational activity) and PDT generate active oxygenated species and lead to a diminution of the intracellular thiol levels [14]. In the case of PDT by ultraviolet-A (UVA), apoptosis of T helper cells is induced by the generation of singlet oxygen, but this effect depends essentially on the initial concentration in photosensitizers (PS) and on the local oxygen concentration [15]. For SDT, as a result of the high energies involved, the cell lysis is the major phenomenon, probably masking other effects on the surviving cells [16]. Classical SDT leading to apoptosis involves specific sensitizing molecules, and requires an electrical power of about 5 W/cm2 and irradiation time of several minutes. In the process described here, we used ultrasound at low energy to induce apoptosis specifically in leukemic cells, in the absence of any chemical agent synergy. Since singlet oxygen and hydroxyl radicals seem implicated in the induction of apoptosis, we have named this technique SLDT: Sonochemical Low-energy Dynamic Therapy.
Materials and methods Cell preparation Human leukemia cell lines (K562, Nalm-6, KG1a, and HL-60) obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) were grown in RPMI-1640 (BioWhittaker, Walkersville, MD, USA) supplemented with 10% fetal calf serum (Gibco, Grand Island, NY, USA) and 1% L-glutamine (Gibco). Leukemic cells were harvested, resuspended in phosphate-buffered saline (PBS, pH 7.2, Gibco), and immediately used for the experiment at a concentration of 106 total. Heparinized venous blood was obtained from healthy volunteers and leukemic patients after informed consent was obtained. Mononuclear cells were separated by Ficoll-Hypaque gradient density centrifugation (International Medical Products, Brussels, Belgium). Ultrasonic treatment The ultrasonic treatment and its specific results are part of patent PCT/BE97/00078 (pending) under the name of Eric Cordemans de Meulenaer et al. A total of 2.5 mL of cell suspension in 13 100-mm disposable plastic tubes (Greiner, Frickenhausen, Germany) was treated in our system with a frequency of 1.8 MHz during various times of exposure. Unless indicated otherwise, the power supplied by the generator to the ceramic disk is 0.22 W/cm2, which represents an acoustical power transmitted to the interior of the test cell of 0.007 W/mL. This calorimetric measurement was made at the University of Coventry, England, under Professor Tim Mason. The irradiation (ON/OFF) cycles are 5.5 ms/3 ms. Cell viability The cell viability was assessed by the trypan blue exclusion test immediately after ultrasonic treatment and after 18 hours culture in the incubator (37C and 5% CO2). Morphological studies Cytocentrifuge preparations were made from the cell suspension and after air drying, cells were stained with May Grünwald Giemsa and analyzed by light microscopy.
Annexin V binding assay Flow cytometric analysis of annexin-V–fluorescein isothiocyanate (FITC)- and propidium iodide (PI)-stained cells was performed using the kit purchased from Biosource International (Camarillo, CA, USA) as recommended by the manufacturer. Data are presented as dot plots showing the change in mean fluorescence intensity of annexin-V–FITC/propidium iodide. DNA fragmentation The level of DNA fragmentation of apoptotic cells was determined using the Apotarget Quick DNA Ladder Detection Kit (Biosource). Cell pellets (106 cells) were resuspended in 20 L of lysis buffer and DNA was extracted according to manufacturer’s instructions. DNA was analyzed after separation by gel electrophoresis (1% agarose). As positive control, cells were irradiated with UV light by placing a plate directly under a UV transilluminator for 10 minutes (intensity of 5 mW/cm2). Cells were then incubated at 37C for 5 and 18 hours before apoptosis was assessed. Quantification of cells with degraded DNA was also performed using a method described by Nicoletti et al. [17]. After permeabilization, cells were incubated with solution containing PI and RNAse (Coulter DNA-prep Reagent). The tubes were placed at 4C in the dark overnight before analysis by flow cytometry to identify the sub-G0 peak corresponding to apoptosis. Mitochondrial damage Mitochondrial potential was estimated by incorporation of the cationic fluorochrome DiOC6 immediately after cell treatment according to a published protocol [18]. Briefly, K562 cells (106/mL) were incubated with 2.5 nmol/L 3,3-dihexyloxacarbocyanine (DiOC6; Molecular Probes, Eugene, OR) for 15 minutes at 37C, followed by flow cytometric analysis. Glutathione determination Cell Tracker green CMFDA (5-chloromethyl fluorescein diacetate; Molecular Probes) was used for determining levels of intracellular glutathione as previously described [19]. Glutathione level was evaluated in viable cells (PI cells). Analysis of caspase-3 activity Caspase-3 was detected by flow cytometric analysis using the phycoerythrin (PE)-conjugated polyclonal rabbit antibody anti-active caspase-3 monoclonal antibody (BD-Pharmingen, San Diego, CA, USA). Cells were fixed and permeabilized using Fix and Perm kit (Caltag, Burlingame, CA) for 15 minutes at room temperature. Cells were then stained with anti-caspase-3 Ab and incubated for 15 minutes. Cells were washed and analyzed by flow cytometry. The enzymatic activity of caspase-3 was determined using the Apotarget caspase-3/cpp32/colorimetric protease assay kit (Biosource), as suggested by the manufacturer. Caspase-3 activation was also indirectly evaluated by PARP cleavage using a rabbit anti-PARP cleavage site AB, FITC conjugate (Biosource). bcl-2 and bax expression After permeabilization, cells were incubated with isotype-matched negative control, FITC-labeled mouse anti-human bcl-2 (Dako, Glostrup, Denmark), and polyclonal rabbit antibodies to bax. Subsequently, a FITC-labeled secondary antibody (Dako) was added to bax. To quantify bcl-2 and bax expression, the cytometer was calibrated using a mixture of beads labeled with known amounts of fluorochrome (Dako). The values of mean fluorescent
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intensity (MFI) were then converted to molecules of equivalent soluble fluorochrome (MESF) using a calibration curve. Clonogenic assay for K562 cell line Colony-forming unit-leukemic (CFU-L) cells were assayed as previously described [20]. Briefly, the culture medium consisted of IMDM supplemented with 20% FCS and methylcellulose at a final concentration of 4%. Cultures were incubated at 37C in 5% CO2 air, and colonies ( 20 cells) were scored after 5 days. The clonogenic efficiency of K562 cell line was 16%. Assay for hematopoietic colony-forming cells Hematopoietic colony-forming cells (CFU-GEMM, CFU-GM, and BFU-E) were assayed using fetal bovine serum (FBS)-free methylcellulose medium supplemented by granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, GCSF, and 3 U/mL EPO (erythropoietin; Methocult H4536, StemCell Technologies, Vancouver, BC, Canada). Cultures were plated in 0.25-mL volumes in 4-well tissue culture plates (Nunclon, Life Technologies, Merelbeke, Belgium). Mononuclear cells were plated at 104 cells/well. After 14 days at 37C in 5% CO2 in air and 100% humidity, the colonies were counted. Statistical analysis The Wilcoxon Signed Ranks test was used to analyze the statistical significance of experimental results.
Results Loss of phosphatidylserine asymmetry during ultrasonic treatment During apoptosis, phosphatidylserine residues flip from the inside to the outside of the plasma membrane and this change can be detected using annexin-FITC, which binds to the PS residues [21]. Figure 1A indicates the changes of phosphatidylserine distribution according to time. Ultrasonic treatment provokes plasma membrane injury in a low percentage of cells, demonstrating that, in our conditions, the necrotic action of ultrasound is very weak. Interestingly, 2 hours posttreatment, an increase in apoptotic cells is observed and 5 hours after the treatment, 35% of cells are annexin-V-positive, demonstrating the ultrasonic induction of apoptosis in K562 cells. Ultrasonic treatment affects the mitochondrial membrane potential ( m) The early disruption of mitochondrial transmembrane potential ( m), preceding advanced DNA fragmentation, has been observed in several models of cell apoptosis [22]. Ultrasonic treatment is accompanied by an increase of cell population displaying a low m (Fig. 1B). Already, 30 minutes posttreatment, a population of cells displaying a reduced DiOC6 incorporation is evidenced. The drop of mitochondrial potential is very clear 5 hours after the ultrasonic treatment with more than 50% of mlow cells.
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DNA fragmentation after ultrasonic treatment DNA fragmentation is assessed by agarose gel electrophoresis and by measuring signals in the hypodiploid region after PI labeling. Classic nucleosomal DNA ladder patterns are observed in DNA samples from cells treated by ultraviolet (positive control) and by ultrasound. Internucleosomal DNA cleavage is barely noticeable at 5 hours post–ultrasonic treatment but becomes evident at 18 hours posttreatment (Fig. 2). Five hours posttreatment, an increase in the number of nuclei with fragmented DNA is observed after PI staining (respectively 2% and 15% for untreated and treated cells) (data not shown). Effect of successive ultrasonic treatments Figure 3A shows the results obtained by a flow cytometry follow-up of K562 cells cultured during 0.5, 2, and 5 hours post–ultrasonic treatment. After one treatment, the level of apoptotic cells observed is thrice that of the control (respectively 26% and 8% after 5 hours of culture for treated and untreated cells). A necrotic effect of 5 to 10% is observed, which is well below that found when using drugs or photodynamic treatment (PDT) treatment. With successive irradiations, under the same conditions (7 mW/mL, 20 seconds) and at different intervals, apoptosis of K562 cells increases to 37 3% (p 0.02) and 49 5% (p 0.02) after respectively 1 and 3 successive treatments (Fig. 3B). Morphological variations (e.g., cell shrinkage, membrane blebbing, chromatin condensation) observed after successive treatments are shown in Figure 3C. Caspase-3 activity and PARP cleavage during ultrasound-induced apoptosis Caspase-3 has been shown to play an important role in chemotherapy-induced apoptosis [23]. To directly address the involvement of caspase-3 in ultrasound-induced apoptosis, caspase activity was determined using flow cytometry and colorimetric assay. As shown in Figure 4 (a and b), ultrasonic treatment leads to activation of caspase-3. Moreover, this protease activity was maximal at 1 hour posttreatment. Activation of caspases leads to cell demise via cleavage of cellular substrates such as actin, gelsolin, or PARP [24]. As shown in Figure 4c, cleavage of PARP was apparent 2 hours after treatment, with 40% of cells stained by the rabbit antiPARP FITC vs 5% for untreated cells. Ultrasound bcl-2/bax ratio modulation Different proteins of the bcl-2 family have been implicated in triggering or preventing apoptosis. Therefore, we have evaluated whether bcl-2 and bax proteins, the two major members of the bcl-2 family, are involved in the induction of apoptosis by ultrasound. As shown in Figure 5, untreated cells express high levels of bcl-2 protein (47 4 103 MESF) and this expression appears as a unimodal peak of fluorescence. One hour post–ultrasonic treatment, the expression of bcl-2 protein is already downregulated (respec-
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Figure 1. (A) Flow cytometric analysis of apoptosis in K562 cells. The cytograms represent the evolution of green (annexin-V) and red (PI) fluorescences for various times post–ultrasonic (US) treatment. In each panel, the upper right quadrants represent the percentage of necrotic cells or late-apoptotic cells (positive for annexin-V binding and for PI uptake). The lower right quadrant contains the apoptotic cells (annexin-V and PI, demonstrating cytoplasmic membrane integrity). (B) The DiOC6 staining of K562 cells treated or not by ultrasound. The histograms represent cell number (counts) vs green fluorescence intensity (FL-1): K562 cells were incubated with DiOC6 30 minutes (A), 2 hours (B), and 5 hours (C) posttreatment followed by immediate cytofluorometric analysis. Results from one representative experiment are shown. After US treatment, a population of cells appeared displaying a reduced DiOC6 incorporation.
tively 40 0.9 and 32 0.9 103 MESF in K562 cells treated by 1 or 3 ultrasonic treatments). Two hours posttreatment, bcl-2 expression appears clearly bimodal, the cells displaying either a bcl-2high (comparable to untreated cells) or bcl-2low phenotype (11 2 103 MESF). In contrast, bax protein level was higher after ultrasonic treatment as compared with the bax level in untreated cells (respectively 85 0.5 and 48 5 103 MESF for treated and untreated K562). The ratio of bcl-2/bax is thus significantly reduced during ultrasonic treatment (0.98 for control cells vs 0.38 for ultrasound-treated cells). Depletion of cellular glutathione content by ultrasonic treatment As shown in Figure 6, directly post–ultrasonic treatment, a subpopulation appears with lower GSH level than that observed in untreated cells (50% of cells displaying a low level of GSH). Successive treatments indicate a larger GSH depletion after 5 hours. The results, expressed as a percent-
age of cells displaying a GSH level comparable to untreated cells, demonstrate clearly that ultrasonic treatment is associated with GSH depletion. Susceptibility of normal and malignant cells to ultrasonic treatment We evaluated the effect of ultrasonic treatment on such other cell lines as KG1a (immature minimally differentiated acute myeloid leukemia blasts), HL-60 (promyelocytic leukemia), and Nalm-6 (ALL cell line). The results presented in Figure 7 demonstrate that the sensitivity to ultrasound seems to depend on cell type, but successive treatments lead to a significant increase in the number of apoptotic cells for all cell lines evaluated. Mononuclear cells from 5 patients (1 refractory anemia with excess of blast cells [RAEB], 1 secondary acute myelogenous leukemia [AML], and 3 cases of AML French-American-British [FAB] classification M3, M4, and M4Eo) are also treated by ultrasound, and blast cells are discriminated from contaminating normal cells on the basis of
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Figure 2. Analysis of ultrasound-induced DNA fragmentation in K562 cells. DNA was extracted from K562 cells treated or not by ultrasound (US) at 5 hours and 18 hours posttreatment. The samples were analyzed by electrophoresis on a 1% agarose gel and stained with ethidium bromide. Lane M contains a 50-bp DNA ladder used as a marker. As positive controls, K562 were treated by ultraviolet radiation.
their CD45 expression as previously described [25]. These cells had also been labeled for their phosphatidylserine exposure by FITC-annexin. It is possible with this method to compare the respective apoptotic behavior of leukemic blast cells and normal cells treated by ultrasound. The results presented in Figure 7 demonstrate that primary leukemic cells are sensitive to ultrasonic treatment with more than 37 18% of apoptotic cells observed 5 hours post–3 treatments. On the contrary, ultrasonic treatment has no effect on normal mononuclear cells isolated from normal subjects and leukemic patients (after CD45 gating). Effect of ultrasonic irradiation on cloning efficiency The ultimate proof for an effect on cell viability is the inability of a cell to multiply and form a colony. As shown in Figure 8, a significant reduction in cloning efficiency of K562 cells is observed after 1 and 3 treatments (respectively 25% and 42% of inhibition), confirming the sensitivity of leukemic cells to ultrasound. In the case of normal hematopoietic progenitors (CFU-GM, BFU-E, and CFU-GEMM), the cloning efficiency is totally unaffected by ultrasound even after 3 treatments (data not shown). Effect of active oxygen scavengers on the induction of apoptosis by ultrasound K562 cells are incubated with L-histidine (10 mM) and/or mannitol (100 mM) and treated or not by ultrasound. Cell apoptosis is detected by annexin-V/PI assay. Our results demonstrate that the ultrasonically induced cell damage is significantly reduced in the presence of histidine and man-
Figure 3. Effect of successive ultrasonic (US) treatments on the viability of K562 cells. (A) Changes of phosphatidylserine distribution according to time and successive US treatments. Results from one representative experiment are expressed as percentage of cells stained with annexin-V-FITC. (B) Percentage of apoptotic K562 cells 5 hours after 1 or 3 ultrasonic treatments. The rate of apoptosis is determined by flow cytometry after annexin-V staining and the results are expressed as mean SEM of 7 independent experiments. (C) Morphological features of May-Grünwald Giemsa-stained K562 cells. (a) untreated cells; (b) 1 treatment; (c) 2 treatments; (d) 3 treatments. Arrows indicate cells with morphological changes (pyknosis, chromatin condensation and fragmentation). Magnification 1000.
nitol (respectively 43% and 47% of inhibition of apoptosis induced by 3 successive treatments) (Table 1). The association of mannitol and histidine leads to more than 60% of inhibition. The effectiveness of these agents on reducing the cell apoptosis induced by ultrasonic treatment may imply that ultrasonically generated singlet oxygen and hydroxyl radicals are important mediators to induce apoptosis. These observations thus suggest a sonochemical mechanism.
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Figure 4. (a) Flow cytometric analysis of caspase-3 activity. K562 cells were treated or not by ultrasound and after 1 hour of culture, cells were fixed, permeabilized, and stained with anti-active caspase-3. A minimum of 10,000 events was analyzed and the dotted line represents the isotypic control. (b) Caspase-3 activity was also determined for times indicated by the colorimetric assay described in Materials and Methods. Results of a representative experiment are shown. (c) PARP cleavage evidenced by flow cytometry in K562 cells treated or not by ultrasound. Two hours posttreatment, cells were washed, permeabilized, and stained by anti-PARP cleavage site AB, FITC conjugate. (— untreated cells — US-treated cells.)
Discussion Apoptosis ensures the homeostasis of tissues during development, host defense, and aging and occurs in response to a large variety of signals including -irradiation and ultraviolet exposure [26–28]. Apoptotic cell death is characterized by early changes in the nuclear membrane and by chromatin condensation, followed by DNA fragmentation [29]. Ultrasound at high power also induces structural and functional changes in sonicated cells [30]. Moreover, ultrasound beams have the potential to treat malignant tumors when combined with sensitizers such as porphyrins [31]. Re-
cently, in vitro studies demonstrated therapeutic ultrasoundinduced apoptosis in cultured cells via the process of cavitation [32]. This therapeutic ultrasound (750 kHz), characterized by a “high-intensity” delivery to generate cavitation leading to apoptotic cell death, was observed in the surviving cells besides a large amount of necrosis (more than 40%). In contrast to the mechanism reported for -irradiation, apoptosis induced by ultrasound seems independent of cell-cycle modifications [33]. In this study, the induction of apoptosis in leukemic cells by a “low-energy” ultrasonic treatment is demonstrated. This treatment leads to a sequence of events recognized as hallmarks of apoptosis: a drop in mitochondrial potential, loss of phosphatidylserine asymmetry, morphological variations, DNA fragmentation, and, finally, loss of plasma membrane. This “low-energy” ultrasound-induced apoptosis involves activation of caspase-3 and is accompanied by the proteolytic degradation of the caspase substrate PARP and by the modulation of bcl-2/bax ratio, demonstrated using flow cytometric analysis. A comparable mechanism has been demonstrated after photodynamic therapy [34–35]. Soon after ultrasonic treatment, an important decrease of intracellular glutathione level is observed, suggesting that oxidative stress may play a role in ultrasound-induced apoptosis. Loss of glutathione and oxidative damage have been suggested to constitute early signaling events in apoptotic cell death [36]. A mechanism coherent with all our observations involves ultrasound-induced sonochemical luminescence triggering photosensitized singlet oxygen production to initiate apoptosis as previously described for direct photoirradiation [37]. In classic ultrasonic irradiation conditions, the direct destructive cavitation effects dominate the sonoluminescence, which is weak in the absence of an air/liquid interface injected into the medium. The fact that there are no ultrasound effects in the absence of bubbles suggests the major role played by 1O2 in our system. Data obtained in the presence of histidine, a quencher of 1 O2, suggest the importance of singlet oxygen in the induction of apoptosis by ultrasound under the “low energy” conditions. However, the fact that mannitol, an inhibitor of hydroxyl radicals, protects against ultrasound-induced apoptosis also implies the intervention of these radicals in our system. Evidence against singlet oxygen formation during sonodynamic therapy has been presented by Miyoshi et al. [38], but these data are only consistent with a long and “highenergy” ultrasound exposure, leading to an accumulation of sensitizer-derived free radicals either by direct pyrolysis or due to reactions with Ho or OHo radicals formed by pyrolysis of the water solvent. In the present study, the supernatant of cells submitted to one or several ultrasonic treatments is unable to induce apoptosis in K562 cells, demonstrating that the effects generated do not originate from chemical species produced by the solvent. At the chosen frequencies, the
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Figure 5. The effect of ultrasonic treatment on bcl-2 and bax protein expression in K562 cells. Histograms showing mean fluorescent intensity for bcl-2 and bax expression in untreated cells and in ultrasound (US)-treated cells (2 hours post–3-US treatment). Black line, expression in untreated K562 cells; gray line, expression in US-treated cells; light gray line, isotype control. MFI mean fluorescence intensity.
“low-energy” ultrasound does not directly generate free radicals such as those originating in the sonolysis of the solvent or those from the addition of molecules such as dimsethylsulfoxide (DMSO) or N, N-dimethylformamide (DMF), which generate a sonodynamic action on leukemia cells [39–40]. Sonoluminescence being in our case the only possible inducing physical phenomenon due to the presence of topical cellular PS, it is normal that these results can be compared to photolytical damages suffered after an exposure to classic ultrasound. The effects obtained with our technique are achieved without the necessity of classical PS. The physiological effects obtained with techniques such as phototherapy depend at the same time on the radiation dose, on the nature of the PS used, on their concentration, and on their localization [41]. The major targets of PDT are cell mem-
Figure 6. Effect of ultrasonic treatment on cellular glutathione levels. Intracellular GSH content was evaluated by the method described in Materials and Methods. Dead cells that had lost the capacity to exclude propidium iodide were gated out from glutathione analysis. Data are expressed in percentage of cells displaying glutathione level comparable to untreated cells. Values are mean SEM of the data from three independent experiments.
branes but, significantly, the technique discussed here is the only one to act directly within the interior of the cells. Within the cells, it is the endogenous photoabsorbing molecules such as the porphyrins and flavoproteins that play the photosensitizing role [42]. Indeed, an implication of endogenous porphyrins in photodynamic DNA damage has been proposed [43]. In our technique, the net effects of the ultrasonic action suggest that endogenous PS may be implicated in the structure where their local concentration is high. Endogenous PS are localized mainly in the membrane structures such as lysosomes, mitochondria, nuclear membranes, and the microsomes of the endoplasmic reticulum, of which the rela-
Figure 7. Effect of ultrasonic treatment on the apoptosis of normal mononuclear cells (MNC) and leukemic cells (Nalm-6, KG1a, HL-60, and primary leukemic cells obtained from 5 patients). Apoptosis was evaluated by annexin/PI assay 5 hours posttreatment directly in the case of cell lines but after a CD45 gating strategy from primary blast cells. Results are mean SEM of 5 independent expseriments.
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References
Figure 8. Effect of irradiation on cloning efficiency of K562 cells. Cells were treated or not by ultrasound, washed, and immediately seeded in methylcellulose medium at a concentration of 1000 cells per plate. After 5 days of culture, the colonies were counted. Results are expressed as mean SEM of 3 independent experiments.
tive surface represents nearly 50% of the cell membrane surface [44]. Under these soft conditions to which they are subjected, healthy cells are much less sensitive to ultrasound than leukemic cells. This difference in behavior between the healthy and leukemic cells cannot be related to a difference in the localization of the endogenous PS but is probably due to a modification of the fundamental cell mechanisms (e.g., p53 status, signaling pathways, resistance to oxidative stress). In conclusion, this “low-energy” ultrasonic treatment (SLDT) induces a similar sequence of events as that induced by PDT: a rapid formation of 1O2, having on the one hand an effect on mitochondrial membranes (drop of mitochondrial potential) and provoking on the other hand a lipidic oxidation of the membrane (decrease of cellular GSH level). Specific tests have confirmed the very rapid induction of apoptosis in the absence of necrosis. This ultrasonic treatment could be a promising tool for the ex vivo elimination of leukemic cells by means of apoptosis.
Table 1. Effects of active oxygen scavengers on the ultrasonically induced cell apoptosis
No agent Histidine Mannitol Histidine Mannitol
No US treatment
1 US treatment
3 US treatments
18 6 15 5 11 4 11 2
42 8 24 8 21 5 16 3
63 5 36 11 30 3 25 5
Results are expressed as percentage of cells displaying phosphatidylserine externalization 5 hours posttreatment (mean SEM from 4 independent experiments).
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