Apoptosis signals in lymphoblasts induced by focused ultrasound

The FASEB Journal express article 10.1096/fj.04-1601fje. Published online July 1, 2004.

Apoptosis signals in lymphoblasts induced by focused ultrasound Amir Abdollahi,* Sophie Domhan,* Juergen W. Jenne,* Mazin Hallaj,‡ Giorgio Dell` Aqua,* Martina Mueckenthaler,† Alexandra Richter,† Heather Martin,‡ Juergen Debus,* Wilhelm Ansorge,† Kullervo Hynynen,‡ and Peter E. Huber* *Department of Radiation Oncology, German Cancer Research Center (DKFZ), D-69120 Heidelberg; †European Molecular Biology Laboratory (EMBL), D-69117 Heidelberg, Germany; and ‡Department of Radiology, Brigham and Womens’ Hospital, Harvard Medical School, Boston, Massachusetts 02115

Corresponding author: Peter E. Huber, M.D., Ph.D., German Cancer Research Center/Deutsches Krebsforschungszentrum (DKFZ), Department of Radiation Oncology, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: [email protected]

ABSTRACT

We investigated the effects of focused ultrasound (FUS) on specific molecular signaling and cellular response in three closely related human Tk6 lymphoblast cell lines that differed only in their p53 status. The applied ultrasound parameters fell between the physical dose range, which is safely used in medical diagnostics (peak pressure<0.1 MPa) and that used for high-energy FUS thermal ablation therapy (peak pressure>10 MPa). Based on cDNA microarrays and protein analysis, we found that FUS at the intermediate peak pressure of 1.5 MPa induced a complex signaling cascade with upregulation of proapoptotic genes [e.g., p53, p21, Thy1 (CD 90)]. Simultaneously, FUS downregulated cellular survival components (e.g., bcl-2, SOD). The p53 status was important for the reaction of the cells to ultrasound. Apoptosis and G1 arrest were induced primarily in p53+ cells, while p53- cells showed less apoptosis but exhibited G2 arrest. Likewise, the proliferation of lymphoblasts was much more strongly inhibited in p53+ than in p53- cells. Microarray analysis further demonstrated an upregulation of genes involved in oxidative stress (e.g., ferritin), suggesting that indirect sonochemical effects via reactive oxygen species play a causative role in the interaction of ultrasound with lymphoblasts. An important characteristic of FUS in therapeutic ultrasound applications is its ability to be administered to the human body in a targeted manner while sparing intermediate tissues. Therefore, our data indicate that this noninvasive, mechanical wave transmission, which is free of ionizing radiation, has the potential to specifically induce localized cell signals and apoptosis.

Key words: FUS ● p53 ● bcl-2

nowledge of the details of molecular signaling in response to mechanical pressure or pressure waves such as ultrasound is limited (1). We were interested in shedding some light on the complex biological events and signaling cascades initiated by structural and

mechanical stimuli subsequent to the application of ultrasound waves. Today, ultrasound primarily serves as a safe diagnostic tool without any apparent adverse effects in nearly all fields

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of medicine. However, ultrasound can be focused through the intact skin and be delivered precisely to a given volume consisting of a few millimeters cubed. When focused in this manner, local energy densities can be achieved that are higher by a factor of 104 when compared with ultrasound diagnostics (kW/cm2, peak pressure p>10 MPa vs. 0.1 W/cm2, peak pressure<0.1 MPa) (2). This ability of high-power, focused ultrasound (FUS) to induce irreversible cell damage by thermal ablation and mechanical destruction has prompted clinical studies in human liver, brain, prostate, and breast cancer (3–7). The biological effects of ultrasound depend on the physical dose parameters applied, which include maximum pressure, intensity and pulse rate.

However, ultrasound bioeffects are not completely understood (8). The nonthermal ultrasound bioeffects are primarily thought to be indirectly caused by either the sonochemical or sonomechanical effects of acoustic cavitation on the cells. A variety of ultrasound-associated effects have been reported in this dose range. These nondestructive ultrasound bioeffects on cells include the following: 1) permeabilization of cell membranes for proteins (9), 2) enhancement of reporter gene expression in benign and malignant cell types both in culture and in vivo (10–14), and 3) induction of apoptosis in cell culture (15–17) as well as in vivo in rabbit brain (18) and human breast cancer (6).

The potential of ultrasound to induce apoptosis is especially interesting. Modulating the expression of key molecular components of the apoptotic processes that comprise cell death is an attractive antineoplastic approach. Apoptosis-modulating therapies are currently undergoing human clinical trials after having demonstrated efficacy in preclinical animal models (19). One of the most important regulatory elements of apoptosis is the tumor suppressor and proapoptotic protein p53. This guardian of the genome that prevents the development of cancer (20) exerts inhibitory effects on the growth of abnormal cells. In contrast, the antiapoptotic, prosurvival bcl-2-like proteins can promote tumorigenesis and tumor progression (21). So far, the most promising approaches for modulating apoptosis target different components of these cell death pathways by using biochemical compounds that must be delivered either locally or systemically (22). In addition to the local limitations of such biological strategies, the question of whether apoptosis can be selectively modulated in one organ or cell type without adversely affecting other key systems has not yet been clearly answered. For example, major obstacles include the delivery of these compounds or the resulting immune response when recombinant proteins are used.

In contrast to the biological strategies, FUS could be used as an alternate, physical method to induce apoptosis safely from outside the body through its application to a defined volume with high spatial precision. However, given the very recent reports of this phenomenon, the molecular signaling pathways involved in the induction of apoptosis by ultrasound are not understood.

We set out to examine the cell signals after FUS therapy in three closely related TK6 human lymphoblast cell lines that differed only in their p53 status. This allowed us to specifically analyze the role played by p53 in response to ultrasound. Using expression profiling of stress response genes coupled with FACS and Western analyses, we detected ultrasound-mediated specific cell signaling that ultimately led to apoptosis and the inhibition of proliferation in human lymphoblast cells.

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MATERIALS AND METHODS

Cell culture

All three cell lines were a gift from John Little, Ph.D., Harvard School of Public Health, Boston, MA. The TK6-E6 5E (5E) cell line was derived from TK6 cells but was stably transduced with the human papilloma virus (HPV) 16 gene E6 (23). This gene encodes a protein that targets p53 for degradation, rendering the 5E cell line p53 null. The TK6 cell line was also stably transduced with a truncated E6 gene, which lacks the activity exhibited by its complete form. This cell line TK6-E6 20C (20C) expresses p53, which is unaffected by the partial E6 gene product and hence acts as a vector control (24). TK6 cells (wild-type p53), TK6-E6-5E (transduced with the E6 gene of HPV 16, abrogated p53), and TK6-E6-20C (vector control) were grown at 37°C in a humidified incubator in RPMI 1640 medium supplemented with 10% heat-inactivated fetal horse serum and 1% penicillin/streptomycin (PS; standard condition). Cell cultures were split periodically to maintain a population density of ~2 × 105 to 2 × 106 cells/ml.

Sonication of cells

We used pulsed, FUS waves (0.68 MHz frequency, range 0-15 MPa peak pressure amplitude in the focus, 10 ms pulse length, 1 Hz pulse frequency, 60 s total exposure time) as a specific probe to study the transduction of ultrasound energy into molecular signaling with emphasis on the cell cycle and apoptosis signaling pathways. Approximately, 4.5 × 105 cells/ml were transferred into each well of a 12-well plate immediately before sonication. We added either Albunex® human albumin microspheres (Mallinckrodt Medical, Inc., St. Louis, MO) at a final concentration of 107 bubbles/ml to increase cavitation activity or an equal volume of phosphate-buffered saline (PBS) to maintain equal cell densities throughout the wells. The wells were filled completely to a final volume of 2.5 ml, and a sterile thin plastic membrane was placed over the entire plate to achieve direct contact between cell suspension and this membrane (Fig. 1). The plate was placed in a device designed to extend the outer walls of the plate 10 cm in the vertical direction. Water at 37°C was then poured on top of the membrane into the apparatus. The plastic membrane on the top of fluid was ~50 µm thick and thus transparent to ultrasound. The ultrasound beam was focused in the fluid with cells and then propagated through the membrane into the water on the other side of the membrane. There is practically no scattering or reflection of the sound when it propagates through the water. A rubber plate as absorbent material was placed in the water ~5 cm above the cell suspension. This plate reflects only ~5% of the incident sound, and the rest propagates into the plate and is absorbed. Since the ultrasound beam is spreading out beyond the focus, the acoustic intensity at the rubber plate was <2% of the intensity at the focus in the cell suspension. Therefore the maximum reflected intensity was <1/1000 of the intensity at the focus resulting in almost complete elimination of standing waves. The ultrasound transducer was calibrated using a membrane hydrophone having a spot size of 0.5 mm. These measurements were performed at low intensities up to ~2 MPa and then extrapolated based on acoustic power measurements. The hydrophones and thermocouples were positioned inside the wells (without the covering membrane) along the geometrical beam axis after the ultrasound focus had been predetermined by visual inspection of the water streaming outside of the wells in the water basin.


Cell proliferation, FACS, ELISA, and Western blotting

Immediately after ultrasound therapy, cells were incubated under standard conditions. At time points 0, 6, 12, 24, and 48 h after therapy, cells were counted under the light microscope using trypan blue exclusion. Furthermore, a quantitative flow cytometric (FACS) determination of apoptosis and a determination of the cell cycle distribution were performed (Becton-Dickinson FACScans, San Jose, CA). Briefly, cells were fixed in Hanks’ solution and 70% ethanol. After the cells were spun in a centrifuge and the supernatant was removed, the cells were washed in PBS. Cells were again spun down, and the supernatant was discarded. Next, the cells were resuspended in the staining solution of PBS, RNase, and propidium iodide. Apoptotic cells are characterized by a shift to the left representing the sub-G1 state of the cells. By using only the forward/sideward scattered cell population on the DNA content histogram, necrotic cells can be excluded. For additional apoptosis measurements, the cells were incubated with 10 µg/ml fluorescent DNA binding dye DAPI (4',6-diamidino-2-phenylindole, Sigma) and images were obtained on a Zeiss Axiovert 10 inverted microscope with a x40 objective. Apoptotic cells are characterized by the presence of condensed and fragmented nuclei, in contrast to the diffuse staining observed in nonapoptotic cells. Additionally, p53 protein was measured using an enzyme-linked immunosorbent assay (ELISA, Boehringer, Mannheim, Germany) and Western blotting for p53, p21 waf, and bcl-2 was performed at 0 h, 10 min, 30 min, 1 h, 4 h, 6h, 12 h, and 24 h after ultrasound application. Antibodies to p53, p21, and bcl-2 were purchased from Calbiochem.

Total RNA isolation and quality control

Total RNA from human dermal microvascular endothelial cells (DMVEC) was isolated using the RNeasy® Kit (Qiagen, Hilden, Germany). RNA quality was insured by lab-on-chip technology according to the instructions of the manufacturer (Agilent, 2100 bioanalyzer in combination with the RNA 6000 lab chip kit, Agilent Technologies, Boeblingen, Germany) and spectrophotometric analysis. The RNA was only used with a 28S:18S rRNA ratio of 2.0 (±0.3).

Design and production of the c-DNA chip

Selection of cDNA clones and preparation of the “iron chip” microarray platform including amplification, spotting, and attachment of the cDNAs was performed as described previously (25, 26). Briefly, 120 human expressed sequence tag (EST) clones that had been sequence verified from both ends were chosen for the iron chip (version 2.0; http://www.embl-heidelberg.de/ExternalInfo/hentze/suppinfo.html). The ESTs were selected to contain the 3′ end of a cDNA (i.e., the polyadenylation signal) and to extend for at least 300 bp toward the 5′ end.

Chip hybridization and data analysis

Fluorescent cDNA probes were synthesized from 40 µg total RNA using a direct labeling protocol, as described previously (25). Cy3 fluorescent dyes and Cy5 fluorescent dyes were incorporated into the cDNA synthesized from the control and the treated samples. Hybridization was performed according to the protocol developed by Richter et al. (25). At least three independent cell culture experiments were performed for each experimental condition tested. All microarrays were scanned on a GenePix 4000B Microarray Scanner (Axon Instruments, Union


City, CA). To analyze the chip images, Chipskipper® microarray data evaluation software (Ansorge Lab. EMBL, Heidelberg, Germany) was used. Intensity values for each spot were calculated by subtraction of the local background surrounding the spot.

Radical measurement

For the quantitative measurement of radical formation in the ultrasound field, we used electron spin resonance (ESR) spectroscopy (27). Briefly, to detect radicals with short lifetimes in ultrasound fields, we used the nonspecific spin trap TMPone (2,2,6,6-tetramethyl-4-piperidone). Hydroxyl radicals or singlet oxygen converts the TMPone into the stable nitroxide radical TMPone (2,2,6,6-tetramethyl-4-piperidone-1-oxyl), which was quantified using a conventional ESR spectrometer (Miniscope MS 100, Magnettech, Berlin, Germany). Samples were sonicated as described but without the addition of cells. The test solution consisted of PBS with 25 µM of the spin trap TMPone. The pH was adjusted to 9.0. For comparison with radicals induced by ionizing radiation, the ESR signal was calibrated using a Cs-137 gamma ray source with a dosage rate of 12.1 Gy/min.

RESULTS AND DISCUSSION

Lymphoblast proliferation, apoptosis, and cell cycle

A broad spectrum of dose-dependent responses to FUS was observed (Fig. 2): low-power ultrasound (range 0-0.5 MPa) had very little effect on the survival of proliferating lymphoblast cells, irrespective of the p53 status or the addition of Albunex. In contrast, high-power ultrasound (≥10 MPa) completely destroyed the lymphoblasts, independently of the p53 status (Fig. 2A and B). Within 12 h of high-power treatment, no intact cells were visible; the suspension appeared nearly empty, and only a small quantity of cell debris could be seen under the microscope. Moreover, apoptosis could not be detected by either FACS or DAPI staining. In contrast, the intermediate dose range (0.5≤peak pressure<10MPa) yielded many visible intact cells, but proliferation was strongly reduced compared with the controls (Fig. 2A and C). At the intermediate focal peak pressure of 1.5 MPa, FUS induced a normalized cell count of 50 ± 4% (means±SD) compared with untreated controls when determined 48 h after sonication (Fig. 2A). Therefore, 1.5 MPa was considered to be the “IC50” dose for this endpoint. Further analyses were carried out at the intermediate dose of 1.5 MPa focal peak pressure.

Notably, at 1.5 MPa the lymphoblast cells with wild-type p53 (Tk6, p53+) showed a greater inhibition of proliferation than p53- cells (Tk6 5E abrogated p53; Fig. 2C), suggesting that p53+ cells are more sensitive to ultrasound than p53- cells. These different effects associated with p53 were less pronounced at lower and higher pressures and were absent below 0.5 MPa and above 10 MPa (Fig. 2B).

When analyzing the rate of apoptotic lymphoblasts by FACS and DAPI, significantly more apoptotic cells (P<0.01) were induced by FUS in p53+ vs. p53- cells (Fig. 3A-D). This functional finding correlated with the protein level since the apoptosis-related p53 protein was found to be strongly upregulated after FUS at 1.5 MPa (Fig. 4A and B). Furthermore, we detected that downstream from p53, the p21/WAF protein was upregulated while the anti-apoptotic protein bcl-2 was downregulated after FUS, especially in p53+ cells (Fig. 4A).


The addition of Albunex, an ultrasound contrast agent, not only increased the anti-proliferative effects of FUS particularly in p53+ cells (Fig. 2C) but also increased the expression of the p53 protein in the ELISA assay (Fig. 4B). This can be attributed to enhanced cavitation activity and possibly to free radical generation (see below) by Albunex.

Because these different effects for p53+/p53- cells were not observed with either high or low ultrasound doses, it appears that FUS in a certain dose range can specifically diminish cellular growth, depending on the p53 status of the human lymphoblast cell lines.

Next, we studied cell cycle changes after FUS at 1.5 MPa, since cell cycle arrest can result from the actions of p53. Accordingly, G1 arrest was measured primarily in the p53 + cells, while p53- cells mainly exhibited a G2 arrest (Fig. 5A and B). It is known that p53 can be activated by cellular stress, for example by ionizing radiation (IR) or ultraviolet light (UV). However, the precise mechanism is not fully understood. The ability of p53 to promote cell cycle arrest is well understood in terms of its ability to transactivate p21/WAF1, which binds to and inactivates cyclin-dependent kinases, arrests cells in G1, and prevents S-phase entry (28). In the same lymphoblast model that we used in the present study, IR or UV also has been reported to induce apoptosis and inhibition of cell growth. An explanation of the effects of IR and UV was postulated to be the induction of physical damage to the DNA strand that stabilizes the p53 protein (20, 29, 30). Our findings made using FUS of upregulated p21/WAF protein in p53+ cells, support a similar model of p53-dependent transactivation. Thus, a p53-dependent response to direct DNA damage could also be a possible mechanism for ultrasound-induced cell growth inhibition.

Cavitation and radical formation

It is an accepted concept that ~2/3 of the bioeffects of ionizing radiation are mediated via the formation of (OH-) free radicals (31). The ability of ultrasound to sonochemically induce radicals in aqueous solutions has been demonstrated, especially in association with acoustic inertial cavitation (32–34). The collapse of cavitation bubbles can cause high local pressures and temperatures (104 K) and additional sonodynamic processes (35). Therefore, a plausible explanation for the existence of oxidative stress would entail the involvement of ultrasound-induced radicals. Here radical formation was determined in cell-free samples filled with PBS using ESR spectrometry calibrated with a Cs-137 gamma source. It was found that increasing ultrasound pressures and increasing sonication times induced increased levels of radicals. Compared with the calibrated gamma-ray source an equivalent dose rate of up to 0.3 Gy/min was induced. The addition of Albunex further increased the quantity of radicals by approximately a factor of 2 and lowered the cavitation threshold from 1 to ~0.5 MPa. If the presence of air above the test samples was permitted, the quantity of radicals increased by ~10-fold. Since 2 Gy is the typical daily dose of standard fractionated radiotherapy for various cancers (at a dose rate of ~1 Gy/min), the ultrasound-induced quantity of radicals falls within a comparable range. However, it remains unclear whether and to what extent these radicals can affect the intracellular structures. Nevertheless, taken together with the results regarding radical formation associated with p53 regulation, this may suggest that ionizing radiation and ultrasonic waves at least partially can trigger apoptosis and cell death via similar molecular pathways.


Chip analysis

To understand more about the FUS-induced differentially expressed genes, we performed cDNA-chip analysis based on comparisons of labeled RNA extracts from TK6 human lymphoblast cell lines 8 h after FUS treatment (intermediate FUS at 1.5 MPa, 60 s) vs. control samples. Genes were considered to be upregulated or downregulated if the observed change was associated with at least a factor of 2 in each of the individual experiments. We used a stepwise approach with the first step being a commercial general gene chip and the second step being a custom-made stress response chip. With use of three commercially available chips (Atlas Glass Human 1.0 Microarray, Clontech, Heidelberg, Germany), we detected an upregulation of p53 (2.6-fold) and p21/waf (2.1-fold) and the downregulation of bcl-2 (0.5-fold). With cytotoxic ultrasound parameters, we detected genes associated with classical apoptosis pathways, emphasizing the p53-related pathway. In contrast, one array-based analysis of ultrasound-related bioeffects (36) used lower, nontoxic ultrasound doses, which may explain why no apoptosis-related genes were found. For detailed analysis of the stress responsive genes, custom-made arrays (iron chip version 2.0) were used and the involvement of several distinct pathways such as SOD, Thy-1, and ribosomal proteins was further investigated (Fig. 6A).

Ultrasound-mediated decrease of ribosomal proteins

Expression profiling data revealed a marked decrease of two ribosomal proteins [RPL32 (0.34-fold) and RPL6 (0.23-fold)] after FUS treatment in the TK6 lymphoblasts (Fig. 6A).

Ribosomal protein (Rp) L6 is also called Taxreb107 (tax responsive element binding protein 107) for its binding activity to the long terminal repeats of human T cell leukemia virus (HTLV)-I in addition to its assembly into ribosomes (37, 38). HTLV-I is an etiological reason for the occurrence of adult T cell leukemia (ATL) as well as some chronic inflammatory disorders such as myelopathy and tropical paraparesis. Tax is a transcription activator encoded by HTLV-I (39). RPL32 and RpL6/Taxreb107 have been shown to be involved in regenerating the liver after partial hepatectomy (40), and the 60 S ribosomal protein L32 mRNA is reportedly associated with the progression of human prostate cancer (41).

A change in the ratio of ribosomes to mRNA can have deleterious effects on the cell (42). Ribosomal proteins mainly participate in protein synthesis, but they are also involved in the neoplastic transformation of cells (43–45). Moreover, targeted disruption of ribosomal genes impairs cell proliferation (46). Altogether, FUS-induced downregulation of RpL6/Taxreb107 and RPL32 expression may have two functional implications: one is to decrease protein synthesis and facilitate the inhibition of TK6-lymphoblast proliferation. Additionally, FUS inhibition of RP L32 and L6 may convert the TK-6 tumor cells into a less aggressive and more sensitive phenotype.

Decrease of β2-microglobulin expression

After FUS exposure, a decrease of β2-microglobulin (β2m) RNA (0.35-fold) was detected in TK6 cells. β2m is best characterized by its function of interacting with and stabilizing the tertiary structure of the MHC class I α-chain (47). The level of β2m is one of the most important independent predictors of survival in hematological cancers such as multiple myeloma and a


general indicator of the tumor burden (48). Therefore, the downregulation of β2m by FUS may indicate a level of FUS-induced cell damage in lymphoblast cells.

Thy-1 and FUS-induced apoptosis

Another potential FUS-meditated mechanism is the upregulation of Thy-1 (CD90) in TK6 lymphoblasts detected 8 h after FUS (2.67-fold; Fig. 6A). Thy-1 is a 25 kDa glycosylphosphatidylinositol-linked membrane glycoprotein originally described as a marker for thymocyte differentiation in mice. It is a multifunctional molecule involved in cell adhesion, signaling, growth, and differentiation with no known ligand (49). The aggregation of Thy-1 glycoprotein has been shown to induce thymocyte apoptosis through activation of CPP32-like proteases and a decrease in bcl-2 and bcl-XLexpression (50). In this context, it is conceivable that the Thy-1 pathway is involved in FUS-mediated apoptosis in TK6 lymphoblasts in conjunction with the detected loss of bcl-2 expression.

Ferritin, SOD, oxidative stress, and apoptosis

Reactive oxygen species (ROS) encompass a variety of diverse chemical species including superoxide (O2

-·) and hydroxyl radicals (OH·) that can lead to the production of hydrogen peroxide (H2O2). Additional hydroxyl radicals are generated in a reaction that either depends on, or is catalyzed by, Fe 2+ ions (51). The accumulation of more iron than can be adequately stored in ferritin creates oxidative stress. Thus, upregulation of ferritin is a mechanism for cells to respond to this environmental change (52). We found that H-ferritin levels were increased (2.42-fold) in TK6 cells after FUS treatment, indicating that FUS-treated TK6 cells experienced increased oxidative stress.

Superoxide and other free radicals can cause cell death by apoptosis and necrosis (53). Cellular defense systems include the essential intracellular enzymes called the superoxide dismutases (SODs) that eliminate the superoxide radical (O2

-·) and protect cells from damage induced by free radicals (53, 54).

On DNA array, we have detected a decreased SOD level 8 h after FUS treatment in TK6 lymphoblasts (0.53-fold).

This finding suggests that FUS can regulate oxidative stress pathways from two directions. First, FUS can directly cause an increase in the amount of dangerous O2

-· in the tumor cells via sonochemical radical formation. More SODs would be needed to mop up the radicals. Second, using a cDNA microarray, we determined that SOD, itself, was a target of ultrasound action. The increased levels of ROS with simultaneous inhibition of SOD could be an interesting approach for killing cancer cells.

The regulation of p53 and bcl-2 together with increased oxidative stress can be integrated into the concept of FUS-mediated apoptosis (Fig. 6B): elevated p53 expression, itself, can result in increased oxidative stress (29, 55), suggesting that an important consequence of oxidant-induced p53 activation is a further increase in the level of oxidative stress that occurs within a positive feedback loop. Wt-p53 has been shown to downregulate SOD gene expression at the promoter


level. In turn an overexpression of SOD decreases the transcription level from p53 promoter and inhibits the p53-mediated induction of apoptosis (56).

Furthermore, the antioxidant enzyme SOD has been shown to be negatively regulated by p53 (57). Therefore, it proved plausible to suggest that in cells with impaired p53 activity (e.g., TK6-E5), the SOD activity is likely to be high and to exhibit an increased antioxidant capacity with less sensitivity to FUS. In fact, we found that Tk6-E5 (p53-) cells were less sensitive to FUS-induced inhibition of proliferation than Tk6 (p53+) (Fig. 2C).

A mechanical means of ultrasound-cell interaction is cavitation-related microstreaming, which can lead to leakiness of the mitochondrial membrane. Released ROS and cytochrome c may then induce apoptosis. Supporting the role of cavitation, we found increased inhibition of cell growth, especially in p53+ cells after the addition of an ultrasound contrast agent. An additional disruption of the normal proportion of bcl-2 and bax, located at the outer mitochondrial membrane, may also occur (22). Accordingly, we found that the antiapoptotic protein bcl-2 was downregulated in lymphoblasts after FUS treatment depending on their p53 status (Fig. 4A and B).

The consequences of this FUS-induced disturbance of the physiological balance between the levels of the pro-oxidant p53 and bcl-2, which has been shown to oppose the action of p53 (58, 59), are an increased level of oxidative cell stress, apoptosis and tumor cell death. Additionally, the observed upregulation of p53 RNA and p53 protein after intermediate FUS treatment could also be responsible for the inhibition of bcl-2 expression resulting in a positive loop effect toward increasing levels of oxidative stress and apoptosis (Fig. 6B).

To exclude the potential effect of FUS-induced heat, we measured the increased temperature both during and after sonication directly at the critical sites on the inner parts of the plates along the geometrical beam axis. The maximum temperature elevation was below 1.5 K. Furthermore, the DNA array only showed a modest regulation of the heat shock protein HSP70 (1.33-fold), which is attributable to the general oxidative cell stress.

CONCLUSION AND POTENTIAL CLINICAL IMPLICATIONS

The ability of ROS to damage cellular components and cause cell death suggests the possibility of exploiting this chemical property to kill cancer cells by means of a free radical-mediated mechanism. This strategy is of therapeutic relevance, since most cancer cells are active in the metabolic production of ROS and are intrinsically under increased oxidative stress. Therefore, they are more susceptible to exogenous free radical insults (60). This can provide a biochemical basis for FUS to preferentially kill cancer cells similar to ionizing radiation or chemotherapeutic drugs that produce free radicals (61, 62).

In summary, FUS, a minimally invasive pressure wave, is described as a means of transducing mechanical energy into specific molecular signaling and cellular response pathways occurring in human lymphoblast cells. We show that ultrasound effectively kills both p53+ and p53- cells but that p53+ cells are more sensitive to ultrasound-induced apoptosis. Therefore, we could demonstrate at a basic level that the primary yet still largely unknown physical ultrasound-cell interaction led to a specific transcriptional response.


Moreover, the potential clinical applications (e.g., in cancer therapy) are shown by the spatially defined induction of apoptosis signals, which could be beneficial in conjunction with other anti-cancer therapies. In principle, blood cell malignancies could be a suitable target for ultrasound therapy by using an extracorporeal “purging” approach. In situ ultrasound could enhance the antineoplastic effects of conventional therapies in ultrasound-accessible tumors.

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Received February 19, 2004; accepted May 11, 2004.


Fig. 1

Figure 1. Experimental setup fiagram of lymphoblast sonication with focused ultrasound (FUS). A 12-plate well with cells was placed in a device designed to minimize ultrasound reflection, scattering, and heat induction at the plastic walls to mimic in vivo conditions. FUS was delivered (at 0.68 MHz center frequency, range 0-15 MPa pressure amplitude, 10 ms pulse length, 1 Hz pulse frequency, 60 s total exposure time) vertically to cells from the bottom through a water tank.


Fig. 2

Figure 2. Proliferation in response to sonication for 3 lymphoblast cell lines that only differ in p53 status (Tk6 with wt p53, Tk6 transduced with 5E abrogated p53, Tk6 20C as vector control). Approximately 4.5 × 105 cells/ml were plated into each well of a 12-well plate immediately before sonication. Either Albunex human albumin microspheres contrast agent was added to facilitate artificial cavitation, or an equal volume of PBS was added. At time points 0, 6, 12, 24, and 48 h after sonication, cells were counted using trypan blue exclusion under light microscope and normalized to each untreated control value. A) Ultrasound dose response of cell count as a function of focal peak pressure 48 h after sonication in TK6 p53 wt lymphoblasts. Probability plot of means ± SD shows an IC 50 at ~1.5 MPa. B) Time course of cell count after treatment with high focal pressure of 12 MPa shows a complete cell death within 6-12 h independently of p53 status. Low power ultrasound at 0.1 MPa has no significant effect on lymphoblast cell count, irrespectively of p53 status or the addition of Albunex (data not shown). C) Time course of cell count after treatment with intermediate ultrasound dose at 1.5 MPa shows a strongly reduced cell count depending on p53 status. P53+ cells were more sensitive to ultrasound treatment than p53- (P<0.01). Addition of Albunex further enhanced the cytotoxic ultrasound effect in p53+ cells (P<0.02) but not in p53- (P>0.8).


Fig. 3

Figure 3. Apoptosis induced by FUS. Tk6 (p53 wt, p53+) and Tk6 5E (abrogated p53 by 5E transduction, p53-) human lymphoblast cells were treated with FUS (1.5 MPa pressure amplitude for 60 s). Photographs of DAPI-stained cells at 24 h after FUS show many apoptotic cells. Nuclear condensations and fragmentations are marked by arrows. Apoptotic fraction was further quantified by PI staining and FACS analysis. A) Tk6, untreated control. B) Tk6 5E treated with FUS. C) TK6 treated with FUS. D) Percentage of apoptotic lymphoblasts in FACS using PI staining.


Fig. 4

Figure 4. Protein analysis. A) Western Blot of bcl-2, p21-waf, p53, and α-tubulin of Tk6 (p53 wt, p53+), Tk6 20 C (vector control, no E5 transduction, p53+), and Tk6 5E (abrogated p53 by 5E transduction, p53-) human lymphoblast cells after treatment with FUS (1.5 MPa pressure amplitude for 60 s) 12 h after therapy. In all cell lines, bcl-2 is expressed after FUS, irrespectively of p53 status. P21 and p53 are expressed in p53-positive cells only. B) P53 protein expression as measured by ELISA 24 h after therapy. FUS-induced p53 in both Tk6 and Tk 5E cells [FUS vs. control, P<0.001 (Tk6); P<0.02 (Tk6 5E)]. Ultrasound-induced p53 amount was higher for Tk6 than for 5E cells (P<0.01). Cavitation enhancement through addition of Albunex further facilitated p53 induction, especially in the p53+ cells (P<0.05). Bars are means ± SD.


Fig. 5

Figure 5. Cell cycle effects of intermediate FUS at 1 MPa in lymphoblasts. A) Cells in G0/G1 cell cycle phase, normalized to untreated control (P<0.05 at 24 and 48 h). B) Cells in G2/M cell cycle phase, normalized to untreated control (P<0.05 at 12, 24, and 48 h). Data points are means ± SD.


Fig. 6

Figure 6. Signaling cascades induced by FUS. A) cDNA microarray on stress chip 8 h after treatment with FUS (1.5 MPa pressure amplitude for 60 s) of Tk6 lymphoblast cells. Green spots are upregulated genes; red spots are downregulated genes; yellow spots are unregulated genes. Iron chip 2.0 was hybridized with 40 µl RNA (labeled with Cy3) and 40 µl RNA (labeled with Cy5). Compensated ratios of a part of Cy3/Cy5 overlay are shown: not regulated (Yellow) – beta-actin, L-ferritin (L-fer), Mobilferrin (Mofer); upregulated – Thy-1 glycoprotein gene (Thy1), H-ferritin (H-fer); downregulated – TAXREB 107, ribosomal protein L32 (L32), beta-2-micoroglobulin (Beta-2-µg), superoxide dismutase (SOD). Attachment controls: Attach. B) Schematic diagram showing FUS-induced molecular signaling concentrating on activation of apoptotic machinery in lymphoblast cells at 1.5 MPa peak pressure.


Apoptosis signals in lymphoblasts induced by focused ultrasound

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