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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^*,1

^* Department of Radiation Oncology, German Cancer Research Center (DKFZ), Heidelberg, Germany;
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany; and
Department of Radiology, Brigham and Womens’ Hospital, Harvard Medical School, Boston, Massachusetts, USA

¹Correspondence: German Cancer Research Center/Deutsches Krebsforschungszentrum (DKFZ), Department of Radiation Oncology, Im Neuenheimer Feld 280, Heidelberg D-69120, Germany. E-mail: p.huber{at}dkfz.de

SPECIFIC AIMS

The purpose of this study was to understand the specific molecular signaling after mechanical stress induced by focused ultrasound in human Tk6 lymphoblasts which differed only in their p53 status (Fig. 1 ). We investigated the ability of focused ultrasound to 1) induce apoptosis and to affect cell cycle in three closely related TK6 human lymphoblast cell lines; 2) analyze transcriptional response by expression profiling and protein analysis; and 3) generate reactive oxygen species.

View larger version (39K): [in this window] [in a new window]   Figure 1. Biological effects of ultrasound depend on physical dose parameters applied. While low power ultrasound is used in medical diagnostics without significant clinical adverse effects, high power focused ultrasound (HIFU) can thermally ablate tissue (e.g., in breast or prostate cancer). The dose range between might offer a therapeutic window for cancer therapy using induction of specific molecular signaling, including induction of localized apoptosis.

PRINCIPAL FINDINGS

1. Focused ultrasound inhibits proliferation and induces apoptosis in lymphoblasts depending on their p53 status
Focused ultrasound potently inhibited lymphoblasts proliferation with greater sensitivity for wild type p53 cells (Tk6, p53+) compared with p53– cells (Tk6 5E, abrogated p53). Similarly, G1 arrest was measured primarily in p53+ cells, while p53– cells showed G2 arrest.

Ultrasound-mediated apoptosis occurred more frequently in p53+ than in p53– cells (Fig. 2 A–D). We found a strong up-regulation of p53 after FUS in TK6 cells which was associated with an increase of p21/WAF protein. In contrast, antiapoptotic protein bcl-2 was markedly down-regulated after FUS, particularly in p53+ cells. These differing effects were observed at focal peak pressures between the range which is safely used in medical diagnostics (<0.1 MPa) and that used for high-energy focused ultrasound thermal ablation therapy (>10 MPa), (e.g., at a peak pressure of 1.5 MPa).

View larger version (54K): [in this window] [in a new window]   Figure 2. Apoptosis induced by focused ultrasound Tk6 (p53 wt, p53+) and Tk6 5E (abrogated p53 by 5E transduction, p53–) human lymphoblast cells were treated with focused ultrasound (1.5 MPa pressure amplitude for 60 s). Typical photographs of DAPI staining showing apoptotic cell death induced by ultrasound. DAPI staining was carried out at 24 h after ultrasound exposure. Nuclear condensations and fragmentation typical of apoptosis are marked by arrows. The apoptotic fraction was further quantified by PI staining and FACS analysis. A) Tk6 control; B) Tk6 5E treated with FUS; C) TK6 treated with FUS; D) % of apoptotic cells in FACS using PI staining.

2. DNA-chip analysis reveals a complex signaling cascade in ultrasound-induced apoptosis
To understand more about FUS-induced differentially expressed genes, we performed cDNA-chip analysis on ultrasound treated (1.5 MPa, 10 ms pulse length, 1 Hz pulse frequency, 60 s total sonification) vs. control lymphoblast samples. FUS treatment up-regulated cellular signaling cascades involved in apoptosis (p53, p21, Thy1 (CD 90) ) and oxidative stress response (ferritin), while antiapoptotic genes such as bcl-2, ribosomal proteins, and radical scavenger superoxide dismutase (SOD) were down-regulated. The result is a unique alignment between the direction of gene regulation and observed cellular and sonochemical effects.

3. Focused ultrasounds generates reactive oxygen species (ROS)
To determine whether sonochemically induced radical formation plays a causative role in ultrasound mediated apoptosis, free radicals were measured by electron spin resonance (ESR). FUS also induced ROS to an extent typically observed after ionizing radiation. Thus radical formation appears to be a potential mechanism by which ultrasound exerts its p53 dependent antiproliferative and proapoptotic effects. These results are consistent with our expression profiling data and in accordance with functional data acquired from TK6 cell lines after ionizing radiation.

CONCLUSIONS AND SIGNIFICANCE

Here we report that mechanical energy by focused ultrasound transmission triggers an intricate molecular signaling which ultimately leads to apoptosis of human lymphoblast cells. It is demonstrated that nonthermal ultrasound-cell interactions may lead to a specific transcriptional response (Fig. 3 ). For example, others have shown that apoptosis can be induced by Thy-1 through activation of proteases and a decrease in bcl-2. In the context of FUS-induced Thy-1 up-regulation in conjunction with detected loss of bcl-2 expression, we identify a possible mechanism for FUS mediated apoptosis in TK6 lymphoblasts. Our findings also suggest mechanisms by which FUS may regulate oxidative stress pathways: 1) direct ultrasonic mechanical effects and indirect sonochemical effects via cavitation-induced reactive oxygen species (ROS) may play a causative role in up-regulation of oxidative stress pathways; 2) FUS down-regulates superoxide dismutases (SOD) that protect cells from damage induced by free radicals; and 3) an important consequence of oxidant-induced p53 activation is that elevated p53 expression, itself, can result in increased oxidative stress within a positive feedback loop. We show that ultrasound effectively killed both p53+ and p53– cells, but p53+ cells are more sensitive to ultrasound-induced apoptosis. Free radical-mediated mechanisms are of therapeutic relevance in the treatment of cancer. They provide a biochemical basis for FUS to preferentially kill cancer cells similar to ionizing radiation or chemotherapeutic drugs that share the ability to produce free radicals.

View larger version (39K): [in this window] [in a new window]   Figure 3. Schematic diagram showing ultrasound (FUS) -induced molecular signaling, concentrating on activation of apoptotic machinery in lymphoblast cells at 1.5 MPa peak pressure.

Because focused ultrasound can be targeted in the human body while sparing intermediate tissue, FUS has potential to noninvasively induce localized apoptosis.

The relevance of our finding for basic research is the suggestion that focused ultrasound may be useful as a powerful instrument to discover how specific pathways are triggered by mechanical signals, such as in cancer, developmental biology, and morphogenesis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1601fje;

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This Article Full Text (PDF) All Versions of this Article: 18/12/1413 04-1601fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ABDOLLAHI, A. Articles by HUBER, P. E. Search for Related Content PubMed PubMed Citation Articles by ABDOLLAHI, A. Articles by HUBER, P. E.